Loganin enhances long-term potentiation and recovers scopolamine-induced learning and memory impairments

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Loganin enhances long-term potentiation and recovers scopolamine-induced learning and memory impairments

Eun-Sang Hwang

^a,1

, Hyun-Bum Kim

^a,1

, Seok Lee

^a

, Min-Ji Kim

^b

, Sung-Ok Lee

^c

, Seung-Moo Han

^d

, Sungho Maeng

^b,

⁎ , Ji-Ho Park

^b,

⁎

aDepartment of East-West Medical Science, Graduate School of East-West Medical Science, Kyung Hee University, Deogyeong-daero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea

bDepartment of East-West Medicine, Graduate School of East-West Medical Science, Kyung Hee University, Deogyeong-daero, Giheung-gu, Yongin 446-701, Republic of Korea

cDepartment of Oriental Medicinal Materials and Processing, College of Life Science, Kyung Hee University, Deogyeong-daero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea

dDepartment of Biomedical Engineering, Kyung Hee University, 1 Seocheon-dong, Giheung-gu, Yongin 446-701, Republic of Korea

H I G H L I G H T S

•loganin can effectively block cholinergic muscarinic receptor blockade.

•loganin may have signiﬁcant therapeutic value for alleviating memory impairments.

•loganin may be expected to have a future use as an agent for the prevention and treatment of Alzheimer's disease.

a b s t r a c t a r t i c l e i n f o

Article history:

Received 25 October 2016

Received in revised form 4 December 2016 Accepted 21 December 2016

Available online 6 January 2017

Although the incidence rate of dementia is rapidly growing in the aged population, therapeutic and preventive reagents are still suboptimal. Various model systems are used for the development of such reagents in which scopolamine is one of the favorable pharmacological tools widely applied.

Loganin is a major iridoid glycoside obtained from Corni fructus (Cornus officinaliset Zucc) and demonstrated to have anti-inflammatory, anti-tumor and osteoporosis prevention effects. It has also been found to attenuate Aβ- induced inflammatory reactions and ameliorate memory deficits induced by scopolamine. However, there has been limited information available on how loganin affects learning and memory both electrophysiologically and behaviorally.

To assess its effect on learning and memory, we investigated the inﬂuence of acute loganin administration on long-term potentiation (LTP) using organotypic cultured hippocampal tissues. In addition, we measured the effects of loganin on the behavior performance related to avoidance memory, short-term spatial navigation memory and long-term spatial learning and memory in the passive avoidance, Y-maze, and Morris water maze learning paradigms, respectively.

Loganin dose-dependently increased the total activity of fEPSP after high frequency stimulation and attenuated scopolamine-induced blockade of fEPSP in the hippocampal CA1 area. In accordance with theseﬁndings, loganin behaviorally attenuated scopolamine-induced shortening of step-through latency in the passive avoidance test, reduced the percent alternation in the Y-maze, and increased memory retention in the Morris water maze test.

These results indicate that loganin can effectively block cholinergic muscarinic receptor blockade -induced deterioration of LTP and memory related behavioral performance. Based on theseﬁndings, loganin may aid in the prevention and treatment of Alzheimer's disease and learning and memory-deﬁcit disorders in the future.

1. Introduction

Alzheimer's disease (AD) is the most common form of dementia with a growing incidence rate in the elderly, especially in the population over 65 years old. AD results from chronic, progressive neurodegenera- tion characterized by the deposition of amyloid-beta (Aβ) plaques, in- ﬂammation and neuronal loss[1].

⁎ Corresponding authors at: Department of East-West Medicine, Graduate School of East-West Medical Science, Kyung Hee University, #1732 Deogyeong-daero, Giheung-gu, Yongin 446-702, Republic of Korea.

E-mail addresses:[email protected](S. Maeng),[email protected](J.-H. Park).

1Eun-Sang Hwang and Hyun-Bum Kim contributed equally to this work and are co-ﬁrst authors.

Contents lists available atScienceDirect

Physiology & Behavior

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / p h b

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Scopolamine is a cholinergic muscarinic receptor antagonist which can impair learning and memory in rodents and humans, particularly during the acquisition step of memory formation[2]. Because of this effect, scopolamine has become a favorable pharmacological tool to model learning and memory impairments.

Loganin is a major iridoid glycoside obtained from Corni fructus (Cornus officinaliset Zucc). Numerous effects of loganin have been demonstrated including anti-inflammatory and anti-tumor proper- ties, along with osteoporosis prevention. Specifically in an AD model, loganin has been found to protect PC12 cells against Aβtox- icity[3]. Loganin may have beneficial effects in treating AD by atten- uating the Aβ-induced inflammatory reaction[3]. Loganin has also been found to further ameliorate memory deficits induced by the muscarinic antagonist scopolamine in mice[4]. However, there has been limited information available on how loganin affects learning and memory electrophysiologically.

Long-term potentiation (LTP) is the enhancement of postsynaptic responses for hours, days or weeks following brief repetitive stimulation of synaptic nerves[5]. Brief trains of high-frequency stimulation (HFS) applied to excitatory afferents in the hippocampus lead to an increase in the strength of synaptic transmission[6]. It is generally accepted that the induction of LTP at this synapse requires activation of post- synapticN-methyl-D-aspartic acid (NMDA) receptors by synaptically released glutamate during adequate postsynaptic depolarization[7].

As memories are thought to be encoded by modiﬁcation of synaptic strength, LTP is widely considered one of the major experimental models for examining the synaptic mechanisms of learning and memory[5]. Moreover, LTP in neuroscience can be used to study neurodegenerative diseases associated with memory such as AD, a degenerative brain disease with memory loss, cognitive decline and learning disabilities[8].

In this study, we evaluated the effects of loganin on learning and memory in organotypic hippocampal tissue. To assess its effect on learning and memory, we investigated the inﬂuence of acute loganin administration on long-term potentiation (LTP). In addition, we investigated the effects of loganin on rat behavior related to avoidance memory, short-term spatial navigation memory and long-term spatial learning and memory. We provide evidence that loganin can block scopolamine-induced deterioration of LTP and memory related behavioral performance.

2. Materials and methods 2.1. Materials

Loganin (536954), HEPES (H4034),L-glutamine (G-8540) and

D-glucose (G-7528) were purchased from Sigma (St. Louis, MO, USA).

Minimum essential medium (MEM, LM 007-01), Hank's balanced salt solution (HBSS, LB 003-01), and horse serum (S 104-01) were purchased from JBI (Daegu, South Korea). Penicillin-streptomycin was obtained from Gibco BRL (LS 202–02, Rockville, MD, USA).

2.2. Animals and experimental groups

All animal procedures complied with the Institutional Care and Use Committee (KHUASP(SE)-15-024) of Kyung Hee University, and were performed in accordance with the guiding principles of the Council of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For behavioral measurements, 8-week-old rats were purchased from Orient Bio (Orient Bio Inc., Seongnam, KR). Before the initiation of behavioral measurements, rats were randomly assigned to control (distilled water p.o. as vehicle), S (scopolamine 1.5 mg/kg, i.p.) and L + S (loganin 40 mg/kg p.o. + scopolamine 1.5 mg/kg i.p.) groups.

2.3. Organotypic hippocampal slice cultures

Interface organotypic hippocampal slice cultures were prepared as described previously[9]. In brief, hippocampi were harvested from 5- to 7-day-old Sprague Dawley rat pups and sectioned trans- versely at a thickness of 350μm using a tissue chopper (Mickle Laboratory Engineering Co., Surrey, UK). Five to six slices were then plated onto each 0.4μm culture insert (Millicell-CM; Millipore, Bedford, MA, USA) and maintained in an incubator at 36 °C with 5%

CO2. Culture medium (MEM; supplemented with 20 mM HEPES, 25% Hank's balanced salt solution, 6 g/lD-glucose, 1 mML-glutamine, 25% horse serum and 1% penicillin-streptomycin, pH = 7.1) was changed every two days and the sections were cultured for 12–14 d before treatment and experiments.

2.4. Preparation of organotypic hippocampal slice tissue on MEA probes

A single stabilized hippocampal slice was carefully removed from a membrane insert with a needle and then placed on an 8 × 8 micro- electrode array (MEA) with 10μm-diameter electrodes spaced 100μm apart (Multi Channel Systems, Reutlingen, Germany) pre- coated with 0.01% polyethylenimine. MEAs consist of a high density electrode array with a stimulator, amplifier, temperature control unit and computer for data acquisition. The slice was stabilized in ar- tificial cerebrospinalfluid (aCSF containing 114 mM NaCl, 26 mM NaHCO3, 10 mM glucose, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, pH 7.4) for 30 min at 33 °C with 95% O2and 5% CO2

gas aeration. Extraneous aCSF was then removed using a pipette.

The MEA containing the hippocampal slice was transferred to an MEA1060 ampliﬁer interface. The solution in the array was grounded using an Ag/AgCl pellet. The stimulating channel was disconnected from the sampling device during stimulation. The solution in the array was grounded using an Ag/AgCl pellet.

2.5. Induction of LTP in hippocampal slices

The MEA system is composed of an array, stimulator (STG1004;

Multi Channel Systems GmbH, Germany), ampliﬁer (MEA1060;

Multi Channel Systems GmbH), temperature control unit (Multi Channel Systems GmbH) and data acquisition software (www.

multichannelsystems.com)[10]. The amplifier was placed in a Fara- day cage and bipolar electrical stimulation was applied to the stra- tum radiatum CA2 region to stimulate the Schaffer collateral (SC) and commissural pathways. The intensity of bipolar test pulse baseline stimulation was set at 100 mA, which is a value optimized to provide 40 to 65% of the maximum tissue response. High-frequency stimulation (HFS) consisted of 300 1-second biphasic pulses at 100 Hz each. Each experiment consisted of 40 min of baseline recording offield excitatory postsynaptic potentials (fEPSPs) at 1 stimulation per minute, 15 min of HFS, 5 min of pre-recording buff- er time, and 50 min of post-HFS fEPSP measurement. Schaffer collateral and commissural pathways were selected on the basis of morphological structure and electrical stimulation response. During experiments, the slices were continuously perfused with fresh aCSF bubbled with 95% O₂and 5% CO₂at a rate of 3 ml/min. Unfiltered data were sampled from 60 recording channels at 25 kHz using Recorder-Rack software (Multi Channel Systems, Reutlingen, Germany).

2.6. Electrophysiology data processing

MC_Rack (v.3.2.1.0, Multi Channel Systems) was used to digitize the analog MEA signal and isolate EPSPs from triggering amplitudes over 40 mV, and a custom MATLAB (v.7.0.1, Mathworks, Inc.) program was used to remove stimulus artifacts and integrate the evokedﬁeld poten- tial trajectory as reported previously[11,12].

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2.7. Passive avoidance test

The passive avoidance test apparatus consisted of an acrylic box di- vided into a light and dark chamber with a guillotine door between the chambers and an electriﬁed stainless steel rodﬂoor. Rats underwent a training trial for acquisition of fear memory and a 24 h test trial to quantify retention of fear memory. Loganin (40 mg/kg, p.o.) was administered an hour before and scopolamine (1.5 mg/kg, i.p.) was administered 30 min before the training trial. In the training trail, the rat was placed in the light chamber and the latency to step into the dark chamber was measured. After the rat entered the dark chamber, an electric foot shock (0.5 mA) was applied for 3 s and the rat was transferred to its home cage after 30 s. For the test trial, the latency to enter the dark chamber measured up to 300 s. If the rat did not enter the dark chamber within 300 s, it was scored with a latency of 300 s.

2.8. Y-maze test

The Y-maze consisted of three identical arms made of acrylic to give pathways separated by 120°. Arm entry was monitored by placing a rat in the center of the Y maze and allowing it to move freely for 8 min. An arm entry was counted when all 4 paws of the rat completely entered an arm. The sequence of arm entry was videotaped to calculate alternation behavior and % alternation. Alternation was deﬁned as a sequential visit to three different arms. Percent spontaneous alternation as a measurement of short-term spatial memory was calculated by the following equation: spontaneous alternation (%) = total observed alternation / (total arm entries−2) × 100.

2.9. Morris water maze

A circular pool 140 cm in diameter and 60 cm in height wasﬁlled with water (20 ± 1 °C, 30 cm in depth) made nontransparent by black ink with visual cues placed on the edge of the pool. A 10 cm diameter platform was submerged 5 cm below the water surface. Four points [east (E), west (W), north (N) and south (S)] on the pool were set as swimming start positions. The training session was scheduled for 4 con- secutive days and each day consisted of 4 trials starting from different positions. During the training, the latency of the rat to locate the platform was measured within 60 s. If a rat failed to reach the platform within 60 s, the rat was guided to the platform and scored with a latency of 60 s. The retention test was performed on theﬁfth day after removing the submerged platform. The time spent and swimming distance in the target quadrant were measured. The latency to reach the hidden platform and the retention time in the target quadrant were later analyzed by Smart System 3.0 videotracking software (Panlab).

2.10. Statistical analysis

The results were expressed as the mean ± S.E.M. Statistical compar- isons were conducted using one-way and two-way ANOVA followed by Duncan's post-hoc multiple comparison test using SPSS 20.0 for Win- dows (SPSS Inc., Chicago, IL, USA). The values were compared with the control using analysis of variance followed by unpaired Student'st- test. Differences were considered to be statistically signiﬁcant at pb0.05.

3. Results

3.1. Loganin potentiated hippocampal CA1 fEPSP

Hippocampal CA1 LTP was initially measured during loganin treatment (doses of 100 ng/ml, 500 ng/ml and 1μg/ml) on hippocampal slices. Both the time dependent change of % fEPSP activity before and after HFS and the mean % of fEPSP from 30 to 40 min after HFS were pooled for analysis. Loganin increased post-HFS stimulated

fEPSP in a dose dependent manner [F(1,39) = 127.4,pb0.001] (Fig.

1A). The average fEPSP activity was signiﬁcantly different among treatment groups 30 to 40 min post-stimulation [F(3,35) = 85.1,pb0.001]

(Fig. 1B). At all doses tested (100 ng/ml, 500 ng/ml and 1μg/ml), loganin increased the average fEPSP activity (n= 3,pb0.001 as compared to control). Not only in case of post-HFS, loganin also increased fEPSP significantly last 20 min before HFS [F(3,76) = 27.338, pb0.001] excluding 1μg/ml (Fig. 1C). Next, loganin was tested to deter- mine if it could improve scopolamine-induced LTP impairment, which is considered an ex vivo model of memory impairment. Repeated measures ANOVA of the measurement of fEPSP during vehicle, 500 ng/ml loganin, 300μM scopolamine, and loganin and scopolamine combined treatment showed a significant activity change [F(1,39) = 110.5, pb0.001] (Fig. 1D). The average fEPSP value 30 to 40 min after HFS in each treatment group was also significantly different [F(3,36) = 302.9,pb0.001] (Fig. 1E). Loganin increased (pb0.001) and scopolamine decreased (pb0.001) the fEPSP relative to the control group.

In the scopolamine and loganin combined treatment group, the mean fEPSP increased relative to the scopolamine only treatment group (pb0.001). Similarly, signiﬁcant difference also appeared among the groups 20 min before HFS [F(3,76) = 55.149]. Scopolamine decreased (pb0.05) and loganin increased (pb0.001) the fEPSP comparing with control group for last 20 min before HFS. When treated loganin and scopolamine together, the fEPSP increased signiﬁcantly (pb0.001) relative to scopolamine only treated group. According to these results, loganin dose-dependently increased hippocampal CA1 LTP and improved LTP suppression induced by scopolamine.

3.2. Loganin protected the disruption in avoidance memory induced by cholinergic neuronal blockade

The effect of scopolamine and loganin on avoidance memory was measured through the passive avoidance paradigm (Fig. 2). Step- through latency was measured immediately before and 24 h after the delivery of electric foot shock. Scopolamine and loganin were administered 30 min and 1 h before the exposure to electric shock, respectively.

Based on the statistical analysis, pre-training latency was not different, however scopolamine induced a signiﬁcant reduction in the latency 24 h after training (pb0.001). Co-treatment of loganin improved the reduction in latency induced by scopolamine (p= 0.024). As a result, loganin considerably blocked the avoidance memory impairment effect of scopolamine.

3.3. Loganin protected the disruption in short-term spatial memory induced by cholinergic neuronal blockade

The effect of scopolamine and loganin on short-term spatial memory was evaluated in the Y-maze (Fig. 3). Scopolamine and loganin were administered 30 min and 1 h before the 8 min session of spontaneous alternation in the maze arms, respectively. The percent alternation was reduced by scopolamine (p= 0.039), however co-treatment with loganin increased the percent alternation in scopolamine treated mice (p= 0.003) (Fig. 3A). Activity levels measured by the total number of arm entries were not different among experimental groups (Fig. 3B).

Based on these results, loganin blocked the short-term spatial memory disruption induced by cholinergic blocking reagents.

3.4. Loganin protected the disruption of long-term spatial memory retention but not spatial learning induced by cholinergic neuronal blockade

The effect of scopolamine and loganin on long-term spatial memory was evaluated in the Morris water maze (Fig. 4). Scopolamine and loganin were administered 30 min and 1 h before the daily water maze training session, respectively. The latency to locate the hidden platform gradually decreased in all experimental groups over the 4 day training session, however the latency improved less in

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scopolamine treated mice (pb0.001) (Fig. 4A). Co-treatment with loganin did not shorten the latency in scopolamine treated mice (p= 0.87). In the probe trial to measure the time spent in the quadrant in which the platform was previously located, the target quadrant time was signiﬁcantly reduced by scopolamine treatment (p= 0.002) (Fig. 4B). In loganin co-treated animals, the target quadrant time signiﬁcantly increased (p= 0.009). As a result, loganin countervailed scopolamine in the disruption of long-term memory retention, but not of spatial learning.

4. Discussion

In this study, we evaluated the cognitive-enhancing activity and al- teration in synaptic transmission of loganin to potentially prevent and treat learning and memory deﬁcits such as those seen in Alzheimer's disease. We examined whether loganin attenuates scopolamine-induced learning and memory impairments using electrophysiological and behavioral measurements. Our results suggest that loganin can attenuate cognitive deﬁcits induced by muscarinic cholinergic blockade.

LTP is widely considered one of the major experimental models for examining the synaptic mechanisms of learning and memory[5]. More- over, LTP in neuroscience can be used to study neurodegenerative diseases associated with memory, such as AD, a degenerative brain disease characterized by memory loss, cognitive decline and learning disabilities[13]. In our experiment, acute loganin treatment increased Fig. 1.Loganin potentiates fEPSP in non-treated and scopolamine treated hippocampal tissue (n= 3 per group). (A) LTP from all recordings in control and loganin treated hippocampus.

There was a significant difference between the fEPSP before and after HFS stimulation (pb0.001) and treatment (pb0.001). (B) Total fEPSP was increased by all given doses of loganin (pb0.001). (C) There were also significant increases in loganin 100 ng/ml (pb0.001) and 500 ng/ml (pb0.001) 20 min before HFS, but not in loganin 1μg/ml. (D) LTP from all recordings in loganin 500 ng/ml, scopolamine 300μM, and loganin + scopolamine treated hippocampus. There was a significant difference between the fEPSP before and after HFS stimulation (pb0.001) and treatment (pb0.001). (E) Total fEPSP was increased by loganin (pb0.001) and decreased by scopolamine (pb0.001) when compared with control. When co-treated, loganin reduced the scopolamine-induced decline in fEPSP (pb0.001). (F) In case of 20 min before HFS, total fEPSP was also increased by loganin (pb0.001) and decreased by scopolamine (pb0.05) significantly comparing with control. In case of co-treated group, loganin reduced scopolamine's effect on total fEPSP (pb0.001) comparing with scopolamine group. *pb0.01, ***pb0.001 vs control,^###pb0.001 vs scopolamine 300μM. ANOVA and repeated measures ANOVA with Tukey's HSD post-hoc test.

Fig. 2.Loganin attenuated scopolamine-induced deﬁcits of avoidance memory in the passive avoidance paradigm. Step-through latency was signiﬁcantly different before and after electric foot shock delivery [F(1,19) = 43.3,pb0.001] and between experimental groups [F(2,19) = 10.9,p= 0.0061]. Latency was reduced in the S group relative to the control group (pb0.001), and was increased in the L + S group relative to the S group (p= 0.024). All data represent average ± S.E.M. Control: received distilled water 60 min before the training session and normal saline 30 min before the training session.

S: received distilled water 60 min before the training session and scopolamine 1.5 mg/kg i.p. before the training session. L + S: received loganin 40 mg/kg p.o. 60 min before the training session and scopolamine 1.5 mg/kg i.p. 30 min before the training session. **pb0.01 vs control group,^#pb0.05 vs S group.

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HFS stimulated LTP in organotypic cultured hippocampal slices dose- dependently in the range of 100 to 1000 ng/ml. Loganin treatment at 500 ng/ml also reversed LTP impairments induced by scopolamine.

Based on theseﬁndings, loganin appears to enhance synaptic strength and recovers the memory impairment induced by scopolamine.

The passive avoidance test is a measurement of cognitive memory based on avoidance of a fear-inducing context. Rodents naturally prefer dark compartments, however receiving an electric shock in the dark compartment causes conflict to this tendency[14]. While there were no differences between experimental groups before the shock delivery, the latency to step into the dark compartment increased in non-treated controls 24 h after shock delivery, but not in mice under cholinergic receptor blockade. This inhibited avoidance memory was substantially improved by loganin cotreatment. It was previously shown that loganin can improve avoidance memory in scopolamine (0.5 mg/kg i.p.) treated mice[15]. Researchers have also shown an improvement in avoidance memory in normal animals after loganin treatment. Similarly, loganin and its herbal origin‘Cornus officinalis’extract improved amnesia induced by scopolamine (1 mg/kg, s.c.) in the passive avoidance test [16]. These results consistently show that loganin can prevent deficien- cies in avoidance memory during cholinergic receptor blockade conditions.

Performance in the Y-maze is a measurement of short-term spatial memory, mainly controlled by the prefrontal cortex[17]. Alternation behavior is measured by the succession of arm visits in an alternative manner and is based on the tendency for rodents to explore a novel area based on short-term spatial memories. As expected, scopolamine caused deﬁcits in spatial memory, but loganin cotreatment attenuated this effect without affecting the exploratory activity indicated by the total number of arm entries.

To examine long-term spatial memory, latency and searching effort tofind the hidden platform were measured using the Morris water maze. This test is known to effectively evaluate spatial memory and de- tects changes in the central cholinergic system[18–21]. We have shown that learning the location of the hidden platform based on memory of environmental cues was impaired by scopolamine, and loganin cotreatment failed to improve this impairment. However, loganin cotreatment reduced impairment in the probe test, in which scopolamine deteriorated long-term spatial recognition. Previously, loganin was shown to improve both scopolamine-induced spatial learning and spatial recognition memory in a dose-dependent manner[15]. This in- consistency may be due to the different animal species (SD rats vs ICR CD-1 mice) that were used in each study. But more possibly, such results may indicate that loganin specifically improves the long-term storage of Fig. 3.Loganin attenuated scopolamine-induced short-term memory deficits in the Y-maze. (A) Percent alternation was significantly different between experimental groups [F(2,19) = 7.6,p= 0.004]. Percent alternation was decreased in the S group relative to the control (p= 0.039), and was increased in the L + S group relative to the S group (p= 0.003). (B) Total arm entry in the Y-maze. There were no significant differences between experimental groups. All data represent average ± S.E.M. Control: received distilled water 60 min before the test and normal saline 30 min before the test. S: received distilled water 60 min before the test and scopolamine 1.5 mg/kg i.p. before the test. L + S: received loganin 40 mg/kg p.o. 60 min before the test and scopolamine 1.5 mg/kg i.p. 30 min before the test. *pb0.05 vs control group,^#pb0.05 vs S group.

Fig. 4.Loganin attenuated scopolamine-induced spatial memory retention deﬁcits but not spatial memory acquisition in the Morris water maze. (A) Latency to locate the hidden platform.

There were signiﬁcant effects by training [F(2.0, 42.4) = 34.9,pb0.001] and treatment [F(1,21) = 1093.5,pb0.001], but not in the training × treatment interaction [F(4.0, 42.4) = 0.11, p= 0.98]. The latency to locate the platform was reduced in the S (pb0.001) and L + S (pb0.001) groups relative to the control group. (B) Time spent in the target quadrant. There were signiﬁcant effects between treatment groups [F(2,15) = 10.2,p= 0.002]. The target quadrant time decreased in the S group relative to the control group (p= 0.002) and increased in the L + S group relative to the S group (p= 0.009). Dashed line: quadrant % time by chance (25%). All data represent average ± S.E.M. Control: received distilled water 60 min before training and normal saline 30 min before training. S: received distilled water 60 min before training and scopolamine 1.5 mg/kg i.p. before training. L + S: received loganin 40 mg/kg p.o. 60 min before training and scopolamine 1.5 mg/kg i.p. 30 min before training. **pb0.01 vs control group,^##pb0.01 vs S group.

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memory through the hippocampus but not the short-term processing of memories dependent on the prefrontal cortex. In the probe trial of the Morris water maze, scopolamine-induced long-term memory deﬁcit was improved by loganin. In contrast, loganin failed to improve the memory performance at the 1st day of training, which only depends on the short-term memory. But on subsequent days of training, which depends on both short-term and long-term memories from the previ- ous day, has constantly improved.

It has been suggested that the improvement in cognitive functions by loganin is associated with the inhibition of acetylcholinesterase activity in the cortex and hippocampus[15,16]. Such inhibition can increase the synaptic concentration of acetylcholine, and may enhance LTP through nicotinic[22]or muscarinic receptors[23]in the hippocampus. Behaviorally, acetylcholinesterase inhibitors were shown to re- verse scopolamine-induced amnesia in the passive avoidance paradigm [24], improve short-term memory deficits in mild cognitive impairments[25], as well as long-term spatial memory deficits[26]. Donepezil, a cholinesterase inhibitor widely prescribed for dementia prevention, can also attenuate scopolamine-induced cognitive dysfunctions via improving the cholinergic system of the central nervous system[27]. Ac- cording to these electrophysiologic and behavioralfindings of loganin, it will be of great interest to directly measure loganin-induced cholinergic synaptic activity changes for the future development of a dementia preventing reagent. In addition to the cholinergic potentiation, inhibition of BACE1, effect associated with the improvement of spatial memory in streptozotocin-induced dementia and anti-apoptotic effect against hydrogen peroxide may be associated with the memory improving mechanisms of loganin[28–30].

In conclusion, our results suggest that loganin can prevent deteriora- tions in LTP, short-term and long-term spatial recognition and avoidance memory induced by cholinergic muscarinic receptor blockade. As activation of the acetylcholine receptor system is suggested as a mech- anism of action, loganin may have signiﬁcant therapeutic value for alleviating memory impairments. Therefore, loganin may be expected to have a future use as an agent for the prevention and treatment of Alzheimer's disease or learning and memory-deﬁcit disorders.

Conﬂicts of interest

The authors declare they have no conﬂicts of interest.

Acknowledgements

This work was supported by the National Research Foundation (NRF) of Korea (2014R1A2A1A09006320).

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