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Ginkgo biloba extract improves spatial memory in rats mainly but not exclusively via a histaminergic mechanism
BR A I N R ES E A RC H 1 1 2 9 ( 2 00 7 ) 1 6 1 –1 65
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Research Report
Ginkgo biloba extract improves spatial memory in rats mainly but not exclusively via a histaminergic mechanism Yasuko Yamamoto, Yutaka Adachi, Yoko Fujii, Chiaki Kamei ⁎ Department of Medicinal Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
In order to clarify the mechanism of Ginkgo biloba extract (GBE) on learning and memory, we
Accepted 26 August 2006
studied the effect of GBE on spatial memory deficits induced by diphenhydramine,
Available online 6 December 2006
pyrilamine and scopolamine using the eight-arm radial maze performance of rats, in comparison with donepezil. Total error (TE), reference memory error (RME) and working
Keywords:
memory error (WME) were used as indices of spatial memory deficits. Both GBE and
Ginkgo biloba extract
donepezil caused a potent antagonistic effect on the increase in TE, RME and WME induced
Scopolamine
by diphenhydramine. GBE and donepezil also antagonized scopolamine-induced spatial
H1-antagonist
memory deficits. Although the antagonistic effect of GBE on pyrilamine-induced spatial
Radial maze
memory deficits was weak, a significant difference was observed with TE and WME.
Reference memory
However, donepezil caused no antagonistic effect on pyrilamine-induced memory deficits.
Working memory
From these findings, we concluded that the effects of GBE are mainly contributable to cholinergic activity and perhaps partly due to a histaminergic mechanism. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Ginkgo biloba extract (GBE) is an ethanol extract obtained from the leaves of the G. biloba. In contrast, EGb-761 is an acetone extract obtained from the leaves of G. biloba, and contains 24% flavonol glycosides, 6% terpene lactone and limited amounts of other substances, including proanthocyanidins and organic acid (DeFeudis and Drieu, 2000; Maclennan et al., 2002). The preparation is an ethanolic extract, to contain the same components as EGb-761 (Yamamoto et al., 2005). GBE has been reported to have several pharmacological actions. GBE can inhibit the neuronal apoptosis that is induced in cultured chick embryonic neuron and in mixed cultures of neuron and astrocytes from neonatal rat hippocampus either by serum deprivation or by staurosporine, both of which lead to increased generation of reactive oxygen species (Ahlemeyer
et al., 1999). Shen et al. (1998) showed that GBE had cardiovascular protective effects by means of adjusting the level of NO and inhibiting oxygen free radicals induced lipid peroxidation in myocardial ischemia–reperfusion injury in vivo. Rapin et al. (1994) reported that GBE had anti-stress effect in rats subjected to a stressful situation. In Europe, GBE is widely used as a herbal medicine or dietary supplement, and it has been also demonstrated that GBE is effective in the facilitation of learning and the recollection of memory in rodents (Winter, 1998). It is well recognized that the brain cholinergic system is closely related to learning and memory. We have reported that histamine also plays an important role in learning and memory via H1 receptors (Kamei et al., 1990, 1993; Kamei and Tasaka, 1991, 1993). In addition, the brain concentration of histamine was demonstrated to decrease in Alzheimer’s patients. For instance, Schneider et al. (1997)
⁎ Corresponding author. Fax: +81 86 2517939. E-mail address: [email protected] (C. Kamei). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.08.102
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showed that brain histamine contents, as well as the histidine decarboxylase activity, were decreased in Alzheimer’s patients. Morisset et al. (1996) also reported that tacrine, an anticholinesterase agent and cognition enhancer, inhibited histamine N-methyltransferase activity. Another attempt revealed that the binding potential of the histamine H1 receptor showed a significant decrease in the frontal and temporal areas of the Alzheimer’s disease affected brain compared to elderly, normal subjects (Higuchi et al., 2000). From these findings, it is clear that the brain cholinergic and histaminergic systems are related to learning and memory. There are some reports about the interaction between brain cholinergic neurons and GBE in learning and memory. For instance, Chopin and Briley (1992) have reported that GBE improved scopolamine-induced memory deficits of passive avoidance performance. However, little is known about the interactions between GBE and histaminergic systems in spatial memory. Therefore, the present study was performed to clarify the effect of GBE on spatial memory deficits induced by H1 antagonists, diphenhydramine and pyrilamine and scopolamine (muscarinic receptor antagonist) using eightarm radial maze performance in comparison with donepezil, inhibitor of acetylcholinesterase, cognitive enhancers.
2.
Results
2.1. Effects of diphenhydramine, pyrilamine and scopolamine on radial maze performance Diphenhydramine at a dose of 10 mg/kg caused no significant effect on total error (TE), reference memory error (RME) or working memory error (WME). On the other hand, at a dose of 20 mg/kg, this drug significantly increased TE, RME and WME. Although pyrilamine at a dose of 20 mg/kg caused no significant effect on TE, RME or WME, at 35 mg/kg the drug significantly increased in TE, RME and WME. Scopolamine at a dose of 0.2 mg/kg caused no significant effect on TE, RME or WME. However, at a dose of 0.5 mg/kg, it caused a significant increase in TE, RME and WME. Therefore, 20 mg/kg in diphenhydramine, 35 mg/kg pyrilamine and 0.5 mg/kg scopolamine were used to study the effects of GBE and donepezil (Table 1).
Table 1 – Effect of diphenhydramine, pyrilamine and scopolamine on spatial memory in the radial performance Drugs Saline Diphenhydramine Pyrilamine Scopolamine
Dose (mg/kg, i.p.)
TE
RME
WME
– 10 20 20 35 0.2 0.5
0.6 ± 0.2 1.1 ± 0.2 3.3 ± 0.3** 1.3 ± 0.3 2.8 ± 0.2** 0.4 ± 0.2 3.1 ± 0.4**
0.5 ± 0.2 0.9 ± 0.2 1.7 ± 0.2* 0.9 ± 0.2 1.7 ± 0.3* 0.1 ± 0.1 1.8 ± 0.3*
0.1 ± 0.1 0.2 ± 0.1 1.1 ± 0.3* 0.4 ± 0.2 1.1 ± 0.3* 0.2 ± 0.1 1.3 ± 0.4*
Drugs were administrated intraperitoneally. Each value represents the mean ± SEM of 16 rats. TE: Total error, RME: Reference memory error, WME: Working memory error. *, **: Significantly different from the control group with p < 0.05 and p < 0.01, respectively.
Fig. 1 – Effects of Ginkgo biloba extract and donepezil on spatial memory deficits induced by diphenhydramine in rats. Diphenhydramine (20 mg/kg, i.p.) was administrated before and G. biloba extract and donepezil were administrated 1 h before the test trial. Each value represents the mean ± SEM of 18–19 rats. TE: Total error, RME: Reference memory error, WME: Working memory error. *,**: Significantly different from control group with p < 0.05 and p < 0.01, respectively.
2.2. Effects of GBE and donepezil on diphenhydramine-induced memory deficits GBE at doses of 30 and 50 mg/kg significantly inhibited the diphenhydramine-induced increase of TE and WME. At 50 mg/kg, this drug also inhibited the diphenhydramineinduced increase in RME. Donepezil at 0.5 mg/kg significantly inhibited the diphenhydramine-induced increase in TE and RME. At 1 mg/kg, it also inhibited the diphenhydramineinduced increase in WME (Fig. 1).
2.3. Effects of GBE and donepezil on pyrilamine-induced memory deficits The effects of GBE and donepezil on pyrilamine-induced spatial memory deficits are shown in Fig. 2. GBE at doses of 20 and 30 mg/kg caused no significant effect on pyrilamineinduced memory deficits, but at a dose of 50 mg/kg it significantly inhibited the pyrilamine-induced increase in TE and WME. On the other hand, donepezil caused no inhibitory effect on the pyrilamine-induced memory deficits, even at a dose of 1 mg/kg.
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163
Although both H1-antagonists caused a significant increase in TE, RME and WME, the effect of diphenhydramine was more potent than that of pyrilamine. Diphenhydramine is well known to have a potent anticholinergic activity, while pyrilamine is a pure histamine H1 antagonist and has a weak anticholinergic effect. That is, diphenhydramine showed moderate affinity for the muscarinic receptor (Ki = 280 ± 50 nM), and H1 receptor antagonism in the Ki value of 14 ± 2 nM. Pyrilamine exhibited a weak antagonism of the muscarinic receptor (Ki = 30,000 ± 2000 nM), and a potent antagonism of H1 receptor (Ki = 0.24 ± 0.03 nM) (Kubo et al., 1987). It is well recognized that anticholinergic properties are responsible for memory deficits in cognition (Watts et al., 1981). As shown in the present study, we confirmed that scopolamine, a representative anticholinergic drug, caused potent spatial memory deficits. This is the reason why diphenhydramine was more effective than pyrilamine in causing spatial memory deficits on radial maze performance. GBE inhibited the diphenhydramine-induced an increase in TE, RME and WME. Almost the same findings were obtained with donepezil. Both drugs also inhibited the scopolamine-
Fig. 2 – Effects of Ginkgo biloba extract and donepezil on spatial memory deficits induced by pyrilamine in rats. Pyrilamine (35 mg/kg, i.p.) was administrated 30 min before and G. biloba extract and donepezil were administrated 1 h before the test trial. Each value represents the mean ± SEM of 10–12 rats. TE: Total error, RME: Reference memory error, WME: Working memory error. *,**: Significantly different from the control group with p < 0.05 and p < 0.01, respectively.
2.4. Effects of GBE and donepezil on scopolamine-induced memory deficits Fig. 3 shows the effects of GBE and donepezil on the spatial memory deficits induced by scopolamine (0.5 mg/kg, i.p.). GBE at doses of 30 and 50 mg/kg significantly inhibited the scopolamine-induced increase in TE and WME. A significant effect was also obtained with the scopolamine-induced increase in RME at a dose of 50 mg/kg. Almost the same results were obtained with donepezil; 0.5 mg/kg of donepezil inhibited the scopolamine-induced increase in TE and WME. The increase in RME induced by scopolamine was inhibited by 1 mg/kg of donepezil.
3.
Discussion
It was found that diphenhydramine, pyrilamine and scopolamine caused a significant increase of TE, RME and WME at doses of 20, 35 and 0.5 mg/kg, respectively. This result confirmed our previous findings that these drugs increased TE, RME and WME at the same dose levels (Taga et al., 2001).
Fig. 3 – Effects of Ginkgo biloba extract and donepezil on spatial memory deficits induced by scopolamine in rats. Scopolamine (0.5 mg/kg, i.p.) was administrated 30 min before and G. biloba extract and donepezil were administrated 1 h before the test trial. Each value represents the mean ± SEM of 12–16 rats. TE: Total error, RME: Reference memory error, WME: Working memory error. *,**: Significantly different from the control group with p < 0.05 and p < 0.01, respectively.
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induced memory deficits. There are many reports that both GBE and donepezil showed an antagonistic effect on scopolamine-induced memory deficits and a cholinergic effect. For instance, Chopin and Briley (1992) reported that GBE improved the scopolamine-induced memory deficits of passive avoidance performance. In addition, Das et al. (2002) showed that GBE caused a dose-dependent inhibition of acetylcholinesterase activity. As for donepezil, Ogura et al. (2000) reported that donepezil alleviates learning deficits in a hypocholinergic model in rats. Misane and Ogren (2003); Staay van der and Bouger (2005) also demonstrated that donepezil attenuates the impairment of passive avoidance in the spatial cone field orientation task in rats. From these findings, it seems likely that the antagonistic effect of GBE on the diphenhydramineinduced inhibition of spatial memory is mainly attributable to its cholinergic activity. The same idea was also applicable to donepezil. As shown in the present data, GBE seems to counteract WME in low doses and only affects RME at high doses in diphenhydramine-induced memory deficits. Whereas no such pattern was observed for donepezil. It is generally accepted that the acquisition of working memory was faster than that of reference memory (Klein et al., 2004). That is, the number of training to require the working memory is less than that of reference memory. From these findings, it seems likely that working memory is more sensitive than reference memory to some drugs. This is the reason why GBE showed more effective in WME than RME. On the other hand, donepezil is also effective in RME. Donepezil is a powerful drug in antagonizing memory deficit induced by diphenhydramine. Therefore, it was thought at the time, that donepezil was effective not only in WME but also in RME. Although GBE caused no antagonism on the pyrilamineinduced increase in RME at a dose of 50 mg/kg, TE and WME were antagonized significantly at a dose of 50 mg/kg. As described previously, pyrilamine only has a weak anticholinergic activity and it is a pure H1 antagonist. However, GBE caused an inhibitory effect on the pyrilamine-induced memory deficits. Therefore, it is reasonable to presume that the improvement of spatial memory deficits induced by GBE may be partly due to a histaminergic mechanism. As for donepezil, it caused no antagonistic effect on pyrilamine-induced memory deficits, even at a high dose. Therefore, it seems likely that there is no interaction between the ameliorating effect of donepezil on memory deficits and a histaminergic mechanism. In fact, there are no reports that donepezil has a histaminergic activity. From these findings, we concluded that GBE participates not only in the cholinergic system, but also the histaminergic system in spatial memory.
4.
Experimental procedures
4.1.
Animals
Male Wistar rats, 6 weeks old (body weight 160–180 g) were purchased from Japan SLC, Shizuoka, Japan. They were maintained in an air conditioned room with controlled temperature (24 ± 2 °C) and humidity (55 ± 15%). Animals were housed in aluminum cages with sawdust and with a 12h light–dark cycle (light on from 7:00 to 19:00 h). Before the
behavioral test, the body weight was maintained to 80–85% of their free-feeding weight, and then the animals were kept on a restricted diet for the rest of the experiment. Water was provided ad libitum. All procedures involving the animals were conducted in accordance with the Guidelines for Animal Experiments at Okayama University Advanced Sciences Research Center.
4.2.
Eight arm radial maze
The apparatus used was described in our previous report (Chen et al., 1999). The procedure was as follows. To familiarize the rat with the radial maze, they received 1 daily habituation session for 3 days prior to training. On the first day, food pellets (45 mg, each, Bio-Serv, A Holton Industries, Frenchtown, NJ, USA) were scattered over the entire maze surface and 3 or 4 rats were simultaneously placed on the radial maze and allowed to take pellets freely. On the next 2 days, a pellet was placed in each of the eight arms in the food cup, and the rat was allowed to explore freely until it had taken all the pellets. After adaptation, all rats were trained with 1 trial per day. In each trial, only four arms were baited, and the sequence was not changed throughout the experiment. The rat was placed on the center platform, which was closed off by a door. After 20 s, the door was opened and the rat was allowed to make an arm choice to obtain food pellets until all four pellets had been eaten or 10 min had elapsed. Rats were trained continually until reaching the criterion of at most 1 error per trial for 5 successive trials. The number of entries into the unbaited arms was scored as the TE. The first entry into never-baited arms was scored as a RME, while a re-entry into arms where the pellet had already been taken was scored as a WME.
4.3.
Drugs
Diphenhydramine hydrochloride (Sigma, St. Louis, MO, USA), pyrilamine maleate (Sigma) and scopolamine hydrobromide (Sigma) were dissolved in saline and injected intraperitoneally 30 min before test trial. GBE (Schwabe Greenwave, Karlsruhe Germany) and donepezil hydrochloride (Aricept®, Eisai Co., Ltd.) were suspended in 5% gum arabic and administered orally 1 h before test trial. Studies for drug effect were carried out once a week. Ten to 19 rats were used in each group and the same rats were repeatedly used for 3 months, and they experienced all doses of either drugs.
4.4.
Data analysis
One-way analysis of variance (ANOVA) with Dunnett’s test was used for the statistical analysis of the results. A difference of p < 0.05 was regarded as being statistically significant. REFERENCES
Ahlemeyer, B., Mowes, A., Krieglstein, J., 1999. Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents. Eur. J. Pharmacol. 367, 423–430.
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Chen, Z., Sugimoto, Y., Kamei, C., 1999. Effects of intracerebroventricular injection of α-fluoromethylhistidine on radial maze performance in rats. Pharmacol. Biochem. Behav. 64, 513–518. Chopin, P., Briley, M., 1992. Effects of four non-cholinergic cognitive enhancers in comparison with tacrine and galanthamine on scopolamine-induced amnesia in rats Psychopharmacology 106, 26–30. Das, A., Shanker, G., Nath, C., Pal, R., Singh, S., Singh, H.K., 2002. A comparative study in rodents of standardized extracts of Bacopa monniera and Ginkgo biloba Anticholinesterase and cognitive enhancing activities. Pharmacol. Biochem. Behav. 73, 893–900. DeFeudis, F.V., Drieu, K., 2000. Ginkgo biloba extract (EGb761) and CNS functions: basic studies and clinical applications. Curr. Drug Targets 1, 25–58. Higuchi, M., Yanai, K., Okamura, N., Meguro, K., Arai, H., Itoh, M., Iwata, R., Ido, T., Watanabe, T., Sasaki, H., 2000. Histamine H1 receptors in patients with Alzheimer’s disease assessed by positron emission tomography. Neuroscience 99, 721–729. Kamei, C., Tasaka, K., 1991. Participation of histamine in the step-through active avoidance response and its inhibition by H1-blockers. Jpn. J. Pharmacol. 57, 473–482. Kamei, C., Tasaka, K., 1993. Effect of histamine on memory retrieval in old rats. Biol. Pharm. Bull. 16, 128–132. Kamei, C., Chung, Y.H., Tasaka, K., 1990. Influence of certain H1-blockers on the step-through active avoidance response in rats. Psychopharmacology 102, 312–318. Kamei, C., Okumura, Y., Tasaka, K., 1993. Influence of histamine depletion on learning and memory recollection in rats. Psychopharmacology 111, 376–382. Klein, S., Hadamitzky, H., Koch, M., Schwabe, K., 2004. Role of glutamate receptors in nucleus accumbens core and shell in spatial behaviour of rats. Neuroscience 28, 229–238. Kubo, N., Shirakawa, O., Kuno, T., Tanaka, C., 1987. Antimuscarinic effects of antihistamines: quantitative evaluation by receptor-binding assay. Jpn. J. Pharmacol. 43, 277–282. Maclennan, K.M., Darlington, C.L., Smith, P.F., 2002. The CNS effects of Ginkgo biloba extracts and ginkgolide B. Prog. Neurobiol. 67, 235–257.
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Misane, I., Ogren, S.O., 2003. Selective 5-HT1A antagonists WAY100635 and NAD-299 attenuate the impairment of passive avoidance caused by scopolamine in the rat. Neuropsychopharmacology 28, 253–264. Morisset, S., Traiffort, E., Schwartz, J.C., 1996. Inhibition of histamine versus acetylcholine metabolism as a mechanism of tacrine activity. Eur. J. Pharmacol. 315, R1–R2. Ogura, H., Kosasa, T., Kuriya, Y., Yamanishi, Y., 2000. Donepezil, a centrally acting acetylcholinesterase inhibitor, alleviates learning deficits in hypocholinergic models in rats. Methods Find Exp. Clin. Pharmacol. 22, 89–95. Rapin, J.R., Lamproglou, I., Drieu, K., DeFeudis, F.V., 1994. Demonstration of the “anti-stress” activity of an extract of Ginkgo biloba (EGb 761) using a discrimination learning task. Gen. Pharmacol. 25, 1009–1016. Schneider, C., Risser, D., Kirchner, L., Kitzmuller, E., Cairns, N., Prast, H., Singewald, N., Lubec, G., 1997. Similar deficits of central histaminergic system in patients with Down syndrome and Alzheimer disease. Neurosci. Lett. 222, 183–186. Shen, J., Wang, J., Zhao, B., Hou, J., Gao, T., Xin, W., 1998. Effects of EGb 761 on nitric oxide and oxygen free radicals, myocardial damage and arrhythmia in ischemia–reperfusion injury in vivo. Biochim. Biophys. Acta 1406, 228–236. Staay van der, F.J., Bouger, P.C., 2005. Effects of the cholinesterase inhibitors donepezil and metrifonate on scopolamine-induced impairments in the spatial cone field orientation task in rats. Behav. Brain Res. 156, 1–10. Taga, C., Sugimoto, Y., Nishiga, M., Fujii, Y., Kamei, C., 2001. Effects of vasopressin on histamine H1 receptor antagonist-induced spatial memory deficits in rats. Eur. J. Pharmacol. 423, 167–170. Watts, J., Stevens, R., Robinson, C., 1981. Effects of scopolamine on radial maze performance in rats. Physiol. Behav. 26, 845–851. Winter, J.C., 1998. The effects of an extract of Ginkgo biloba, EGb761, on cognitive behavior and longevity in the rat. Physiol. Behav. 63, 425–433. Yamamoto, Y., Adachi, Y., Fujii, Y., Kamei, C., 2005. Effect of Ginkgo biloba extract on memory deficits in radial maze performance induced by some drugs in rats. Jpn. J. Neuropsychopharmacol. 25, 85–90 (Abstract in English).
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Ginkgo biloba extract improves spatial memory in rats mainly but not exclusively via a histaminergic mechanism Yasuko Yamamoto, Yutaka Adachi, Yoko Fujii, Chiaki Kamei ⁎ Department of Medicinal Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
In order to clarify the mechanism of Ginkgo biloba extract (GBE) on learning and memory, we
Accepted 26 August 2006
studied the effect of GBE on spatial memory deficits induced by diphenhydramine,
Available online 6 December 2006
pyrilamine and scopolamine using the eight-arm radial maze performance of rats, in comparison with donepezil. Total error (TE), reference memory error (RME) and working
Keywords:
memory error (WME) were used as indices of spatial memory deficits. Both GBE and
Ginkgo biloba extract
donepezil caused a potent antagonistic effect on the increase in TE, RME and WME induced
Scopolamine
by diphenhydramine. GBE and donepezil also antagonized scopolamine-induced spatial
H1-antagonist
memory deficits. Although the antagonistic effect of GBE on pyrilamine-induced spatial
Radial maze
memory deficits was weak, a significant difference was observed with TE and WME.
Reference memory
However, donepezil caused no antagonistic effect on pyrilamine-induced memory deficits.
Working memory
From these findings, we concluded that the effects of GBE are mainly contributable to cholinergic activity and perhaps partly due to a histaminergic mechanism. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Ginkgo biloba extract (GBE) is an ethanol extract obtained from the leaves of the G. biloba. In contrast, EGb-761 is an acetone extract obtained from the leaves of G. biloba, and contains 24% flavonol glycosides, 6% terpene lactone and limited amounts of other substances, including proanthocyanidins and organic acid (DeFeudis and Drieu, 2000; Maclennan et al., 2002). The preparation is an ethanolic extract, to contain the same components as EGb-761 (Yamamoto et al., 2005). GBE has been reported to have several pharmacological actions. GBE can inhibit the neuronal apoptosis that is induced in cultured chick embryonic neuron and in mixed cultures of neuron and astrocytes from neonatal rat hippocampus either by serum deprivation or by staurosporine, both of which lead to increased generation of reactive oxygen species (Ahlemeyer
et al., 1999). Shen et al. (1998) showed that GBE had cardiovascular protective effects by means of adjusting the level of NO and inhibiting oxygen free radicals induced lipid peroxidation in myocardial ischemia–reperfusion injury in vivo. Rapin et al. (1994) reported that GBE had anti-stress effect in rats subjected to a stressful situation. In Europe, GBE is widely used as a herbal medicine or dietary supplement, and it has been also demonstrated that GBE is effective in the facilitation of learning and the recollection of memory in rodents (Winter, 1998). It is well recognized that the brain cholinergic system is closely related to learning and memory. We have reported that histamine also plays an important role in learning and memory via H1 receptors (Kamei et al., 1990, 1993; Kamei and Tasaka, 1991, 1993). In addition, the brain concentration of histamine was demonstrated to decrease in Alzheimer’s patients. For instance, Schneider et al. (1997)
⁎ Corresponding author. Fax: +81 86 2517939. E-mail address: [email protected] (C. Kamei). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.08.102
162
BR A I N R ES E A RC H 1 1 2 9 ( 2 00 7 ) 1 6 1 –16 5
showed that brain histamine contents, as well as the histidine decarboxylase activity, were decreased in Alzheimer’s patients. Morisset et al. (1996) also reported that tacrine, an anticholinesterase agent and cognition enhancer, inhibited histamine N-methyltransferase activity. Another attempt revealed that the binding potential of the histamine H1 receptor showed a significant decrease in the frontal and temporal areas of the Alzheimer’s disease affected brain compared to elderly, normal subjects (Higuchi et al., 2000). From these findings, it is clear that the brain cholinergic and histaminergic systems are related to learning and memory. There are some reports about the interaction between brain cholinergic neurons and GBE in learning and memory. For instance, Chopin and Briley (1992) have reported that GBE improved scopolamine-induced memory deficits of passive avoidance performance. However, little is known about the interactions between GBE and histaminergic systems in spatial memory. Therefore, the present study was performed to clarify the effect of GBE on spatial memory deficits induced by H1 antagonists, diphenhydramine and pyrilamine and scopolamine (muscarinic receptor antagonist) using eightarm radial maze performance in comparison with donepezil, inhibitor of acetylcholinesterase, cognitive enhancers.
2.
Results
2.1. Effects of diphenhydramine, pyrilamine and scopolamine on radial maze performance Diphenhydramine at a dose of 10 mg/kg caused no significant effect on total error (TE), reference memory error (RME) or working memory error (WME). On the other hand, at a dose of 20 mg/kg, this drug significantly increased TE, RME and WME. Although pyrilamine at a dose of 20 mg/kg caused no significant effect on TE, RME or WME, at 35 mg/kg the drug significantly increased in TE, RME and WME. Scopolamine at a dose of 0.2 mg/kg caused no significant effect on TE, RME or WME. However, at a dose of 0.5 mg/kg, it caused a significant increase in TE, RME and WME. Therefore, 20 mg/kg in diphenhydramine, 35 mg/kg pyrilamine and 0.5 mg/kg scopolamine were used to study the effects of GBE and donepezil (Table 1).
Table 1 – Effect of diphenhydramine, pyrilamine and scopolamine on spatial memory in the radial performance Drugs Saline Diphenhydramine Pyrilamine Scopolamine
Dose (mg/kg, i.p.)
TE
RME
WME
– 10 20 20 35 0.2 0.5
0.6 ± 0.2 1.1 ± 0.2 3.3 ± 0.3** 1.3 ± 0.3 2.8 ± 0.2** 0.4 ± 0.2 3.1 ± 0.4**
0.5 ± 0.2 0.9 ± 0.2 1.7 ± 0.2* 0.9 ± 0.2 1.7 ± 0.3* 0.1 ± 0.1 1.8 ± 0.3*
0.1 ± 0.1 0.2 ± 0.1 1.1 ± 0.3* 0.4 ± 0.2 1.1 ± 0.3* 0.2 ± 0.1 1.3 ± 0.4*
Drugs were administrated intraperitoneally. Each value represents the mean ± SEM of 16 rats. TE: Total error, RME: Reference memory error, WME: Working memory error. *, **: Significantly different from the control group with p < 0.05 and p < 0.01, respectively.
Fig. 1 – Effects of Ginkgo biloba extract and donepezil on spatial memory deficits induced by diphenhydramine in rats. Diphenhydramine (20 mg/kg, i.p.) was administrated before and G. biloba extract and donepezil were administrated 1 h before the test trial. Each value represents the mean ± SEM of 18–19 rats. TE: Total error, RME: Reference memory error, WME: Working memory error. *,**: Significantly different from control group with p < 0.05 and p < 0.01, respectively.
2.2. Effects of GBE and donepezil on diphenhydramine-induced memory deficits GBE at doses of 30 and 50 mg/kg significantly inhibited the diphenhydramine-induced increase of TE and WME. At 50 mg/kg, this drug also inhibited the diphenhydramineinduced increase in RME. Donepezil at 0.5 mg/kg significantly inhibited the diphenhydramine-induced increase in TE and RME. At 1 mg/kg, it also inhibited the diphenhydramineinduced increase in WME (Fig. 1).
2.3. Effects of GBE and donepezil on pyrilamine-induced memory deficits The effects of GBE and donepezil on pyrilamine-induced spatial memory deficits are shown in Fig. 2. GBE at doses of 20 and 30 mg/kg caused no significant effect on pyrilamineinduced memory deficits, but at a dose of 50 mg/kg it significantly inhibited the pyrilamine-induced increase in TE and WME. On the other hand, donepezil caused no inhibitory effect on the pyrilamine-induced memory deficits, even at a dose of 1 mg/kg.
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163
Although both H1-antagonists caused a significant increase in TE, RME and WME, the effect of diphenhydramine was more potent than that of pyrilamine. Diphenhydramine is well known to have a potent anticholinergic activity, while pyrilamine is a pure histamine H1 antagonist and has a weak anticholinergic effect. That is, diphenhydramine showed moderate affinity for the muscarinic receptor (Ki = 280 ± 50 nM), and H1 receptor antagonism in the Ki value of 14 ± 2 nM. Pyrilamine exhibited a weak antagonism of the muscarinic receptor (Ki = 30,000 ± 2000 nM), and a potent antagonism of H1 receptor (Ki = 0.24 ± 0.03 nM) (Kubo et al., 1987). It is well recognized that anticholinergic properties are responsible for memory deficits in cognition (Watts et al., 1981). As shown in the present study, we confirmed that scopolamine, a representative anticholinergic drug, caused potent spatial memory deficits. This is the reason why diphenhydramine was more effective than pyrilamine in causing spatial memory deficits on radial maze performance. GBE inhibited the diphenhydramine-induced an increase in TE, RME and WME. Almost the same findings were obtained with donepezil. Both drugs also inhibited the scopolamine-
Fig. 2 – Effects of Ginkgo biloba extract and donepezil on spatial memory deficits induced by pyrilamine in rats. Pyrilamine (35 mg/kg, i.p.) was administrated 30 min before and G. biloba extract and donepezil were administrated 1 h before the test trial. Each value represents the mean ± SEM of 10–12 rats. TE: Total error, RME: Reference memory error, WME: Working memory error. *,**: Significantly different from the control group with p < 0.05 and p < 0.01, respectively.
2.4. Effects of GBE and donepezil on scopolamine-induced memory deficits Fig. 3 shows the effects of GBE and donepezil on the spatial memory deficits induced by scopolamine (0.5 mg/kg, i.p.). GBE at doses of 30 and 50 mg/kg significantly inhibited the scopolamine-induced increase in TE and WME. A significant effect was also obtained with the scopolamine-induced increase in RME at a dose of 50 mg/kg. Almost the same results were obtained with donepezil; 0.5 mg/kg of donepezil inhibited the scopolamine-induced increase in TE and WME. The increase in RME induced by scopolamine was inhibited by 1 mg/kg of donepezil.
3.
Discussion
It was found that diphenhydramine, pyrilamine and scopolamine caused a significant increase of TE, RME and WME at doses of 20, 35 and 0.5 mg/kg, respectively. This result confirmed our previous findings that these drugs increased TE, RME and WME at the same dose levels (Taga et al., 2001).
Fig. 3 – Effects of Ginkgo biloba extract and donepezil on spatial memory deficits induced by scopolamine in rats. Scopolamine (0.5 mg/kg, i.p.) was administrated 30 min before and G. biloba extract and donepezil were administrated 1 h before the test trial. Each value represents the mean ± SEM of 12–16 rats. TE: Total error, RME: Reference memory error, WME: Working memory error. *,**: Significantly different from the control group with p < 0.05 and p < 0.01, respectively.
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induced memory deficits. There are many reports that both GBE and donepezil showed an antagonistic effect on scopolamine-induced memory deficits and a cholinergic effect. For instance, Chopin and Briley (1992) reported that GBE improved the scopolamine-induced memory deficits of passive avoidance performance. In addition, Das et al. (2002) showed that GBE caused a dose-dependent inhibition of acetylcholinesterase activity. As for donepezil, Ogura et al. (2000) reported that donepezil alleviates learning deficits in a hypocholinergic model in rats. Misane and Ogren (2003); Staay van der and Bouger (2005) also demonstrated that donepezil attenuates the impairment of passive avoidance in the spatial cone field orientation task in rats. From these findings, it seems likely that the antagonistic effect of GBE on the diphenhydramineinduced inhibition of spatial memory is mainly attributable to its cholinergic activity. The same idea was also applicable to donepezil. As shown in the present data, GBE seems to counteract WME in low doses and only affects RME at high doses in diphenhydramine-induced memory deficits. Whereas no such pattern was observed for donepezil. It is generally accepted that the acquisition of working memory was faster than that of reference memory (Klein et al., 2004). That is, the number of training to require the working memory is less than that of reference memory. From these findings, it seems likely that working memory is more sensitive than reference memory to some drugs. This is the reason why GBE showed more effective in WME than RME. On the other hand, donepezil is also effective in RME. Donepezil is a powerful drug in antagonizing memory deficit induced by diphenhydramine. Therefore, it was thought at the time, that donepezil was effective not only in WME but also in RME. Although GBE caused no antagonism on the pyrilamineinduced increase in RME at a dose of 50 mg/kg, TE and WME were antagonized significantly at a dose of 50 mg/kg. As described previously, pyrilamine only has a weak anticholinergic activity and it is a pure H1 antagonist. However, GBE caused an inhibitory effect on the pyrilamine-induced memory deficits. Therefore, it is reasonable to presume that the improvement of spatial memory deficits induced by GBE may be partly due to a histaminergic mechanism. As for donepezil, it caused no antagonistic effect on pyrilamine-induced memory deficits, even at a high dose. Therefore, it seems likely that there is no interaction between the ameliorating effect of donepezil on memory deficits and a histaminergic mechanism. In fact, there are no reports that donepezil has a histaminergic activity. From these findings, we concluded that GBE participates not only in the cholinergic system, but also the histaminergic system in spatial memory.
4.
Experimental procedures
4.1.
Animals
Male Wistar rats, 6 weeks old (body weight 160–180 g) were purchased from Japan SLC, Shizuoka, Japan. They were maintained in an air conditioned room with controlled temperature (24 ± 2 °C) and humidity (55 ± 15%). Animals were housed in aluminum cages with sawdust and with a 12h light–dark cycle (light on from 7:00 to 19:00 h). Before the
behavioral test, the body weight was maintained to 80–85% of their free-feeding weight, and then the animals were kept on a restricted diet for the rest of the experiment. Water was provided ad libitum. All procedures involving the animals were conducted in accordance with the Guidelines for Animal Experiments at Okayama University Advanced Sciences Research Center.
4.2.
Eight arm radial maze
The apparatus used was described in our previous report (Chen et al., 1999). The procedure was as follows. To familiarize the rat with the radial maze, they received 1 daily habituation session for 3 days prior to training. On the first day, food pellets (45 mg, each, Bio-Serv, A Holton Industries, Frenchtown, NJ, USA) were scattered over the entire maze surface and 3 or 4 rats were simultaneously placed on the radial maze and allowed to take pellets freely. On the next 2 days, a pellet was placed in each of the eight arms in the food cup, and the rat was allowed to explore freely until it had taken all the pellets. After adaptation, all rats were trained with 1 trial per day. In each trial, only four arms were baited, and the sequence was not changed throughout the experiment. The rat was placed on the center platform, which was closed off by a door. After 20 s, the door was opened and the rat was allowed to make an arm choice to obtain food pellets until all four pellets had been eaten or 10 min had elapsed. Rats were trained continually until reaching the criterion of at most 1 error per trial for 5 successive trials. The number of entries into the unbaited arms was scored as the TE. The first entry into never-baited arms was scored as a RME, while a re-entry into arms where the pellet had already been taken was scored as a WME.
4.3.
Drugs
Diphenhydramine hydrochloride (Sigma, St. Louis, MO, USA), pyrilamine maleate (Sigma) and scopolamine hydrobromide (Sigma) were dissolved in saline and injected intraperitoneally 30 min before test trial. GBE (Schwabe Greenwave, Karlsruhe Germany) and donepezil hydrochloride (Aricept®, Eisai Co., Ltd.) were suspended in 5% gum arabic and administered orally 1 h before test trial. Studies for drug effect were carried out once a week. Ten to 19 rats were used in each group and the same rats were repeatedly used for 3 months, and they experienced all doses of either drugs.
4.4.
Data analysis
One-way analysis of variance (ANOVA) with Dunnett’s test was used for the statistical analysis of the results. A difference of p < 0.05 was regarded as being statistically significant. REFERENCES
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