Biol. Pharm. Bull. 32(5): 850-855 (2009)

850 Biol. Pharm. Bull. 32(5) 850—855 (2009) Vol. 32, No. 5

Inﬂuence of Memantine on Brain Monoaminergic Neurotransmission Parameters in Mice: Neurochemical and Behavioral Study

a b ,a c d,1) Hiroshi ONOGI, Seiichiro ISHIGAKI, Osamu NAKAGAWASAI,* Yumiko ARAI-KATO, Yuichiro ARAI, a e a a Hiromi WATANABE, Atsushi MIYAMOTO, Koichi TAN-NO, and Takeshi TADANO a Department of Pharmacology, Tohoku Pharmaceutical University; 4–4–1 Komatsushima, Aoba-ku, Sendai 981–8558, Japan: b Department of Neurology, School of Medicine, Showa University; Tokyo 142–8666, Japan: c Japan Pharmaceutical Information Center; Tokyo 150–0002, Japan: d Division of Pharmacology, School of Pharmaceutical Sciences, Ohu University; Koriyama 963–8611, Japan: and e Division of Pharmaceutical Health Care and Sciences, Sapporo Medical University; Sapporo 060–8543, Japan. Received October 16, 2008; accepted January 30, 2009; published online February 6, 2009

Memantine, a non-competitive antagonist of NMDA receptors, has recently been used in Alzheimer’s disease. The inﬂuences of memantine on behavioral changes, monoamine oxidase (MAO) activity and reuptake of both serotonin (5-HT) and dopamine in mice were examined in the present study. Memantine dose-dependently increased locomotor activity. This effect was inhibited by intraperitoneal (i.p.) administration of haloperidol. Furthermore, administration [intracerebroventricular (i.c.v.)] of memantine did not induce the head-twitch response (HTR). However, the 5-HT-induced HTR was potentiated by the combined administration of memantine.

The enhanced HTR was inhibited by i.p. administration of haloperidol or 5-HT2A antagonist ketanserin. Meman- tine at 1 mM inhibited both MAO-A and MAO-B activities in mouse forebrain homogenates to 37% and 64% of controls, respectively. Lineweaver–Burk plots analysis revealed competitive inhibition with both MAO-A and MAO-B. The inhibitions were also reversible. Memantine inhibited the reuptake of both 5-HT and dopamine into mouse forebrain synaptosomes. 5-HT and dopamine reuptakes were inhibited to 2% and 16% of controls, respectively, with 1 mM memantine. These ﬁndings suggest that the increased locomotor activity and enhanced 5- HT-induced HTR by memantine may result from the reuptake and turnover inhibitions of 5-HT and dopamine. Key words memantine; monoamine oxidase; serotonin; dopamine; locomotor activity; head-twitch response

Alzheimer’s disease (AD), a chronic neurodegenerative tral nervous system disorders such as AD and Parkinson’s disorder characterized clinically by progressive loss of cogni- disease by oxidative stress.17) The two types of MAO tive and behavioral function, is the most common form of de- (EC.1.4.3.4), MAO-A and MAO-B, exhibit differences in mentia in the world.2) The underlying pathogenesis of AD is sensitivity towards their selective inhibitors as well as in their caused by neuronal loss related to the abnormal extracellular amino acid sequences.18—21) MAOs play an important role in accumulation of amyloid-beta peptide in oligomeric form the inactivation of monoamine neurotransmitters in the cen- and in neuritic plaques, as well as the intraneuronal aggrega- tral and peripheral nervous systems. MAO-A metabolizes tion of hyperphosphorylated tau in the form of neurofibril- serotonin (5-HT, 5-hydroxytryptamine) and noradrenaline lary tangles.3) There are several other factors that contribute while MAO-B metabolizes benzylamine and b-phenylethyl- to neuronal degeneration, including inflammation, oxidative amine. Tyramine and dopamine (DA) are metabolized by stress, and glutamatergic dysfunction. both MAO-A and MAO-B. MAO-A is selectively and irre- The pathogenesis of AD is multifactorial. It has been pro- versibly inhibited by clorgyline, and selegiline is known to be posed that inappropriate activation of N-methyl-D-aspartate a selective and irreversible inhibitor of MAO-B. Conversely, (NMDA) receptors is responsible for part of the neuronal moclobemide is a selective but reversible MAO-A inhibitor toxicity and cognitive impairment observed in AD.4,5) Abnor- whereas lazabemide is a selective and reversible MAO-B in- malities in glutamatergic signaling associated with AD have hibitor.22,23) We reported earlier that amantadine enhanced the been linked to excitotoxicity caused by the excessive influx inhibition of reuptake of both 5-HT and DA, weakly inhibits of Ca through the NMDA receptor calcium channel during MAO activity in mice brain, and that 5-HT induced the head- sustained low-level stimulation of glutamatergic neurons.6) twitch response (HTR).24) The numbers of HTR induced by Cumulative evidence indicates that glutamate-related alter- 5-HT in mice is an effective method to evaluate serotonergic ation in AD can be corrected to some extent by NMDA re- effects of drugs in vivo.25) It has been generally accepted that ceptor antagonists such as memantine.7—9) numbers of HTR represent the level of 5-HT in the synapses. In humans, it is known that brain monoamine oxidase On the other hand, memantine (1-amino-3,5-dimethyl- (MAO)-B activity is higher in AD and normal aging whereas adamantan) is an analogous compound of amantadine. Me- it is lower in psychiatric disorders such as depression, cy- mantine is known to elicit DA action indirectly26,27) as well as cloidal psychosis, etc.10—12) In psychiatric patients, it is also direct stimulation of DA receptors that cause hyperactivity known that brain MAO-B activity is correlated with platelet and circling.28,29) However, there are no reports on the influ- MAO activity.10,13) The increase in brain MAO-B is most ences of memantine on MAO activity and reuptake of likely due to transcriptional elevation of MAO-B protein14) monoamines. Thus, we sought to determine the influence of and predominant in plaque-associated astrocytes in neu- memantine on brain MAO activity and reuptake of both 5- ropathologically verified AD brains.15,16) MAO-B inhibitors HT and DA, as well as its effect on some pharmacological such as selegiline and rasagiline are effective in some cen- behaviors including HTR and DA-related behavior locomo-

∗ To whom correspondence should be addressed. e-mail: [email protected] © 2009 Pharmaceutical Society of Japan May 2009 851 tor activity in mice. We report here the different effects ob- male mice weighing about 22 g were rapidly removed after tained with memantine compared to our previous results decapitation, and dissected on an ice-cold glass plate. The using amantadine. forebrains were weighed then homogenized in ice-cold 0.32 M sucrose into 10% homogenates, and stored at 20 °C MATERIALS AND METHODS until use (referred to as enzyme preparations). MAO activi- 14 ties were determined by radiometry with C-5-HT (0.1 mM) 14 All experiments were performed according to the Guide as the substrate for MAO-A and C-benzylamine (0.1 mM) for Care and Use of Laboratory Animals at Showa University as the substrate for MAO-B. The incubations (20 ml of sub- and Tohoku Pharmaceutical University. strate, 20 ml of enzyme preparation, 40 ml of water and 20 ml Behavioral Measurements Male ddY mice (4 weeks of 0.05 M phosphate buffer, pH 7.4) were carried out at 37 °C old), weighing about 22 g, were housed in plastic cages for 5 min. To determine any influence of memantine on MAO (251813 cm) with free access to food and water under activity, 20 ml of various concentrations of memantine as in- conditions of constant temperature (231 °C), humidity hibitor were substituted for 20 ml water in the incubation (555%), and light/dark cycle (9—21/21—9 h). All behav- medium. The incubations were terminated by the addition of ioral studies were performed between 10:00 a.m. and 5:00 20 ml of 3 N HCl, followed by organic solvents (toluene : ethyl p.m. Each mouse was used only once for each experiment. acetate1 : 1, saturated with water). The metabolites deami- The technique employed here for intracerebroventricular nated by MAO were extracted into organic solvents. The ra- (i.c.v.) administration was the same as that originally de- dioactivity in each organic layer was measured in a liquid scribed by Brittain and Handley.30) ICV administration of scintillation counter and the MAO activities calculated from memantine and/or 5-HT in mice was administered by a dis- the radioactivities and expressed as nmol/mg protein/min, as posal 27-G needle which was inserted into the lateral ventri- previously described.31) Protein contents were assayed by the cle. Memantine and/or 5-HT were administered in a total method of Markwell et al.32) with bovine serum albumin used volume of 5 ml at a constant rate of 1 ml/2 s attached to a as standard. 50 ml Hamilton microsyringe. The number of head twitches 5-HT and Dopamine Uptake Assays The estimation of (rapid movements of the head with little or no involvement of 5-HT and DA uptake into synaptosomes was carried out es- the trunk) was counted for 30 min after the i.c.v. administra- sentially by a previously described method.25) Briefly, the re- tion of memantine and/or 5-HT. The effects of memantine on moved mouse forebrain was immediately homogenized in locomotor activity were evaluated by the multichannel activ- 0.32 M sucrose and the homogenate was preincubated for ity-counting system Supermex (Muromachi Kikai, Tokyo, 5 min at 37 °C in Krebs–Hensleit’s bicarbonate solution, con- 3 8 Japan). This instrument can monitor even minute movements taining 110 to 110 M memantine, or without meman- in all three planes of motion (sagittal, coronal and horizontal) tine as control. Subsequently, 14C-5-HT or 3H-DA was added as one movement, since its infrared sensor with multiple to the incubation medium to achieve a concentration of Fresnel lenses that can be moved close enough to the cage 100 nM, and the reactions were terminated after 2 min by the can capture multidirectional locomotor alterations in a single addition of cocaine (50 m M). Then, the incubation medium mouse. The Supermex instrument was connected to a behav- was filtrated on a glass filter using a cell-harvester and the ra- ioural analyzing system (CompACT AMS) (Muromachi dioactivities collected on the glass filter were measured in a Kikai), which can interpret each movement as one count. liquid scintillation counter. The uptake rates of 5-HT and DA Therefore, vertical movements such as jumping, as well as into synaptosomes were calculated from the radioactivities horizontal movements such as walking and running, could be and expressed as pmol/mg protein/min. counted. On the day of the experiment the mice were individ- Statistical Analysis Results of experiments are ex- ually placed in activity cages and were allowed 15 min to pressed as meansS.E.M. Statistical significance of differ- adapt to the new environment. The number of activity counts ences between two means were estimated using the Student’s was recorded for the total locomotor activity over a 90 min two-tailed t-test. Comparisons were made by one-way or two- period. way analysis of variance (ANOVA) combined with Scheffe’s Reagents Memantine and 5-HT hydrochloride were pur- test. p0.05 represented a significant difference. chased from Sigma-Aldrich, Inc., St. Louis, MO, U.S.A., and they were dissolved in ringer solution. Haloperidol was RESULTS bought from RBI, Natick, Mass., U.S.A. and it was dissolved in 0.5% Tween 80. Ketanserin (RBI) was dissolved in saline. Effects of Memantine on Locomotor Activity and Haloperidol or ketanserin was injected intraperitoneally (i.p.) Head-Twitch Response When injected i.c.v. at the doses in a volume of 10 ml/kg of the mice body weight. Haloperi- of 0.625, 2.5 or 10 mg/mouse, memantine increased the loco- dol (0.1 mg/kg) or ketanserin (1 mg/kg) was injected i.p. motor activity in a dose-dependent manner (Fig. 1). This in- 30 min before memantine or 5-HT plus memantine adminis- crease was blocked by the DA antagonist haloperidol (Fig. tration. 5-[2-14C]-Hydroxytryptamine binoxalate (1.70 GBq/ 2). The in vivo pharmacological behavior of memantine was mmol) was purchased from PerkinElmer Life Sciences Inc., studied in mice. Similarly to amantadine, administration Boston, MA, U.S.A. and [7-14C]-benzylamine hydrochloride (i.c.v.) of memantine at high doses induced behaviors such as (2.11 GBq/mmol) and [7,8-3H] dopamine (1.52 TBq/mmol) head-twitch response (HTR), penis licking, and irritability were purchased from Amersham Pharmacia Biotech, Lon- (data not shown). Among these, we focused on HTR to eval- don, U.K. All chemicals used in the present study were of the uate serotonergic or dopaminergic effects of memantine. As highest grade commercially available. shown in Fig. 3, the number of HTR were counted following Assay of MAO Activity Forebrains of 4-week-old ddY i.c.v. administration to mice of memantine (40 mg/mouse), 5- 852 Vol. 32, No. 5

Fig. 1. Effect of Memantine on Locomotor Activity in Mice Fig. 4. Inhibition of MAO-A and MAO-B Activities with Memantine in Each column shows the total number of locomotor activities during 90 min. Error Mouse Forebrain Homogenates bars show the S.E.M. (n10). ∗p0.05 vs. Ringer-treated mice. Ordinate shows the inhibition as the percentage of control MAO activity. Abscissa shows the concentration of memantine in negative logarithm. Error bars show the S.E.M. (n4).

Fig. 2. Effect of Haloperidol on Memantine-Induced Hyperlocomotor Ac- tivity in Mice Fig. 5. Inhibition of MAO-A Activity in Mouse Forebrains with Meman- tine Each column shows the total number of locomotor activities during 90 min. Error bars show the S.E.M. (n10). ∗∗p0.01 vs. Saline/Ringer-treated mice. ## p0.01 vs. (A) Lineweaver–Burk plots. Ordinate shows 1/velocity of MAO-A activity. Abscissa Saline/Memantine-treated mice. shows 1/5-HT concentration. (B) Dixon plots for Ki. Ordinate shows Vmax/Km ratio. Ab- scissa shows memantine concentration. Ki value is 0.09 mM. Error bars show the S.E.M. (n4).

HT (20 mg/mouse), or memantine plus 5-HT. The HTR was monitored during 30 min post-injections. As illustrated in Fig. 3, virtually no HTR was observed when memantine was administered alone whereas a small effect, which peaked around 15 to 20 min, was observed following 5-HT injection and disappeared after 25 min. However, when memantine and 5-HT were co-administered, the HTR was enhanced and peaked between 10 and 15 min. This HTR still occurred at 30 min post-injection, albeit was much smaller. 5-HT plus

memantine-induced HTR was inhibited by 5-HT2A antagonist ketanserin or haloperidol. Effects of Memantine on Mouse Forebrain MAO Activ- ity, 5-HT and Dopamine Uptakes Memantine-induced inhibition of MAO activity was ﬁrst determined. The inﬂuence of memantine at concentrations ranging from 1108 to 3 110 M on MAO-A and MAO-B activities in mice forebrain homogenates are shown in Fig. 4. Memantine inhibited both MAO-A and MAO-B activities. We show that in the presence of 1 mM memantine, MAO-A and MAO-B activities decreased to 37% and 64% of control activity, respectively. The Lineweaver–Burk plots for the inhibition of MAO-A and MAO-B in mice forebrain by memantine were then determined. As shown in Figs. 5 and 6, the patterns of meman- Fig. 3. Effect of Memantine on 5-HT-Induced Head-Twitch Response tine-induced inhibition of MAO-A and MAO-B with 5-HT (HTR) in Mice and benzylamine as substrates, respectively, showed competi- (A) Each point represents the mean number of HTR for a 5 min period. (B) Each col- tive inhibition. The Ki values of inhibition for MAO-A and umn shows the total number of HTR during 30 min. Error bars show the S.E.M. (n10). ∗p0.05 vs. 5-HT-treated mice. # p0.05 and ## p0.01 vs. 5-HTmeman- MAO-B by memantine were calculated by re-plotting from tine-treated mice. the Lineweaver–Burk plots; the Ki value was 0.09 mM for May 2009 853

the striatum and nucleus accumbens.26,27,39) It is well known that DA receptors in the nucleus accumbens play an important role in locomotor activity. Indeed, the accumbal core region contributes to the generation of motor activity, whereas the accumbal shell region is involved in emotional and moti- vational mechanisms.40) The present study also showed that administration of the DA antagonist haloperidol inhibited the memantine-induced hyperlocomotion. Recently, it has been reported that the memantine has a DA agonist action at rat 41) striatal D2 receptor. These results thus suggest that the nucleus accumbens core D2 receptors could be involved in the hyperlocomotion. Fig. 6. Inhibition of MAO-B in Mouse Forebrains with Memantine The memantine and amantadine compounds are based on (A) Lineweaver–Burk plots. Ordinate shows as 1/velocity of MAO-B activity. Ab- the chemical structure of aminoadamantane derivatives. scissa shows 1/benzylamine concentration. (B) Dixon plots for Ki. Ordinate shows Vmax/Km ratio. Abscissa shows memantine concentration. Ki value is 0.74 mM. Error Amantadine is also known to increase DA release in the bars show the S.E.M. (n4). striatum by antagonism at NMDA receptors.42) We reported earlier that amantadine inhibited both MAO-A (Ki 1.85 mM) and MAO-B (Ki 1.42 mM) activities in the mouse brain, and 5-HT and DA uptakes inhibited to 2% and 19% of control 24) with 1 mM amantadine. Here we showed that memantine competitively inhibited both MAO-A (Ki 0.09 mM) and MAO-B (Ki 0.74 mM), and that memantine also inhibited the reuptakes of both 5-HT and DA into synaptosomes. Thus, MAO inhibitions of memantine were stronger than those of amantadine previously reported using identical experimental conditions. The increase of monoamine contents in the brain, especially 5-HT, is known to cause some abnormal behaviours in mice, such as HTR, head-weaving, etc.43) It is also Fig. 7. Inhibition of 5-HT and Dopamine (DA) Uptake into Synaptosomes known that HTR is induced by stimulation of 5-HT recep- in Mouse Forebrain Homogenates with Memantine 2A tors in the prefrontal cortex.44) Moreover, NMDA receptor Ordinate shows the inhibition as the percentage of control uptake of 5-HT and DA. Abscissa shows the concentration of memantine in negative logarithm. Error bars show antagonists were reported to markedly enhance 5-HT-induced the S.E.M. (n6). selective serotonergic behaviours, such as HTR, whereas NMDA itself inhibits 5-HT-induced HTR in mice.43) In the

MAO-A and 0.74 mM for MAO-B. For reference, the Ki val- present study, HTR was observed when 5-HT plus meman- ues of MAO-A inhibitor clorgyline and MAO-B inhibitor de- tine were co-administered to mice but not when 5-HT or me- 3 prenyl were 0.0510 mM for MAO-A and 0.06 mM for mantine were administered alone, as shown in Fig. 3. In addi- 3 MAO-B, and 0.04 mM for MAO-A and 110 mM for tion, the HTR was inhibited by ketanserin and haloperidol MAO-B, respectively.33) (Fig. 3). Previous studies have demonstrated that haloperidol

Next, we examined the influence of memantine on 5-HT significantly attenuates 5-HT2A/2C agonist DOI-induced HTR and DA uptakes into mouse forebrain synaptosomes. As in mice.45,46) That haloperidol may act as an antagonist at 5- 45,47) shown in Fig. 7, memantine inhibited 5-HT uptake (IC50 HT2 receptors has been reported. Thus, the 5-HT release 0.1 mM) into synaptosomes to 50% and 2% of controls at following NMDA receptor inhibition by memantine appears concentrations of 0.1 mM and 1 mM, respectively. Memantine to be insufficient to evoke HTR. In agreement, the level of 5- also inhibited DA uptake (IC50 0.2 mM) to 60% and 16% of HT in rat prefrontal cortex was reported not to change during 27) controls at concentrations of 0.1 mM and 1 mM, respectively 60 min after systemic administration of memantine. On the

(Fig. 7). For reference,the IC50 values of selective 5-HT up- other hand, another report stated that HTR is induced by en- take inhibitor citalopram34) and DA uptake inhibitor bupro- hancement of dopaminergic function.48) Memantine has been 35) 3 27) pion were 0.016 mM for 5-HT uptake and 1.310 mM for shown to increase DA levels in the prefrontal cortex. Fur- DA uptake, respectively. ther, as shown in Fig. 7, memantine inhibited both the reuptake and turnover of 5-HT and DA which, together with in- DISCUSSION creased DA release, might potentiate the HTR induced by 5- HT plus memantine. The increased locomotor activity fol- In the present study, i.c.v. administration of memantine to lowing memantine also might involve these inhibitions of mice increased locomotor activity in comparison with vehi- DA reuptake and turnover. Based on these findings, it is hy- cle. Several other non-competitive NMDA receptor antago- pothesized that the abnormal behaviours induced by meman- nists, such as MK-801 and phencyclidine, were previously tine are the result of an increment in DA and 5-HT contents shown to increase locomotion in various experimental para- by the additive synergistic effects of NMDA receptor antago- 36,37) digms in contrast to competitive antagonists, such as D- nism, D2 receptor agonism, MAO inhibition and the inhibi- 2-amino-5-phosphonovaleric acid, at least at moderate tion of reuptake of 5-HT and DA. doses.38) In addition, non-competitive NMDA receptor antag- Memantine is often administered to patients with onists increased DA biosynthesis, its turnover and release in Alzheimer’s disease; the doses administered are 20mg/d de- 854 Vol. 32, No. 5 pending on the case.49) It is known that the plasma concentra- M., Booker L., Oremus M., Ann. Intern. Med., 148, 379—397 (2008). 10) Oreland L., Arai Y., Stenstrom A., Fowler C. J., Mod. Probl. Pharma- tion of memantine reaches over 150 ng/ml as Cmax following the administration of 20 mg memantine to patients.50) This copsychiatry, 19, 246—254 (1983). 11) Kennedy B. P., Ziegler M. G., Alford M., Hansen L. A., Thal L. J., means that the plasma concentration of memantine might be Masliah E., J. Neural Transm., 110, 789—801 (2003). above 0.7 m M. Although memantine rapidly crosses the 12) Youdim M. B., Edmondson D., Tipton K. F., Nat. Rev. Neurosci., 7, blood–brain barrier,51) it is thought that this concentration 295—309 (2006). might be too low to inhibit MAO activity. However, it is 13) Oreland L., Neurotoxicology, 25, 79—89 (2004). known that other monoamines such as 5-HT, DA and nor- 14) Nakamura S., Kawamata T., Akiguchi I., Kameyama M., Nakamura N., Kimura H., Acta Neuropathol. (Berl.), 80, 419—425 (1990). adrenaline can be actively taken up into synaptosomes by the 15) Jossan S. S., Gillberg P. G., Gottfries C. G., Karlsson I., Oreland L., monoamine transporter where they are concentrated about Neuroscience, 45, 1—12 (1991). 200-fold.52) In support of this report, we hypothesize that, in 16) Saura J., Luque J. M., Cesura A. M., Da Prada M., Chan-Palay V., the brain, memantine may be concentrated in a similar man- Huber G., Loffler J., Richards J. G., Neuroscience, 62, 15—30 (1994). 17) Youdim M. B., Bakhle Y. S., Br. J. Pharmacol., 147, S287—S296 ner and thereby reach effective concentrations. Thus, it is (2006). plausible that peripheral administration of memantine may 18) Johnston J. P., Biochem. Pharmacol., 17, 1285—1297 (1968). inhibit MAO activity in the human brain, as well as having 19) Knoll J., Magyar K., Adv. Biochem. Psychopharmacol., 5, 393—408 effects through NMDA receptor antagonism. (1972). It was reported that memantine inhibits in vitro DA reup- 20) Tsugeno Y., Ito A., J. Biol. Chem., 272, 14033—14036 (1997). 29) 21) Shih J. C., Chen K., Ridd M. J., Annu. Rev. Neurosci., 22, 197—217 take into synaptosomes of mouse brain (IC50 0.21 mM). (1999). This result has shown similar our data (IC50 0.2 mM). This 22) Fulton B., Benfield P., Drugs, 52, 450—474 (1996). inhibition might be important as an anti-Alzheimer agent in 23) Berlin I., Aubin H. J., Pedarriosse A. M., Rames A., Lancrenon S., La- order to increase the DA contents in the synaptic cleft in the grue G., Addiction, 97, 1347—1354 (2002). prefrontal cortex and striatum.53,54) In Alzheimer’s disease, it 24) Ishigaki S., Arai Y., Nakagawasai O., Arai Y., Tadano T., Yasuhara H., Biogenic Amines, 20, 71—80 (2006). is known that DA content is lower and DA release is also 25) Nakagawasai O., Tadano T., Arai Y., Hozumi S., Oba A., Tan-No K., 55) lower in the brain. The loss of D2 receptors in the striatum Yasuhara H., Kisara K., Oreland L., Neuroscience, 117, 1017—1023 of Alzheimer’s disease cases was also observed,56) and this (2003). loss was associated with impaired cognitive function and 26) Peeters M., Maloteaux J. M., Hermans E., Neurosci. Lett., 343, 205— motor function.57,58) According to our results on the reuptake 209 (2003). 27) Shearman E., Rossi S., Szasz B., Juranyi Z., Fallon S., Pomara N., Ser- inhibition of DA by memantine, it seems that its concentra- shen H., Lajtha A., Brain Res. Bull., 69, 204—213 (2006). tion is sufficient to inhibit the DA reuptake when considering 28) Costall B., Naylor R. J., Psychopharmacologia, 43, 53—61 (1975). 29) Dunn J. P., Henkel J. G., Gianutsos G., J. Pharm. Pharmacol., 38, Cmax in memantine administered to humans. From this, it is suggested that DA reuptake inhibition is additionally effec- 353—356 (1986). 30) Brittain R. T., Handley S. L., J. Physiol., 192, 805—813 (1967). tive against Alzheimer’s disease. Thus, although memantine 31) Arai Y., Biogenic Amines, 8, 413—420 (1992). weakly inhibits DA reuptake and MAO activity in the present 32) Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E., Anal. study, their synergistic effects of memantine might be useful Biochem., 87, 206—210 (1978). against impaired cognitive and motor functions in Alz- 33) Tipton K. F., Fowler Ch. J., “Neuropharmacology ’85,” ed. by Kelemen heimer’s disease. K., Magyar K., Vizi E. S., Akademiai Kiado, Budapest, 1985, pp. 3— 8. In conclusion, these results suggest that the increased lo- 34) Yanez M., Fraiz N., Cano E., Orallo F., Eur. J. Pharmacol., 542, 54— comotor activity and HTR induced by memantine may be 60 (2006). due to reuptake and turnover inhibitions of 5-HT and DA. 35) Sitges M., Reyes A., Chiu L. M., J. Neurosci. Res., 39, 11—22 (1994). 36) Danysz W., Essmann U., Bresink I., Wilke R., Pharmacol. Biochem. Acknowledgements We are grateful to Dr. Marc Danik Behav., 48, 111—118 (1994). 37) Fredriksson A., Archer T., Amino Acids, 23, 111—132 (2002). of the McGill University (Montreal, QC, Canada) for con- 38) Loscher W., Annies R., Honack D., Eur. J. Pharmacol., 242, 263—274 structive comments on the manuscript. This study was sup- (1993). ported in part by Grants-in-Aid for High Technology Re- 39) Ault D. T., Werling L. L., Eur. J. Pharmacol., 386, 145—153 (1999). search Program from the Ministry of Education, Culture, 40) Weiner I., Gal G., Rawlins J. N., Feldon J., Behav. Brain Res., 81, Sports, Science and Technology of Japan. 123—133 (1996). 41) Seeman P., Caruso C., Lasaga M., Synapse, 62, 149—153 (2008). 42) Wesemann W., Ekenna O., Arzneim.-Forsch., 32, 1241—1243 (1982). REFERENCES 43) Kim H. S., Park I. S., Park W. K., Life Sci., 63, 2305—2311 (1998). 44) Nakagawasai O., Arai Y., Satoh S.-E., Satoh N., Neda M., Hozumi M., 1) Tokyo Ariake University of Medical and Health Sciences; Tokyo Oka R., Hiraga H., Tadano T., Neurotoxicology, 25, 223—232 (2004). 135–0063, Japan. 45) Dursun S. M., Handley S. L., Psychopharmacology, 128, 198—205 2) Sadowski M., Wisniewski T., Curr. Pharmaceut. Des., 13, 1943— (1996). 1954 (2007). 46) Wettstein J. G., Host M., Hitchcock J. M., Prog. Neuropsychopharma- 3) Blennow K., De Leon M. J., Zetterberg H., Lancet, 368, 387—403 col. Biol. Psychiatry, 23, 533—544 (1999). (2006). 47) Yamada J., Sugimoto Y., Horisaka K., Biol. Pharm. Bull., 11, 1580— 4) Hynd M. R., Scott H. L., Dodd P. R., Neurochem. Int., 45, 583—595 1583 (1995). (2004). 48) Nichols D. E., Pharmacol. Ther., 101, 131—181 (2004). 5) Chohan M. O., Iqbal K., J. Alzheimers Dis., 10, 81—87 (2006). 49) Witt A., Macdonald N., Kirkpatrick P., Nat. Rev. Drug Discov., 3, 6) Parsons C. G., Danysz W., Quack G., Neuropharmacology, 38, 735— 109—110 (2004). 767 (1999). 50) Almeida A. A., Campos D. R., Bernasconi G., Calafatti S., Barros F. 7) Banerjee P. K., Lahiri D. K., Tanila H., Miguel-Hidalgo J. J., Iqbal K., A., Eberlin M. N., Meurer E. C., Paris E. G., Pedrazzoli J., J. Chro- Biol. Psychiatry, 57, 173S—174S (2005). matogr. B, 848, 311—316 (2007). 8) Robinson D. M., Keating G. M., Drugs, 66, 1515—1534 (2006). 51) Ametamey S. M., Bruehlmeier M., Kneifel S., Kokic M., Honer M., 9) Raina P., Santaguida P., Ismaila A., Patterson C., Cowan D., Levine Arigoni M., Buck A., Burger C., Samnick S., Quack G., Schubiger P. May 2009 855

A., Nucl. Med. Biol., 29, 227—231 (2002). 56) Piggott M. A., Marshall E. F., Thomas N., Lloyd S., Court J. A., Jaros 52) Holz R. W., Coyle J. T., Mol. Pharmacol., 10, 746—758 (1974). E., Burn D., Johnson M., Perry R. H., McKeith I. G., Ballard C., Perry 53) Spanagel R., Eilbacher B., Wilke R., Eur. J. Pharmacol., 262, 21—26 E. K., Brain, 122, 1449—1468 (1999). (1994). 57) Volkow N. D., Gur R. C., Wang G. J., Fowler J. S., Moberg P. J., Ding 54) Hesselink M. B., De Boer A. G., Breimer D. D., Danysz W., J. Neural Y. S., Hitzemann R., Smith G., Logan J., Am. J. Psychiatry, 155, 344— Transm., 106, 803—818 (1999). 349 (1988). 55) McNeill T. H., Koek L. L., Haycock J. W., Peptides, 5, 263—268 58) Kaasinen V., Nagren K., Hietala J., Oikonen V., Vilkman H., Farde L., (1984). Halldin C., Rinne J. O., Neurology, 54, 1482—1487 (2000).