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Luteolin promotes long-term potentiation and improves cognitive functions in chronic cerebral hypoperfused rats
European Journal of Pharmacology 627 (2010) 99–105
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Luteolin promotes long-term potentiation and improves cognitive functions in chronic cerebral hypoperfused rats Bei Xu, Xiao-Xiu Li, Guo-Rong He, Juan-Juan Hu, Xin Mu, Shuo Tian, Guan-Hua Du ⁎ National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China
a r t i c l e
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Article history: Received 22 March 2009 Received in revised form 26 September 2009 Accepted 14 October 2009 Available online 24 October 2009 Keywords: Luteolin Alzheimer's disease Synaptic plasticity Long-term potentiation Basal synaptic transmission Learning and memory Chronic cerebral hypoperfusion 2-vessel occlusion
a b s t r a c t Processes of synaptic plasticity, such as long-term potentiation (LTP), has been considered a cellular correlate of learning and memory and many neurological disorders accompanied by cognitive deficits exhibit abnormal synaptic function. This emerging concept is exemplified by Alzheimer's disease. Mounting evidence suggests that Alzheimer's disease begins with subtle alterations of hippocampal synaptic efficacy prior to frank neuronal degeneration, which make it critical to identify LTP enhancers to slow down or stop the progression of Alzheimer's disease. In this study, we found flavonoid luteolin could enhance basal synaptic transmission and facilitate the induction of LTP by high frequency stimulation in the dental gyrus of rat hippocampus. Furthermore, we investigated the effects of luteolin on chronic cerebral hypoperfusioninduced spatial learning dysfunction and LTP impairment in rat. The results showed chronic cerebral hypoperfusion produced by 2-vessel occlusion significantly impaired spatial learning and memory, and luteolin reversed the learning and memory deficit. 2-vessel occlusion resulted in dramatic inhibition of LTP formation in the hippocampus and luteolin significantly rescued the LTP impairment. These results demonstrate that luteolin not only directly modulates LTP formation, but also protects synapses from the detrimental effects of chronic cerebral hypoperfusion on LTP formation, which may contribute to the protective effects of luteolin on learning and memory. By immunoblotting, we found the effects of luteolin on LTP and memory may due to the activation of cAMP response element-binding protein (CREB). Therefore, flavonoid luteolin shows great potential as a novel treatment agent for protecting synaptic function and enhancing memory in neurodegenerative disorders. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Neurodegenerative disorders, such as Alzheimer's disease, characterized by loss of cells in the brain and spinal cord, are the most common diseases of modern society. The current standard of care for mild to moderate Alzheimer's disease includes treatment with acetylcholinesterase (AChE) inhibitors to improve cognitive function (Lleo et al., 2006), and a N-methyl-D-aspartate (NMDA) receptor antagonist, memantine (Lipton, 2006), has recently been approved for the treatment of advanced Alzheimer's disease in the United States. Currently-used treatments offer a small symptomatic benefit; no treatments to delay or halt the progression of the disease are as yet available. Disease-modifying therapies that target the underlying pathogenic mechanisms represent one of the most exciting approaches to novel drug development for Alzheimer's disease (Citron, 2004; Melnikova, 2007; Skovronsky et al., 2006). Disappointingly, over the past year, two highly anticipated investigational drugs that might have ⁎ Corresponding author. National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 1 Xian Nong Tan Street, Beijing 100050, PR China. Tel./fax: +86 10 63165184. E-mail address: [email protected] (G.-H. Du). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.10.038
begun to meet this need have failed (Becker and Greig, 2008). Although the identification of the molecular constituents of plaques and tangles, amyloid β (Aβ) and tau protein, and the discovery of mutations in their genes in Alzheimer's disease have helped to elucidate the origin of the disease, an explanation of their neurotoxic mechanisms is still required (Sorrentino and Bonavita, 2007). Processes of synaptic plasticity, such as long-term potentiation (LTP), has been considered a cellular correlate of learning and memory and many neurological disorders accompanied by cognitive deficits exhibit abnormal synaptic function (Martin et al., 2000). In its earliest clinical phase, Alzheimer's disease characteristically produces a remarkably pure impairment of memory. Mounting evidence suggests that this syndrome begins with subtle alterations of hippocampal synaptic efficacy prior to frank neuronal degeneration, and that the synaptic dysfunction is caused by diffusible oligomeric assemblies of the Aβ protein (Selkoe, 2002; Shankar et al., 2008). Since it is now evident that synaptic plasticity impairment in hippocampus underlie the earliest symptoms of Alzheimer's disease and Alzheimer's disease is believed to be a synaptic failure, it becomes critical to identify the LTP enhancers to slow down or stop the progression of Alzheimer's disease (Marcello et al., 2008; Small, 2007).
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Flavonoids comprise the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants. Emerging evidence suggests that dietary-derived flavonoids have the potential to improve human memory and neuro-cognitive performance (Cazarolli et al., 2008; Spencer, 2008). In this study, our preliminary results show that flavonoid luteolin promote the induction of LTP by tetanic stimulation in the dental gyrus of rat hippocampus. So we hypothesize that flavonoids exert effects on LTP, and consequently memory and cognitive performance, through their interactions with signalling pathways in LTP. The present study explores the potential of flavonoid luteolin to promote memory and learning in neurodegenerative diseases through their interactions with neuronal signalling pathways pivotal in controlling LTP. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing 250–300 g were bought from National Institute for the Control of Pharmaceutical and Biological Products. Rats were housed in a temperature and humidity-controlled room (temperature: 22± 1 °C, humidity: 60%) with free access to food and water. The rats were kept on a 12-h light/dark cycle and adapted to these conditions for at least 7 days before experiments. All experiments were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of our institute.
Field responses evoking a frequency of 0.033 Hz and 50 ms in duration were acquired, amplified, monitored by a VC-11 memory oscilloscope (Nihon Kohden, Japan) and analyzed with a custom program ‘hippocampus LTP signal collecting and processing system’ (Wu Jiajin). The depth of recording and stimulating electrodes was gently adjusted to maximize the amplitude of the excellular population spike. The stimulation intensity for the tests and train pulses was set to 33% of the maximum population spike amplitude, as determined before the test pulse series. A single test stimulus (100 μs) was delivered at 30 s intervals, and the averaged responses of 5 times were measured every 5 min throughout the experiment. Basal synaptic transmission and high-frequency stimulation induced LTP were tested in normal male Sprague-Dawley rats. Each group of animals were subjected to the following procedure: 45 min recovery, 30 min of baseline recording, i.c.v. injection of 5 μl vehicle or luteolin, 10 min elapsed time, with high-frequency stimulation followed or not, and 60 min of fEPSP recording. High-frequency stimulation consisted of 10 trains at 5 Hz each composed of 5 pulses at 200 Hz. The percentage ratio of absolute population spike amplitude to baseline value was used to describe the population spike amplitude level variation. Chronic cerebral hypoperfused rats receive no treatment during electrophysiological recordings. High-frequency stimulation was exerted right away after base line recording. It was defined as a successful induction of LTP if the amplitude of population spike change exceeded 20%. 2.4. Morris water maze test
Experimental cerebral hypoperfusion was imposed on the animals by permanent bilateral occlusion of the common carotid arteries (2vessel occlusion) as described previously (Annahazi et al., 2007). Prior to surgery, the animals were anesthetized by 400 mg/kg chloralhydrate i.p. The common carotid arteries were exposed via a ventral cervical incision, and separated from their sheaths and vagal nerves. Silk sutures were used for the ligation. The same procedure was performed on the sham group without the actual ligation. Survival rate of the surgery was about 80%. Two days after surgery, the hypoperfused rats were randomly divided into 5 groups. Each group consisted of 12 to 16 animals with identical mean body weights. Daily oral administration of luteolin (50, 150 and 450 mg/kg) or vehicle (0.5% carboxymethylcellulose) started on day 2 post surgery, and it lasted for 5 weeks. During the last 6 days of drug administration, spatial learning and memory was assessed in all rats. During the behavioral test, drugs were administered 40 min before the water maze training.
The spatial memory performances were evaluated using a Morris water maze (Morris, 1984) 31 days after induction of hypoperfusion. The swimming pool of the Morris water maze was a circular water tank, 120 cm in diameter and 50 cm deep. The pool was divided into four quadrants with four starting locations called north (N), east (E), south (S) and west (W) at equal distances on the rim. A platform, 10 cm in diameter, was placed in the center of the northeast quadrant, its top surface being 1.5 cm below the surface of the water. The pool was surrounded by many cues external to the maze and filled with water at 22± 1 °C. Each rat received two trials everyday for 5 consecutive days (maximum trial duration 60 s, 15 s reinforcement on the platform, 30 s recovery period between trials). The starting position for each trial was pseudorandomly chosen and counterbalanced across all experimental groups. Latency to escape onto the hidden platform was recorded. The rats were given a maximum of 60 s to find the hidden platform. If the rat failed to find the platform within 60 s, the training was terminated and a maximum score of 60 s was assigned. On the sixth training day, each rat was subjected to a 60 s probe trial in which the platform was removed and the time spent in the northeast quadrant was recorded. Swimming activities were monitored by a video camera linked to a computer-based image analyzer.
2.3. Electrophysiological recordings
2.5. Morphology
Rats were anesthetized by urethane (1.2 g/kg i.p.) and the body temperature was monitored with a rectal thermometer and maintained at 37 °C by an electrically shielded heating pad. The animals were fixed in a stereotaxic frame (SN-3, Narishige, Japan) according to bregma and lambda in the same horizontal plane. A stainless steel bipolar stimulating electrode was inserted into the perforant path (7.5 mm posterior to bregma, 4.2 mm lateral to midline, and 3.0 mm vertical to dura). A recording electrode was placed into dental gyrus (DG) (3.8 mm posterior to bregma, 2.5 mm lateral to midline and 3.5 mm vertical to dura). Two screws in the occipital bone were used as reference and ground. When the intracerebroventricular (i.c.v.) injections of vehicle or luteolin were needed, a third hole was made to insert a cannula into the lateral ventricle (0.8 mm posterior to the bregma, 1.5 mm lateral to the midline, 3.5 mm from the cranial theca). Recording and stimulating electrodes as cannula were always on the same side of the brain.
Rats were anesthetized with chloral hydrate (350 mg/kg, IP), and then perfused transcardially with normal saline followed by 4% paraformaldehyde. All brains were then fixed in the same fixative at 4 ºC, dehydrated and then embedded in paraffin blocks. Coronal sections of 5 mm were stained with hematoxylin-eosin (HE).
2.2. Chronic cerebral hypoperfusion and luteolin treatment
2.6. Immunoblotting For analysis of cAMP response element-binding protein (CREB) phosphorylation, the hippocampus were defrosted and solubilized by sonication, and equal amounts of protein were analyzed by SDS/PAGE and immunoblotting. Equal loading and transfer of the samples were confirmed by staining the nitrocellulose with Ponceau S. Transfers were blocked for 4 h at room temperature with 5% (wt/vol) nonfat milk in TBS/0.1% Tween 20, and then they were incubated overnight at
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4 °C in the primary antibody phospho-CREB and total CREB diluted in 5% (wt/vol) BSA in TBS/0.1% Tween 20. The primary antibodies used were: phospho-CREB (9198, 1/1000), and total CREB (9197, 1/1000) from Cell Signaling (Beverly, MA). The transfers were rinsed with TBS/0.1% Tween 20 and incubated for 1 h at room temperature in horseradish peroxidase/goat anti-rabbit (Immunology Consultants Laboratory, Newberg, OR), diluted 1/1000 in 5% (wt/vol) BSA in TBS/0.1% Tween 20. The immunoblots were developed with super ECL plus kit (Applygen, china).
2.7. Statistical analysis The data are presented as mean± S.E.M. Group differences of population spike amplitude in electrophysiological recordings and the escape latency in the water maze were analyzed using analysis of variance (ANOVA) with repeated measures followed by Tukey-Kramer test or Dunnett's test. One-way analysis of variance (ANOVA) followed by the Duncan multiple group comparison was used to analyze group differences of probe trials. Statistical significance was accepted at P < 0.05. Statistical analysis was performed using SPSS 11.5 for windows.
Fig. 2. Effects of luteolin on LTP in DG of hippocampus. Rats were injected i.c.v. with 2 nmol (n = 7) and 20 nmol luteolin (n = 7) or vehicle (n = 6) after 30 min of baseline recording followed by high-frequency stimulation 10 min later. Changes in the population spike (PS) amplitude are expressed as the percentage of the mean baseline value (A) and the averages of the population spike amplitude during 60 min after the i. c.v. injection of luteolin or vehicle were calculated as an index of LTP magnitude (B). Asterisks indicate significant differences (P < 0.05) compare to the vehicle group. All data are the mean ± S.E.M.
3. Results 3.1. Effects of luteolin on basal synaptic transmission in normal rats Basal synaptic transmission was promoted by luteolin treatment in the perforant-DG path in normal Sprague-Dawley rats. I.c.v. administration of 2 nmol and 20 nmol luteolin caused a significant increase in basal synaptic transmission with respect to vehicle controls and it persisted for at least 60 min (Fig. 1A). In terms of the population spike height, the average percentage ratios of post- to pre-treatment were 113.68 ± 7.17% for vehicle group, 182.74± 8.55% for 2 nmol luteolin group and 152.71 ± 11.02% for 20 nmol luteolin group (Fig. 1B). There was no significant difference between the 2 nmol and 20 nmol luteolin treatment group (P > 0.05). 3.2. Effects of luteolin on LTP in normal rats
Fig. 1. Effects of luteolin on basal synaptic transmission in DG of hippocampus. Rats were injected i.c.v. with 2 nmol (n = 7) and 20 nmol luteolin (n = 9) or vehicle (n = 6) after 30 min of baseline recording. Time course of changes in the population spike (PS) amplitude is expressed as the percentage of the mean baseline value (A) and the averages of the population spike amplitude during 60 min after the i.c.v. injection of luteolin or vehicle were calculated as an index of basal transmission efficacy (B). Asterisks indicate significant differences (P < 0.05) compare to the vehicle group. All data are the mean ± S.E.M.
LTP is considered to be a good model of how memory is formed at the cellular level (Bliss and Collingridge, 1993). I.c.v. injection of 2 nmol and 20 nmol luteolin facilitated the induction of LTP in perforant pathDG cell synapses in normal Sprague-Dawley rats and the effect last more than 60 min (Fig. 2A). Injection of luteolin 10 min prior to highfrequency stimulation produced a significant stronger LTP (2 nmol: 274.87 ± 18.71%, 20 nmol: 227.87 ± 6.33%) compared to that seen for vehicle group (197.77± 7.34%) (Fig. 2B). No significant difference was
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detected between the 2 nmol and 20 nmol luteolin treatment group (P > 0.05). 3.3. Effects of luteolin on LTP in chronic cerebral hypoperfused Sprague-Dawley rats LTP was blocked following bilateral ligation of the common carotid arteries. Luteolin (150 and 450 mg/kg) restored LTP to the similar extent as that observed in the sham-operated (Fig. 3A). In terms of the population spike height, the average percentage ratios of pre- to posthigh frequency stimulation in sham, ischemia, and ischemia plus luteolin at 50, 150 and 450 mg/kg groups were 195.41 ± 13.58%, 140.37 ± 10.20%, 151.06 ± 5.71%, 185.60 ± 10.57% and 193.22 ± 11.08%, respectively (Fig. 3B). There were differences among these groups (sham-operated and ischemia, 150 mg/kg luteolin and ischemia, 450 mg/kg luteolin and ischemia). 3.4. Effect of luteolin on learning and memory in water maze tests in hypoperfused rats Learning and memory retention of Morris water maze were used to evaluate spatial memory in rats. In the acquisition trials, the shamoperated animals gradually learned the location of the hidden platform, indicated by shorter latencies throughout the test period (Fig. 4A). Chronic cerebral hypoperfusion induced by 2-vessel occlusion resulted in a significant impairment in spatial learning compared to sham-operated controls (P < 0.01, Fig. 4A). 150 mg/kg and 450 mg/kg luteolin-treated animals had shorter mean daily latencies to locate the platform compared to ischemia group (P < 0.05 or < 0.01, Fig. 4A). Luteolin at the dose of 50 mg/kg showed a marginal improvement in learning the task but there was no difference Fig. 4. Effects of luteolin on chronic cerebral hypoperfusion-induced Morris water maze performance deficits in rats. The task was performed with 2 trials per day during 5 days for the acquisition test (A), and performed with 2 trials on day 6 without the platform for retention test (B). Rats were administrated with luteolin (oral administration of luteolin 50, 150 and 450 mg/kg, once per day for 30 days, n = 12–16) or treated with only vehicle (saline 10 ml/kg, n = 14) after 2-vessel occlusion surgery. The shamoperated group (Sham, n = 11) was treated with only saline without induction of hypoperfusion. Significance with Tukey's test following a repeated ANOVA is indicated as *P < 0.05 or **P < 0.01 versus vehicle-treated 2-vessel occlusion group and ##P < 0.01 versus Sham. Vertical lines indicate S.E.M.
compared to 2-vessel occlusion group. In the probe trials, the swimming time percentage in probe quadrant was used to evaluate the retention performance. The sham-operated group and the luteolin-treated group (150 mg/kg and 450 mg/kg) swam longer in target quadrant than 2-vessel occlusion group (P < 0.05 or P < 0.01, Fig. 4B). Swimming speed (path length/escape latency) was used to assess the motor activity of rats in this task and the result showed swimming speed did not differ between groups (data not shown), indicating that locomotor activity did not influence performance in the water-maze task. 3.5. Effect of luteolin on neuron morphology in hypoperfused rats
Fig. 3. Effects of luteolin on LTP in the perforant path-DG synapses of rats with chronic hypoperfusion after 2-vessel occlusion. Changes in the population spike (PS) amplitude are expressed as the percentage of the value of base line for 60 min after the high frequency stimulation (A). Significantly higher levels of average population spike amplitudes in luteolin-treated groups (150 and 450 mg/kg) than those in vehicletreated ischemia group were observed (B). #P < 0.05 when compared to sham-operated group; *P < 0.05 when compared to vehicle-treated 2-vessel occlusion group. All data are the mean ± S.E.M.
In order to see if the chronic cerebral hypoperfusion caused any direct pathological alterations in rats, HE stain examination was used. Marked morphological changes were visualized in the hippocampus of chronic hypoperfused group: neuronal cell loss, nuclei shrinkage, and dark staining of neurons. Relative long-term treatment in hypoperfused rats with luteolin (150 and 450 mg/kg) markedly reduced these pathological changes (Fig. 5). 3.6. Flavonoid luteolin induces CREB phosphorylation in rat hippocampus The results we showed before have demonstrated that luteolin not only directly modulates LTP formation, but also protects synapses from
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Fig. 5. In vivo effect of luteolin on morphologic changes induced by chronic hypoperfusion ischemia in hippocampal regions of rats. Sections from three rats in each group were examined. Vehicle-treated 2-vessel occlusion group showing neuronal loss, shrinkage and marked vacuolar changes in the CA1 areas of the hippocampus comparing to sham rats; luteolin 150 and 450 mg/kg-treated group showed long-term administration of luteolin attenuated these morphological alterations. (Magnification = 400×).
the detrimental effects of chronic cerebral hypoperfusion on LTP formation. This effect of luteolin could translate into a facilitation of learning and memory. CREB, a transcription factor which binds to the promoter regions of many genes associated with memory and synaptic plasticity, has been shown to play a crucial role in the long-term memory formation and LTP induction (Josselyn and Nguyen, 2005). In current study, by immunoblotting we found that luteolin not only facilitated the phosphorylation of CREB in hippocampus of normal rat (Fig. 6A) but also rescued the chronic cerebral hypoperfusion-induced impairment of CREB activation (Fig. 6B). The activation of CREB by luteolin may be the mechanism underlying its effects on LTP facilitation and memory enhancement. 4. Discussion Flavonoids can be classified into flavonols, flavanones, flavones, isoflavones, and anthocyanidins. A number of physiological and pharmacological benefits have been attributed to flavonoids, including protection from cancer, cardiovascular disease, and neurodegenerative disorders (Havsteen, 2002; Middleton et al., 2000). The neuroprotective potential of flavonoids is well reported and dietary supplementation studies, in human and animals, using flavonoid-rich plant or food extracts have highlighted their potentials to influence cognition and
Fig. 6. Luteolin activates CREB in rat hippocampus. (A) Rats were injected i.c.v. with 2 nmol or 20 nmol luteolin and sacrificed 60 min later to remove the hippocampus. (B) Rats were administrated with luteolin (oral administration of luteolin 50, 150 and 450 mg/kg, once per day for 30 days) or treated with only vehicle (saline 10 ml/kg, n = 14) after 2-vessel occlusion surgery. The sham-operated group was treated with only saline without induction of hypoperfusion. Hippocampus from 2 rats in each group was dissected and frozen for Western blotting. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho-CREB along with antibodies to the unphosphorylated form of the protein, demonstrating no changes in overall protein levels. Similar results were obtained in two independent experiments.
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learning (Haque et al., 2006; Wang et al., 2006; Youdim and Joseph, 2001), presumably by protecting vulnerable neurons, enhancing existing neuronal function or by stimulating neuronal regeneration (Luo et al., 2002; Spencer, 2008). In this report we revealed that longterm administration of luteolin improves spatial cognition deficits and neuronal damage induced by chronic cerebral hypoperfusion in rats, which further confirmed the cognitive improving and neuroprotective effects of flavonoids. Evidence has shown that cerebral blood flow reduction correlates with an increasing degree of cognitive impairment in Alzheimer's disease patient (Komatani et al., 1988). In addition, reduced cerebral blood flow has been suggested as an indicator for the progression of Alzheimer's disease (Encinas et al., 2003; Nobili et al., 2001). Experimental findings indicate that decreased cerebral blood flow can lead to cognitive impairment and neuronal injury (Farkas and Luiten, 2001). Although originally described as a rat model for vascular dementia, Chronic cerebral hypoperfusion induced by permanent bilateral common carotid artery occlusion in the rat, is relevant to Alzheimer's disease, since there is also a slowly developing reduction in cerebral blood flow in this disease (Farkas et al., 2007). The ability of luteolin to attenuate memory deficits in 2-vessel occlusion model indicated its therapeutic potential in Alzhermer's disease. Many neurological disorders accompanied by cognitive deficits exhibit abnormal synaptic function. This emerging concept can be exemplified by Alzheimer's disease as Aβ oligomers have been described as the earliest effectors to adversely affect synaptic structure and function (Marcello et al., 2008; Walsh et al., 2002). Mounting evidence suggests that this syndrome begins with subtle alterations of hippocampal synaptic efficacy prior to frank neuronal degeneration (Selkoe, 2002). To analyses the earliest stages in the disorder, methods that can reveal synaptic dysfunction rather than just structural changes are necessary. Two assays for measuring synaptic function are most commonly used: measurement of the magnitude of the fEPSPs after evoked stimulation, which is often referred to as “basal synaptic transmission”, and measurement of the activity-dependent changes in synaptic strength, namely synaptic plasticity, with the most studied experimental form being LTP (Neves et al., 2008). In this study, we found that i.c.v. injection of flavonoid luteolin could enhance both the basal synaptic transmission and the induction of LTP by high-frequency stimulation in the dental gyrus of rat hippocampus. In 2-vessel occlusion model, chronic cerebral hypoperfusion blocked the induction of LTP in the hippocampus of rat. It has been shown that 2-vessel occlusion results in progressive accumulation of Aβ peptides in brain of rats and reproduces features of Aβ biogenesis characteristic of sporadic Alzheimer's disease (Bennett et al., 2000), which may partially account for the impaired synaptic plasticity in this model of neurodegenerative diseases. Long-term administration of luteolin restored LTP to the similar extent as that observed in the sham-operated, demonstrating that luteolin might serve as a LTP enhancer for the treatment of Alzheimer's disease. LTP is widely considered to be one of the major mechanisms underlying memory acquisition, consolidation and storage in the brain and is known to be controlled at the molecular level by the activation of a number of neuronal signalling pathways (Lynch, 2004). Our results showed luteolin induced CREB phosphorylation both in normal rat and experimental rat model of neurodegenerative diseases. Evidence from Aplysia, Drosophila, mice, and rats shows that CREB-dependent transcription is required for the cellular events underlying LTP and long-term memory (Silva et al., 1998). CREB loss-of-function mutants have impairments in long-term memory, whereas CREB gain-offunction mutants show enhanced long-term memory. CREB activation may represent a point of convergence since it has been shown to be activated downstream of ERK, PKA, PKC, and CaMKII. Based on our results, luteolin might be considered as a CREB-based memory enhancers. Animal models of Aβ pathology show that memory defects develop well before any signs of neurodegeneration. As neurodegenera-
tion proceeds, however, individual neurons die more or less randomly within particular brain regions. In the initial stages of Alzheimer's disease, then, CREB-based memory enhancer luteolin might be a particularly effective treatment by improving the Aβ-impaired LTP induction. The clinical outcome of such treatment would most likely be a delay in progressive memory loss, thereby delaying the duration of the period during which patients require assisted living. Here again, quality of life would be extended, and economic savings could be significant. In conclusion, our results demonstrated that luteolin could attenuate cognitive deficit and synaptic plasticity impairment induced by chronic cerebral hypoperfusion and the activation of the signaling pathways implicated in the development LTP might be involved in luteolininduced neuroprotection. 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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Luteolin promotes long-term potentiation and improves cognitive functions in chronic cerebral hypoperfused rats Bei Xu, Xiao-Xiu Li, Guo-Rong He, Juan-Juan Hu, Xin Mu, Shuo Tian, Guan-Hua Du ⁎ National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 22 March 2009 Received in revised form 26 September 2009 Accepted 14 October 2009 Available online 24 October 2009 Keywords: Luteolin Alzheimer's disease Synaptic plasticity Long-term potentiation Basal synaptic transmission Learning and memory Chronic cerebral hypoperfusion 2-vessel occlusion
a b s t r a c t Processes of synaptic plasticity, such as long-term potentiation (LTP), has been considered a cellular correlate of learning and memory and many neurological disorders accompanied by cognitive deficits exhibit abnormal synaptic function. This emerging concept is exemplified by Alzheimer's disease. Mounting evidence suggests that Alzheimer's disease begins with subtle alterations of hippocampal synaptic efficacy prior to frank neuronal degeneration, which make it critical to identify LTP enhancers to slow down or stop the progression of Alzheimer's disease. In this study, we found flavonoid luteolin could enhance basal synaptic transmission and facilitate the induction of LTP by high frequency stimulation in the dental gyrus of rat hippocampus. Furthermore, we investigated the effects of luteolin on chronic cerebral hypoperfusioninduced spatial learning dysfunction and LTP impairment in rat. The results showed chronic cerebral hypoperfusion produced by 2-vessel occlusion significantly impaired spatial learning and memory, and luteolin reversed the learning and memory deficit. 2-vessel occlusion resulted in dramatic inhibition of LTP formation in the hippocampus and luteolin significantly rescued the LTP impairment. These results demonstrate that luteolin not only directly modulates LTP formation, but also protects synapses from the detrimental effects of chronic cerebral hypoperfusion on LTP formation, which may contribute to the protective effects of luteolin on learning and memory. By immunoblotting, we found the effects of luteolin on LTP and memory may due to the activation of cAMP response element-binding protein (CREB). Therefore, flavonoid luteolin shows great potential as a novel treatment agent for protecting synaptic function and enhancing memory in neurodegenerative disorders. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Neurodegenerative disorders, such as Alzheimer's disease, characterized by loss of cells in the brain and spinal cord, are the most common diseases of modern society. The current standard of care for mild to moderate Alzheimer's disease includes treatment with acetylcholinesterase (AChE) inhibitors to improve cognitive function (Lleo et al., 2006), and a N-methyl-D-aspartate (NMDA) receptor antagonist, memantine (Lipton, 2006), has recently been approved for the treatment of advanced Alzheimer's disease in the United States. Currently-used treatments offer a small symptomatic benefit; no treatments to delay or halt the progression of the disease are as yet available. Disease-modifying therapies that target the underlying pathogenic mechanisms represent one of the most exciting approaches to novel drug development for Alzheimer's disease (Citron, 2004; Melnikova, 2007; Skovronsky et al., 2006). Disappointingly, over the past year, two highly anticipated investigational drugs that might have ⁎ Corresponding author. National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 1 Xian Nong Tan Street, Beijing 100050, PR China. Tel./fax: +86 10 63165184. E-mail address: [email protected] (G.-H. Du). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.10.038
begun to meet this need have failed (Becker and Greig, 2008). Although the identification of the molecular constituents of plaques and tangles, amyloid β (Aβ) and tau protein, and the discovery of mutations in their genes in Alzheimer's disease have helped to elucidate the origin of the disease, an explanation of their neurotoxic mechanisms is still required (Sorrentino and Bonavita, 2007). Processes of synaptic plasticity, such as long-term potentiation (LTP), has been considered a cellular correlate of learning and memory and many neurological disorders accompanied by cognitive deficits exhibit abnormal synaptic function (Martin et al., 2000). In its earliest clinical phase, Alzheimer's disease characteristically produces a remarkably pure impairment of memory. Mounting evidence suggests that this syndrome begins with subtle alterations of hippocampal synaptic efficacy prior to frank neuronal degeneration, and that the synaptic dysfunction is caused by diffusible oligomeric assemblies of the Aβ protein (Selkoe, 2002; Shankar et al., 2008). Since it is now evident that synaptic plasticity impairment in hippocampus underlie the earliest symptoms of Alzheimer's disease and Alzheimer's disease is believed to be a synaptic failure, it becomes critical to identify the LTP enhancers to slow down or stop the progression of Alzheimer's disease (Marcello et al., 2008; Small, 2007).
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Flavonoids comprise the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants. Emerging evidence suggests that dietary-derived flavonoids have the potential to improve human memory and neuro-cognitive performance (Cazarolli et al., 2008; Spencer, 2008). In this study, our preliminary results show that flavonoid luteolin promote the induction of LTP by tetanic stimulation in the dental gyrus of rat hippocampus. So we hypothesize that flavonoids exert effects on LTP, and consequently memory and cognitive performance, through their interactions with signalling pathways in LTP. The present study explores the potential of flavonoid luteolin to promote memory and learning in neurodegenerative diseases through their interactions with neuronal signalling pathways pivotal in controlling LTP. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing 250–300 g were bought from National Institute for the Control of Pharmaceutical and Biological Products. Rats were housed in a temperature and humidity-controlled room (temperature: 22± 1 °C, humidity: 60%) with free access to food and water. The rats were kept on a 12-h light/dark cycle and adapted to these conditions for at least 7 days before experiments. All experiments were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of our institute.
Field responses evoking a frequency of 0.033 Hz and 50 ms in duration were acquired, amplified, monitored by a VC-11 memory oscilloscope (Nihon Kohden, Japan) and analyzed with a custom program ‘hippocampus LTP signal collecting and processing system’ (Wu Jiajin). The depth of recording and stimulating electrodes was gently adjusted to maximize the amplitude of the excellular population spike. The stimulation intensity for the tests and train pulses was set to 33% of the maximum population spike amplitude, as determined before the test pulse series. A single test stimulus (100 μs) was delivered at 30 s intervals, and the averaged responses of 5 times were measured every 5 min throughout the experiment. Basal synaptic transmission and high-frequency stimulation induced LTP were tested in normal male Sprague-Dawley rats. Each group of animals were subjected to the following procedure: 45 min recovery, 30 min of baseline recording, i.c.v. injection of 5 μl vehicle or luteolin, 10 min elapsed time, with high-frequency stimulation followed or not, and 60 min of fEPSP recording. High-frequency stimulation consisted of 10 trains at 5 Hz each composed of 5 pulses at 200 Hz. The percentage ratio of absolute population spike amplitude to baseline value was used to describe the population spike amplitude level variation. Chronic cerebral hypoperfused rats receive no treatment during electrophysiological recordings. High-frequency stimulation was exerted right away after base line recording. It was defined as a successful induction of LTP if the amplitude of population spike change exceeded 20%. 2.4. Morris water maze test
Experimental cerebral hypoperfusion was imposed on the animals by permanent bilateral occlusion of the common carotid arteries (2vessel occlusion) as described previously (Annahazi et al., 2007). Prior to surgery, the animals were anesthetized by 400 mg/kg chloralhydrate i.p. The common carotid arteries were exposed via a ventral cervical incision, and separated from their sheaths and vagal nerves. Silk sutures were used for the ligation. The same procedure was performed on the sham group without the actual ligation. Survival rate of the surgery was about 80%. Two days after surgery, the hypoperfused rats were randomly divided into 5 groups. Each group consisted of 12 to 16 animals with identical mean body weights. Daily oral administration of luteolin (50, 150 and 450 mg/kg) or vehicle (0.5% carboxymethylcellulose) started on day 2 post surgery, and it lasted for 5 weeks. During the last 6 days of drug administration, spatial learning and memory was assessed in all rats. During the behavioral test, drugs were administered 40 min before the water maze training.
The spatial memory performances were evaluated using a Morris water maze (Morris, 1984) 31 days after induction of hypoperfusion. The swimming pool of the Morris water maze was a circular water tank, 120 cm in diameter and 50 cm deep. The pool was divided into four quadrants with four starting locations called north (N), east (E), south (S) and west (W) at equal distances on the rim. A platform, 10 cm in diameter, was placed in the center of the northeast quadrant, its top surface being 1.5 cm below the surface of the water. The pool was surrounded by many cues external to the maze and filled with water at 22± 1 °C. Each rat received two trials everyday for 5 consecutive days (maximum trial duration 60 s, 15 s reinforcement on the platform, 30 s recovery period between trials). The starting position for each trial was pseudorandomly chosen and counterbalanced across all experimental groups. Latency to escape onto the hidden platform was recorded. The rats were given a maximum of 60 s to find the hidden platform. If the rat failed to find the platform within 60 s, the training was terminated and a maximum score of 60 s was assigned. On the sixth training day, each rat was subjected to a 60 s probe trial in which the platform was removed and the time spent in the northeast quadrant was recorded. Swimming activities were monitored by a video camera linked to a computer-based image analyzer.
2.3. Electrophysiological recordings
2.5. Morphology
Rats were anesthetized by urethane (1.2 g/kg i.p.) and the body temperature was monitored with a rectal thermometer and maintained at 37 °C by an electrically shielded heating pad. The animals were fixed in a stereotaxic frame (SN-3, Narishige, Japan) according to bregma and lambda in the same horizontal plane. A stainless steel bipolar stimulating electrode was inserted into the perforant path (7.5 mm posterior to bregma, 4.2 mm lateral to midline, and 3.0 mm vertical to dura). A recording electrode was placed into dental gyrus (DG) (3.8 mm posterior to bregma, 2.5 mm lateral to midline and 3.5 mm vertical to dura). Two screws in the occipital bone were used as reference and ground. When the intracerebroventricular (i.c.v.) injections of vehicle or luteolin were needed, a third hole was made to insert a cannula into the lateral ventricle (0.8 mm posterior to the bregma, 1.5 mm lateral to the midline, 3.5 mm from the cranial theca). Recording and stimulating electrodes as cannula were always on the same side of the brain.
Rats were anesthetized with chloral hydrate (350 mg/kg, IP), and then perfused transcardially with normal saline followed by 4% paraformaldehyde. All brains were then fixed in the same fixative at 4 ºC, dehydrated and then embedded in paraffin blocks. Coronal sections of 5 mm were stained with hematoxylin-eosin (HE).
2.2. Chronic cerebral hypoperfusion and luteolin treatment
2.6. Immunoblotting For analysis of cAMP response element-binding protein (CREB) phosphorylation, the hippocampus were defrosted and solubilized by sonication, and equal amounts of protein were analyzed by SDS/PAGE and immunoblotting. Equal loading and transfer of the samples were confirmed by staining the nitrocellulose with Ponceau S. Transfers were blocked for 4 h at room temperature with 5% (wt/vol) nonfat milk in TBS/0.1% Tween 20, and then they were incubated overnight at
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4 °C in the primary antibody phospho-CREB and total CREB diluted in 5% (wt/vol) BSA in TBS/0.1% Tween 20. The primary antibodies used were: phospho-CREB (9198, 1/1000), and total CREB (9197, 1/1000) from Cell Signaling (Beverly, MA). The transfers were rinsed with TBS/0.1% Tween 20 and incubated for 1 h at room temperature in horseradish peroxidase/goat anti-rabbit (Immunology Consultants Laboratory, Newberg, OR), diluted 1/1000 in 5% (wt/vol) BSA in TBS/0.1% Tween 20. The immunoblots were developed with super ECL plus kit (Applygen, china).
2.7. Statistical analysis The data are presented as mean± S.E.M. Group differences of population spike amplitude in electrophysiological recordings and the escape latency in the water maze were analyzed using analysis of variance (ANOVA) with repeated measures followed by Tukey-Kramer test or Dunnett's test. One-way analysis of variance (ANOVA) followed by the Duncan multiple group comparison was used to analyze group differences of probe trials. Statistical significance was accepted at P < 0.05. Statistical analysis was performed using SPSS 11.5 for windows.
Fig. 2. Effects of luteolin on LTP in DG of hippocampus. Rats were injected i.c.v. with 2 nmol (n = 7) and 20 nmol luteolin (n = 7) or vehicle (n = 6) after 30 min of baseline recording followed by high-frequency stimulation 10 min later. Changes in the population spike (PS) amplitude are expressed as the percentage of the mean baseline value (A) and the averages of the population spike amplitude during 60 min after the i. c.v. injection of luteolin or vehicle were calculated as an index of LTP magnitude (B). Asterisks indicate significant differences (P < 0.05) compare to the vehicle group. All data are the mean ± S.E.M.
3. Results 3.1. Effects of luteolin on basal synaptic transmission in normal rats Basal synaptic transmission was promoted by luteolin treatment in the perforant-DG path in normal Sprague-Dawley rats. I.c.v. administration of 2 nmol and 20 nmol luteolin caused a significant increase in basal synaptic transmission with respect to vehicle controls and it persisted for at least 60 min (Fig. 1A). In terms of the population spike height, the average percentage ratios of post- to pre-treatment were 113.68 ± 7.17% for vehicle group, 182.74± 8.55% for 2 nmol luteolin group and 152.71 ± 11.02% for 20 nmol luteolin group (Fig. 1B). There was no significant difference between the 2 nmol and 20 nmol luteolin treatment group (P > 0.05). 3.2. Effects of luteolin on LTP in normal rats
Fig. 1. Effects of luteolin on basal synaptic transmission in DG of hippocampus. Rats were injected i.c.v. with 2 nmol (n = 7) and 20 nmol luteolin (n = 9) or vehicle (n = 6) after 30 min of baseline recording. Time course of changes in the population spike (PS) amplitude is expressed as the percentage of the mean baseline value (A) and the averages of the population spike amplitude during 60 min after the i.c.v. injection of luteolin or vehicle were calculated as an index of basal transmission efficacy (B). Asterisks indicate significant differences (P < 0.05) compare to the vehicle group. All data are the mean ± S.E.M.
LTP is considered to be a good model of how memory is formed at the cellular level (Bliss and Collingridge, 1993). I.c.v. injection of 2 nmol and 20 nmol luteolin facilitated the induction of LTP in perforant pathDG cell synapses in normal Sprague-Dawley rats and the effect last more than 60 min (Fig. 2A). Injection of luteolin 10 min prior to highfrequency stimulation produced a significant stronger LTP (2 nmol: 274.87 ± 18.71%, 20 nmol: 227.87 ± 6.33%) compared to that seen for vehicle group (197.77± 7.34%) (Fig. 2B). No significant difference was
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detected between the 2 nmol and 20 nmol luteolin treatment group (P > 0.05). 3.3. Effects of luteolin on LTP in chronic cerebral hypoperfused Sprague-Dawley rats LTP was blocked following bilateral ligation of the common carotid arteries. Luteolin (150 and 450 mg/kg) restored LTP to the similar extent as that observed in the sham-operated (Fig. 3A). In terms of the population spike height, the average percentage ratios of pre- to posthigh frequency stimulation in sham, ischemia, and ischemia plus luteolin at 50, 150 and 450 mg/kg groups were 195.41 ± 13.58%, 140.37 ± 10.20%, 151.06 ± 5.71%, 185.60 ± 10.57% and 193.22 ± 11.08%, respectively (Fig. 3B). There were differences among these groups (sham-operated and ischemia, 150 mg/kg luteolin and ischemia, 450 mg/kg luteolin and ischemia). 3.4. Effect of luteolin on learning and memory in water maze tests in hypoperfused rats Learning and memory retention of Morris water maze were used to evaluate spatial memory in rats. In the acquisition trials, the shamoperated animals gradually learned the location of the hidden platform, indicated by shorter latencies throughout the test period (Fig. 4A). Chronic cerebral hypoperfusion induced by 2-vessel occlusion resulted in a significant impairment in spatial learning compared to sham-operated controls (P < 0.01, Fig. 4A). 150 mg/kg and 450 mg/kg luteolin-treated animals had shorter mean daily latencies to locate the platform compared to ischemia group (P < 0.05 or < 0.01, Fig. 4A). Luteolin at the dose of 50 mg/kg showed a marginal improvement in learning the task but there was no difference Fig. 4. Effects of luteolin on chronic cerebral hypoperfusion-induced Morris water maze performance deficits in rats. The task was performed with 2 trials per day during 5 days for the acquisition test (A), and performed with 2 trials on day 6 without the platform for retention test (B). Rats were administrated with luteolin (oral administration of luteolin 50, 150 and 450 mg/kg, once per day for 30 days, n = 12–16) or treated with only vehicle (saline 10 ml/kg, n = 14) after 2-vessel occlusion surgery. The shamoperated group (Sham, n = 11) was treated with only saline without induction of hypoperfusion. Significance with Tukey's test following a repeated ANOVA is indicated as *P < 0.05 or **P < 0.01 versus vehicle-treated 2-vessel occlusion group and ##P < 0.01 versus Sham. Vertical lines indicate S.E.M.
compared to 2-vessel occlusion group. In the probe trials, the swimming time percentage in probe quadrant was used to evaluate the retention performance. The sham-operated group and the luteolin-treated group (150 mg/kg and 450 mg/kg) swam longer in target quadrant than 2-vessel occlusion group (P < 0.05 or P < 0.01, Fig. 4B). Swimming speed (path length/escape latency) was used to assess the motor activity of rats in this task and the result showed swimming speed did not differ between groups (data not shown), indicating that locomotor activity did not influence performance in the water-maze task. 3.5. Effect of luteolin on neuron morphology in hypoperfused rats
Fig. 3. Effects of luteolin on LTP in the perforant path-DG synapses of rats with chronic hypoperfusion after 2-vessel occlusion. Changes in the population spike (PS) amplitude are expressed as the percentage of the value of base line for 60 min after the high frequency stimulation (A). Significantly higher levels of average population spike amplitudes in luteolin-treated groups (150 and 450 mg/kg) than those in vehicletreated ischemia group were observed (B). #P < 0.05 when compared to sham-operated group; *P < 0.05 when compared to vehicle-treated 2-vessel occlusion group. All data are the mean ± S.E.M.
In order to see if the chronic cerebral hypoperfusion caused any direct pathological alterations in rats, HE stain examination was used. Marked morphological changes were visualized in the hippocampus of chronic hypoperfused group: neuronal cell loss, nuclei shrinkage, and dark staining of neurons. Relative long-term treatment in hypoperfused rats with luteolin (150 and 450 mg/kg) markedly reduced these pathological changes (Fig. 5). 3.6. Flavonoid luteolin induces CREB phosphorylation in rat hippocampus The results we showed before have demonstrated that luteolin not only directly modulates LTP formation, but also protects synapses from
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Fig. 5. In vivo effect of luteolin on morphologic changes induced by chronic hypoperfusion ischemia in hippocampal regions of rats. Sections from three rats in each group were examined. Vehicle-treated 2-vessel occlusion group showing neuronal loss, shrinkage and marked vacuolar changes in the CA1 areas of the hippocampus comparing to sham rats; luteolin 150 and 450 mg/kg-treated group showed long-term administration of luteolin attenuated these morphological alterations. (Magnification = 400×).
the detrimental effects of chronic cerebral hypoperfusion on LTP formation. This effect of luteolin could translate into a facilitation of learning and memory. CREB, a transcription factor which binds to the promoter regions of many genes associated with memory and synaptic plasticity, has been shown to play a crucial role in the long-term memory formation and LTP induction (Josselyn and Nguyen, 2005). In current study, by immunoblotting we found that luteolin not only facilitated the phosphorylation of CREB in hippocampus of normal rat (Fig. 6A) but also rescued the chronic cerebral hypoperfusion-induced impairment of CREB activation (Fig. 6B). The activation of CREB by luteolin may be the mechanism underlying its effects on LTP facilitation and memory enhancement. 4. Discussion Flavonoids can be classified into flavonols, flavanones, flavones, isoflavones, and anthocyanidins. A number of physiological and pharmacological benefits have been attributed to flavonoids, including protection from cancer, cardiovascular disease, and neurodegenerative disorders (Havsteen, 2002; Middleton et al., 2000). The neuroprotective potential of flavonoids is well reported and dietary supplementation studies, in human and animals, using flavonoid-rich plant or food extracts have highlighted their potentials to influence cognition and
Fig. 6. Luteolin activates CREB in rat hippocampus. (A) Rats were injected i.c.v. with 2 nmol or 20 nmol luteolin and sacrificed 60 min later to remove the hippocampus. (B) Rats were administrated with luteolin (oral administration of luteolin 50, 150 and 450 mg/kg, once per day for 30 days) or treated with only vehicle (saline 10 ml/kg, n = 14) after 2-vessel occlusion surgery. The sham-operated group was treated with only saline without induction of hypoperfusion. Hippocampus from 2 rats in each group was dissected and frozen for Western blotting. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho-CREB along with antibodies to the unphosphorylated form of the protein, demonstrating no changes in overall protein levels. Similar results were obtained in two independent experiments.
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learning (Haque et al., 2006; Wang et al., 2006; Youdim and Joseph, 2001), presumably by protecting vulnerable neurons, enhancing existing neuronal function or by stimulating neuronal regeneration (Luo et al., 2002; Spencer, 2008). In this report we revealed that longterm administration of luteolin improves spatial cognition deficits and neuronal damage induced by chronic cerebral hypoperfusion in rats, which further confirmed the cognitive improving and neuroprotective effects of flavonoids. Evidence has shown that cerebral blood flow reduction correlates with an increasing degree of cognitive impairment in Alzheimer's disease patient (Komatani et al., 1988). In addition, reduced cerebral blood flow has been suggested as an indicator for the progression of Alzheimer's disease (Encinas et al., 2003; Nobili et al., 2001). Experimental findings indicate that decreased cerebral blood flow can lead to cognitive impairment and neuronal injury (Farkas and Luiten, 2001). Although originally described as a rat model for vascular dementia, Chronic cerebral hypoperfusion induced by permanent bilateral common carotid artery occlusion in the rat, is relevant to Alzheimer's disease, since there is also a slowly developing reduction in cerebral blood flow in this disease (Farkas et al., 2007). The ability of luteolin to attenuate memory deficits in 2-vessel occlusion model indicated its therapeutic potential in Alzhermer's disease. Many neurological disorders accompanied by cognitive deficits exhibit abnormal synaptic function. This emerging concept can be exemplified by Alzheimer's disease as Aβ oligomers have been described as the earliest effectors to adversely affect synaptic structure and function (Marcello et al., 2008; Walsh et al., 2002). Mounting evidence suggests that this syndrome begins with subtle alterations of hippocampal synaptic efficacy prior to frank neuronal degeneration (Selkoe, 2002). To analyses the earliest stages in the disorder, methods that can reveal synaptic dysfunction rather than just structural changes are necessary. Two assays for measuring synaptic function are most commonly used: measurement of the magnitude of the fEPSPs after evoked stimulation, which is often referred to as “basal synaptic transmission”, and measurement of the activity-dependent changes in synaptic strength, namely synaptic plasticity, with the most studied experimental form being LTP (Neves et al., 2008). In this study, we found that i.c.v. injection of flavonoid luteolin could enhance both the basal synaptic transmission and the induction of LTP by high-frequency stimulation in the dental gyrus of rat hippocampus. In 2-vessel occlusion model, chronic cerebral hypoperfusion blocked the induction of LTP in the hippocampus of rat. It has been shown that 2-vessel occlusion results in progressive accumulation of Aβ peptides in brain of rats and reproduces features of Aβ biogenesis characteristic of sporadic Alzheimer's disease (Bennett et al., 2000), which may partially account for the impaired synaptic plasticity in this model of neurodegenerative diseases. Long-term administration of luteolin restored LTP to the similar extent as that observed in the sham-operated, demonstrating that luteolin might serve as a LTP enhancer for the treatment of Alzheimer's disease. LTP is widely considered to be one of the major mechanisms underlying memory acquisition, consolidation and storage in the brain and is known to be controlled at the molecular level by the activation of a number of neuronal signalling pathways (Lynch, 2004). Our results showed luteolin induced CREB phosphorylation both in normal rat and experimental rat model of neurodegenerative diseases. Evidence from Aplysia, Drosophila, mice, and rats shows that CREB-dependent transcription is required for the cellular events underlying LTP and long-term memory (Silva et al., 1998). CREB loss-of-function mutants have impairments in long-term memory, whereas CREB gain-offunction mutants show enhanced long-term memory. CREB activation may represent a point of convergence since it has been shown to be activated downstream of ERK, PKA, PKC, and CaMKII. Based on our results, luteolin might be considered as a CREB-based memory enhancers. Animal models of Aβ pathology show that memory defects develop well before any signs of neurodegeneration. As neurodegenera-
tion proceeds, however, individual neurons die more or less randomly within particular brain regions. In the initial stages of Alzheimer's disease, then, CREB-based memory enhancer luteolin might be a particularly effective treatment by improving the Aβ-impaired LTP induction. The clinical outcome of such treatment would most likely be a delay in progressive memory loss, thereby delaying the duration of the period during which patients require assisted living. Here again, quality of life would be extended, and economic savings could be significant. In conclusion, our results demonstrated that luteolin could attenuate cognitive deficit and synaptic plasticity impairment induced by chronic cerebral hypoperfusion and the activation of the signaling pathways implicated in the development LTP might be involved in luteolininduced neuroprotection. 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