Sweet Dream Liquid Chinese Medicine Ameliorates Learning and Memory Deficit in a Rat Model of Paradoxical Sleep Deprivation through the ERK/CREB Signaling Pathway

Abstract

Sweet dream oral liquid (SDOL), a traditional Chinese herbal compound contains 17 traditional Chinese medicines. It has various pharmacological effects, such as improving brain dysfunction and increasing sleeping quality. This study investigated the neuroprotective effect and the underlying mechanisms of SDOL-impaired hippocampus learning and memory-induced paradoxical sleep deprivation (PSD) in rats. Sixty Male Wistar rats were randomly divided into six groups. Before PSD, SDOL treatment group rats were intragastrically administered SDOL for 25 days at dose of 2.1, 4.2, and 8.4 mL/kg body weight per day. Normal control group, large platform control group, and PSD groups were treated with normal saline instead of SDOL. After 25 days treatment, PSD and SDOL groups were deprived of paradoxical sleep for 72 h. Then two behavioral studies were conducted to test the spatial learning and memory ability using the open field test and Morris water maze test. Expression of the c-fos, c-jun, cyclic AMP response element binding protein (CREB), extracellular signal-regulated protein kinase (ERK), mitogen-activated protein kinases (MAPK)/ERK kinase (MEK), and p-CREB, p-ERK, and p-MEK in the hippocampus were also assayed by western blot. In this study, PSD decreased the levels of p-CREB, p-ERK, p-MEK, c-fos, and c-jun. However, SDOL treatment increased expressions of these proteins. Our results showed that SDOL improved 72-h PSD-induced cognitive impairment. These affects may be mediated by increasing the contents of c-fos, c-jun, and p-CREB/ERK signaling.

Introduction

Sleep plays an important role in growth, development, and cognitive functions, such as learning and memory. Electroencephalograh studies of eye movement and muscle tension during sleep have revealed that sleep is divided into nonrapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS, also called paradoxical sleep).¹ Recently, there is a growing body of evidence showing a strong correlation between sleep, especially paradoxical sleep, and learning and memory. For example, paradoxical sleep deprivation (PSD) reduces learning ability and impairs memory in humans and experimental animals.^2,3 With the acceleration of pace of life and increased society competition, the incidence of sleep disorder or sleep-related diseases has increased year by year, and increasing numbers of people are in a chronic PSD state. Sleep deprivation, especially PSD has been shown to impair central nervous system function, including reductions in the activity, learning ability, and spatial memory.^4,5

A large body of evidence suggests that a critical step of learning and memory is the phosphorylation of cyclic AMP response element binding protein (CREB).^6,7 It has also been reported that the decrease of p-CREB is one of the mechanisms of PSD-induced learning and memory impairment. CREB is a transcription factor that regulates its immediate downstream early genes transcription such as c-fos and c-jun. ⁸ C-fos and c-jun proteins encoded by immediate early genes are closely related to learning and memory and may play important roles in the regulation of these processes.⁹

Various signaling pathways could regulate the phosphorylation of CREB, and among them, the extracellular signal-regulated protein kinase (ERK) signaling pathway is a critical step, which can directly phosphorylate CREB.

ERK is one group of such protein kinases that belong to a larger family of mitogen-activated protein kinases (MAPK).¹⁰ ERK is also activated by learning and memory.^11
–13 A large body of studies have confirmed that ERK is necessary for long-term memory in different tasks across different species.¹⁴ Given the importance of sleep and ERK for long-term memory, we hypothesize that ERK is involved in phosphorylation of CREB.

Drug therapy is an important method for treating sleep disorder. The typical sedative–hypnotic and anticonvulsant drugs, which are mostly obtained by chemical synthesis, are the main drugs used for sleep disorders. However, these drugs have many side effects like low safety margins, addiction, and dependence.

Chinese medicine has a long history of preventing and treating brain-related disease.^15,16 These medicines have well-documented curative effects and are deemed to be safe due to low toxicity and few side effects.^17,18 Sweet dream oral liquid (SDOL; Rong Chang Pharmaceutical Company Limited) is a traditional Chinese herbal compound prepared as an oral solution that contains 17 traditional Chinese medicines (Table 1). It has various pharmacological effects, such as ameliorating brain dysfunction, coronary vascular disease, appeasing neural system, relieving fatigue, improving sleep, and increasing sleep quality step by step. Moreover, in clinical practice, it has been used for the treatment of neurasthenia and cerebral embolism.^19,20 In previous studies, Radix Et Rhizoma Caulis acanthopanacis Senticosi improves spatial learning and depressive behavior in depressed rats and Herba Epimedii significantly improves the learning and memory ability in AD mouse model induced by Aβ_1–40 lateral ventricle injection.^21,22

Table 1.

The Component of Sweet Dream Oral Liquid

English name	Chinese name	Part used	Collected place	Amounts used (g)
Radix Et Rhizoma Caulis acanthopanacis Senticosi	ciwujia	Root or Rhizome	Heilongjiang	53
Bombyx	cane	Dry body	Sichuan	13
Rhizoma Polygonati	huangjing	Rhizome	Hunan	67
Radix Codonopsis	dangshen	Root	Gansu	40
Fructus Mori	sangshen	Dry fruit	Anhui	33
Radix Astragali	huangqi	Root	Gansu	40
Fructus Amomi	sharen	Dry fruit	Hainan	5
Fructus Lycii	gouqi	Dry fruit	Ningxia	40
Fructus Crataegi	shanzha	Dry fruit	Shandong	160
Radix Rehmanniae Praeparata	shudihuang	Root	Henan	27
Herba Epimedii	zhiyinyanghuo	Rhizome	Gansu	27
Poria	fuling	Dry body	Anhui	27
Pericarpium Citri reticulatae	Chenpi	Pericarp	Guangdong	27
Rhizoma Pinelliae Praeparatum	fabanxia	Rhizome	Guizhou	1.3
Semen Strychni Praeparatum	zhimaqianzi	Dry seed	Guangxi	27
Rhizoma Dioscoreae	Shanyao	Rhizome	Henan	27
Rhizoma Alismatis	zexie	Rhizome	Sichuan	40

Although this oral liquid has been used frequently, the effect and mechanism of SDOL for PSD-induced cognition impairment is still unknown. In this study, we studied the role of SDOL in modulating hippocampus-dependent spatial learning and memory through the Morris water maze (MWM) test, excitability and exploration through an open field test (OFT), and by examining changes in c-fos, c-jun, and ERK/CREB expression in the rat hippocampus in the SDOL-treated group after 72 h of PSD.

Materials and Methods

Preparation of SDOL

Seventeen traditional Chinese medicines (Table 1) were carefully authenticated by Rong Chang of the Pharmaceutical Company Limited Quality Inspection department, according to Chinese pharmacopoeia (The Pharmacopoeia Commission of PRC, 2010). All herbs were decocted with distilled water twice in proportion, the first time was for a period of 1.5 h and the second was for 1 h. After that, the supernatants were combined, filtered, and concentrated to a relative density of 1.18–1.20. Then 65% ethanol was added and allowed to stand for 10 min. The supernatant was concentrated to a relative density of 1.16–1.20, 2 g Potassium sorbate was added, and the final volume brought to 1000 mL with distilled water. Quality Inspection department determined that the icariin concentration was not less than 1.6 mg per gram of Herba Epimedii Praeparatum and not less than 25 μg per mL of SDOL. The entire process was supervised according to the policy of the State Food and Drug Administration of P.R. China.

Analysis of chemical contents of SDOL by HPLC

SDOL contained many effective constituents, but icariin (C₃₃H₄₀O₁₅) is a characteristic stable compound, and in Chinese pharmacopoeia, icariin is used as the quality evaluation standard of SDOL. To determine the identification and quantification of compounds of SDOL, high performance liquid chromatography (HPLC) analysis was performed using a method reported previously. A sample of the SDOL extract (10 mL) was precision measured into the 25 mL of solvent (methanol:H₂O = 1:1). 20 μg icariin dissolved in 1 mL of 70% methanol as standard sample icariin solution. The HPLC system consisted of a Waters 600 Binary pump, Waters 486 detector, HW-2000 chromatography analytical workstation, and Hedera ODS-3 C₁₈ column (4.6 × 250 mm 5 μm). The mobile phase consisted of a mixture of acetonitrile-0.05% and phosphoric acid water (75:25, v/v) at a flow rate of 1.0 mL/min and 30°C temperature. The detector wavelength was set at 270 nm.

Since SDOL is a compounded medicine with a complex composition, we prepared two batches of SDOL extracts under the same conditions and analyzed the main components in SDOL by HPLC. Then, we found that all the main chromatographic peaks detected in batch I were consistent with batch II, demonstrating a good reproducibility of SDOL chemical composition. Figure 1 shows the HPLC fingerprint of the two batches of SDOL extracts.

FIG. 1.

Chromatograms showing bioactive compounds in standard components of sweet dream oral liquid (SDOL). (A_1–2) SDOL extract, (B) standard sample icariin (Icariin, Retention time (tR) = 12.259 min).

Animals and drug treatment

Sixty healthy male Wistar rats (body weight 200–220 g) were purchased from Shandong Lukang Pharmaceutical Company Limited. They were randomly divided into six groups of 10 rats each: normal control (NC); large platform control (LPC); PSD; SDOL low; middle; and high groups (2.1, 4.2, and 8.4 mL/kg). They were given free access to feed and water and housed under standard laboratory conditions at automatic 12-h light/12-h dark cycle, constant temperature (22°C ± 2°C), and humidity of 50–60%. The experimental protocols followed the Guide for the Care and Use of Laboratory Animals of our institute and were carried out according to international guidelines on the ethical use of laboratory animals. Care was taken to minimize the number of animals used and their suffering during the experiment. Every rat was given intragastric gavage with 0.9% NaCl (8.4 mL/kg body weight) and SDOL (2.1, 4.2, and 8.4 mL/kg body weight) once a day for 32 days, respectively.

REMS deprivation

After 25 days of intragastric administration, PSD and SDOL group rats were deprived of REMS for 72 h using modified multiple platform method (MMPM). Experimental protocol and timeline are listed in Figure 2. The rats were placed into a water tank (170 × 70 × 50 cm), containing 12 circular platforms (6.5 cm diameter, 10 cm height), and the water level reached up to 1 cm beneath the platform levels. When the rats reached the REMS phase, they fell into the water and were awakened because of muscle atonia.²³ To exclude the effects of water stress and activity restrictions, the LPC group rats were placed into a similar water tank, containing six circular platforms (18 cm, diameter), so they were too large for the rats to fall into the water. During the sleep deprivation period, the rats were given free access to food and water. The water in the tanks was changed daily.

FIG. 2.

Experimental protocol and timeline depicting the two behavioral tests.

Open field test

The OFT provides a novel environment in which animal locomotion, exploration, and anxiety are measured. This test was carried out at the end of 72 h of PSD. Briefly, the Open Field apparatus consisted of a square plastic arena (80 × 80 × 40 cm) with black walls and floor, which is divided into 25 squares of equal areas.²⁴ In the test, every rat was placed in the center of the field and allowed to explore freely for 3 min. Distances of crossings and numbers of rearings are usually used as measures of locomotor and exploration activity.^14,25 The higher number of crossing and rearing indicates the stronger excitability and exploration. This apparatus was cleaned with 5% ethanol after the test of every rat to avoid residual odors of rats.

MWM test

To evaluate the effect of SDOL on memory and learning functions of PSS rats, all rats were tested, using the MWM, for spatial learning and memory. The water maze apparatus consisted of a black circular pool 200 cm in diameter, which was divided into four quadrants of equal size. The water in the apparatus was kept at 23°C ± 1°C and a depth of 35 cm. A hidden 10-cm circular platform was placed 2 cm below the surface of the water and kept in a constant position and a constant quadrant of the pool throughout the trials. The trials lasted for 5 days. In each trial, every rat was released into three other quadrants (excluding the platform-containing quadrant) with its face toward the wall of the water maze. The rat was allowed to swim for a maximal duration of 120 sec in each trial to find the platform. When a rat mounted the platform, it was kept there for 30 sec. If the rat did not find the platform within the stipulated time (120 sec), it was guided on the platform and kept there for 30 sec. The escape latency to find the hidden platform was recorded automatically using the Video Tracking and Analysis System. On the 6th day, a probe trial was conducted, which consisted of a single trial in which the rats were allowed to swim for 120 sec, with the platform removed from its position. The time that a rat spent in the target quadrant and the number of crossing over at the original platform position were measured.²⁶

Western blotting

Hippocampal tissues of all the rats in each group were isolated and then homogenized in 1% sodium dodecyl sulfate (SDS) containing 1 mM of phenylmethylsulfonyl-fluoride (PMSF). The homogenates were allowed to stand for 30 min and then centrifuged for 20 min at 8000 g. The supernatant was used for protein concentration assay (BCA Protein Assay Reagent; Beyotime Biotechnology) and loading sample preparation. The whole tissue homogenates (50 μg of total protein) were heated at 95°C for 10 min and resolved on 10% gels using SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 7% skimmed milk for 2 h at room temperature and incubated with primary antibody CREB (1:1000; Cell Signaling Technology, Inc.), p-CREB (1:1000; Cell Signaling Technology, Inc.), ERK (1:1000; Cell Signaling Technology, Inc.), p-ERK (1:2000; Cell Signaling Technology, Inc.), MAPK/ERK kinase (MEK) (1:1000; Cell Signaling Technology, Inc.), p-MEK (1:1000; Cell Signaling Technology, Inc.), c-fos (1:1000; Cell Signaling Technology, Inc.), and c-jun (1:1000; Cell Signaling Technology, Inc.) in TBS plus 0.1% Tween-20 (TBST) with 5% bovine serum albumin overnight at 4°C, respectively. The secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000; BioWordle) was applied for 1 h after rinsing with a buffer. Proteins were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) on a FluorChem SP Imaging System (Alpha Innotech). The intensities of the bands were quantified using scanning densitometry.

Statistical analysis

Data were expressed as mean ± standard deviation and analyzed by one-way ANOVA. Statistical analysis was done by software SPSS 13.0 for windows (SPSS, Inc. ). P < .05 was considered significant.

Results

HPLC analyses of SDOL

SDOL exact and the most important component, icariin (C₃₃H₄₀O₁₅), were determined to be as shown in Figure 2.

Behavioral experiments

Effects of SDOL on locomotor and exploration activity in the OFT

Crossings and rearings are commonly used as measures of exploration and locomotor activity.²⁷ In Figure 3, after 72-h PSD, there was a significant reduction in the number of crossings and rearings in rats, compared to the controls. However, SDOL treatment groups showed higher numbers of crossings and rearings in a dose-dependent manner (P < .05 or P < .01) when compared to the PSD group.

FIG. 3.

Effect of paradoxical sleep deprivation (PSD) and/or SDOL treatment (2.1, 4.2, and 8.4 mL/kg body weight per day) on the numbers of rearings (A) and crossings (B) in the Open Field test in rats. ^## P < .01 compared with the control group; **P < .01 compared with the SDOL groups.

Effects of SDOL treatment on PSD-induced impairment of learning in MWM test

As shown in Figure 4A, escape latency of all groups of rats were significantly reduced on days 3, 4, and 5 of training. PSD markedly impaired learning function, which is indicated by the observation that animals in the PSD groups had significantly increased escape latencies, compared to the control group. On the other hand, treatment with SDOL at daily doses of 2.1, 4.2, and 8.4 mL/kg body weight significantly reduced the escape latency compared to PSD rats (P < .05 or P < .01). However, there is no significant difference between the NC and LPC groups (Fig. 4B, C).

FIG. 4.

Performance of rats in the Morris water maze test during spatial learning and memory phase. (A) Results of spatial learning. (B) Mean escape latency to find the hidden platform. (C) Results of memory test. In the memory phase test, the platform was removed from the target quadrant, and number of times and the time the rats spent in the target quadrant during 120 sec were recorded. ^## P < .01 compared with the control group; **P < .01 compared with the SDOL-treated PSD group.

Effects of SDOL treatment on PSD-induced impairment of long-term memory

The probe trail revealed a considerable decrease in preference for the target quadrant and the number of platform crossings in PSD rats when compared to the control group rats. Rats administered with SDOL also spent maximum time in the target quadrant and crossed the most number of times into the platform after their release into the maze compared to PSD rats. In summary, PSD-impaired learning and long-term memory and SDOL prevent these effects.

Effects of SDOL on CREB and p-CREB protein levels in hippocampus

The levels of p-CREB and total CREB in the hippocampus were detected by Western blot (Fig. 5). The phosphorylated form of CREB was measured so that the active state of the protein could be analyzed. In this study, Western blot revealed that there was a significant decrease of p-CREB/CREB protein level in the hippocampus of PSD rats compared to the control group. However, SDOL treatment at daily doses of 4.2 and 8.4 mL/kg prevented the effects of sleep deprivation on the protein levels of P-CREB/CREB, which were dose dependently increased.

FIG. 5.

Effect of SDOL treatment on the levels of cyclic AMP response element binding protein (CREB) (phosphorylated and total) in the hippocampus. There was no significant difference in total CREB among all groups (A). PSD caused a significant decrease in the levels of p-CREB compared to the control groups (B). SDOL treatment (especially at the doses of 4.2 and 8.4 mL/kg) increased the content of p-CREB. ^# P < .05 and ^## P < .01 compared with the control group; *P < .05 and **P < .01 compared with the SDOL group.

Effects of SDOL on ERK, p-ERK, MEK, and p-MEK protein levels in hippocampus

Figure 6 showed effects of SDOL on ERK, p-ERK, MEK, and p-MEK protein levels in the hippocampus. p-ERK (p-ERK1/2) and p-MEK were decreased in the hippocampus of PSD rats, compared to control rats (P < .01). In comparison, SDOL treatment dose dependently prevented the decrease of ERK1/2 and MEK phosphorylation in the hippocampus (P < .01). However, there were no significant differences in total-ERK and MEK among all groups.

FIG. 6.

Effects of SDOL on p-ERK1/2, p-MEK and total extracellular signal-regulated protein kinase (ERK), and mitogen-activated protein kinases (MAPK)/ERK kinase (MEK) in the hippocampus. There was no change of the total of ERK1/2 and MEK. p-ERK1/2 and p-MEK significantly decreased after PSD, but they were higher in the SDOL treatment group. ^# P < .05 and ^## P < .01 compared with the control group; *P < .05 and **P < .01 compared with the PSD group.

Effects of SDOL on c-fos and c-jun protein levels in hippocampus

The c-fos and c-jun genes, which are the downstream targets of CREB, may play important roles in learning and long-term memory (Fig. 7). C-fos and c-jun protein were detected by Western blot. In this study, the protein levels of c-jun and c-fos significantly decreased in the PSD group compared to that of control group. However, in the SDOL treatment group, especially at doses of 4.2 and 8.4 mL/kg, there was a significant increase in the levels of c-fos and c-jun.

FIG. 7.

Effects of SDOL treatment on the levels of c-fos and c-jun in the hippocampus. The total c-fos (A) and c-jun (B) after PSD treatment were significantly lower, but SDOL treatment prevented the decrease. ^# P < .05 and ^## P < .01 compared with the control group; *P < .05 and **P < .01 compared with the SDOL-treated PSD group.

Discussion

Numerous studies have reached a consensus that PSD produces negative effects on cognitive function such as learning and memory.²⁸ Our data showed that spatial learning and memory were impaired, which were caused by the 72-h PSD. Animals with PSD displayed a decreased locomotor activity in the OFT, and impairment of spatial learning and memory in the MWM task after the 72-h PSD. However, the mechanism of sleep deprivation on cognitive function impairment is not fully understood.

SDOL is an oral solution composed of 17 traditional Chinese herbs and can improve brain function. However, whether SDOL could improve cognitive function of PSD rats and the action mechanism are not clear. In this study, we adopted an MMPM to advance PSD.

The MMPM is a widely used model in PSD to overcome many limits of single platform studies such as moving restriction of rats, separating one rat from the groups' results in stress.²⁹ Our data indicated that the numbers of rearings and crossings in OFT increased in SDOL treatment groups, especially with doses of 4.2 and 8.4 mL/kg, showing that SDOL has an effect on exploration and attention ability. Moreover, compared to the control group, PSD rats showed an increased escape latency and decreased numbers of platform crossings and time in the target quadrant in the water maze task. In other words, these results indicated that PSD induced memory and learning ability deficits, and SDOL decreased escape latency and increased the numbers and time into the target quadrant, revealing that SDOL could improve the cognitive ability in PSD rats.

In recent years, we have found that many genes are involved in learning and memory. Immediate early genes c-fos and c-jun become more active.³⁰ In this study, the contents of their expression products, c-fos and c-jun protein, were detected by Western blotting. After 72-h PSD, the protein levels of c-fos and c-jun were decreased in the hippocampus compared to the control group. SDOL treatment, especially at the doses of 4.2 and 8.4 mL/kg, increased c-fos and c-jun protein levels.

The immediate early genes, c-fos and c-jun, are considered to be two of the CREB targets. C-fos and c-jun are CREB downstream targets, and their transcription depends on CREB phosphorylation at Ser133.³¹ Several studies indicate that p-CREB is abundant and plays an important role in learning and memory in the hippocampus.³² CREB mediates synthesis of proteins that are important for long-term synaptic plasticity and memory.³³ Our data showed that the levels of p-CREB are decreased in the PSD group, but not the total CREB. This indicates that the phosphorylated form of CREB is the critical factor related to learning and memory after PSD. The decrease in p-CREB was prevented by SDOL treatment. Therefore, we speculated that SDOL might partially reverse the reduction of p-CREB expression in the hippocampus induced by PSD and mitigate PSD-induced learning and memory impairment.

The upstream modulators of CREB include extracellular signal-regulated kinase (ERK), calcium/calmodulin-dependent protein kinase IV (CaMK IV), and cyclic adenosine monophosphate-protein kinase A (cAMP-PKA).

ERK is involved in long-term synaptic plasticity and synaptic plasticity-related gene expression.^34

–38 Many articles have confirmed that decreased ERK activation in the hippocampus was involved in sleep deprivation-induced spatial learning and memory impairment.³⁹ In this study, the reduction of ERK and MEK phosphorylation, but not the total ERK or MEK in the hippocampus after the 72-h PSD, was consistent with the above reported studies, suggesting that the PSD-induced reduction of ERK phosphorylation in the hippocampus is sufficient to cause memory impairment. SDOL at doses of 2.1, 4.2, and 8.4 mL/kg reversed PSD-induced reduction of p-ERK1/2 and p-MEK in the hippocampus. Therefore, we speculated that SDOL might partially reverse the reduction of p-CREB expression by the ERK1/2 signaling pathway in hippocampus induced by PSD, and mitigate PSD-induced memory impairment. Thus, we concluded that it might be one of the mechanisms of the SDOL protective function in improving learning and memory impairment.

Conclusion

In this study, SDOL improved learning and memory function in a PSD rat model, especially at the doses of 4.2 and 8.4 mL/kg. The effect may be due to its ability to increase p-CREB and its downstream genes c-fos and c-jun, by the ERK signaling pathway.

Footnotes

Acknowledgments

This study gained support from the National Natural Science Foundation of China (Grant No. 81102828, 81273037) and the Natural Science Foundation of Shandong province of China (No. 2015ZRB14548). The help from the project of Taishan Scholars Construction Engineering to Hanfang must been acknowledged too.

Author Disclosure Statement

No competing financial interests exist.

References

Benington

, Heller

: Does the function of REM sleep concern non-REM sleep or waking?. Prog Neurobiol, 1994; 44:433–449.

Walker

: Cognitive consequences of sleep and sleep loss. Sleep Med, 2008; 9:29–34.

Diekelmann

, Wilhelm

, Born

: The whats and whens of sleep-dependent memory consolidation. Sleep Med Rev, 2009; 13:309–321.

Hagewoud

, Havekes

, Novati

, Keijser

, Van der Zee

, Meerlo

: Sleep deprivation impairs spatial working memory and reduces hippocampal AMPA receptor phosphorylation. J Sleep Res, 2010; 19:280–288.

Aleisa

, Helal

, Alhaider

, et al.: Acute nicotine treatment prevents REM sleep deprivation-induced learning and memory impairment in rat. Hippocampus, 2011; 21:899–909.

Cirelli

, Tononi

: Differential expression of plasticity-related genes in waking and sleep and their regulation by the noradrenergic system. J Neurosci, 2009; 20:9187–9194.

, Gao

, Fang

, Zhou

, Jiang

: The mechanism and characterization of learning and memory impairment in sleep-deprived mice. Cell Biochem Biophys, 2010; 58:137–140.

Alhaider

, Aleisa

, Tran

, Alkadhi

: Sleep deprivation prevents stimulation-induced increases of levels of P-CREB and BDNF: Protection by caffeine. Mol Cell Neurosci, 2011; 46:742–751.

Tischmeyer

, Kaczmarek

, Strauss

: Accumulation of c-fos mRNA in rat hippocampus during acquisition of a brightness discrimination. Behav Neural Biol, 1990; 54:165–171.

10.

Dong

, Huang

, Yu

, Hu

, Mu

, Liu

: Effect of Tenuifoliside A isolated from Polygala tenuifolia on the ERK and PI3K pathways in C6 glioma cells. Phytomedicine, 2014; 21:1451–1457.

11.

Josselyn

: Continuing the search for the engram: Examining the mechanism of fear memories. J Psychiatry Neurosci, 2010; 35:221–228.

12.

Matsuyama

, Taniguchi

, Kadoyama

, Matsumoto

: Long-term potentiation-like facilitation through GABAA receptor blockade in the mouse dentate gyrus in vivo. Neuroreport, 2008; 19:1809–1813.

13.

Grahnstedt

, Ursin

: Platform sleep deprivation affects deep slow wave sleep in addition to REM sleep. Behav Brain Res, 1985; 18:233–239.

14.

Silva

, Abílio

, Kameda

, et al.: Effects of 3-nitropropionic acid administration on memory and hippocampal lipid peroxidation in sleep-deprived mice. Prog Neuropsychopharmacol Biol Psychiatry, 2007; 31:65–70.

15.

Chen

, Zhang

, Wang

, Ye

, Huang

: Formononetin promotes proliferation that involves a feedback loop of microRNA-375 and estrogen receptor alpha in estrogen receptor-positive cells. Mol Carcinog, 2015; [Epub ahead of print]; DOI: 10.1002/mc.22282.

16.

Chen

, Zhao

, Ye

, Wang

, Tian

: Estrogen receptor beta-mediated proliferative inhibition and apoptosis in human breast cancer by calycosin and formononetin. Cell Physiol Biochem, 2013; 32:1790–1797.

17.

Chen

, Zhao

, He

, et al.: Improvement in the neural stem cell proliferation in rats treated with modified “Shengyu” decoction may contribute to the neurorestoration. J Ethnopharmacol, 2015; 165:9–19.

18.

Huang

: Chinese medicine treatment of cardio-cerebrovascular disease gradually have highlight social advantage. J China Prescription Drug, 2009; 92:54–55.

19.

Xing

, Sun

, Huang

, et al.: Influence of α-asarone on open field behavior of mice. Anat Res, 2009; 31:170–172.

20.

Zeng

, Cui

, Wang

: Clinical observation about the therapy of sweet dream oral liquid to anxiety neurosis and cerebral hypofunction resulted from cerebral infarction. Clin Focus, 2004; 19:489–501.

21.

Wan

, Wang

, Weng

, Wang

, Liu

: Effect of radix acanthopanacis Senticosi on behevior, spatial learning and memory in rat depression model. Chin J Psychiatry, 2008; 41:53–56.

22.

, Song

, Ai

, Li

: Effects of epimedium flavanoids on learning and memory ability in Alzheimer's disease mouse model. Chin J Behav Med Brain Sci, 2009; 18:769–771.

23.

Fortes

, Almeida

, Mendonça-Júnior

, et al.: Anxiolytic properties of new chemical entity, 5TIO1. Neurochem Res, 2013; 38:726–731.

24.

Prut

, Belzung

: The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: A review. Eur J Pharmacol, 2003; 463:3–33.

25.

Netto

, Hodges

, Sinden

, et al.: Effects of fetal hippocampal grafts on ischemic-induced deficits in spatial navigation in the water maze. Neuroscience, 1993; 54:69–72.

26.

Wang

, Zhang

, Guo

, Zhou

, Teng

, Chen

: Anhedonia and activity deficits in rats: Impact of post-stroke depression. J Psychopharmacol, 2009; 23:295–304.

27.

Williams

, El Mohsen

, Vauzour

, et al.: Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels. Free Radic Biol Med, 2008; 45:295–305.

28.

Casu

, Pisu

, Sanna

, Tambaro

, Spada

, Mongeau

: Effect of delta-terahydrocannabinol on phosphorylated CREB in rat cerebellum: An immunohistochemical study. Brain Res, 2005; 1048:41–47.

29.

Jerry

, Jay

, Hendrikus

, Lemmens

: Lean body weight scalar for the anesthetic induction dose of propofol in morbidly obese subjects. Anesth Analg, 2010; 113:57–62.

30.

Sheng

, Greenberg

: The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron, 1990; 4:477–485.

31.

Miyamoto

: Molecular mechanism of neuronal plasticity: Induction and maintenance of long-term potentiation in the hippocampus. J Pharmacol Sci, 2006; 100:433–442.

32.

, Liu

, et al.: Effect of Kai Xin San on learning and memory in a rat model of paradoxical sleep deprivation. J Med Food, 2013; 16:280–287.

33.

Guzman-Marin

, Ying

, Suntsova

: Suppression of hippocampal plasticity-related gene expression by sleep deprivation in rats. J Physiol, 2006; 575:807–819.

34.

Watabe

, Zaki

, O'Dell

: Coactivation of β-adrenergic and cholinergic receptors enhances the induction of long-term potentiation and synergistically activates mitogen-activated protein kinase in the hippocampal CA1 region. J Neurosci, 2000; 20:5924–5931.

35.

, Lu

, Chang

, Gean

: Involvement of mitogen-activated protein kinase in hippocampal long-term potentiation. J Biomed Sci, 1999; 96:409–417.

36.

, Deisseroth

, Tsien

: Spaced stimuli stabilize MAPK pathway activation and its effects on dendritic morphology. Nat Neurosci, 2001; 4:151–158.

37.

Adams

, Roberson

, English

, Selcher

, Sweatt

: MAPK regulation of gene expression in the central nervous system. Acta Neurobiol Exp (Wars), 2000; 60:377–394.

38.

Davis

, Vanhoutte

, Pagès

, Caboche

, Laroche

: The MAPK/ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo. J Neurosci, 2000; 20:4563–4572.

39.

Andersen

, Perry

, Tufik

: Acute cocaine effects in paradoxical sleep deprived male rats. Prog Neuropsychopharmacol Biol Psychiatry, 2005; 29:245–251.