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The multi-herbal formula Guibi-tang enhances memory and increases cell proliferation in the rat hippocampus
Neuroscience Letters 379 (2005) 205–208
The multi-herbal formula Guibi-tang enhances memory and increases cell proliferation in the rat hippocampus Myung Sook Oha , Youngbuhm Huhb , Hyunsu Baec , Duk Kyun Ahnd , Seong Kyu Parka,∗ a
Department of Prescriptionology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea b Department of Anatomy, College of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea c Department of Physiology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea d Jaseng Research Institute of Bio-technology and Bioscience, Jin Deuk Building 202, 636-10 Sinsa-dong, Kangnam-gu, Seoul 135-896, Republic of Korea Received 23 August 2004; received in revised form 25 November 2004; accepted 28 December 2004
Abstract Guibi-tang (GBT) is a multi-herbal medicinal formula that has been used to treat amnesia in the traditional Korean medicine system for hundreds of years. In this study, we investigated the effects of GBT on learning and memory function in Wistar rats by using the foot shock passive avoidance test. In addition, cell proliferation was examined using 5-bromo-2-deoxyuridine (BrdU) uptake and immunostaining in the dentate gyrus of the rat hippocampus after oral administration of GBT. In the passive avoidance test, the retention time of the GBT-treated group was significantly longer (almost 2.5-fold) than that of the control group. The number of BrdU-immunoreactive cells in the dentate gyrus was significantly higher in the GBT-treated group (almost twofold) than in the control group. These results suggest that administration of GBT to rats not only improves learning and memory but also increases the proliferation of cells in the dentate gyrus of the hippocampus. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Guibi-tang; Learning and memory; Passive avoidance test; Cell proliferation; Dentate gyrus
Memory is the process by which facts and events are recorded and made available for later use and is thus one of our most valuable health assets. From this point of view, memory enhancement is a matter of general concern, although it is of particular importance to patients with Alzheimer’s disease and other progressive neurodegenerative diseases involving memory loss. It is well known that the hippocampus plays a critical role in memory and learning. The memory consolidation process involves the hippocampus, the amygdala, the entorhinal cortex, and the parietal cortex [10]. The hippocampus and the entorhinal cortex both participate in the formation and expression of memory in the one-trial step-down inhibitory avoidance task in rats [3]. Learning mediated by the hippocampus may be related to the generation of new neurons in the adult dentate gyrus [16]. These new cells express neuronal markers, differentiate into granule neurons, and have projections ∗
Corresponding author. Tel.: +82 2 961 0330; fax: +82 2 961 0536. E-mail address: [email protected] (S.K. Park).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.12.077
called mossy fibers that extend to the CA3 hippocampal region [21]. Many plants are used for improvement of memory and cognitive function in traditional medicine [8]. Generally, in traditional medicine in Asian countries, many herbal drugs are combined in the form of a multi-herbal formula to enhance cognitive function. The herbal constituents are selected to accentuate the therapeutic actions and enhance the activity of the component compounds in clinical applications or to reduce the toxicity or side effects of compounds from other herbal species in the mixture [1]. Recently, research concerning the memory-enhancing effects of some formulas such as Kami-Untan-To [15] and S-113m [17] has been reported. Guibi-tang (GBT), also called Qui-Pi-Tang or Kihi-To, is a multi-herbal traditional Korean medicinal formula that has been used to treat amnesia, poor memory or forgetfulness, fatigue, insomnia, anemia, palpitation, and neurosis for several hundred years. However, its neural effects have not yet been well investigated. In this study, we investigated the effects of the administration of water extract of GBT to rats on learning
206
M.S. Oh et al. / Neuroscience Letters 379 (2005) 205–208
Table 1 Composition of GBT Botanical name
Family
Part used
Ratio (g)
Panax ginseng MEYER Astragalus membranaceus BUNGE Atractylodes macrocephala KOIDZ· Poria cocos WOLF· Zizyphus jujuba MILL· Euphoria Longan STEUD· Angelica sinensis DIELS Polygala tenuifolia WILLD· Saussurea lappa CLARKE Glycyrrhiza uralensis FISCH·
Araliaceae Leguminosae
Root Root
60 60
Compositae
Rhizome
60
Polyporaceae Rhamnaceae Sapindaceae Umbelliferae Polygalaceae Compositae Leguminosae
Sclerotium Seed Aril Root Root Root Root
60 60 60 60 60 30 18
and memory function using passive avoidance tests and on cell proliferation in the dentate gyrus of the hippocampus using BrdU uptake and immunohistochemical staining. GBT was prepared by extraction of 10 dried herbs, listed in Table 1. A total of 528 g of ingredients was boiled with 10 L of water for 2 h at 100 ◦ C; the extract was then filtered and lyophilized to yield 112 g of powder. Six-week-old male Wistar rats (Japan SLC, Shizaoka, Japan) were housed under temperature- and humiditycontrolled, pathogen-free conditions with a 12:12 h light:dark cycle and access to food and water ad libitum. After 10 days of adaptation to the environment, the rats (n = 11) were given GBT (2.0 g/kg/day, p.o.) for 16 days. The control rats (n = 11) received vehicle only. The day after the end of treatment, a modified passive avoidance test was used to assess the effect of GBT on learning and memory in the treated rats [2]. The step-through passive avoidance apparatus consisted of one illuminated and one dark chamber, separated by a guillotine door. The rats were placed in the light chamber with the door open. When a rat entered the dark chamber, the guillotine door was closed immediately. After being allowed to explore the dark chamber for 1 min, the rat was removed to its cage. The training was repeated until the rat entered the dark chamber within 20 s of being placed in the illuminated section. Animals not entering the dark chamber were excluded from the experiment. The next day, 24 h after training, the rat was placed in the light chamber. Immediately after the rat entered the dark chamber with all four paws, the door was closed and an electrical foot shock (2.0 mA) was delivered for 5 s. To evaluate memory retention, the rat was again placed in the light chamber 24 h later, and the latency time before entering the dark compartment with all four paws was measured. No shock was delivered. If the rat did not enter the dark chamber within the 300-s cut-off period, it was assigned a value of 300 s as its latency time. Raw data from the passive avoidance test were analyzed by the non-parametric Mann–Whitney test. To evaluate the effect of GBT on proliferation of cells in the hippocampus, BrdU uptake was assessed by immunohistochemistry [18] after the retention test. Rats were injected with BrdU (50 mg/kg, i.p.) 14 and 2 h before they
were sacrificed to prepare brain tissue. The rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The brains were removed and post-fixed in the same fixative overnight. Coronal sections of 40 m were then cut with a microtome cryostat (CM3000; Leica, Wetzlar, Germany). For the immunohistochemical detection of BrdU, every 10th section of tissue containing the dentate gyrus of the hippocampal formation was used. Free-floating brain sections were pretreated in 50% formamide in 2× SSC at 65 ◦ C for 2 h, incubated at 37 ◦ C for 30 min in 2N HCl, and rinsed for 10 min at 25 ◦ C in 0.1 M boric acid (pH 8.5). The sections were then incubated overnight at 4 ◦ C with anti-BrdU antibody (Roche Diagnostics, Mannheim, Germany) diluted 1:1000 in phosphate-buffered saline (PBS) with 0.3% Triton X-100, 0.5 mg/mL bovine serum albumin, and 1.5% normal goat serum. The sections were then incubated with horse anti-mouse secondary antibody (1:100; Vector, Burlingame, CA, USA) for 90 min and finally incubated with an avidin–biotin-peroxidase complex (1:100; Vector) for 1 h at room temperature. The sections were reacted with 0.02% 3,3 -diaminobenzidine tetrahydrochloride, 0.01% H2 O2 , and 0.04% NiCl2 for about 3 min. After each incubation step, the sections were washed three times with PBS for a total of 15 min. The sections were mounted on gelatin-coated slides and counterstained with cresyl violet. The numbers of BrdU-positive cells in the granule cell layer of every 10th section throughout the entire dentate gyrus were counted under ×400 magnification. Total dentate gyrus volume was determined using Cavalieri’s principle [14], and the cross-sectional areas were determined using an image-analysis system (Multiscan, Fullerton, CA, USA). No changes in the volumes of the granule cell layer of the dentate gyrus were observed in any of the experiments conducted. Hence, the data are expressed as estimates of the total number of BrdU-positive cells. At this point, each sample was given a coded identification number to allow the data to be analyzed in a “blind” manner. The immunohistochemistry data were analyzed using Student’s t test. In this study, we evaluated the memory-enhancing effects of GBT in rats using the passive avoidance test. We observed that the mean response latency of GBT-treated rats (175.82 ± 34.95 s) was significantly increased compared with that of control rats (71.55 ± 32.06 s; p < 0.05; Fig. 1A). GBT treatment also induced cell proliferation in the dentate gyrus. The number of BrdU-labeled cells was significantly higher in the GBT-treated group (1872.81 ± 217.47 per dentate gyrus) than in the control group (938.41 ± 166.61 per dentate gyrus; p < 0.01; Fig. 1B). There were no differences in food intake or body weight between groups after GBT treatment. Moreover, no noticeable changes in actions were observed. Memory has been shown to improve in several laboratory animal models not only after administration of drugs such as taurine [9] and calcineurin activator [13] but also after
M.S. Oh et al. / Neuroscience Letters 379 (2005) 205–208
207
Fig. 1. Effects of GBT treatment on response latency in the passive avoidance test and on cell proliferation in the rat dentate gyrus. The control group (n = 11) received vehicle only and the GBT group (n = 11) was treated with GBT orally (2.0 g/kg/day). The response latency of GBT-treated rats was significantly (p < 0.05) greater than that of control rats (A). There were significantly (p < 0.01) more BrdU-labeled cells in the GBT-treated group than in the control group (B). Representative photographs of the dentate gyrus from the control (C) and GBT (D) groups are shown. The density of BrdU-labeled cells (black puncta) was enhanced in the GBT group.
administration of herbal medicines such as Gingko biloba [22] and Heteropterys aphrodisiaca [6]. GBT is composed of several memory-enhancing herbs. Poria and Ginseng radix have been shown to promote hippocampal long-term potentiation [20]. Polygalae radix alleviates scopolamineinduced impairment of the passive avoidance response in mice [5] and has some protective effects against neuronal death and cognitive impairments in Alzheimer’s disease or other neurodegenerative diseases [19]. Astragali radix has protective effects against anoxic damage to cultured neurons [7]. Angelicae sinensis radix and Ziziphi semen have CNS effects at the receptor level [12]. These constituents of GBT may be responsible for its neural effect. Various factors have been found to modulate the rate of new cell proliferation in the dentate gyrus of rodents, such as physical activity [24], an enriched environment [11], and hippocampal-dependent learning [23]. In addition to cell proliferation, another important aspect of adult neurogenesis is the migration of new cells and their differentiation into neurons and eventual integration into a functional network. Newly generated cells in the adult mouse hippocampus are reported to have neuronal morphology and can display
passive membrane properties, action potentials, and functional synaptic inputs similar to those found in mature dentate granule cells. These findings demonstrate that newly generated cells mature into functional neurons in the adult mammalian brain [25]. Moreover, approximately 9000 new cells are generated in the dentate gyrus each day in young adult rats and within 5–12 days 50% of these cells can be double-labeled with neuron-specific markers [4]. In this regard, the newly proliferated cells stimulated by GBT may differentiate into neurons, but further research is required to determine the neuronal function of GBT. In conclusion, our results show that treatment of rats with GBT not only improves learning and memory but also increases the proliferation of cells in the dentate gyrus of the hippocampus. References [1] D. Bansky, R. Barolet, Chinese Herbal Medicine Formulas and Strategies, Eastland Press, Seattle, 1990, pp. 7–8. [2] O.F. Bueno, M.G. Oliveira, A.C. Pomarico, E.B. Gugliano, A dissociation between the proactive ECS effects on inhibitory avoidance
208
[3] [4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
M.S. Oh et al. / Neuroscience Letters 379 (2005) 205–208 learning and on classical fear conditioning, Behav. Neural. Biol. 59 (1993) 180–185. L. Cahill, J.L. McGaugh, Mechanisms of emotional arousal and lasting declarative memory, Trends Neurosci. 21 (1998) 294–299. H.A. Cameron, R.D. McKay, Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus, J. Comp. Neurol. 435 (2001) 406–417. N. Egashira, M. Yuzurihara, N. Hattori, I. Sakakibara, A. Ishige, Ninjin-yoei-to (Ren-Shen-Yang-Rong-Tang) and Polygalae radix improves scopolamine-induced impairment of passive avoidance response in mice, Phytomedicine 10 (2003) 467–473. S.M. Galvao, L.C. Marques, M.G. Oliveira, E.A. Carlini, Heteropterys aphrodisiaca (extract BST0298): a Brazilian plant that improves memory in aged rats, J. Ethnopharmacol. 79 (2002) 305–311. X. He, C. Li, S. Yu, Protective effects of radix astragali against anoxic damages to in vitro cultured neurons, J. Tongji Med. Univ. 20 (2000) 126–127. M.J. Howes, P.J. Houghton, Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function, Pharmacol. Biochem. Behav. 75 (2003) 513–527. J.D. Hu, Q.H. Gao, D.G. Yu, X.T. Xu, The improvement of taurine in learning and memory ability of rats exposed to lead, Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 21 (2003) 413–416. I. Izquierdo, J.A. Quillfedt, M.S. Zanatta, J. Quevedo, E. Schaeffer, P.K. Schmitz, J.H. Medina, Sequential role of hippocampus and amygdala, the entorhinal cortex and the posterior parietal cortex in formation and retrieval of memory for inhibitory avoidance in rats, Eur. J. Neurosci. 9 (1997) 786–793. G. Kempermann, E.P. Brandon, F.H. Gage, Environmental stimulation of 129/SvJ mice causes increased cell proliferation and neurogenesis in the adult dentate gyrus, Curr. Biol. 8 (1998) 939–942. J.F. Liao, Y.M. Jan, S.Y. Huang, H.H. Wang, L.L. Yu, C.F. Chen, Evaluation with receptor binding assay on the water extracts of ten CNS-active Chinese herbal drugs, Proc. Natl. Sci. Counc. Repub. China B 19 (1995) 151–158. J. Luo, J.H. Yin, Q. Wei, The effect of calcineurin activator, extracted from Chinese herbal medicine, on memory and immunity in mice, Pharmacol. Biochem. Behav. 75 (2003) 749–754. R.P. Michel, L.M. Cruz-Orive, Application of the Cavalieri principle and vertical sections method to lung: estimation of volume and pleural surface area, J. Microsc. 150 (1988) 117–136.
[15] O. Nakagawasai, F. Yamadera, K. Iwasaki, H. Arai, R. Taniguchi, K. Tan-No, H. Sasaki, T. Tadano, Effect of kami-untan-to on the impairment of learning and memory induced by thiamine-deficient feeding in mice, Neuroscience 125 (2004) 233–241. [16] M. Nilsson, E. Perfilieva, U. Johansson, O. Orwar, P.S. Eriksson, Enriched environment increases neurogenesis in the adult rat dentate gyrus and improves spatial memory, J. Neurobiol. 39 (1999) 569–578. [17] N. Nishiyama, P.J. Chu, H. Saito, An herbal prescription, S-113m, consisting of biota, ginseng and schizandra, improves learning performance in senescence accelerated mouse, Biol. Pharm. Bull. 19 (1996) 388–393. [18] C. Park, M. Kang, Y.K. Kim, J. Chung, H. Ahn, Y. Huh, Inhibition of neuronal nitric oxide synthase enhances cell proliferation in the dentate gyrus of the adrenalectomized rat, Neurosci. Lett. 309 (2001) 9–12. [19] C.H. Park, S.H. Choi, J.W. Koo, J.H. Seo, H.S. Kim, S.J. Jeong, Y.H. Suh, Novel cognitive improving and neuroprotective activities of Polygala tenuifolia Willdenow extract, BT-11, J. Neurosci. Res. 70 (2002) 484–492. [20] M. Smriga, H. Saito, N. Nishiyama, Hoelen (Poria cocos Wolf) and ginseng (Panax ginseng C. A. Meyer), the ingredients of a Chinese prescription DX-9386, individually promote hippocampal long-term potentiation in vivo, Biol. Pharm. Bull. 18 (1995) 518– 522. [21] B.B. Stanfield, J.E. Trice, Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections, Exp. Brain Res. 72 (1988) 399–406. [22] S. Stoll, K. Scheuer, O. Pohl, W.E. Muller, Ginkgo biloba extract (EGb 761) independently improves changes in passive avoidance learning and brain membrane fluidity in the aging mouse, Pharmacopsychiatry 29 (1996) 144–149. [23] L.R. Squire, Memory and the hippocampus: a synthesis from findings with rats, monkeys, and humans, Psychol. Rev. 99 (1992) 195– 231. [24] H. Van Praag, G. Kempermann, F.H. Gage, Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus, Nat. Neurosci. 2 (1999) 266–720. [25] H. van Praag, A.F. Schinder, B.R. Christie, N. Toni, T.D. Palmer, F.H. Gage, Functional neurogenesis in the adult hippocampus, Nature 415 (2002) 1030–1034.
The multi-herbal formula Guibi-tang enhances memory and increases cell proliferation in the rat hippocampus Myung Sook Oha , Youngbuhm Huhb , Hyunsu Baec , Duk Kyun Ahnd , Seong Kyu Parka,∗ a
Department of Prescriptionology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea b Department of Anatomy, College of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea c Department of Physiology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea d Jaseng Research Institute of Bio-technology and Bioscience, Jin Deuk Building 202, 636-10 Sinsa-dong, Kangnam-gu, Seoul 135-896, Republic of Korea Received 23 August 2004; received in revised form 25 November 2004; accepted 28 December 2004
Abstract Guibi-tang (GBT) is a multi-herbal medicinal formula that has been used to treat amnesia in the traditional Korean medicine system for hundreds of years. In this study, we investigated the effects of GBT on learning and memory function in Wistar rats by using the foot shock passive avoidance test. In addition, cell proliferation was examined using 5-bromo-2-deoxyuridine (BrdU) uptake and immunostaining in the dentate gyrus of the rat hippocampus after oral administration of GBT. In the passive avoidance test, the retention time of the GBT-treated group was significantly longer (almost 2.5-fold) than that of the control group. The number of BrdU-immunoreactive cells in the dentate gyrus was significantly higher in the GBT-treated group (almost twofold) than in the control group. These results suggest that administration of GBT to rats not only improves learning and memory but also increases the proliferation of cells in the dentate gyrus of the hippocampus. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Guibi-tang; Learning and memory; Passive avoidance test; Cell proliferation; Dentate gyrus
Memory is the process by which facts and events are recorded and made available for later use and is thus one of our most valuable health assets. From this point of view, memory enhancement is a matter of general concern, although it is of particular importance to patients with Alzheimer’s disease and other progressive neurodegenerative diseases involving memory loss. It is well known that the hippocampus plays a critical role in memory and learning. The memory consolidation process involves the hippocampus, the amygdala, the entorhinal cortex, and the parietal cortex [10]. The hippocampus and the entorhinal cortex both participate in the formation and expression of memory in the one-trial step-down inhibitory avoidance task in rats [3]. Learning mediated by the hippocampus may be related to the generation of new neurons in the adult dentate gyrus [16]. These new cells express neuronal markers, differentiate into granule neurons, and have projections ∗
Corresponding author. Tel.: +82 2 961 0330; fax: +82 2 961 0536. E-mail address: [email protected] (S.K. Park).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.12.077
called mossy fibers that extend to the CA3 hippocampal region [21]. Many plants are used for improvement of memory and cognitive function in traditional medicine [8]. Generally, in traditional medicine in Asian countries, many herbal drugs are combined in the form of a multi-herbal formula to enhance cognitive function. The herbal constituents are selected to accentuate the therapeutic actions and enhance the activity of the component compounds in clinical applications or to reduce the toxicity or side effects of compounds from other herbal species in the mixture [1]. Recently, research concerning the memory-enhancing effects of some formulas such as Kami-Untan-To [15] and S-113m [17] has been reported. Guibi-tang (GBT), also called Qui-Pi-Tang or Kihi-To, is a multi-herbal traditional Korean medicinal formula that has been used to treat amnesia, poor memory or forgetfulness, fatigue, insomnia, anemia, palpitation, and neurosis for several hundred years. However, its neural effects have not yet been well investigated. In this study, we investigated the effects of the administration of water extract of GBT to rats on learning
206
M.S. Oh et al. / Neuroscience Letters 379 (2005) 205–208
Table 1 Composition of GBT Botanical name
Family
Part used
Ratio (g)
Panax ginseng MEYER Astragalus membranaceus BUNGE Atractylodes macrocephala KOIDZ· Poria cocos WOLF· Zizyphus jujuba MILL· Euphoria Longan STEUD· Angelica sinensis DIELS Polygala tenuifolia WILLD· Saussurea lappa CLARKE Glycyrrhiza uralensis FISCH·
Araliaceae Leguminosae
Root Root
60 60
Compositae
Rhizome
60
Polyporaceae Rhamnaceae Sapindaceae Umbelliferae Polygalaceae Compositae Leguminosae
Sclerotium Seed Aril Root Root Root Root
60 60 60 60 60 30 18
and memory function using passive avoidance tests and on cell proliferation in the dentate gyrus of the hippocampus using BrdU uptake and immunohistochemical staining. GBT was prepared by extraction of 10 dried herbs, listed in Table 1. A total of 528 g of ingredients was boiled with 10 L of water for 2 h at 100 ◦ C; the extract was then filtered and lyophilized to yield 112 g of powder. Six-week-old male Wistar rats (Japan SLC, Shizaoka, Japan) were housed under temperature- and humiditycontrolled, pathogen-free conditions with a 12:12 h light:dark cycle and access to food and water ad libitum. After 10 days of adaptation to the environment, the rats (n = 11) were given GBT (2.0 g/kg/day, p.o.) for 16 days. The control rats (n = 11) received vehicle only. The day after the end of treatment, a modified passive avoidance test was used to assess the effect of GBT on learning and memory in the treated rats [2]. The step-through passive avoidance apparatus consisted of one illuminated and one dark chamber, separated by a guillotine door. The rats were placed in the light chamber with the door open. When a rat entered the dark chamber, the guillotine door was closed immediately. After being allowed to explore the dark chamber for 1 min, the rat was removed to its cage. The training was repeated until the rat entered the dark chamber within 20 s of being placed in the illuminated section. Animals not entering the dark chamber were excluded from the experiment. The next day, 24 h after training, the rat was placed in the light chamber. Immediately after the rat entered the dark chamber with all four paws, the door was closed and an electrical foot shock (2.0 mA) was delivered for 5 s. To evaluate memory retention, the rat was again placed in the light chamber 24 h later, and the latency time before entering the dark compartment with all four paws was measured. No shock was delivered. If the rat did not enter the dark chamber within the 300-s cut-off period, it was assigned a value of 300 s as its latency time. Raw data from the passive avoidance test were analyzed by the non-parametric Mann–Whitney test. To evaluate the effect of GBT on proliferation of cells in the hippocampus, BrdU uptake was assessed by immunohistochemistry [18] after the retention test. Rats were injected with BrdU (50 mg/kg, i.p.) 14 and 2 h before they
were sacrificed to prepare brain tissue. The rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The brains were removed and post-fixed in the same fixative overnight. Coronal sections of 40 m were then cut with a microtome cryostat (CM3000; Leica, Wetzlar, Germany). For the immunohistochemical detection of BrdU, every 10th section of tissue containing the dentate gyrus of the hippocampal formation was used. Free-floating brain sections were pretreated in 50% formamide in 2× SSC at 65 ◦ C for 2 h, incubated at 37 ◦ C for 30 min in 2N HCl, and rinsed for 10 min at 25 ◦ C in 0.1 M boric acid (pH 8.5). The sections were then incubated overnight at 4 ◦ C with anti-BrdU antibody (Roche Diagnostics, Mannheim, Germany) diluted 1:1000 in phosphate-buffered saline (PBS) with 0.3% Triton X-100, 0.5 mg/mL bovine serum albumin, and 1.5% normal goat serum. The sections were then incubated with horse anti-mouse secondary antibody (1:100; Vector, Burlingame, CA, USA) for 90 min and finally incubated with an avidin–biotin-peroxidase complex (1:100; Vector) for 1 h at room temperature. The sections were reacted with 0.02% 3,3 -diaminobenzidine tetrahydrochloride, 0.01% H2 O2 , and 0.04% NiCl2 for about 3 min. After each incubation step, the sections were washed three times with PBS for a total of 15 min. The sections were mounted on gelatin-coated slides and counterstained with cresyl violet. The numbers of BrdU-positive cells in the granule cell layer of every 10th section throughout the entire dentate gyrus were counted under ×400 magnification. Total dentate gyrus volume was determined using Cavalieri’s principle [14], and the cross-sectional areas were determined using an image-analysis system (Multiscan, Fullerton, CA, USA). No changes in the volumes of the granule cell layer of the dentate gyrus were observed in any of the experiments conducted. Hence, the data are expressed as estimates of the total number of BrdU-positive cells. At this point, each sample was given a coded identification number to allow the data to be analyzed in a “blind” manner. The immunohistochemistry data were analyzed using Student’s t test. In this study, we evaluated the memory-enhancing effects of GBT in rats using the passive avoidance test. We observed that the mean response latency of GBT-treated rats (175.82 ± 34.95 s) was significantly increased compared with that of control rats (71.55 ± 32.06 s; p < 0.05; Fig. 1A). GBT treatment also induced cell proliferation in the dentate gyrus. The number of BrdU-labeled cells was significantly higher in the GBT-treated group (1872.81 ± 217.47 per dentate gyrus) than in the control group (938.41 ± 166.61 per dentate gyrus; p < 0.01; Fig. 1B). There were no differences in food intake or body weight between groups after GBT treatment. Moreover, no noticeable changes in actions were observed. Memory has been shown to improve in several laboratory animal models not only after administration of drugs such as taurine [9] and calcineurin activator [13] but also after
M.S. Oh et al. / Neuroscience Letters 379 (2005) 205–208
207
Fig. 1. Effects of GBT treatment on response latency in the passive avoidance test and on cell proliferation in the rat dentate gyrus. The control group (n = 11) received vehicle only and the GBT group (n = 11) was treated with GBT orally (2.0 g/kg/day). The response latency of GBT-treated rats was significantly (p < 0.05) greater than that of control rats (A). There were significantly (p < 0.01) more BrdU-labeled cells in the GBT-treated group than in the control group (B). Representative photographs of the dentate gyrus from the control (C) and GBT (D) groups are shown. The density of BrdU-labeled cells (black puncta) was enhanced in the GBT group.
administration of herbal medicines such as Gingko biloba [22] and Heteropterys aphrodisiaca [6]. GBT is composed of several memory-enhancing herbs. Poria and Ginseng radix have been shown to promote hippocampal long-term potentiation [20]. Polygalae radix alleviates scopolamineinduced impairment of the passive avoidance response in mice [5] and has some protective effects against neuronal death and cognitive impairments in Alzheimer’s disease or other neurodegenerative diseases [19]. Astragali radix has protective effects against anoxic damage to cultured neurons [7]. Angelicae sinensis radix and Ziziphi semen have CNS effects at the receptor level [12]. These constituents of GBT may be responsible for its neural effect. Various factors have been found to modulate the rate of new cell proliferation in the dentate gyrus of rodents, such as physical activity [24], an enriched environment [11], and hippocampal-dependent learning [23]. In addition to cell proliferation, another important aspect of adult neurogenesis is the migration of new cells and their differentiation into neurons and eventual integration into a functional network. Newly generated cells in the adult mouse hippocampus are reported to have neuronal morphology and can display
passive membrane properties, action potentials, and functional synaptic inputs similar to those found in mature dentate granule cells. These findings demonstrate that newly generated cells mature into functional neurons in the adult mammalian brain [25]. Moreover, approximately 9000 new cells are generated in the dentate gyrus each day in young adult rats and within 5–12 days 50% of these cells can be double-labeled with neuron-specific markers [4]. In this regard, the newly proliferated cells stimulated by GBT may differentiate into neurons, but further research is required to determine the neuronal function of GBT. In conclusion, our results show that treatment of rats with GBT not only improves learning and memory but also increases the proliferation of cells in the dentate gyrus of the hippocampus. References [1] D. Bansky, R. Barolet, Chinese Herbal Medicine Formulas and Strategies, Eastland Press, Seattle, 1990, pp. 7–8. [2] O.F. Bueno, M.G. Oliveira, A.C. Pomarico, E.B. Gugliano, A dissociation between the proactive ECS effects on inhibitory avoidance
208
[3] [4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
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