Melatonin Improves Cognitive Deficits via Restoration of Cholinergic

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Melatonin improves cognitive deficits via restoration of cholinergic dysfunction in a mouse model of scopolamine-induced amnesia. Bai Hui Chen, Joon Ha Park, Dae Won Kim, Jinseu Park, Soo Young Choi, In Hye Kim, Jeong Hwi Cho, Tae-Kyeong Lee, Jae Chul Lee, Choong-Hyun Lee, In Koo Hwang, YoungMyeong Kim, Bing Chun Yan, Il-Jun Kang, Bich Na Shin, Yun Lyul Lee, Myoung Cheol Shin, Jun Hwi Cho, Young Joo Lee, Yong Hwan Jeon, Moo-Ho Won, and Ji Hyeon Ahn ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00278 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017

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ACS Chemical Neuroscience

Shin, Myoung Cheol; Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 24341, South Korea Cho, Jun Hwi; Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 24341, South Korea Lee, Young Joo; Department of Emergency Medicine, Seoul Hospital, College of Medicine, Sooncheonhyang University, Seoul 04401, South Korea. Jeon, Yong Hwan ; Department of Radiology, School of Medicine, Kangwon National University, Chuncheon 24289, South Korea Won, Moo-Ho; Kangwon National University, Department of Neurobiology, School of Medicine, Ahn, Ji Hyeon; Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chuncheon 24252, South Korea

ACS Paragon Plus Environment


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Melatonin improves cognitive deficits via restoration of cholinergic dysfunction in a mouse model of scopolamine-induced amnesia

Bai Hui Chen1, Joon Ha Park2, Dae Won Kim3, Jinseu Park2, Soo Young Choi2, In Hye Kim4, Jeong Hwi Cho4, Tae-Kyeong Lee4, Jae Chul Lee4, Choong-Hyun Lee5, In Koo Hwang6, Young-Myeong Kim7, Bing Chun Yan8, Il Jun Kang9, Bich Na Shin10, Yun Lyul Lee10, Myoung Cheol Shin11, Jun Hwi Cho11, Young Joo Lee12, Yong Hwan Jeon13, Moo-Ho Won4*, Ji Hyeon Ahn2*

1

Department of Histology and Embryology, Institute of Neuroscience, Wenzhou Medical University, Wenzhou,

Zhejiang 325035, P.R. China 2

Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University,

Chuncheon 24252, South Korea 3

Department of Biochemistry and Molecular Biology, and Research Institute of Oral Sciences, College of Dentistry,

Kangnung-Wonju National University, Gangneung 25457, South Korea 4

Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 24341, South Korea

5

Department of Pharmacy, College of Pharmacy, Dankook University, Cheonan 31116, South Korea

6

Department of Anatomy and Cell Biology, College of Veterinary Medicine, and Research Institute for Veterinary

Science, Seoul National University, Seoul 08826, South Korea 7

Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University,

Chuncheon, South Korea 8

Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment

of Senile Diseases, Yangzhou 225001, People’s Republic of China 9

Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, South Korea

10

Department of Physiology, College of Medicine, and Institute of Neurodegeneration and Neuroregeneration,

Hallym University, Chuncheon 24252, South Korea 11

Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 24341,

South Korea 12

Department of Emergency Medicine, Seoul Hospital, College of Medicine, Sooncheonhyang University, Seoul

04401, South Korea. 13

Department of Radiology, School of Medicine, Kangwon National University, Chuncheon 24289, South Korea


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ABSTRACT: Melatonin is known to improve cognitive deficits and its functions have been studied in various disease models, including Alzheimer's disease. In this study, we investigated effects of melatonin on cognition and the cholinergic system of the septum and hippocampus in a mouse model of scopolamine-induced amnesia. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were administered intraperitoneally to mice for 2 and 4 weeks. The Morris water maze and passive avoidance tests revealed that both treatments of scopolamine significantly impaired spatial learning and memory; however, 2- and 4-week melatonin treatments significantly improved the spatial learning and memory. In addition, scopolamine treatments significantly decreased protein levels and immunoreactivities of choline acetyltransferase (ChAT), high-affinity choline transporter (CHT), vesicular acetylcholine transporter (VAChT), and muscarinic acetylcholine receptor M1 (M1R) in the septum and hippocampus. However, the treatments with melatonin resulted in increased ChAT-, CHT-, VAChT- and M1Rimmunoreactivities and their protein levels in the septum and hippocampus. Our results demonstrate that melatonin treatment is effective in improving the cognitive deficits via restoration of the cholinergic system in the septum and hippocampus of a mouse model of scopolamine-induced amnesia.

KEYWOARDS: cholinergic degeneration; cognitive deficits; hippocampus; medial septum; melatonin; neurohormone



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■ INTRODUCTION

Alzheimer's disease (AD), a chronic neurodegenerative disease and a major global health problem, causes deficits in cognition including memory and attention 1,2, the degree of which is associated to the severity of the disease 3,4. So far, its mechanisms remain unclear. The cholinergic system in the hippocampus plays a crucial role in memory formation and cognition, and a reduction in its function is correlated with AD, resulting in cognitive impairment 5-8. Cholinergic neurons, which use the neurotransmitter acetylcholine (ACh), are within the nucleus basalis and septal diagonal band complex of the basal forebrain, and provide the main source of cholinergic innervation to the hippocampus as well as the cerebral cortex, playing a key role in attention and memory function 9,10. Neurotransmitters transmit signals from neurons of the basal forebrain to those of the hippocampus; this process includes synthesis, packaging, secretion, and removal of neurotransmitters

11

. Among the

neurotransmitters, newly synthesized ACh from choline and acetylcoenzyme A by choline acetyltransferase (ChAT) is transported into synaptic vesicles from the cytoplasm by vesicular acetylcholine transporter (VAChT), and the ACh stored in synaptic vesicles is released into the synaptic cleft when a neuron fires. Next, some ACh is hydrolyzed by acetylcholinesterase (AChE) to yield choline and acetate, and the choline is taken up by highaffinity choline transporter (CHT) at the presynaptic plasma membrane and is again used for ACh synthesis by ChAT 12. Hence, an abnormality in any one of these processes may lead to cholinergic hypofunction. For example, the dysfunction of the cholinergic system in the basal forebrain, cortical regions, and hippocampus is an important facet of AD 13. Scopolamine, a blocker of muscarinic ACh receptors, induces the dysregulation of cholinergic signals in the central nervous system, leading to cognitive deficits 14. Scopolamine-induced models of amnesia are widely used to explore memory dysfunction. Scopolamine administration in rats for 14 days decreases the expression of cholinergic transmitters and causes memory impairment after 14 days of scopolamine administration in rats 15. In addition, scopolamine influences reactive oxygen species (ROS) levels, antioxidant capacity, and neurogenesis in the hippocampus 16. Melatonin (N-acetyl-5-methoxytryptamine), a neurohormone produced by the pineal gland and extrapineal tissues such as the retina and liver 17, influences circadian rhythms and sleep homeostasis, and plays key roles in the regulation of behavioral modulation and aging deficits

20

18,19

. Furthermore, melatonin is known to improve cognitive

and to ameliorate cholinergic neuron degeneration in the hippocampus


19

. In addition, it has been

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reported that melatonin improves memory and synaptic function by reducing AChE activity which is related to enhancement of cholinergic activity 21, and by regulating the EPACs/miR-124/Egr1 pathway, which is involved in synaptic remodeling and spine restructuring after scopolamine treatment 22,23. Furthermore, melatonin displays a pronounced positive effect on behavior in AD 24. However, few published studies have addressed the effects of melatonin on scopolamine-induced cholinergic degeneration, particularly its long-term positive effects. The present study therefore undertook the effects of melatonin treatment on cognitive deficits and cholinergic system hypofunction in the mouse medial septum and hippocampus in a scopolamine-induced mouse model of amnesia.

■ RESULTS AND DISCUSSION

Cognition, and learning and memory. To elucidate cognitive function, we performed Morris water maze test and passive avoidance test (Fig. 1). First, in the Morris water maze test, one-way ANOVA and post-hoc test revealed significant melatonin effects on the escape latency (F (4, 100) = 331.93, p < 0.0001) (Fig. 1a, b). Escape latency in the 2 wk-sco group was significantly increased by 33.5 % compared with that in the control group. However, escape latency in the 2 wk-sco-mela group was significantly decreased by 20.6 % compared with that in the 2 wk-sco group. In the 4-sco group, escape latency was significantly increased by 20.7 % compared with that in the 2-sco group. However, in the 4 wk-sco-mela group, escape latency was significantly decreased by 36.2 % compared with that in the 4-sco group; the escape latency was similar to that in the 2 wk-sco-mela group. Second, in the passive avoidance test, one-way ANOVA and post-hoc test revealed significant melatonin effects on the latency (F (4, 29) = 273.94, p < 0.0001) (Fig. 1c). Latency time in the 2 wk-sco group was significantly decreased by 74.7 % compared with that in the control group; however, in the 2 wk-sco-mela group, latency time was significantly increased by 40.6 % compared with that in the 2 wk-sco group. In the 4 wk-sco group, latency time was significantly decreased by 21.3 % compared with that in the 2 wk-sco group; however, latency time in the 4 wk-sco-mela group was significantly increased by 56.8 % compared with that in the 4 wk-sco group; the latency time was similar to that in the 2 wk-sco-mela group (Fig. 1c). In brief, in this study, the animals treated with scopolamine for 2 and 4 weeks exhibited longer escape latency in the Morris water maze test and a shorter latency in passive avoidance test, as compared with the animals in the control group; however, melatonin treatment for 2 and 4 weeks significantly rescued behavioral ability. Previously,



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animal models of scopolamine-induced amnesia have been used to explore memory dysfunction

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14

. Recently,

Pandareesh et al. (2015) reported that scopolamine-treated animals showed a lower discrimination index in the novel object recognition test, a higher transfer latency in the elevated plus-maze test, and a longer latency in the Morris water maze test

25

. It has been demonstrated in animal models that melatonin ameliorates cognitive

disturbances in various forms of dementia, including AD

22,26,27

. Based on the above-mentioned and our results,

melatonin must exert positive effects on cognitive performance in amnesia.

ChAT, CHT, VAChT, and M1R protein levels and immunohistochemistry. To elucidate the levels of ChAT, CHT, VAChT, and M1R proteins and immunoreactivities in the septum and the hippocampus, we performed western blat assay and immunohistochemistry. ChAT, CHT, VAChT, and M1R protein levels: Septum: One-way ANOVA and post-hoc test revealed significant melatonin effects on levels of ChAT (F (4, 10) = 43.773, p < 0.0001), CHT (F (4, 10) = 45.435, p < 0.0001) and VAChT (F (4, 10) = 102.29, p < 0.0001) proteins in western blot analysis (Fig. 2). In the 2 wk- and 4 wk-sco groups, the levels of ChAT, CHT and VAChT proteins were significantly decreased compared with those in the control group; however, in the 2 wk- and 4 wk-sco-mela groups, the levels of ChAT, CHT and VAChT were significantly increased compared with those in the 2 wk- and 4wk-sco groups (Figs. 2a and 2b). Hippocampus: One-way ANOVA and post-hoc test revealed significant melatonin effects on levels of ChAT (F (4, 10) = 39.029, p < 0.0001), CHT (F (4, 10) = 17.568, p < 0.0001) and VAChT (F (4, 10) = 58.774, p < 0.0001) and M1R (F (4, 10) = 20.270, p < 0.0001) proteins in western blot analysis (Fig. 3). In the 2 wk- and 4 wk-sco groups, ChAT, CHT VAChT and M1R levels were significantly decreased compared with those in the control group; however, in the 2 wk- and 4 wk-sco-mela groups, ChAT, CHT, VAChT and M1R levels were significantly increased compared with those in the 2 wk- and 4 wk-sco groups (Figs. 3a and 3b). ChAT immunohistochemistry: Septum: One-way ANOVA and post-hoc test revealed significant melatonin effects on ChAT immunoreactivity (F (4, 30) = 43.240, p < 0.0001) (Fig. 4a). Many ChAT-immunoreactive (ChAT+) cells were easily detected in the septum of the control group (Fig. 4a A). In the 2 wk-sco group, ChAT+ cells were significantly reduced compared with those in the control group (Fig. 4a B and 4c); in the 2 wk-sco-mela group, ChAT+ cells were significantly increased compared with those in the 2 wk-sco group (Fig. 4a C and 4c). In the 4 wk-sco group, ChAT+ cells were more decreased compared with those in the 2 wk-sco group (Fig. 4a D and 4c); however, in the 4 wk-sco-mela group, ChAT+ cells were significantly increased compared with those in the 4 wk-sco group (Fig. 4a E and 4c). Hippocampus: One-way ANOVA and post-hoc test revealed significant


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melatonin effects on ChAT immunoreactivity (F (14, 90) = 40.790, p < 0.0001) (Fig. 4b). In the control group, ChAT+ cells were found in the stratum pyramidale of the hippocampus proper (CA1-3 areas) and in the granule cell layer of the dentate gyrus, and ChAT+ fibers were distributed throughout neuropil of the hippocampus (Figs. 4b A1-A4). In the 2 wk-sco group, ChAT immunoreactivity in the cells and neuropil was significantly decreased in all areas compared with that in the control group (Figs. 4b B1-B4 and 4d); in the 2 wk-sco-mela group, ChAT immunoreactivity was significantly increased compared with that in the 2 wk-sco group (Figs. 4b C1-C4 and 4d). In the 4 wk-sco group, ChAT immunoreactivity in all areas was more decreased compared with that in the 2 wksco group (Figs. 4b D1-D4 and 4d); however, in the 4 wk-sco-mela group, ChAT immunoreactivity was similar to that in the 2 wk-sco-mela group (Figs. 4b E1-E4 and 4d). CHT immunohistochemistry: Septum: One-way ANOVA and post-hoc test revealed significant melatonin effects on CHT immunoreactivity (F (4, 30) = 72.526, p < 0.0001) (Fig. 5a). CHT+ cells were abundantly found in the septum of the control group (Fig. 5a A). In the 2 wk-sco group, numbers of CHT+ cells were significantly decreased compared with those in the control group (Fig. 5a B and 5c); however, they were significantly increased in the 2 wk-sco-mela group compared with those in the 2 wk-sco group (Fig. 5a C and 5c). In the 4 wk-sco group, the number of CHT+ cells was less than that in the 2 wk-sco group (Fig. 5a D and 5c); however, the number in the 4 wk-sco-mela group was significantly increased compared with that in the 4 wk-sco group (Fig. 5a E and 5c). Hippocampus: One-way ANOVA and post-hoc test revealed significant melatonin effects on CHT immunoreactivity (F (14, 90) = 6.836, p < 0.0001) (Fig. 5b). CHT+ fibers were distributed throughout all the hippocampal subregions (CA1-3 areas and dentate gyrus) in the control group (Figs. 5b A1-A4). In the 2 wk-sco group, the density of CHT+ fibers was decreased in the CA1-3 areas, not in the dentate gyrus, compared with that in the control group (Figs. 5b B1-B4 and 5d); however, the density in the CA1-3 areas was significantly increased compared with that in the 2 wk-sco group (Figs. 5b C1-C4 and 5d). In the 4 wk-sco group, CHT+ fibers were more decreased in the CA1-3 areas compared with those in the 2 wk-sco group (Figs. 5b D1-D4 and 5d); however, in the 4 wk-sco-mela group, they were increased compared with those in the 4 wk-sco group (Figs. 5b E1-E4 and 5d). VAChT immunohistochemistry: Septum: One-way ANOVA and post-hoc test revealed significant melatonin effects on VAChT immunoreactivity (F (4, 30) = 88.076, p < 0.0001) (Fig. 6a). VAChT+ cells were distributed throughout the septum in the control group; their distribution pattern in the control septum was similar to the findings in ChAT and CHT immunohistochemistry (Fig. 6a A). In the 2 wk-sco group, VAChT+ cells were significantly decreased compared with those in the control group (Fig. 6a B and 6c); however, VAChT+ cells were



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significantly increased in the 2 wk-sco-mela group compared with those in the 2 wk-sco group (Fig. 6a C and 6c). In the 4 wk-sco group, VAChT+ cells were more reduced compared with those in the 2 wk-sco (Fig. 6a D and 6c); however, in the 4 wk-sco-mela group, VAChT+ cells were significantly increased compared with those in the 4 wk-sco group (Fig. 6a E and 6c). Hippocampus: One-way ANOVA and post-hoc test revealed significant melatonin effects on VAChT immunoreactivity (F (14, 90) = 31.965, p < 0.0001) (Fig. 6b). The distribution pattern of VAChT+ structures in the control group was similar to that of ChAT+ cells and fibers, namely, VAChT immunoreactivity was generally found in pyramidal and granule cells as well as in most neuropil (Figs. 6b A1A4). In the 2 wk-sco group, VAChT immunoreactivity in the cells and neuropil was significantly decreased compared with that in the control group (Figs. 6b B1-B4 and 6d); however, in the 2 wk-sco-mela group, VAChT immunoreactivity was significantly increased compared with that in the 2 wk-sco group (Figs. 6b C1-C4 and 6d). VAChT immunoreactivity in the cells and neuropil in the 4 wk-sco group was similar to that in the 2 wk-sco group (Figs. 6b D1-D4 and 6d); however, in the 4 wk-sco-mela group, VAChT immunoreactivity in the hippocampal subregions was significantly increased compared with that in the 4 wk-sco group (Figs. 6b E1-E4 and 6d). M1R immunohistochemistry: One-way ANOVA and post-hoc test revealed significant melatonin effects on M1R immunoreactivity (F (14, 90) = 82.204, p < 0.0001) (Fig. 7a). M1R immunoreactivity was detected in the hippocampus of the control group, namely, M1R immunoreactivity was generally shown in pyramidal cells (Figs. 7a A-A3). In the 2 wk-sco group, M1R immunoreactivity in pyramidal cells was significantly decreased compared with that in the control group (Figs. 7a B-B3 and 7b); however, in the 2 wk-sco-mela group, M1R immunoreactivity in pyramidal cells was increased compared with that in the 2 wk-sco group (Figs. 7a C-C3 and 7b). In the 4 wk-sco group, M1R immunoreactivity was similar to that in the 2 wk-sco group (Figs. 7a D-D3 and 7b); in the 4 wk-sco-mela group, M1R immunoreactivity in pyramidal cells was also increased compared with that in the 4 wk-sco group (Figs. 7a E-E3 and 7b). Our present study showed that the 2- or 4-week administration of scopolamine caused severe damage to the cholinergic system, namely ChAT-, CHT-, VAChT- and M1R-immunoreactivities and their protein levels in the medial septum and hippocampus were significantly decreased compared with those in the control group. The damage was particularly severe after scopolamine treatment for 4 weeks. In agreement with our present findings, previous studies report that scopolamine crosses the blood brain barrier and induces cognitive dysfunction, which may be attributable to alterations of the cholinergic pathway, in particular, the levels of ChAT, AChE, and other signaling molecules are reduced in the cortex and hippocampus following scopolamine treatment 15,28,29,.


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Our current study shows that melatonin treatment increased immunoreactivities and protein levels of ChAT, CHT and VAChT in the medial septum and hippocampus treated with scopolamine. The cholinergic degeneration of the hippocampus, which is closely connected with the medial septum, as the septo-hippocampal cholinergic system, is a key factor determining the extent of cognitive impairments

5,30

. Recently, Holmstrand et al. (2014)

reported that the administration of melatonin improved spatial learning and memory and attenuated cholinergic degeneration via increasing ChAT in a mouse model of Down syndrome 19. Zhu et al. (2007) demonstrated that melatonin acts as an AChE inhibitor, namely, melatonin was involved in inhibiting AChE activity by regulating the chelation of intracellular free calcium

31

. It was reported that melatonin, as an AChE targeted drug, was

administered for anti-amnesic treatment and enhanced the cholinergic activity through decreasing AChE activity in the brain of amnesic mice 32. Thus, based on the above-mentioned reports and our present findings, it is likely that melatonin improves the ACh synthesis through increasing ChAT, CHT and VAChT, and decreasing AChE activity. It is also reported that short-term (3-4 days) scopolamine treatment acutely increased the acetylcholine release, and thereby increased cholinergic effects on nicotinic receptor systems, but inhibited the release of muscarinic autoreceptors in mice

33

. Recently, Lee et al. (2014) reported that long-term (14 days) administration of

scopolamine significantly decreased M1R in the rat hippocampus 15. Similar to the above findings, we observed in the present study that scopolamine administration significantly decreased M1R immunoreactivity and protein level in the mouse hippocampus. In addition, we found that long-term melatonin treatment restored the scopolamine-induced decrease of M1R expression in the mouse hippocampus. Tsukagoshi et al. (2000) showed that a long-term (7 days) treatment of clomipramine, a tricyclic antidepressant, reduced behavioral sensitivity to scopolamine via up-regulation of muscarinic acetylcholine receptors, and suggested that such a change is responsible for the decreased sensitivity to the muscarinic antagonist scopolamine 34. In this regard, it is likely that long-term treatment of melatonin improves the scopolamine-induced cognitive impairment by up-regulation of M1R in the hippocampus.

■ CONCLUSION Melatonin has ability to improve spatial learning and memory impairments through preventing cholinergic degeneration in the medial septum and hippocampus in a mouse model of scopolamine-induced amnesia.



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■ METHODS

Experimental animals. Male ICR mice (8-week-old) were purchased from Orient Bio Inc. (Seongnam, South Korea) and used after 7 days of acclimation. The procedures for animal handling and care adhered to guidelines that are in compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, The National Academies Press, 8th Ed., 2011), and all the experimental protocols in the present study was reviewed and approved based on ethical procedures and scientific care by the Kangwon National UniversityInstitutional Animal Care and Use Committee (approval no. KW-160802-3). All the experiments were conducted to minimize the number of animals used and the suffering caused by the procedures used in the present study.

Experimental groups and drug treatment. Experimental mice were divide into 5 groups (n = 14 at each point in time per group): 1) control group, which was treated with saline; 2) 2 wk-sco group, which was treated with scopolamine for 2 weeks; 3) 2 wk-sco-mela group, which was treated with scopolamine plus melatonin for 2 weeks; 4) 4 wk-sco group, which was treated with scopolamine for 4 weeks; 5) 4-sco-mela group. The 1 mg/kg scopolamine and/or 10 mg/kg melatonin were dissolved in normal saline and intraperitoneally injected once a day for 2 and 4 weeks 35,36. Mice were sacrificed 24 h after final administration.

Behavioral performance. Morris water maze test: Morris water maze test for spatial memory was performed 30 min after vehicle or scopolamine or scopolamine plus melatonin administration for the last 4 days before sacrifice according to a published procedure 20. In brief, the apparatus consisted of a circular steel tank 90 cm in diameter and 50 cm high, which was filled with water (25 ± 0.5°C) to a depth of 20 cm. Three different visual cues were placed around the inside wall of the tank at a level that would be visible to the mice. A circular platform 15 cm in diameter remained in a fixed location, placed in the center of one quadrant of the pool, 15 cm from the apparatus wall and was 1 cm below the surface of the water. Each mouse was given 3 daily trials with a 5-min intertrial interval for 3 consecutive days to find a platform, and individually placed in the apparatus at one of 3 preselected locations and allowed 120 secs to escape to the hidden platform. The mice not finding the platform after 50 secs were guided to it by the experimenter. The mice were allowed to remain on the platform for 10 secs and were then returned to their holding cage until the next trial. The test was performed at the last day before sacrifice. The time to find the platform was recorded within 120 sec (latency time) in each mouse, and the whole process was monitored by a digital camera and a computer system. Passive avoidance test: Passive avoidance test for short-


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term memory was performed 30 min after vehicle or scopolamine or scopolamine plus melatonin administration for the last 3 days before sacrifice according to a published procedure 37. In brief, the Gemini Avoidance System (GEM 392, San Diego Instruments), which consists of two compartments (light and dark) with a grid floor, was used for this experiment. The training for the test was performed for 2 days, namely, each mouse was allowed to explore the environments in the A- and B-dark compartments for 1 min, at which point the grid floor was opened. Then the light was turned on only in the A-compartment, and the mouse was allowed to explore the environments in both A- and B-compartments for 1 min. When the mouse entered the B-compartment, the grid floor was closed an inescapable foot-shock (0.5 mA for 5 s). The test was performed at the last day before sacrifice. Namely, each mouse was placed in the A-compartment, the light of the A-compartment was turned on, and the grid floor was opened. Latency time for the mouse to enter the B-compartment was recorded within 180 s.

Western blot analysis. Western blotting for ChAT, CHT and VAChT in the medial septum and hippocampus (n = 7 in each group) was performed using our published procedure 38. In brief, after the tissues were homogenized and centrifuged, the supernatants were subjected to western blot analysis. Goat anti-choline acetyltransferase (ChAT) (1:200, Santa Cruz), goat anti-choline transporter (CHT) (1:200, Frontier Institute), goat anti-vesicular ACh transporter (VAChT) (1:200, Santa Cruz) and rabbit anti-M1R (1:200, Frontier Institute) were used as primary antibody. The result of western blot analysis was scanned, and densitometric analysis for the quantification of the bands was done using NIH Image J software (National Institutes of Health, Bethesda, MD), which was used to count relative optical density (ROD): A ratio of the ROD was calibrated as %, with the control group designated as 100 %. In addition, the value of each protein was normalized to that of β-actin.

Immunohistochemistry. Immunohistochemistry for ChAT, CHT and VAChT in the medial septum and ChAT, CHT, VAChT, and M1R in the hippocampus (n = 7 in each group) was carried out according to our published method

38

. Briefly, mice were anesthetized with 30 mg/kg Zoletil 50 (Virbac, Carros, France) and perfused

transcardially with 0.1 m phosphate buffered saline (PBS, pH 7.4) followed by 4 % paraformaldehyde in 0.1 m phosphate buffer (PB, pH 7.4). The brain tissues were cryoprotected and serially sectioned on a cryostat (Leica, Wetzlar, Germany) into 30-μm coronal sections. The sections were incubated with diluted goat anti-ChAT (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-CHT (1:200, Frontier Institute, Hokkaido, Japan), goat antiVAChT (1:200, Santa Cruz), rabbit anti-M1R (1:200, Frontier Institute) overnight and subsequently exposed to biotinylated goat anti-rabbit (Vector, Burlingame, CA) or rabbit anti-goat IgG and streptavidin peroxidase complex



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(1:200, Vector). In order to establish the specificity of the immunostaining, a negative control resulted in the absence of immunoreactivity in all structures. Digital images of ChAT, CHT and VAChT and M1R immunoreactive structures were captured with an AxioM1 light microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. The density of each immunoreactive structures was evaluated on the basis of optical density (OD), which was obtained after the transformation of the mean gray level using the formula: OD = log (256/mean gray level). After the background was subtracted, a ratio of the OD of image file was calibrated as % (relative optical density, ROD) using Adobe Photoshop version 8.0 and NIH Image J software (National Institutes of Health, Bethesda, MD). The mean value of the OD of the control group was designated as 100 %, and the ROD of each group was calibrated and expressed as % of the control group.

Statistical analysis. The data shown here represent the means ± SEM. All comparisons were tested for normality and variance homogeneity using SPSS 17.0 software (IBM, New York, USA). In this study, ROD of the immunoreactive structures was statistically analyzed by one-way analysis of variance (ANOVA) with Duncan’s post hoc test in order to elucidate melatonin-related differences among experimental groups. Statistical significance was considered at p < 0.05.

■ AUTHOR INFORMATION Corresponding authors Professor Moo-Ho Won, DVM, PhD: Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 24341, South Korea. TEL: +82-33-250-8891; FAX: +82-33-256-1614. E-mail: [email protected]

Ji Hyeon Ahn, PhD: Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chuncheon 24252, South Korea. TEL: +82-33-248-3202; FAX: +82-33-248-3201; E-mail: [email protected]

ORCID Moo-Ho Won: 0000-0002-7178-6501 Ji Hyeon Ahn: 0000-0002-5304-0714


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Author Contributions Bai Hui Chen1 and Joon Ha Park2 have contributed equally to this article. Moo-Ho Won, Bai Hui Chen and Ji Hyeon Ahn were responsible for experimental design, data collection, data analysis, and manuscript writing. Dae Won Kim, Joon Ha Park, In Hye Kim, Jeong Hwi Cho, Tae-Kyeong Lee and Bich Na Shin performed the experiments. Jinseu Park, Soo Young Choi, Jae Chul Lee, Choong-Hyun Lee, In Koo Hwang, Young-Myeong Kim, Bing Chun Yan, Il Jun Kang, Yun Lyul Lee, Myoung Cheol Shin, Jun Hwi Cho, Young Joo Lee, and Yong Hwan Jeon performed data analysis and critical comments on the whole process of this study. All authors have read and approved the final manuscript.

Funding This research was supported by the Bio-Synergy Research Project (NRF-2015M3A9C4076322) of the Ministry of Science, ICT and Future Planning through the National Research Foundation, by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2015M3A9B6066835), and by a Priority Research Centers Program grant (NRF-2009-0093812) through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning.

Note The authors declare no competing financial interest.



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■ REFERENCES

(1) Palit, P., Mukherjee, D. and Mandal, S.C. (2015) Reconstituted mother tinctures of Gelsemium sempervirens L. improve memory and cognitive impairment in mice scopolamine-induced dementia model. Journal of ethnopharmacology 159, 274-284. (2) Soreq, H. (2015) Checks and balances on cholinergic signaling in brain and body function. Trends in neurosciences 38, 448-458. (3) Moosavi, M., Khales, G.Y., Abbasi, L., Zarifkar, A. and Rastegar, K. (2012) Agmatine protects against scopolamine-induced water maze performance impairment and hippocampal ERK and Akt inactivation. Neuropharmacology 62, 2018-2023. (4) Howes, M.J., Perry, N.S. and Houghton, P.J. (2003) Plants with traditional uses and activities, relevant to the management of Alzheimer's disease and other cognitive disorders. Phytotherapy research : PTR 17, 1-18. (5) Schliebs, R. and Arendt, T. (2011) The cholinergic system in aging and neuronal degeneration. Behavioural brain research 221, 555-563. (6) Hernandez, C.M. and Dineley, K.T. (2012) alpha7 nicotinic acetylcholine receptors in Alzheimer's disease: neuroprotective, neurotrophic or both? Current drug targets 13, 613-622. (7) Rosales-Corral, S.A., Acuna-Castroviejo, D., Coto-Montes, A., Boga, J.A., Manchester, L.C., Fuentes-Broto, L., Korkmaz, A., Ma, S., Tan, D.X. and Reiter, R.J. (2012) Alzheimer's disease: pathological mechanisms and the beneficial role of melatonin. Journal of pineal research 52, 167-202. (8) Pereira, H.A., Benassi, S.K. and Mello, L.E. (2005) Plastic changes and disease-modifying effects of scopolamine in the pilocarpine model of epilepsy in rats. Epilepsia 46 Suppl 5, 118-124. (9) Mufson, E.J., Ginsberg, S.D., Ikonomovic, M.D. and DeKosky, S.T. (2003) Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction. Journal of chemical neuroanatomy 26, 233-242. (10) Auld, D.S., Kornecook, T.J., Bastianetto, S. and Quirion, R. (2002) Alzheimer's disease and the basal forebrain cholinergic system: relations to beta-amyloid peptides, cognition, and treatment strategies. Progress in neurobiology 68, 209-245. (11) Sun, J., Pan, C.Q., Chew, T.W., Liang, F., Burmeister, M. and Low, B.C. (2015) BNIP-H Recruits the Cholinergic Machinery to Neurite Terminals to Promote Acetylcholine Signaling and Neuritogenesis. Developmental cell 34, 555-568. (12) Taylor, P. and Brown, J.H. (1999) Synthesis, storage and release of acetylcholine.


Page 15 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13) Kása, P., Rakonczay, Z. and Gulya, K. (1997) The cholinergic system in Alzheimer's disease. Progress in neurobiology 52, 511-535. (14) Klinkenberg, I. and Blokland, A. (2010) The validity of scopolamine as a pharmacological model for cognitive impairment: a review of animal behavioral studies. Neuroscience and biobehavioral reviews 34, 1307-1350. (15) Lee, B., Sur, B., Shim, J., Hahm, D.H. and Lee, H. (2014) Acupuncture stimulation improves scopolamineinduced cognitive impairment via activation of cholinergic system and regulation of BDNF and CREB expressions in rats. BMC complementary and alternative medicine 14, 338. (16) Lee, J.S., Kim, H.G., Lee, H.W., Han, J.M., Lee, S.K., Kim, D.W., Saravanakumar, A. and Son, C.G. (2015) Hippocampal memory enhancing activity of pine needle extract against scopolamine-induced amnesia in a mouse model. Scientific reports 5, 9651. (17) Acuña-Castroviejo, D., Escames, G., Venegas, C., Díaz-Casado, M.E., Lima-Cabello, E., López, L.C., Rosales-Corral, S., Tan, D.-X. and Reiter, R.J. (2014) Extrapineal melatonin: sources, regulation, and potential functions. Cellular and molecular life sciences 71, 2997-3025. (18) Hardeland, R., Cardinali, D.P., Srinivasan, V., Spence, D.W., Brown, G.M. and Pandi-Perumal, S.R. (2011) Melatonin--a pleiotropic, orchestrating regulator molecule. Progress in neurobiology 93, 350-384. (19) Corrales, A., Martinez, P., Garcia, S., Vidal, V., Garcia, E., Florez, J., Sanchez-Barcelo, E.J., Martinez-Cue, C. and Rueda, N. (2013) Long-term oral administration of melatonin improves spatial learning and memory and protects against cholinergic degeneration in middle-aged Ts65Dn mice, a model of Down syndrome. Journal of pineal research 54, 346-358. (20) Yoo, D.Y., Kim, W., Lee, C.H., Shin, B.N., Nam, S.M., Choi, J.H., Won, M.H., Yoon, Y.S. and Hwang, I.K. (2012) Melatonin improves D-galactose-induced aging effects on behavior, neurogenesis, and lipid peroxidation in the mouse dentate gyrus via increasing pCREB expression. Journal of pineal research 52, 2128. (21) Agrawal, R., Tyagi, E., Shukla, R. and Nath, C. (2008) Effect of insulin and melatonin on acetylcholinesterase activity in the brain of amnesic mice. Behavioural brain research 189, 381-386. (22) Wang, X., Wang, Z.H., Wu, Y.Y., Tang, H., Tan, L., Gao, X.Y., Xiong, Y.S., Liu, D., Wang, J.Z. and Zhu, L.Q. (2013) Melatonin attenuates scopolamine-induced memory/synaptic disorder by rescuing EPACs/miR124/Egr1 pathway. Molecular neurobiology 47, 373-381.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 26

(23) Woolfrey, K.M., Srivastava, D.P., Photowala, H., Yamashita, M., Barbolina, M.V., Cahill, M.E., Xie, Z., Jones, K.A., Quilliam, L.A., Prakriya, M. and Penzes, P. (2009) Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines. Nature neuroscience 12, 1275-1284. (24) Feng, Z., Cheng, Y. and Zhang, J.T. (2004) Long-term effects of melatonin or 17 beta-estradiol on improving spatial memory performance in cognitively impaired, ovariectomized adult rats. Journal of pineal research 37, 198-206. (25) Pandareesh, M.D., Anand, T. and Khanum, F. (2015) Cognition Enhancing and Neuromodulatory Propensity of Bacopa monniera Extract Against Scopolamine Induced Cognitive Impairments in Rat Hippocampus. Neurochemical research (26) Stefanova, N.A., Maksimova, K.Y., Kiseleva, E., Rudnitskaya, E.A., Muraleva, N.A. and Kolosova, N.G. (2015) Melatonin attenuates impairments of structural hippocampal neuroplasticity in OXYS rats during active progression of Alzheimer's disease-like pathology. Journal of pineal research 59, 163-177. (27) Ali, T., Badshah, H., Kim, T.H. and Kim, M.O. (2015) Melatonin attenuates D-galactose-induced memory impairment, neuroinflammation and neurodegeneration via RAGE/NF-K B/JNK signaling pathway in aging mouse model. Journal of pineal research 58, 71-85. (28) Hiramatsu, M., Murasawa, H., Nabeshima, T. and Kameyama, T. (1998) Effects of U-50,488H on scopolamine-, mecamylamine- and dizocilpine-induced learning and memory impairment in rats. The Journal of pharmacology and experimental therapeutics 284, 858-867. (29) Yamada, M., Chiba, T., Sasabe, J., Terashita, K., Aiso, S. and Matsuoka, M. (2008) Nasal Colivelin treatment ameliorates memory impairment related to Alzheimer's disease. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 33, 2020-2032. (30) Millan Sanchez, M., Heyn, S.N., Das, D., Moghadam, S., Martin, K.J. and Salehi, A. (2012) Neurobiological elements of cognitive dysfunction in down syndrome: exploring the role of APP. Biological psychiatry 71, 403-409. (31) Zhu, H., Gao, W., Jiang, H., Jin, Q.H., Shi, Y.F., Tsim, K.W. and Zhang, X.J. (2007) Regulation of acetylcholinesterase expression by calcium signaling during calcium ionophore A23187- and thapsigargininduced apoptosis. The international journal of biochemistry & cell biology 39, 93-108. (32) Agrawal, R., Tyagi, E., Shukla, R. and Nath, C. (2008) Effect of insulin and melatonin on acetylcholinesterase activity in the brain of amnesic mice. Behavioural brain research 189, 381-386.


Page 17 of 26

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(33) Kuribara, H. and Tadokoro, S. (1983) Development of tolerance to ambulation-increasing effect of scopolamine dependent on environmental factors in mice. Japanese journal of pharmacology 33, 1041-1048. (34) Tsukagoshi, H., Morita, T., Hitomi, S., Saito, S., Kadoi, Y., Uchihashi, Y., Kuribara, H. and Goto, F. (2000) Long-term clomipramine treatment upregulates forebrain acetylcholine muscarinic receptors, and reduces behavioural sensitivity to scopolamine in mice. The Journal of pharmacy and pharmacology 52, 87-92. (35) Yan, B.C., Park, J.H., Chen, B.H., Cho, J.H., Kim, I.H., Ahn, J.H., Lee, J.C., Hwang, I.K., Lee, Y.L., Kang, I.J. and Won, M.H. (2014) Long-term administration of scopolamine interferes with nerve cell proliferation, differentiation and migration in adult mouse hippocampal dentate gyrus, but it does not induce cell death. Neural regeneration research 9, 1731-1739. (36) Onaolapo, O.J., Onaolapo, A.Y., Abiola, A.A. and Lillian, E.A. (2014) Central depressant and nootropic effects of daytime melatonin in mice. Annals of neurosciences 21, 90-96. (37) Ahn, J.H., Choi, J.H., Park, J.H., Kim, I.H., Cho, J.-H., Lee, J.-C., Koo, H.-M., Hwangbo, G., Yoo, K.-Y. and Lee, C.H. (2016) Long-term exercise improves memory deficits via restoration of myelin and microvessel damage, and enhancement of neurogenesis in the aged gerbil hippocampus after ischemic stroke. Neurorehabilitation and neural repair 30, 894-905. (38) Chen, B.H., Yan, B.C., Park, J.H., Ahn, J.H., Lee, D.H., Kim, I.H., Cho, J.H., Lee, J.C., Kim, S.K., Lee, B., Won, M.H. and Lee, Y.L. (2013) Aripiprazole, an atypical antipsychotic drug, improves maturation and complexity of neuroblast dendrites in the mouse dentate gyrus via increasing superoxide dismutases. Neurochemical research 38, 1980-1988.



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Figure legends

Fig. 1 Cognition, and learning and memory tests using Morris water maze (a, b) and passive avoidance tests (c). In Morris water maze test, escape latency in all the sco-mela groups is significantly decreased compared with those in the sco groups (a, b). In passive avoidance test, latency time in all the sco-mela groups is significantly increased compared with that in the sco groups (c) (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM.

Fig. 2 Western blot analysis of ChAT, CHT and VAChT in the septum of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups (a). Relative optical density (ROD) expressed as percentage of immunoblot band in the septum is presented (b) (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM.

Fig. 3 Western blot analysis of ChAT, CHT, VAChT and M1R in the hippocampus of the control, 2 wk-sco, 2 wksco-mela, 4 wk-sco and 4 wk-sco-mela groups (a). Relative optical density (ROD) expressed as percentage of immunoblot band in the hippocampus is presented (b) (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM.

Fig. 4 ChAT immunohistochemistry in the medial septum (a) and hippocampus (b) of the control, 2 wk-sco, 2 wksco-mela, 4 wk-sco and 4 wk-sco-mela groups. In the septum, ChAT+ cells are significantly increased in all the sco-mela groups compared with those in all the sco groups. In the hippocampus, ChAT+ cells and fibers are significantly increased in all the sco-mela groups compared with the sco groups. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scar bars: 100 (A-E), 200 (A1-E1) and 50 μm (A2-E4). ROD expressed as percentage of ChAT immunoreactivity in the septum (c) and the hippocampus (d). (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM.


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Fig. 5 CHT immunohistochemistry in the medial septum (a) and hippocampus (b) of the control, 2 wk-sco, 2 wksco-mela, 4 wk-sco and 4 wk-sco-mela groups. In the septum, CHT+ cells in all the sco-mela groups are increased compared with the sco groups. In the hippocampus, CHT+ fibers are significantly increased in the CA1-3 areas of all the sco-mela groups compared with the sco groups. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scar bars: 100 (A-E), 200 (A1-E1) and 50 μm (A2-E4). Relative optical density (ROD) expressed as percentage of CHT immunoreactivity in the medial septum (c) and the hippocampus (d). (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM.

Fig. 6 VAChT immunohistochemistry in the medial septum (a) and hippocampus (b) of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups. In the septum, VAChT+ cells are significantly increased in all the sco groups compared with those in the sco groups. In the hippocampus, VAChT+ cells and fibers in all the scomela groups are increased compared with those in the sco groups. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scar bars: 100 (A-E), 200 (A1-E1) and 50 μm (A2-E4). ROD expressed as percentage of CHT immunoreactivity in the medial septum (c) and the hippocampus (d). (n = 7 per group; *P < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco-treated group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM.

Fig. 7 M1R immunohistochemistry in the hippocampus (a) of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups. M1R immunoreactivity is found in pyramidal cells in the control group. M1R immunoreactivity is significantly decreased in all the sco groups compared with the sco groups; however, M1R immunoreactivity is significantly increased in all the sco-mela groups compared with all the sco groups. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scar bars: 200 (A-E) and 50 μm (A1-E3). ROD expressed as percentage of M1R immunoreactivity in the CA1, CA2/3, and dentate gyrus regions of the hippocampus (b). (n = 7 per group; *

p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco-treated group). The bars indicate the means

± SEM.



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Fig. 1 Cognition, and learning and memory tests using Morris water maze (a, b) and passive avoidance tests (c). In Morris water maze test, escape latency in all the sco-mela groups is significantly decreased compared with those in the sco groups (a, b). In passive avoidance test, latency time in all the sco-mela groups is significantly increased compared with that in the sco groups (c) (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM. 254x61mm (300 x 300 DPI)


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Fig. 2 Western blot analysis of ChAT, CHT and VAChT in the septum of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups (a). Relative optical density (ROD) expressed as percentage of immunoblot band in the septum is presented (b) (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM. 170x145mm (300 x 300 DPI)



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Fig. 3 Western blot analysis of ChAT, CHT, VAChT and M1R in the hippocampus of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups (a). Relative optical density (ROD) expressed as percentage of immunoblot band in the hippocampus is presented (b) (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM. 170x164mm (300 x 300 DPI)


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Fig. 4 ChAT immunohistochemistry in the medial septum (a) and hippocampus (b) of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups. In the septum, ChAT+ cells are significantly increased in all the sco-mela groups compared with those in all the sco groups. In the hippocampus, ChAT+ cells and fibers are significantly increased in all the sco-mela groups compared with the sco groups. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scar bars: 100 (A-E), 200 (A1-E1) and 50 µm (A2-E4). ROD expressed as percentage of ChAT immunoreactivity in the septum (c) and the hippocampus (d). (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM. 173x192mm (300 x 300 DPI)



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Fig. 5 CHT immunohistochemistry in the medial septum (a) and hippocampus (b) of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups. In the septum, CHT+ cells in all the sco-mela groups are increased compared with the sco groups. In the hippocampus, CHT+ fibers are significantly increased in the CA1-3 areas of all the sco-mela groups compared with the sco groups. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scar bars: 100 (A-E), 200 (A1-E1) and 50 µm (A2-E4). Relative optical density (ROD) expressed as percentage of CHT immunoreactivity in the medial septum (c) and the hippocampus (d). (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM. 173x193mm (300 x 300 DPI)


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Fig. 6 VAChT immunohistochemistry in the medial septum (a) and hippocampus (b) of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups. In the septum, VAChT+ cells are significantly increased in all the sco groups compared with those in the sco groups. In the hippocampus, VAChT+ cells and fibers in all the sco-mela groups are increased compared with those in the sco groups. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scar bars: 100 (A-E), 200 (A1-E1) and 50 µm (A2-E4). ROD expressed as percentage of CHT immunoreactivity in the medial septum (c) and the hippocampus (d). (n = 7 per group; *P < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco-treated group; #p < 0.05, vs. the respective pre-time point group). The bars indicate the means ± SEM. 173x192mm (300 x 300 DPI)



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Fig. 7 M1R immunohistochemistry in the hippocampus (a) of the control, 2 wk-sco, 2 wk-sco-mela, 4 wk-sco and 4 wk-sco-mela groups. M1R immunoreactivity is found in pyramidal cells in the control group. M1R immunoreactivity is significantly decreased in all the sco groups compared with the sco groups; however, M1R immunoreactivity is significantly increased in all the sco-mela groups compared with all the sco groups. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scar bars: 200 (A-E) and 50 µm (A1-E3). ROD expressed as percentage of M1R immunoreactivity in the CA1, CA2/3, and dentate gyrus regions of the hippocampus (b). (n = 7 per group; *p < 0.05, vs. the control group; †p < 0.05, vs. the same time point sco-treated group). The bars indicate the means ± SEM. 173x156mm (300 x 300 DPI)


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Graphical Abstract 69x39mm (300 x 300 DPI)


Melatonin Improves Cognitive Deficits via Restoration of Cholinergic

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