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Effects of cordycepin on spontaneous alternation behavior and adenosine receptors expression in hippocampus
Physiology & Behavior 184 (2018) 135–142
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Effects of cordycepin on spontaneous alternation behavior and adenosine receptors expression in hippocampus
T
Zhi-Ping Caoa,1, Dan Daia,1, Peng-Ju Weia, Yuan-Yuan Hana, Yan-Qing Guana, Han-Hang Lia, ⁎ Wen-Xiao Liua, Peng Xiaoa, Chu-Hua Lia,b, a b
School of Life Science, South China Normal University, Guangzhou 510631, China Brain Science Institute, South China Normal University, Guangzhou 510631, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Cordycepin Spontaneous alternation behavior Hippocampus Adenosine Adenosine A2A receptor Adenosine A1 receptor
Cordycepin, an adenosine analogue, has been reported to improve cognitive function. Important roles on learning and memory of adenosine and its receptors, such as adenosine A1 and A2A receptors (A1R and A2AR), also have been shown. Therefore, we assume that the improvement of learning and memory induced by cordycepin is likely related to hippocampal adenosine content and adenosine receptor density. Here we investigated the effects of cordycepin on the short-term spatial memory by using a spontaneous alternation behavior (SAB) test in Y-maze, and then examined hippocampal adenosine content and A1R and A2AR densities. We found that orally administrated cordycepin (at dosages of 5 and 10 mg/kg twice daily for three weeks) significantly increased the percent of relative alternation of mice in SAB but not altered body weight, hippocampus weight and hippocampal adenosine content. Furthermore, cordycepin decreased A2AR density in hippocampal subareas; however, cordycepin only reduced the A1R density in DG but not CA1 or CA3 region. Our results suggest that cordycepin exerts a nootropic role possibly through modulating A2AR density of hippocampus, which further support the concept that it is mostly A2AR rather than A1R to control the adaptive processes of memory performance. These findings would be helpful to provide a new window into the pharmacological properties of cordycepin for cognitive promotion.
1. Introduction Cordycepin (3′-deoxyadenosine), first isolated from the fermented broth of medicinal mushroom Cordyceps militaris, is widely used as a traditional Chinese medicine and exhibits a variety of clinical effects such as immunomodulatory, antioxidant, anti-inflammatory and antimicrobial activities [1–3]. Cordycepin is deaminated quickly by adenosine deaminase and metabolized rapidly to an inactive metabolite, 3′-deoxyhypoxanthinosine in vivo. The half-life and bioavailability of cordycepin by oral administration are 2.1 h and 19.2 μmol h/L (area under concentration-time curve), respectively [4]. In recent studies, cordycepin has been reported to improve cognitive function in mice and protect against cell death induced by cerebral ischemia injury [5,6]. Adenosine is mainly through its action on adenosine A1 receptor (A1R) and adenosine A2A receptor (A2AR) to control and integrate cognition and memory [7]. Adenosine deficiency is able to result in the impairment of synaptic plasticity [8,9]. In the brain, A1R and A2AR are mainly located in synapses, controlling the release of neurotransmitters,
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1
such as glutamate (Glu) and acetylcholine (ACh) that are involved in memory and other cognitive processes [10]. Adenosine usually relies on a balance on the activation of inhibitory A1R and facilitatory A2AR [11]. The activation of A1R prevents neuronal damage [12], while the activation of A2AR plays an important effect on the associative learning process and its relevant hippocampal circuits [13]. Previous studies have shown that synaptic levels of adenosine could control synaptic transmission and plasticity by acting on synaptic A1R and A2AR [14,15]. It is worth mentioning that cordycepin, as an adenosine analogue, is structurally similar to adenosine. A previous study has shown that the anti-apoptotic effects of cordycepin are partially dependent on the activation of A1R [16], and cordycepin increases theta waves power density via nonspecific adenosine receptor in rats [17]. These researches indicated that cordycepin may exert potentially biological effects as a nonspecific (or partial) agonist of adenosine receptor. Given that adenosine is mainly through its action on A1R and A2AR to control and integrate cognition functions [7], therefore, we assume that the
Correspondence author at: School of Life Science, South China Normal University, Guangzhou 510631, China. E-mail address: [email protected] (C.-H. Li). Both authors contributed equally to the paper.
https://doi.org/10.1016/j.physbeh.2017.11.026 Received 5 May 2017; Received in revised form 18 October 2017; Accepted 21 November 2017 Available online 23 November 2017 0031-9384/ © 2017 Elsevier Inc. All rights reserved.
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recorded if the mouse went back to either of the two arms just previously visited. The percentage of relative alternation during a period of 5 min (from 3 to 8 min) observation was calculated from the ratio of the number of alternations divided by the number of total entries [21,22]. The value was multiplied by 100. Experiments and statistical analysis were double blinded. At the end of behavioral test, mice were randomly divided into two parts: HPLC and immunohistochemistry. All mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and then carried out the next experiments.
improvement on learning and memory by cordycepin is likely related to the densities of A1R and A2AR. In order to provide a new window into the pharmacological properties of cordycepin, in the present study, we examined the roles of cordycepin on short-term spatial memory using a spontaneous alternation behavior (SAB) test in Y-maze, and investigated adenosine content and A1R and A2AR densities in hippocampal subregions. 2. Materials and methods 2.1. Drugs and chemicals
2.4. HPLC procedure
Cordycepin (> 98% purity) was obtained by using a column chromatographic method to perform extraction and separation [18]. Adenosine standard and other chemicals were purchased from Sigma Ltd. (Louis, USA). Antibodies of rabbit anti-adenosine receptor A1 (bs6649R) and rabbit anti-adenosine receptor A2A (bs-1456R), and SP kit (rabbit, SP-0023) were bought from Bioss Company (Beijing, China). The constitution of the secondary kit is biotin-labeled goat anti-rabbit IgG.
Anesthetized mice were sacrificed by rapid decapitation. The brains were removed and carefully rinsed in ice-cold saline to remove any blood contaminants. Bilateral hippocampus was dissected quickly to ensure bioactivity of tissues. After weigh and homogenization in 2.0 mL Eppendorf tubes, the sample was thawed and minced in 0.4 mmol/L HClO4 (200 μL) for 2 min. The homogenate was centrifuged at 15000 rpm for 15 min at 4 °C. The supernatant was transferred and filtered through a 0.22 μm pore size Milipore filter for HPLC analysis. Analysis was carried out by using Waters-HPLC system (Waters, Massachusetts, USA) in 240 nm. The sample was separated with an XTerra MS C18 column (5 μm, 4.6 × 250 mm column, USA) manufactured by water. The mobile phase was composed of a mixture of acetonitrile-water solution (0.6 mL/min, 5: 95, v/v) filtered through a 0.45 μm nylon filter and degassed under ultrasound and vacuum for 30 min, with injecting 10 μL solution into the equilibrated HPLC system every time. Adenosine standard was dissolved in distilled water in concentrations of 2, 4, 6, 8, 10 and 20 μg/mL and then established a standard curve. The content of adenosine in hippocampus was calculated by dividing hippocampal weight.
2.2. Animals, ethics statements and groups All studies were reported in accordance with the ARRIVE guidelines for all the experiments involving animals. Animal procedures undertaken were approved by the Committee on Animal Care and Usage of South China Normal University and every effort was made to minimize animal suffering. Kunming mice (an outbred mouse stock deriving from Swiss albino mice with a high ratio of gene heterozygosis, 25–28 g, 6 weeks of age) were obtained from the animal facility at the Sun Yatsen University (Guangzhou, China) [19]. The animals were housed in the animal facility of South China Normal University in standard conditions. Food and water were supplied ad libitum. Room temperature was 23–25 °C, relative humidity was 40–50%, and the day/night cycle was set at 12 h/12 h. All animals were acclimatized for 3 days before starting any procedures. Mice were group housed (6 mice per cage) in conventional laboratory rodent cages (ZS Dichuang Co., Beijing, China) of dimensions 18.7 (W) × 15.5 (H) × 29.5 (L) cm. Animals were selected randomly from all cages and divided into groups. All analyses were carried out without prior knowledge of the treatments. Animals received a numerical code throughout the whole experiment. The current study included two experiments: a preliminary experiment and a formal experiment. In the preliminary experiment, mice were divided into male (n = 31) and female (n = 28) groups and tested to obtain an appropriate detection time period. According to the results of the preliminary experiment, 84 male mice were randomly divided into three groups in the formal experiment: control, 5 and 10 mg/kg (0.1 mL/10 g body weight) cordycepin groups. Cordycepin was administered intragastrically twice daily (in the morning and afternoon) for 3 weeks. Animals in the control group were treated with an equal volume of double distilled water. Mice were evaluated using the SAB test in a Y-maze at least 24 h after the final drug treatments.
2.5. Immunohistochemistry Briefly, mice were anesthetized followed by perfusion with 0.9% NaCl and then 4% paraformaldehyde in 0.01 M phosphate buffer, pH 7.4. The brains were removed and post fixed for 1 h at 4 °C refrigerator, and then stored in 30% sucrose in phosphate buffer for cryoprotection. Ceyostat sections in 30 μm thickness were received and stored at 4 °C in 0.01 M PBS and transferred to sodium citrate for microwave repair (medium heat, 10 min for 2 times). After washed by 0.01 M PBS for 2 times and treated with 3% H202 for 20 min to eliminate influence of endogenous peroxidase, monoclonal antibodies of A1R and A2AR were used at a dilution of 1: 200 at 4 °C refrigerator for 24 h and then placed in 37 °C incubator for 30 min. Washing by 0.01 M PBS and secondary antibodies treatment, sections were processed with horseradish peroxidase-biotin-avidin complex for 20 min by DAB for 5 min, and then ended the reaction with 0.01 M PBS for 3 times. Sections finishing gradient ethanol dehydration were mounted on gelatinated slides and plated on coverslips. Hippocampal CA1, CA3 and DG areas were photographed under 20 × objectives with light microscopy. Image-pro Plus Microsoft (Media Cybernetics, USA) was used for optical density analysis.
2.3. SAB test As described previously [20], SAB, a hippocampus-dependent behavior test to assess attention toward novelty and spatial memory, was performed in a Y-maze with identical dimensions (45 cm long × 14 cm wide × 16 cm high with arms) at a 120° angle from each other. Each mouse was placed at the Y-maze center and allowed to move freely for 8 min (30 min in the preliminary experiment). Over the course of multiple arm entries, the animal typically shows a tendency to enter a less recently visited arm. The numbers of arm entries and alternation (triads) were recorded, respectively. An entry occurred when all four limbs were within the arm. Alternation was defined if mouse's arm entry was different from the two previously entered arms; an error was
2.6. Statistical analysis All data were expressed as mean ± SEM and then analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. Origin 7.5 software (Northampton, MA, USA) and SPSS 17.0 package (Chicago, IL, USA) were employed for graphing and data analysis. The level of statistical significance was set at p < 0.05.
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Fig. 1. Effects of test time and sex on the SAB test in mice. (A) The number of arm entries in the SAB test appeared a downward trend during a period of 30 min. (B) The percent of relative alternation in the SAB test appeared a down trend during a period of 30 min. (C) The total number of arm entries from 3 to 8 min in female mice was significant increased as compared with male mice. (D) The percent of relative alternation from 3 to 8 min in female mice was no significant difference between female and male mice. N = 59 for total mice, including 31 male and 28 female. Data were expressed as mean ± SEM, one-way ANOVA test. *p < 0.05 and N.S. p > 0.05.
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3. Results
application didn't affect the exploratory ability (locomotor activity) or curiosity of mice. However, cordycepin treatments significantly increased the percent of relative alternation, and 10 mg/kg was better than that of 5 mg/kg (one-way ANOVA, F (2, 46) = 5.957, p = 0.005; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.026; control vs. 10 mg/kg, p = 0.003), as shown in Fig. 2B. These results suggest that intragastric cordycepin treatments may improve the short-term spatial memory but not locomotor activity in mice in the Y-maze SAB test.
3.1. Effects of testing time and sex on the SAB test in mice As shown in Fig. 1A and B, the number of arm entries and the percent of relative alternation in the SAB test showed a downward trend during a period of 30 min recordings of behavioral test in Y-maze. Mice displayed the higher percent of relative alternation and the stable exploration in the period of 3–8 min as compared to that in 9–30 min. Therefore, the period of 3–8 min was used to evaluate the behavioral response of mice. This was supported by the literature showing that 5, 6 or 10 min intervals was usually set as the test time in the SAB test but no certain standard [21,22]. In the SAB test, the percent of relative alternation always stands for an index of working memory performance, while the total number of arm entries represents both animal's exploratory ability (locomotor activity) and curiosity [20]. We found that there was an obvious difference in locomotor activity between male and female mice, as shown in Fig. 1C and D. The total number of arm entries from 3 to 8 min in the female mice was significant increased as compared with the male mice (one-way ANOVA, F (1, 52) = 5.68, p = 0.021), but there was no significant difference in the percent of relative alternation between the two groups (one-way ANOVA, F (1, 52) = 1.75, p = 0.192). These data demonstrate that there were important sex differences of mice in the SAB test, which was similar with those previous studies showing that there were sex differences in spontaneous alternation both in rats [23] and hamsters due to habituation differences [24]. We thus selected male mice to carry out the next experiments.
3.3. Effects of cordycepin on body weight, hippocampal weight and adenosine content As shown in Fig. 3A, we observed that there were no significant differences in the body weight between the control and the drug groups after oral cordycepin treatments for 3 weeks (one-way ANOVA, F (2, 21) = 2.22, p = 0.113; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 1.0; control vs. 10 mg/kg, p = 0.144). Consistently, there was no significant changes in the hippocampal weight between the control and the drug groups (one-way ANOVA, F (2, 24) = 0.071, p = 0.931; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.999; control vs. 10 mg/kg, p = 0.935). Although a slight increase in the hippocampal adenosine content was observed with cordycepin applications for 3 weeks but without obvious differences, as shown in Fig. 3C (one-way ANOVA, F (2, 24) = 0.254, p = 0.778; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.937; control vs. 10 mg/kg, p = 0.886). These findings show that oral cordycepin treatment for 3-week has no influence in body weight, hippocampal weight and hippocampal adenosine content in mice.
3.2. Effects of cordycepin on the SAB test
3.4. Effects of cordycepin on A1R and A2AR densities in hippocampal subregions
As shown in Fig. 2A, we observed that the total number of arm entries (3–8 min) in Y-maze had not marked difference between the control and the drug groups (one-way ANOVA, F (2, 46) = 0.96, p = 0.39; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.287; control vs. 10 mg/kg, p = 0.675), suggesting that oral cordycepin
The results about A1R immunoreactivity was showed in Fig. 4A and B, in which we observed that cordycepin with 5 and 10 mg/kg did not change the densities of A1R in the hippocampal CA1 and CA3 areas as compared to the control group (CA1: one-way ANOVA, F (2, 15) 137
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Fig. 2. Effects of cordycepin on behavioral exploration and relative alternation in the SAB test. (A) Oral cordycepin application at 5 and 10 mg/kg didn't alter the total number of arm entry of mice in the SAB test. (B) Oral cordycepin treatment at 5 and 10 mg/kg for 3 weeks significantly increased the percent of relative alternation in the SAB test. N = 28 for each group. Data were expressed as mean ± SEM, one-way ANOVA test and followed by Dunnett's post-hoc test for comparison between the control and drug groups. **p < 0.01, *p < 0.05 and N.S. p > 0.05.
= 2.72, p = 0.098; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.129; control vs. 10 mg/kg, p = 0.094; CA3: one-way ANOVA, F (2, 15) = 0.328, p = 0.725; Dunnett's post-hoc test: control vs. 5 mg/ kg, p = 0.689; control vs. 10 mg/kg, p = 0.736). However, in the DG area, the immunoreactivity of A1R with 10 mg/kg cordycepin but not 5 mg/kg was significantly decreased as compared with the control animals (one-way ANOVA, F (2, 15) = 4.57, p = 0.028; Dunnett's posthoc test: control vs. 5 mg/kg, p = 0.772; control vs. 10 mg/kg, p = 0.022). Interestingly, 10 mg/kg cordycepin markedly reduced the density of A2AR in the hippocampal DG (one-way ANOVA, F (2, 15) = 4.29, p = 0.034, Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.733; control vs. 10 mg/kg, p = 0.025), CA1 (one-way ANOVA, F (2,15) = 9.91, p = 0.002, Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.019; control vs. 10 mg/kg, p = 0.001) and CA3 (one-way ANOVA, F (2, 15) = 3.74, p = 0.048, Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.145; control vs. 10 mg/kg, p = 0.032), as shown in Fig. 5A and B. However, the density of A2AR in 5 mg/kg cordycepin was decreased only in the hippocampal CA1 region but did not alter in DG or CA3 region as compared with the control mice. These results show that the reduction of A2AR immunoreactivity in hippocampal subregions induced by cordycepin may have a concentration-dependent manner, which was consistent with the improvement of cordycepin in
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Fig. 3. Effects of cordycepin on body weight, hippocampal weight and adenosine content in mice. (A) Cordycepin at 5 and 10 mg/kg exerted no significant differences on body weight after oral treatments. N = 28 for each group. (B) Oral cordycepin treatments at 5 and 10 mg/kg had no significant change in the hippocampal weight during the three weeks. N = 12 for each group. (C) Cordycepin applications did not alter the content of adenosine in hippocampus. N = 12 for each group. Data were expressed as mean ± SEM, one-way ANOVA test and followed by Dunnett's post-hoc test for comparison between the control and drug groups. N.S. p > 0.05.
short-term spatial memory in the SAB test. Our experiments demonstrate that the improvement of cordycepin in spatial memory was possibly, at least partially, related to the down-regulating density of A2AR in hippocampus.
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Fig. 4. Effects of cordycepin on hippocampal A1R immunoreactivity. (A) Representative images of hippocampal CA1, CA3 and DG regions from control, 5 and 10 mg/kg cordycepin-treated groups. Boxes indicated the area for statistical analysis. Bar = 100 μm. (B) Cordycepin with 5 and 10 mg/kg did not change the density of A1R in the hippocampal CA1 and CA3 areas but only 10 mg/kg significantly decreased the density of A1R in DG area as compared to the control group. N = 8 mice for each group. Data were expressed as mean ± SEM, one-way ANOVA test and followed by Dunnett's post-hoc test for comparison between the control and drug groups. *p < 0.05 and N.S. p > 0.05.
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4. Discussion
avoiding the need for extensive training and the use of conventional reinforcers [26]. In the present study, we found that the behavioral activity of mice in the SAB test was closely related to the test time and the sex. With the extension of test time, mice alternation was lowered, as well as an increase in instability. We observed that the percent of relative alternation (short-time memory) and the total number of arm entries (locomotor activity) in mice were well-distributed and stable from 3 to 8 min. Our findings were consistent with the literature showing that 5, 8 or 10 min in the SAB test were usually set as the test time [21,22]. Therefore, we recorded the behavioral response from 3 to 8 min as the test time in our behavioral experiment. We also found that the female mice showed more exploration (locomotor activity) and curiosity in the novel environment as compared with the male mice, suggesting that estrogen may be helpful for the novel exploration but does not alter the short-term spatial memory.
The present study found that oral cordycepin treatment improved the percent of relative alternation in the SAB test but did not affect body weight, hippocampus weight and adenosine content in mice. Furthermore, intragastric administration of 10 mg/kg cordycepin only decreased the density of A1R in hippocampal DG region, but significantly reduced the density of A2AR in hippocampal DG, CA1 and CA3 areas. SAB always stands for the tendency for rats, mice and other animals to alternate their choices on Y- or T-maze. It has been ascribed to the operation of a variety of mechanism including stimulus satiation, action decrement, curiosity and habituation to novelty and spatial working memory [25]. In recent years, the SAB test has been widely accepted as a quick and relatively simple measure of memory retention because of 139
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Fig. 5. Effects of cordycepin on hippocampal A2AR immunoreactivity. (A) Representative images of hippocampal CA1, CA3 and DG regions from control, 5 and 10 mg/kg cordycepin-treated groups. Boxes indicated the area for statistical analysis. Bar = 100 μm. (B) Cordycepin with 10 mg/kg markedly reduced the density of A2AR in hippocampal DG, CA1 and CA3 regions, however, 5 mg/kg was only decreased the A2AR density in DG area. N = 8 mice for each group. Data were expressed as mean ± SEM, one-way ANOVA test and followed by Dunnett's post-hoc test for comparison between the control and drug groups. **p < 0.01, *p < 0.05 and N.S. P > 0.05.
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DG Our results showed that oral administration of cordycepin, in the concentrations of 5 and 10 mg/kg for 3 weeks, exerted no influence on body weight and hippocampus weight in mice. Although some reports have pointed out that cordycepin had toxic effects because it may be potential for inducing cell death and retarding cell growth [32,33], cordycepin administrated orally as a pellet at daily dose of 20 mg/kg for 4 months could protect the liver, kidneys, heart and lungs of aged rats from oxidative stress [34]. This inconsistency may result from the different method of cordycepin application. Basically, cordycepin is deaminated quickly by adenosine deaminase and metabolized rapidly to an inactive metabolite, 3′-deoxyhypoxanthinosine, and the half-life of cordycepin is as brief as 1.6 min with intravenous treatment [35] but can extend to 2.1 h by oral administration [4]. To some extent, the metabolic cycle of cordycepin is related with its method of
Similarly, the improvement of exploratory behavior in female mice had been observed [27]. Moreover, a previous study has shown that mice, proestrous compared to diestrous wildtype, but not estrogen receptor beta knockout, have better performance in the spontaneous alternation [28]. Although it has been largely proved that estrogen could improve learning and memory [29,30], the role of estrogen in the modulating the performance on tasks of learning and memory is complex because it exerts enhancing effects on some tasks and impairing effects on others [31]. Hypotheses have been offered to explain these varied results including differentiating the effects of estrogen on cognitive processes required to complete tasks and analyzing the influence of fluctuating levels of estrogen on the strategies selected by animals to solve tasks. Collectively, it is necessary to further measure the level of estrogen in mice to explain the roles of estrogen in the SAB test in future. 140
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transmission by acting presynaptic A1R [55]. Postsynaptic A1R density can influence the responsiveness to excitatory stimuli by a simultaneous control of N-type calcium channels and NMDA receptors. Selective A1R agonists or antagonists have been reported to impair or facilitate learning and memory, respectively [56,57]. However, our results showed that cordycepin reduced the density of A1R in the hippocampal DG area of mice only at dose of 10 mg/kg rather than 5 mg/kg, suggesting that the improvement of cordycepin on short-term spatial memory may play a very small part in reducing A1R density in the hippocampus.
administration; therefore, the elimination half-life cordycepin varies with different method of drug treatment [35]. In our experiments, cordycepin was dissolved in distilled water and administrated orally twice daily using a feeding needle. Overall, our results displayed that intragastric administration of cordycepin at twice daily dosage of 10 mg/kg for 3 weeks is safe and effective. Furthermore, cordycepin improved Y-maze learning and memory in both healthy and ischemic mice has been reported [5]. Similarly, our results further confirmed that cordycepin could improve short-term spatial memory. Adenosine, as an extracellular signaling molecule, is important for modulating physiological functions in the central nervous system. Extracellular adenosine usually originates from the extracellular metabolism of nucleotides and the release of cyclic AMP [36]. Under pathological states, extracellular adenosine is released via carrier-mediated mechanisms as a result of the hypoxia-induced massive breakdown of intracellular nucleotides; on the other hand, extracellular ATP is inactivated by a surface-located enzyme chain that results in the extracellular formation of adenosine [37]. Endogenous extracellular adenosine level was unstable in an activity-dependent manner and modulated synaptic transmission as well as short-term plasticity [38]. It is worthy to note that adenosine always regulates the release of various neurotransmitters as a neuromodulator through presynaptic mechanism, and then exerts different roles [7]. For instance, adenosine is able to modulate ACh release from nerve terminals, and then regulates learning and memory [39]. Therefore, we assume that the improvement of cordycepin (an adenosine analogue) on the short-term memory is possibly involved in modulating the release of neurotransmitters. Usually, adenosine acts on the four adenosine receptors: A1R, A2AR, A2BR and A3R. Following the higher density of A1R and A2AR in brain, the impact of adenosine on brain function might mainly be dependent on the actions of A1R and A2AR [39]. A2AR was shown to display a widespread distribution in brain and mostly located in synapses [40]. Presynaptic A2AR facilitated hippocampal synaptic transmission by promoting glutamate release, and showed a major role in shutting down the profound A1R-mediated synaptic transmission inhibition [41]. Postsynaptic A2AR could control NMDA receptors which plays a vital role in long-term potentiation [42]. Blockade of A2AR could attenuate long-term potentiation at excitatory synapses, as reported in the hippocampus, accumbens, striatum, amygdala [43,44]. Furthermore, A2AR doesn't affect memory performance but A2AR blockade prevents memory deterioration in early AD, diabetic encephalopathy, childhood epilepsy, cerebrospinal ataxia, early stress and aggregated α-synuclein PD model [45–48]. But the overexpression, as well as the over-activation of A2AR in naïve animals is sufficient to trigger memory deficits [49]. Initial studies characterizing A2AR in the hippocampus showed that the density of A2AR is circa 15–20 times lower than that of A1R [50]. And later studies with antibodies carefully validated in knockout mice pointed out the inability to detect A2AR in the hippocampus using immunohistochemical approaches while EM is only allowed visualizing A2AR in the hippocampal sections [51]. However, a presence of low level A2AR immunoreactivity that we observed is supported by a variety of studies. For example, binding sites for A2AR (~ 25% of striatal levels) and mRNA for the receptor were found in CA1, CA3, and DG; remaining discrepancies may be due to limits of detectability and persistent and inherent difficulty in measuring G-protein coupled receptors whose level of expression is quite low [52]. In our study, we found that cordycepin significantly lowered the density of A2AR in the hippocampus subareas, suggesting that cordycepin-related improvement of short-term spatial memory may involve in the decrease of A2AR density. The most widely recognized effects of adenosine are operated through inhibiting A1R, one of the most abundant G protein-coupled receptors in brain tissue [53]. A1R are located presynaptically, postsynaptically and nonsynaptically [54]. Effect of adenosine in neuronal circuits of adult mammals is to selectively depress excitatory
5. Conclusion In summary, cordycepin improves short-term spatial memory in the SAB test, in which the potential mechanism is mostly through downregulating the density of A2AR in hippocampus. Our results further support the concept that it is mostly A2AR rather than A1R to control the adaptive processes of memory performance. These findings also strongly recommend a nootropic role of cordycepin. Conflict of interest The authors declare no competing financial interests. Acknowledgements Funding for this research was provided by the Science and Technology Foundation of Guangzhou (201510010171) and China Education Ministry for Scholarship Foundation (20131205). References [1] H.S. Tuli, A.K. Sharma, S.S. Sandhu, D. Kashyap, Cordycepin: a bioactive metabolite with therapeutic potential, Life Sci. 93 (2013) 863–869. [2] S. Shin, S. Lee, J. Kwon, S. Moon, S.J. Lee, C.K. Lee, K. Cho, N.J. Ha, K. Kim, Cordycepin suppresses expression of diabetes regulating genes by inhibition of lipopolysaccharide-induced inflammation in macrophages, Immune Netw. 9 (2009) 98–105. [3] Z.Q. Liu, P.T. Li, D. Zhao, H.L. Tang, J.Y. Guo, Protective effect of extract of cordyceps sinensis in middle cerebral artery occlusion-induced focal cerebral ischemia in rats, Behav. Brain Funct. 6 (2010) 61. [4] H.P. Wei, X.L. Ye, Z. Chen, Y.J. Zhong, P.M. Li, S.C. Pu, X.G. Li, Synthesis and pharmacokinetic evaluation of novel N-acyl-cordycepin derivatives with a normal alkyl chain, Eur. J. Med. Chem. 44 (2009) 665–669. [5] Z.L. Cai, C.Y. Wang, Z.J. Jiang, H.H. Li, W.X. Liu, L.W. Gong, P. Xiao, C.H. Li, Effects of cordycepin on Y-maze learning task in mice, Eur. J. Pharmacol. 714 (2013) 249–253. [6] Z. Cheng, W. He, X. Zhou, Q. Lv, X. Xu, S. Yang, C. Zhao, L. Guo, Cordycepin protects against cerebral ischemia/reperfusion injury in vivo and in vitro, Eur. J. Pharmacol. 664 (2011) 20–28. [7] S.M. Thompson, H.L. Haas, B.H. Gähwiler, Comparison of the actions of adenosine at pre- and postsynaptic receptors in the rat hippocampus in vitro, J. Physiol. 451 (1992) 347–363. [8] R. Ricciarelli, D. Puzzo, O. Bruno, E. Canepa, E. Gardella, D. Rivera, L. Privitera, C. Domenicotti, B. Marengo, U.M. Marinari, A. Palmeri, M.A. Pronzato, O. Arancio, E. Fedele, A novel mechanism for cyclic adenosine monophosphate-mediated memory formation: role of amyloid beta, Ann. Neurol. 75 (2014) 602–607. [9] U.S. Sandau, M. Colino-Oliveira, A. Jones, B. Saleumvong, S.Q. Coffman, L. Liu, C. Miranda-Lourenço, C. Palminha, V.L. Batalha, Y. Xu, Y. Huo, M.J. Diógenes, A.M. Sebastião, D. Boison, Adenosine kinase deficiency in the brain results in maladaptive synaptic plasticity, J. Neurosci. 36 (2016) 12117–12128. [10] J.A. Ribeiro, A.M. Sebastião, A. de Mendonça, Adenosine receptors in the nervous system: pathophysiological implications, Prog. Neurobiol. 68 (2002) 377–392. [11] C.V. Gome, M.P. Kaster, A.R. Tomé, P.M. Aqostinho, R.A. Cunha, Adenosine receptors and brain diseases: neuroprotection and neurodegeneration, Biochim. Biophys. Acta 1808 (2011) 1380–1399. [12] de Mendonca, A.M. Sebastião, J.A. Ribeiro, Adenosine: does it have a neuroprotective role after all? Brain Res. Rev. 33 (2000) 258–274. [13] B.M. Fontinha, J.M. Delqado-Garcia, N. Madroñal, J.A. Ribeiro, A.M. Sebastião, A. Gruart, Adenosine A(2A) receptor modulation of hippocampal CA3-CA1 synapse plasticity during associative learning in behaving mice, Neuropsychopharmacology 34 (2009) 1865–1874. [14] R.A. Cunha, Adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles, different sources and different receptors, Neurochem. Int. 38 (2001) 107–125. [15] R.A. Cunha, Different cellular sources and different roles of adenosine: A1 receptor-
141
Physiology & Behavior 184 (2018) 135–142
Z.-P. Cao et al.
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24] [25]
[26] [27]
[28]
[29]
[30]
[31] [32] [33]
[34]
[35]
[36]
[37] G.R. Dubyak, C. El-Moatassim, Signal transduction via P2-purinergic receptors for extracellular ATP and the other nucleotides, Am. J. Phys. 265 (1993) C577–606. [38] N.M. Bannon, M. Chistiakova, J.Y. Chen, M. Bazhenov, M. Volqushev, Adenosine shifts plasticity regimes between associative and homeostatic by modulating heterosynaptic changes, J. Neurosci. 37 (2017) 1439–1452. [39] R.J. Rodrigues, P.M. Canas, L.V. Lopes, C.R. Oliveira, R.A. Cunha, Modification of adenosine modulation of acetylcholine release in the hippocampus of aged rats, Neurobiol. Aging 29 (2008) 1597–1601. [40] L.V. Lopes, R.A. Cunha, B. Kull, B.B. Fredholm, J.A. Ribeiro, Adenosine A(2A) receptor facilitation of hippocampal synaptic transmission is dependent on tonic A(1) receptor inhibition, Neuroscience 112 (2012) 319–329. [41] K. Wirkner, Z. Gerevich, T. Krause, A. Günther, L. Köles, D. Schneider, W. Nörenberg, P. Illes, Adenosine A2A receptor-induced inhibition of NMDA and GABAA receptor-mediated synaptic currents in a subpopulation of rat striatal neurons, Neuropharmacology 46 (2004) 994–1007. [42] N. Rebola, R. Lujan, R.A. Cunha, C. Mulle, Adenosine A2A receptors are essential for long-term potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses, Neuron 57 (2008) 121–134. [43] A.P. Simõe, N.J. Machado, N. Gonçalves, M.P. Kaster, A.T. Simões, R.A. Cunha, Adenosine A2A receptors in the amygdala control synaptic plasticity and contextual fear memory, Neuropsychopharmacology 41 (2016) 2862–2871. [44] S. Viana da Silva, M.G. Haberl, P. Zhang, P. Bethge, C. Lemos, C. Mulle, Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors, Nat. Commun. 7 (2016) 11915. [45] G.P. Cognato, P.M. Agostinho, J. Hockemeyer, C.E. Müller, D.O. Souza, R.A. Cunha, Caffeine and an adenosine A(2A) receptor antagonist prevent memory impairment and synaptotoxicity in adult rats triggered by a convulsive episode in early life, J. Neurochem. 112 (2010) 453–462. [46] N. Goncalves, A.T. Simões, R.A. Cunha, L.P. de Almeida, Caffeine and adenosine A(2A) receptor inactivation decrease striatal neuropathology in a lentiviral-based model of Machado-Joseph disease, Ann. Neurol. 73 (2013) 655–666. [47] D.G. Ferreira, V.L. Batalha, H. Vicente, J.E. Coelho Miranda, R. Gomes, L.V. Lopes, Adenosine A2A receptors modulate α-synuclein aggregation and toxicity, Cereb. Cortex 27 (2017) 718–730. [48] J.L. Huang, Y. Huo, L.L. Wan, Q.J. Yang, J. Li, C. Guo, CERK might contribute to inflammatory pain; Comments on Yu WL, Sun. Y (2015). CERK inhibition might be a good potential therapeutic target for diseases, Br. J. Pharmacol. 173 (2016) 1248–1249. [49] P. Li, D. Rial, P.M. Canas, J.H. Yoo, W. Li, X. Zhou, R.A. Cunha, J. Qu, J.F. Chen, Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory, Mol. Psychiatry 20 (2015) 1339–1349. [50] R.A. Cunha, B. Johansson, M.D. Constantino, A.M. Sebastião, Evidence for highaffinity binding sites for the adenosine A2A receptor agonist [3H] CGS 21680 in the rat hippocampus and cerebral cortex that are different from striatal A2A receptors, Naunyn Schmiedeberg's Arch. Pharmacol. 353 (1996) 261–271. [51] L.V. Lopes, L. Halldner, N. Rebola, B. Johansson, C. Ledent, Binding of the prototypical adenosine A(2A) receptor agonist CGS 21680 to the cerebral cortex of adenosine A(1) and A(2A) receptor knockout mice, Br. J. Pharmacol. 141 (2004) 1006–1014. [52] B. Johansson, B.B. Fredholm, Further characterization of the binding of the adenosine receptor agonist [3H] CGS 21680 to rat brain using autoradiography, Neuropharmacology 34 (1995) 393–403. [53] T.V. Dunwiddie, S.A. Masino, The role and regulation of adenosine in the central nervous system, Annu. Rev. Neurosci. 24 (2001) 31–55. [54] B.B. Fredholm, J.F. Chen, R.A. Cunha, P. Svenningsson, J.M. Vaugeois, Adenosine and brain function, Int. Rev. Neurobiol. 63 (2005) 191–270. [55] D.A. Prince, C.F. Stevens, Adenosine decreases neurotransmitter release at central synapses, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 8586–8590. [56] F. Suzuki, J. Shimada, S. Shiozaki, S. Ichikawa, A. Ishii, Adenosine A1 antagonists. 3. Structure-activity relationships on amelioration against scopolamine- or N6-((R)phenylisopropyl) adenosine-induced cognitive disturbance, J. Med. Chem. 36 (1993) 2508–2518. [57] D.K. Von Lubitz, I.A. Paul, R.T. Bartus, K.A. Jacobson, Effects of chronic administration of adenosine A1 receptor agonist and antagonist on spatial learning and memory, Eur. J. Pharmacol. 249 (1993) 271–280.
mediated inhibition through astrocytic-driven volume transmission and synapserestricted A2A receptor-mediated facilitation of plasticity, Neurochem. Int. 52 (2008) 65–72. M.L. Jin, S.Y. Park, Y.H. Kim, J.I. Oh, S.J. Lee, G. Park, The neuroprotective effects of cordycepin inhibit glutamate-induced oxidative and ER stress-associated apoptosis in hippocampal HT22 cells, Neurotoxicology 41 (2014) 102–111. Z. Hu, C.L. lee, V.K. Shah, E.H. Oh, J.Y. Han, J.R. Bae, K. Lee, M.S. Chong, J.T. Hong, K.W. Oh, Cordycepin increases nonrapid eye movement sleep via adenosine receptors in rats, Evid. Based Complement. Alternat. Med. 2013 (2013) 840134. H. Ni, X.H. Zhou, H.H. Li, W.F. Huang, Column chromatographic extraction and preparation of cordycepin from cordyceps militaris waster medium, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877 (2009) 2135–2141. Z.B. Deng, A.W. Yuan, W. Luo, N.D. Wang, Q.L. Gong, X.L. Yu, L.Q. Xue, Transmission of porcine circovirus type 2b (PCV2b) in Kunming mice, Acta Vet. Hung. 61 (2013) 234–243. E.A. Wappler, G. Szilágyi, A. Gál, J. Skopál, C. Nyakas, Z. Nagy, Adopted cognitive tests for gerbils: validation by studying ageing and ischemia, Physiol. Behav. 97 (2009) 107–114. D. Beracochea, J. Micheau, R. Jaffard, Memory deficits following chronic alcohol consumption in mice: relationships with hippocampal and cortical cholinergic activities, Pharmacol. Biochem. Behav. 42 (1992) 749–753. D.F. Wozniak, K.A. Diggs-Andrews, S. Conyers, C.M. Yuede, J.T. Dearborn, J.A. Brown, K. Tokuda, Y. Izumi, C.F. Zorumski, D.H. Gutmann, Motivational disturbances and effects of L-dopa administration in neurofibromatosis-1 model mice, PLoS One 8 (2013) e66024. R.E. Ulloa, H. Nicolini, A. Fernández-Guasti, Sex differences on spontaneous alternation in prepubertal rats: implications for an animal model of obsessive-compulsive disorder, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 28 (2004) 687–692. R.N. Hughes, Sex differences in spontaneous alternation and open-field behavior of hamsters: habituation differences, Curr. Psychol. 8 (1989) 144–150. R.N. Hughes, The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory, Neurosci. Biobehav. Rev. 28 (2004) 497–505. C.L. Richman, W.N. Dember, P. Kim, Spontaneous alternation behavior in animals: a review, Curr. Psychol. Res. Rev. 5 (1987) 358–391. S.A. Williams, E. Jasarevic, G.M. Vandas, D.A. Warzak, D.C. Geary, M.R. Ellersieck, R.M. Robert, C.S. Rosenfeld, Effects of developmental bisphenol A exposure on reproductive-related behaviors in California mice (Peromyscus californicus): a monogamous animal model, PLoS One 8 (2013) e55698. A.A. Walf, C. Koonce, K. Manley, C.A. Frye, Proestrous compared to diestrous wildtype, but not estrogen receptor beta knockout, mice have better performance in the spontaneous alternation and object recognition tasks and reduced anxiety-like behavior in the elevated plus and mirror maze, Behav. Brain Res. 196 (2009) 254–260. K.S. Ervin, J.M. Lymer, R. Matta, A.E. Clipperton, M. Kavaliers, E. Choleris, Estrogen involvement in social behavior in rodents: rapid and long-term actions, Horm. Behav. 74 (2015) 53–76. B. Li, L. Wang, Y. Liu, Y. Chen, Z. Zhang, J. Zhang, Jujube promotes learning and memory in a rat model by increasing estrogen levels in the blood and nitric oxide and acetylcholine levels in the brain, Exp. Ther. Med. 5 (2013) 1755–1759. M. Daniel, Effects of oestrogen on cognition: what have we learned from basic research? J. Neuroendocrinol. 18 (2006) 787–795. L.S. Chen, C.M. Stellrecht, V. Gandhi, RNA-directed agent, cordycepin, induces cell death in multiple myeloma cells, Br. J. Haematol. 140 (2008) 682–691. X. Zhou, C.U. Meyer, P. Schmidtke, F. Zepp, Effect of cordycepin on interleukin-10 production of human peripheral blood mononuclear cells, Eur. J. Pharmacol. 453 (2002) 309–317. T. Ramesh, S.K. Yoo, S.W. Kim, S.Y. Hwang, S.H. Sohn, I.W. Kim, S.K. Kim, Cordycepin (3′-deoxyadenosine) attenuates age-related oxidative stress and ameliorates antioxidant capacity in rats, Exp. Gerontol. 47 (2012) 979–987. Y.J. Tsai, L.C. Lin, T.H. Tsai, Pharmacokinetics of adenosine and cordycepin, a bioactive constituent of Cordyceps sinensis in rat, J. Agric. Food Chem. 58 (2010) 4638–4643. S. Latini, F. Pedata, Adenosine in the central nervous system: release mechanisms and extracellular concentrations, J. Neurchem. 79 (2001) 463–484.
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Effects of cordycepin on spontaneous alternation behavior and adenosine receptors expression in hippocampus
T
Zhi-Ping Caoa,1, Dan Daia,1, Peng-Ju Weia, Yuan-Yuan Hana, Yan-Qing Guana, Han-Hang Lia, ⁎ Wen-Xiao Liua, Peng Xiaoa, Chu-Hua Lia,b, a b
School of Life Science, South China Normal University, Guangzhou 510631, China Brain Science Institute, South China Normal University, Guangzhou 510631, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Cordycepin Spontaneous alternation behavior Hippocampus Adenosine Adenosine A2A receptor Adenosine A1 receptor
Cordycepin, an adenosine analogue, has been reported to improve cognitive function. Important roles on learning and memory of adenosine and its receptors, such as adenosine A1 and A2A receptors (A1R and A2AR), also have been shown. Therefore, we assume that the improvement of learning and memory induced by cordycepin is likely related to hippocampal adenosine content and adenosine receptor density. Here we investigated the effects of cordycepin on the short-term spatial memory by using a spontaneous alternation behavior (SAB) test in Y-maze, and then examined hippocampal adenosine content and A1R and A2AR densities. We found that orally administrated cordycepin (at dosages of 5 and 10 mg/kg twice daily for three weeks) significantly increased the percent of relative alternation of mice in SAB but not altered body weight, hippocampus weight and hippocampal adenosine content. Furthermore, cordycepin decreased A2AR density in hippocampal subareas; however, cordycepin only reduced the A1R density in DG but not CA1 or CA3 region. Our results suggest that cordycepin exerts a nootropic role possibly through modulating A2AR density of hippocampus, which further support the concept that it is mostly A2AR rather than A1R to control the adaptive processes of memory performance. These findings would be helpful to provide a new window into the pharmacological properties of cordycepin for cognitive promotion.
1. Introduction Cordycepin (3′-deoxyadenosine), first isolated from the fermented broth of medicinal mushroom Cordyceps militaris, is widely used as a traditional Chinese medicine and exhibits a variety of clinical effects such as immunomodulatory, antioxidant, anti-inflammatory and antimicrobial activities [1–3]. Cordycepin is deaminated quickly by adenosine deaminase and metabolized rapidly to an inactive metabolite, 3′-deoxyhypoxanthinosine in vivo. The half-life and bioavailability of cordycepin by oral administration are 2.1 h and 19.2 μmol h/L (area under concentration-time curve), respectively [4]. In recent studies, cordycepin has been reported to improve cognitive function in mice and protect against cell death induced by cerebral ischemia injury [5,6]. Adenosine is mainly through its action on adenosine A1 receptor (A1R) and adenosine A2A receptor (A2AR) to control and integrate cognition and memory [7]. Adenosine deficiency is able to result in the impairment of synaptic plasticity [8,9]. In the brain, A1R and A2AR are mainly located in synapses, controlling the release of neurotransmitters,
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1
such as glutamate (Glu) and acetylcholine (ACh) that are involved in memory and other cognitive processes [10]. Adenosine usually relies on a balance on the activation of inhibitory A1R and facilitatory A2AR [11]. The activation of A1R prevents neuronal damage [12], while the activation of A2AR plays an important effect on the associative learning process and its relevant hippocampal circuits [13]. Previous studies have shown that synaptic levels of adenosine could control synaptic transmission and plasticity by acting on synaptic A1R and A2AR [14,15]. It is worth mentioning that cordycepin, as an adenosine analogue, is structurally similar to adenosine. A previous study has shown that the anti-apoptotic effects of cordycepin are partially dependent on the activation of A1R [16], and cordycepin increases theta waves power density via nonspecific adenosine receptor in rats [17]. These researches indicated that cordycepin may exert potentially biological effects as a nonspecific (or partial) agonist of adenosine receptor. Given that adenosine is mainly through its action on A1R and A2AR to control and integrate cognition functions [7], therefore, we assume that the
Correspondence author at: School of Life Science, South China Normal University, Guangzhou 510631, China. E-mail address: [email protected] (C.-H. Li). Both authors contributed equally to the paper.
https://doi.org/10.1016/j.physbeh.2017.11.026 Received 5 May 2017; Received in revised form 18 October 2017; Accepted 21 November 2017 Available online 23 November 2017 0031-9384/ © 2017 Elsevier Inc. All rights reserved.
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recorded if the mouse went back to either of the two arms just previously visited. The percentage of relative alternation during a period of 5 min (from 3 to 8 min) observation was calculated from the ratio of the number of alternations divided by the number of total entries [21,22]. The value was multiplied by 100. Experiments and statistical analysis were double blinded. At the end of behavioral test, mice were randomly divided into two parts: HPLC and immunohistochemistry. All mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and then carried out the next experiments.
improvement on learning and memory by cordycepin is likely related to the densities of A1R and A2AR. In order to provide a new window into the pharmacological properties of cordycepin, in the present study, we examined the roles of cordycepin on short-term spatial memory using a spontaneous alternation behavior (SAB) test in Y-maze, and investigated adenosine content and A1R and A2AR densities in hippocampal subregions. 2. Materials and methods 2.1. Drugs and chemicals
2.4. HPLC procedure
Cordycepin (> 98% purity) was obtained by using a column chromatographic method to perform extraction and separation [18]. Adenosine standard and other chemicals were purchased from Sigma Ltd. (Louis, USA). Antibodies of rabbit anti-adenosine receptor A1 (bs6649R) and rabbit anti-adenosine receptor A2A (bs-1456R), and SP kit (rabbit, SP-0023) were bought from Bioss Company (Beijing, China). The constitution of the secondary kit is biotin-labeled goat anti-rabbit IgG.
Anesthetized mice were sacrificed by rapid decapitation. The brains were removed and carefully rinsed in ice-cold saline to remove any blood contaminants. Bilateral hippocampus was dissected quickly to ensure bioactivity of tissues. After weigh and homogenization in 2.0 mL Eppendorf tubes, the sample was thawed and minced in 0.4 mmol/L HClO4 (200 μL) for 2 min. The homogenate was centrifuged at 15000 rpm for 15 min at 4 °C. The supernatant was transferred and filtered through a 0.22 μm pore size Milipore filter for HPLC analysis. Analysis was carried out by using Waters-HPLC system (Waters, Massachusetts, USA) in 240 nm. The sample was separated with an XTerra MS C18 column (5 μm, 4.6 × 250 mm column, USA) manufactured by water. The mobile phase was composed of a mixture of acetonitrile-water solution (0.6 mL/min, 5: 95, v/v) filtered through a 0.45 μm nylon filter and degassed under ultrasound and vacuum for 30 min, with injecting 10 μL solution into the equilibrated HPLC system every time. Adenosine standard was dissolved in distilled water in concentrations of 2, 4, 6, 8, 10 and 20 μg/mL and then established a standard curve. The content of adenosine in hippocampus was calculated by dividing hippocampal weight.
2.2. Animals, ethics statements and groups All studies were reported in accordance with the ARRIVE guidelines for all the experiments involving animals. Animal procedures undertaken were approved by the Committee on Animal Care and Usage of South China Normal University and every effort was made to minimize animal suffering. Kunming mice (an outbred mouse stock deriving from Swiss albino mice with a high ratio of gene heterozygosis, 25–28 g, 6 weeks of age) were obtained from the animal facility at the Sun Yatsen University (Guangzhou, China) [19]. The animals were housed in the animal facility of South China Normal University in standard conditions. Food and water were supplied ad libitum. Room temperature was 23–25 °C, relative humidity was 40–50%, and the day/night cycle was set at 12 h/12 h. All animals were acclimatized for 3 days before starting any procedures. Mice were group housed (6 mice per cage) in conventional laboratory rodent cages (ZS Dichuang Co., Beijing, China) of dimensions 18.7 (W) × 15.5 (H) × 29.5 (L) cm. Animals were selected randomly from all cages and divided into groups. All analyses were carried out without prior knowledge of the treatments. Animals received a numerical code throughout the whole experiment. The current study included two experiments: a preliminary experiment and a formal experiment. In the preliminary experiment, mice were divided into male (n = 31) and female (n = 28) groups and tested to obtain an appropriate detection time period. According to the results of the preliminary experiment, 84 male mice were randomly divided into three groups in the formal experiment: control, 5 and 10 mg/kg (0.1 mL/10 g body weight) cordycepin groups. Cordycepin was administered intragastrically twice daily (in the morning and afternoon) for 3 weeks. Animals in the control group were treated with an equal volume of double distilled water. Mice were evaluated using the SAB test in a Y-maze at least 24 h after the final drug treatments.
2.5. Immunohistochemistry Briefly, mice were anesthetized followed by perfusion with 0.9% NaCl and then 4% paraformaldehyde in 0.01 M phosphate buffer, pH 7.4. The brains were removed and post fixed for 1 h at 4 °C refrigerator, and then stored in 30% sucrose in phosphate buffer for cryoprotection. Ceyostat sections in 30 μm thickness were received and stored at 4 °C in 0.01 M PBS and transferred to sodium citrate for microwave repair (medium heat, 10 min for 2 times). After washed by 0.01 M PBS for 2 times and treated with 3% H202 for 20 min to eliminate influence of endogenous peroxidase, monoclonal antibodies of A1R and A2AR were used at a dilution of 1: 200 at 4 °C refrigerator for 24 h and then placed in 37 °C incubator for 30 min. Washing by 0.01 M PBS and secondary antibodies treatment, sections were processed with horseradish peroxidase-biotin-avidin complex for 20 min by DAB for 5 min, and then ended the reaction with 0.01 M PBS for 3 times. Sections finishing gradient ethanol dehydration were mounted on gelatinated slides and plated on coverslips. Hippocampal CA1, CA3 and DG areas were photographed under 20 × objectives with light microscopy. Image-pro Plus Microsoft (Media Cybernetics, USA) was used for optical density analysis.
2.3. SAB test As described previously [20], SAB, a hippocampus-dependent behavior test to assess attention toward novelty and spatial memory, was performed in a Y-maze with identical dimensions (45 cm long × 14 cm wide × 16 cm high with arms) at a 120° angle from each other. Each mouse was placed at the Y-maze center and allowed to move freely for 8 min (30 min in the preliminary experiment). Over the course of multiple arm entries, the animal typically shows a tendency to enter a less recently visited arm. The numbers of arm entries and alternation (triads) were recorded, respectively. An entry occurred when all four limbs were within the arm. Alternation was defined if mouse's arm entry was different from the two previously entered arms; an error was
2.6. Statistical analysis All data were expressed as mean ± SEM and then analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. Origin 7.5 software (Northampton, MA, USA) and SPSS 17.0 package (Chicago, IL, USA) were employed for graphing and data analysis. The level of statistical significance was set at p < 0.05.
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B Number of arm entry (times)
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Fig. 1. Effects of test time and sex on the SAB test in mice. (A) The number of arm entries in the SAB test appeared a downward trend during a period of 30 min. (B) The percent of relative alternation in the SAB test appeared a down trend during a period of 30 min. (C) The total number of arm entries from 3 to 8 min in female mice was significant increased as compared with male mice. (D) The percent of relative alternation from 3 to 8 min in female mice was no significant difference between female and male mice. N = 59 for total mice, including 31 male and 28 female. Data were expressed as mean ± SEM, one-way ANOVA test. *p < 0.05 and N.S. p > 0.05.
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application didn't affect the exploratory ability (locomotor activity) or curiosity of mice. However, cordycepin treatments significantly increased the percent of relative alternation, and 10 mg/kg was better than that of 5 mg/kg (one-way ANOVA, F (2, 46) = 5.957, p = 0.005; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.026; control vs. 10 mg/kg, p = 0.003), as shown in Fig. 2B. These results suggest that intragastric cordycepin treatments may improve the short-term spatial memory but not locomotor activity in mice in the Y-maze SAB test.
3.1. Effects of testing time and sex on the SAB test in mice As shown in Fig. 1A and B, the number of arm entries and the percent of relative alternation in the SAB test showed a downward trend during a period of 30 min recordings of behavioral test in Y-maze. Mice displayed the higher percent of relative alternation and the stable exploration in the period of 3–8 min as compared to that in 9–30 min. Therefore, the period of 3–8 min was used to evaluate the behavioral response of mice. This was supported by the literature showing that 5, 6 or 10 min intervals was usually set as the test time in the SAB test but no certain standard [21,22]. In the SAB test, the percent of relative alternation always stands for an index of working memory performance, while the total number of arm entries represents both animal's exploratory ability (locomotor activity) and curiosity [20]. We found that there was an obvious difference in locomotor activity between male and female mice, as shown in Fig. 1C and D. The total number of arm entries from 3 to 8 min in the female mice was significant increased as compared with the male mice (one-way ANOVA, F (1, 52) = 5.68, p = 0.021), but there was no significant difference in the percent of relative alternation between the two groups (one-way ANOVA, F (1, 52) = 1.75, p = 0.192). These data demonstrate that there were important sex differences of mice in the SAB test, which was similar with those previous studies showing that there were sex differences in spontaneous alternation both in rats [23] and hamsters due to habituation differences [24]. We thus selected male mice to carry out the next experiments.
3.3. Effects of cordycepin on body weight, hippocampal weight and adenosine content As shown in Fig. 3A, we observed that there were no significant differences in the body weight between the control and the drug groups after oral cordycepin treatments for 3 weeks (one-way ANOVA, F (2, 21) = 2.22, p = 0.113; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 1.0; control vs. 10 mg/kg, p = 0.144). Consistently, there was no significant changes in the hippocampal weight between the control and the drug groups (one-way ANOVA, F (2, 24) = 0.071, p = 0.931; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.999; control vs. 10 mg/kg, p = 0.935). Although a slight increase in the hippocampal adenosine content was observed with cordycepin applications for 3 weeks but without obvious differences, as shown in Fig. 3C (one-way ANOVA, F (2, 24) = 0.254, p = 0.778; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.937; control vs. 10 mg/kg, p = 0.886). These findings show that oral cordycepin treatment for 3-week has no influence in body weight, hippocampal weight and hippocampal adenosine content in mice.
3.2. Effects of cordycepin on the SAB test
3.4. Effects of cordycepin on A1R and A2AR densities in hippocampal subregions
As shown in Fig. 2A, we observed that the total number of arm entries (3–8 min) in Y-maze had not marked difference between the control and the drug groups (one-way ANOVA, F (2, 46) = 0.96, p = 0.39; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.287; control vs. 10 mg/kg, p = 0.675), suggesting that oral cordycepin
The results about A1R immunoreactivity was showed in Fig. 4A and B, in which we observed that cordycepin with 5 and 10 mg/kg did not change the densities of A1R in the hippocampal CA1 and CA3 areas as compared to the control group (CA1: one-way ANOVA, F (2, 15) 137
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= 2.72, p = 0.098; Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.129; control vs. 10 mg/kg, p = 0.094; CA3: one-way ANOVA, F (2, 15) = 0.328, p = 0.725; Dunnett's post-hoc test: control vs. 5 mg/ kg, p = 0.689; control vs. 10 mg/kg, p = 0.736). However, in the DG area, the immunoreactivity of A1R with 10 mg/kg cordycepin but not 5 mg/kg was significantly decreased as compared with the control animals (one-way ANOVA, F (2, 15) = 4.57, p = 0.028; Dunnett's posthoc test: control vs. 5 mg/kg, p = 0.772; control vs. 10 mg/kg, p = 0.022). Interestingly, 10 mg/kg cordycepin markedly reduced the density of A2AR in the hippocampal DG (one-way ANOVA, F (2, 15) = 4.29, p = 0.034, Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.733; control vs. 10 mg/kg, p = 0.025), CA1 (one-way ANOVA, F (2,15) = 9.91, p = 0.002, Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.019; control vs. 10 mg/kg, p = 0.001) and CA3 (one-way ANOVA, F (2, 15) = 3.74, p = 0.048, Dunnett's post-hoc test: control vs. 5 mg/kg, p = 0.145; control vs. 10 mg/kg, p = 0.032), as shown in Fig. 5A and B. However, the density of A2AR in 5 mg/kg cordycepin was decreased only in the hippocampal CA1 region but did not alter in DG or CA3 region as compared with the control mice. These results show that the reduction of A2AR immunoreactivity in hippocampal subregions induced by cordycepin may have a concentration-dependent manner, which was consistent with the improvement of cordycepin in
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short-term spatial memory in the SAB test. Our experiments demonstrate that the improvement of cordycepin in spatial memory was possibly, at least partially, related to the down-regulating density of A2AR in hippocampus.
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Fig. 4. Effects of cordycepin on hippocampal A1R immunoreactivity. (A) Representative images of hippocampal CA1, CA3 and DG regions from control, 5 and 10 mg/kg cordycepin-treated groups. Boxes indicated the area for statistical analysis. Bar = 100 μm. (B) Cordycepin with 5 and 10 mg/kg did not change the density of A1R in the hippocampal CA1 and CA3 areas but only 10 mg/kg significantly decreased the density of A1R in DG area as compared to the control group. N = 8 mice for each group. Data were expressed as mean ± SEM, one-way ANOVA test and followed by Dunnett's post-hoc test for comparison between the control and drug groups. *p < 0.05 and N.S. p > 0.05.
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avoiding the need for extensive training and the use of conventional reinforcers [26]. In the present study, we found that the behavioral activity of mice in the SAB test was closely related to the test time and the sex. With the extension of test time, mice alternation was lowered, as well as an increase in instability. We observed that the percent of relative alternation (short-time memory) and the total number of arm entries (locomotor activity) in mice were well-distributed and stable from 3 to 8 min. Our findings were consistent with the literature showing that 5, 8 or 10 min in the SAB test were usually set as the test time [21,22]. Therefore, we recorded the behavioral response from 3 to 8 min as the test time in our behavioral experiment. We also found that the female mice showed more exploration (locomotor activity) and curiosity in the novel environment as compared with the male mice, suggesting that estrogen may be helpful for the novel exploration but does not alter the short-term spatial memory.
The present study found that oral cordycepin treatment improved the percent of relative alternation in the SAB test but did not affect body weight, hippocampus weight and adenosine content in mice. Furthermore, intragastric administration of 10 mg/kg cordycepin only decreased the density of A1R in hippocampal DG region, but significantly reduced the density of A2AR in hippocampal DG, CA1 and CA3 areas. SAB always stands for the tendency for rats, mice and other animals to alternate their choices on Y- or T-maze. It has been ascribed to the operation of a variety of mechanism including stimulus satiation, action decrement, curiosity and habituation to novelty and spatial working memory [25]. In recent years, the SAB test has been widely accepted as a quick and relatively simple measure of memory retention because of 139
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Fig. 5. Effects of cordycepin on hippocampal A2AR immunoreactivity. (A) Representative images of hippocampal CA1, CA3 and DG regions from control, 5 and 10 mg/kg cordycepin-treated groups. Boxes indicated the area for statistical analysis. Bar = 100 μm. (B) Cordycepin with 10 mg/kg markedly reduced the density of A2AR in hippocampal DG, CA1 and CA3 regions, however, 5 mg/kg was only decreased the A2AR density in DG area. N = 8 mice for each group. Data were expressed as mean ± SEM, one-way ANOVA test and followed by Dunnett's post-hoc test for comparison between the control and drug groups. **p < 0.01, *p < 0.05 and N.S. P > 0.05.
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DG Our results showed that oral administration of cordycepin, in the concentrations of 5 and 10 mg/kg for 3 weeks, exerted no influence on body weight and hippocampus weight in mice. Although some reports have pointed out that cordycepin had toxic effects because it may be potential for inducing cell death and retarding cell growth [32,33], cordycepin administrated orally as a pellet at daily dose of 20 mg/kg for 4 months could protect the liver, kidneys, heart and lungs of aged rats from oxidative stress [34]. This inconsistency may result from the different method of cordycepin application. Basically, cordycepin is deaminated quickly by adenosine deaminase and metabolized rapidly to an inactive metabolite, 3′-deoxyhypoxanthinosine, and the half-life of cordycepin is as brief as 1.6 min with intravenous treatment [35] but can extend to 2.1 h by oral administration [4]. To some extent, the metabolic cycle of cordycepin is related with its method of
Similarly, the improvement of exploratory behavior in female mice had been observed [27]. Moreover, a previous study has shown that mice, proestrous compared to diestrous wildtype, but not estrogen receptor beta knockout, have better performance in the spontaneous alternation [28]. Although it has been largely proved that estrogen could improve learning and memory [29,30], the role of estrogen in the modulating the performance on tasks of learning and memory is complex because it exerts enhancing effects on some tasks and impairing effects on others [31]. Hypotheses have been offered to explain these varied results including differentiating the effects of estrogen on cognitive processes required to complete tasks and analyzing the influence of fluctuating levels of estrogen on the strategies selected by animals to solve tasks. Collectively, it is necessary to further measure the level of estrogen in mice to explain the roles of estrogen in the SAB test in future. 140
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transmission by acting presynaptic A1R [55]. Postsynaptic A1R density can influence the responsiveness to excitatory stimuli by a simultaneous control of N-type calcium channels and NMDA receptors. Selective A1R agonists or antagonists have been reported to impair or facilitate learning and memory, respectively [56,57]. However, our results showed that cordycepin reduced the density of A1R in the hippocampal DG area of mice only at dose of 10 mg/kg rather than 5 mg/kg, suggesting that the improvement of cordycepin on short-term spatial memory may play a very small part in reducing A1R density in the hippocampus.
administration; therefore, the elimination half-life cordycepin varies with different method of drug treatment [35]. In our experiments, cordycepin was dissolved in distilled water and administrated orally twice daily using a feeding needle. Overall, our results displayed that intragastric administration of cordycepin at twice daily dosage of 10 mg/kg for 3 weeks is safe and effective. Furthermore, cordycepin improved Y-maze learning and memory in both healthy and ischemic mice has been reported [5]. Similarly, our results further confirmed that cordycepin could improve short-term spatial memory. Adenosine, as an extracellular signaling molecule, is important for modulating physiological functions in the central nervous system. Extracellular adenosine usually originates from the extracellular metabolism of nucleotides and the release of cyclic AMP [36]. Under pathological states, extracellular adenosine is released via carrier-mediated mechanisms as a result of the hypoxia-induced massive breakdown of intracellular nucleotides; on the other hand, extracellular ATP is inactivated by a surface-located enzyme chain that results in the extracellular formation of adenosine [37]. Endogenous extracellular adenosine level was unstable in an activity-dependent manner and modulated synaptic transmission as well as short-term plasticity [38]. It is worthy to note that adenosine always regulates the release of various neurotransmitters as a neuromodulator through presynaptic mechanism, and then exerts different roles [7]. For instance, adenosine is able to modulate ACh release from nerve terminals, and then regulates learning and memory [39]. Therefore, we assume that the improvement of cordycepin (an adenosine analogue) on the short-term memory is possibly involved in modulating the release of neurotransmitters. Usually, adenosine acts on the four adenosine receptors: A1R, A2AR, A2BR and A3R. Following the higher density of A1R and A2AR in brain, the impact of adenosine on brain function might mainly be dependent on the actions of A1R and A2AR [39]. A2AR was shown to display a widespread distribution in brain and mostly located in synapses [40]. Presynaptic A2AR facilitated hippocampal synaptic transmission by promoting glutamate release, and showed a major role in shutting down the profound A1R-mediated synaptic transmission inhibition [41]. Postsynaptic A2AR could control NMDA receptors which plays a vital role in long-term potentiation [42]. Blockade of A2AR could attenuate long-term potentiation at excitatory synapses, as reported in the hippocampus, accumbens, striatum, amygdala [43,44]. Furthermore, A2AR doesn't affect memory performance but A2AR blockade prevents memory deterioration in early AD, diabetic encephalopathy, childhood epilepsy, cerebrospinal ataxia, early stress and aggregated α-synuclein PD model [45–48]. But the overexpression, as well as the over-activation of A2AR in naïve animals is sufficient to trigger memory deficits [49]. Initial studies characterizing A2AR in the hippocampus showed that the density of A2AR is circa 15–20 times lower than that of A1R [50]. And later studies with antibodies carefully validated in knockout mice pointed out the inability to detect A2AR in the hippocampus using immunohistochemical approaches while EM is only allowed visualizing A2AR in the hippocampal sections [51]. However, a presence of low level A2AR immunoreactivity that we observed is supported by a variety of studies. For example, binding sites for A2AR (~ 25% of striatal levels) and mRNA for the receptor were found in CA1, CA3, and DG; remaining discrepancies may be due to limits of detectability and persistent and inherent difficulty in measuring G-protein coupled receptors whose level of expression is quite low [52]. In our study, we found that cordycepin significantly lowered the density of A2AR in the hippocampus subareas, suggesting that cordycepin-related improvement of short-term spatial memory may involve in the decrease of A2AR density. The most widely recognized effects of adenosine are operated through inhibiting A1R, one of the most abundant G protein-coupled receptors in brain tissue [53]. A1R are located presynaptically, postsynaptically and nonsynaptically [54]. Effect of adenosine in neuronal circuits of adult mammals is to selectively depress excitatory
5. Conclusion In summary, cordycepin improves short-term spatial memory in the SAB test, in which the potential mechanism is mostly through downregulating the density of A2AR in hippocampus. Our results further support the concept that it is mostly A2AR rather than A1R to control the adaptive processes of memory performance. These findings also strongly recommend a nootropic role of cordycepin. Conflict of interest The authors declare no competing financial interests. Acknowledgements Funding for this research was provided by the Science and Technology Foundation of Guangzhou (201510010171) and China Education Ministry for Scholarship Foundation (20131205). References [1] H.S. Tuli, A.K. Sharma, S.S. Sandhu, D. Kashyap, Cordycepin: a bioactive metabolite with therapeutic potential, Life Sci. 93 (2013) 863–869. [2] S. Shin, S. Lee, J. Kwon, S. Moon, S.J. Lee, C.K. Lee, K. Cho, N.J. Ha, K. Kim, Cordycepin suppresses expression of diabetes regulating genes by inhibition of lipopolysaccharide-induced inflammation in macrophages, Immune Netw. 9 (2009) 98–105. [3] Z.Q. Liu, P.T. Li, D. Zhao, H.L. Tang, J.Y. Guo, Protective effect of extract of cordyceps sinensis in middle cerebral artery occlusion-induced focal cerebral ischemia in rats, Behav. Brain Funct. 6 (2010) 61. [4] H.P. Wei, X.L. Ye, Z. Chen, Y.J. Zhong, P.M. Li, S.C. Pu, X.G. Li, Synthesis and pharmacokinetic evaluation of novel N-acyl-cordycepin derivatives with a normal alkyl chain, Eur. J. Med. Chem. 44 (2009) 665–669. [5] Z.L. Cai, C.Y. Wang, Z.J. Jiang, H.H. Li, W.X. Liu, L.W. Gong, P. Xiao, C.H. Li, Effects of cordycepin on Y-maze learning task in mice, Eur. J. Pharmacol. 714 (2013) 249–253. [6] Z. Cheng, W. He, X. Zhou, Q. Lv, X. Xu, S. Yang, C. Zhao, L. Guo, Cordycepin protects against cerebral ischemia/reperfusion injury in vivo and in vitro, Eur. J. Pharmacol. 664 (2011) 20–28. [7] S.M. Thompson, H.L. Haas, B.H. Gähwiler, Comparison of the actions of adenosine at pre- and postsynaptic receptors in the rat hippocampus in vitro, J. Physiol. 451 (1992) 347–363. [8] R. Ricciarelli, D. Puzzo, O. Bruno, E. Canepa, E. Gardella, D. Rivera, L. Privitera, C. Domenicotti, B. Marengo, U.M. Marinari, A. Palmeri, M.A. Pronzato, O. Arancio, E. Fedele, A novel mechanism for cyclic adenosine monophosphate-mediated memory formation: role of amyloid beta, Ann. Neurol. 75 (2014) 602–607. [9] U.S. Sandau, M. Colino-Oliveira, A. Jones, B. Saleumvong, S.Q. Coffman, L. Liu, C. Miranda-Lourenço, C. Palminha, V.L. Batalha, Y. Xu, Y. Huo, M.J. Diógenes, A.M. Sebastião, D. Boison, Adenosine kinase deficiency in the brain results in maladaptive synaptic plasticity, J. Neurosci. 36 (2016) 12117–12128. [10] J.A. Ribeiro, A.M. Sebastião, A. de Mendonça, Adenosine receptors in the nervous system: pathophysiological implications, Prog. Neurobiol. 68 (2002) 377–392. [11] C.V. Gome, M.P. Kaster, A.R. Tomé, P.M. Aqostinho, R.A. Cunha, Adenosine receptors and brain diseases: neuroprotection and neurodegeneration, Biochim. Biophys. Acta 1808 (2011) 1380–1399. [12] de Mendonca, A.M. Sebastião, J.A. Ribeiro, Adenosine: does it have a neuroprotective role after all? Brain Res. Rev. 33 (2000) 258–274. [13] B.M. Fontinha, J.M. Delqado-Garcia, N. Madroñal, J.A. Ribeiro, A.M. Sebastião, A. Gruart, Adenosine A(2A) receptor modulation of hippocampal CA3-CA1 synapse plasticity during associative learning in behaving mice, Neuropsychopharmacology 34 (2009) 1865–1874. [14] R.A. Cunha, Adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles, different sources and different receptors, Neurochem. Int. 38 (2001) 107–125. [15] R.A. Cunha, Different cellular sources and different roles of adenosine: A1 receptor-
141
Physiology & Behavior 184 (2018) 135–142
Z.-P. Cao et al.
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24] [25]
[26] [27]
[28]
[29]
[30]
[31] [32] [33]
[34]
[35]
[36]
[37] G.R. Dubyak, C. El-Moatassim, Signal transduction via P2-purinergic receptors for extracellular ATP and the other nucleotides, Am. J. Phys. 265 (1993) C577–606. [38] N.M. Bannon, M. Chistiakova, J.Y. Chen, M. Bazhenov, M. Volqushev, Adenosine shifts plasticity regimes between associative and homeostatic by modulating heterosynaptic changes, J. Neurosci. 37 (2017) 1439–1452. [39] R.J. Rodrigues, P.M. Canas, L.V. Lopes, C.R. Oliveira, R.A. Cunha, Modification of adenosine modulation of acetylcholine release in the hippocampus of aged rats, Neurobiol. Aging 29 (2008) 1597–1601. [40] L.V. Lopes, R.A. Cunha, B. Kull, B.B. Fredholm, J.A. Ribeiro, Adenosine A(2A) receptor facilitation of hippocampal synaptic transmission is dependent on tonic A(1) receptor inhibition, Neuroscience 112 (2012) 319–329. [41] K. Wirkner, Z. Gerevich, T. Krause, A. Günther, L. Köles, D. Schneider, W. Nörenberg, P. Illes, Adenosine A2A receptor-induced inhibition of NMDA and GABAA receptor-mediated synaptic currents in a subpopulation of rat striatal neurons, Neuropharmacology 46 (2004) 994–1007. [42] N. Rebola, R. Lujan, R.A. Cunha, C. Mulle, Adenosine A2A receptors are essential for long-term potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses, Neuron 57 (2008) 121–134. [43] A.P. Simõe, N.J. Machado, N. Gonçalves, M.P. Kaster, A.T. Simões, R.A. Cunha, Adenosine A2A receptors in the amygdala control synaptic plasticity and contextual fear memory, Neuropsychopharmacology 41 (2016) 2862–2871. [44] S. Viana da Silva, M.G. Haberl, P. Zhang, P. Bethge, C. Lemos, C. Mulle, Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors, Nat. Commun. 7 (2016) 11915. [45] G.P. Cognato, P.M. Agostinho, J. Hockemeyer, C.E. Müller, D.O. Souza, R.A. Cunha, Caffeine and an adenosine A(2A) receptor antagonist prevent memory impairment and synaptotoxicity in adult rats triggered by a convulsive episode in early life, J. Neurochem. 112 (2010) 453–462. [46] N. Goncalves, A.T. Simões, R.A. Cunha, L.P. de Almeida, Caffeine and adenosine A(2A) receptor inactivation decrease striatal neuropathology in a lentiviral-based model of Machado-Joseph disease, Ann. Neurol. 73 (2013) 655–666. [47] D.G. Ferreira, V.L. Batalha, H. Vicente, J.E. Coelho Miranda, R. Gomes, L.V. Lopes, Adenosine A2A receptors modulate α-synuclein aggregation and toxicity, Cereb. Cortex 27 (2017) 718–730. [48] J.L. Huang, Y. Huo, L.L. Wan, Q.J. Yang, J. Li, C. Guo, CERK might contribute to inflammatory pain; Comments on Yu WL, Sun. Y (2015). CERK inhibition might be a good potential therapeutic target for diseases, Br. J. Pharmacol. 173 (2016) 1248–1249. [49] P. Li, D. Rial, P.M. Canas, J.H. Yoo, W. Li, X. Zhou, R.A. Cunha, J. Qu, J.F. Chen, Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory, Mol. Psychiatry 20 (2015) 1339–1349. [50] R.A. Cunha, B. Johansson, M.D. Constantino, A.M. Sebastião, Evidence for highaffinity binding sites for the adenosine A2A receptor agonist [3H] CGS 21680 in the rat hippocampus and cerebral cortex that are different from striatal A2A receptors, Naunyn Schmiedeberg's Arch. Pharmacol. 353 (1996) 261–271. [51] L.V. Lopes, L. Halldner, N. Rebola, B. Johansson, C. Ledent, Binding of the prototypical adenosine A(2A) receptor agonist CGS 21680 to the cerebral cortex of adenosine A(1) and A(2A) receptor knockout mice, Br. J. Pharmacol. 141 (2004) 1006–1014. [52] B. Johansson, B.B. Fredholm, Further characterization of the binding of the adenosine receptor agonist [3H] CGS 21680 to rat brain using autoradiography, Neuropharmacology 34 (1995) 393–403. [53] T.V. Dunwiddie, S.A. Masino, The role and regulation of adenosine in the central nervous system, Annu. Rev. Neurosci. 24 (2001) 31–55. [54] B.B. Fredholm, J.F. Chen, R.A. Cunha, P. Svenningsson, J.M. Vaugeois, Adenosine and brain function, Int. Rev. Neurobiol. 63 (2005) 191–270. [55] D.A. Prince, C.F. Stevens, Adenosine decreases neurotransmitter release at central synapses, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 8586–8590. [56] F. Suzuki, J. Shimada, S. Shiozaki, S. Ichikawa, A. Ishii, Adenosine A1 antagonists. 3. Structure-activity relationships on amelioration against scopolamine- or N6-((R)phenylisopropyl) adenosine-induced cognitive disturbance, J. Med. Chem. 36 (1993) 2508–2518. [57] D.K. Von Lubitz, I.A. Paul, R.T. Bartus, K.A. Jacobson, Effects of chronic administration of adenosine A1 receptor agonist and antagonist on spatial learning and memory, Eur. J. Pharmacol. 249 (1993) 271–280.
mediated inhibition through astrocytic-driven volume transmission and synapserestricted A2A receptor-mediated facilitation of plasticity, Neurochem. Int. 52 (2008) 65–72. M.L. Jin, S.Y. Park, Y.H. Kim, J.I. Oh, S.J. Lee, G. Park, The neuroprotective effects of cordycepin inhibit glutamate-induced oxidative and ER stress-associated apoptosis in hippocampal HT22 cells, Neurotoxicology 41 (2014) 102–111. Z. Hu, C.L. lee, V.K. Shah, E.H. Oh, J.Y. Han, J.R. Bae, K. Lee, M.S. Chong, J.T. Hong, K.W. Oh, Cordycepin increases nonrapid eye movement sleep via adenosine receptors in rats, Evid. Based Complement. Alternat. Med. 2013 (2013) 840134. H. Ni, X.H. Zhou, H.H. Li, W.F. Huang, Column chromatographic extraction and preparation of cordycepin from cordyceps militaris waster medium, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877 (2009) 2135–2141. Z.B. Deng, A.W. Yuan, W. Luo, N.D. Wang, Q.L. Gong, X.L. Yu, L.Q. Xue, Transmission of porcine circovirus type 2b (PCV2b) in Kunming mice, Acta Vet. Hung. 61 (2013) 234–243. E.A. Wappler, G. Szilágyi, A. Gál, J. Skopál, C. Nyakas, Z. Nagy, Adopted cognitive tests for gerbils: validation by studying ageing and ischemia, Physiol. Behav. 97 (2009) 107–114. D. Beracochea, J. Micheau, R. Jaffard, Memory deficits following chronic alcohol consumption in mice: relationships with hippocampal and cortical cholinergic activities, Pharmacol. Biochem. Behav. 42 (1992) 749–753. D.F. Wozniak, K.A. Diggs-Andrews, S. Conyers, C.M. Yuede, J.T. Dearborn, J.A. Brown, K. Tokuda, Y. Izumi, C.F. Zorumski, D.H. Gutmann, Motivational disturbances and effects of L-dopa administration in neurofibromatosis-1 model mice, PLoS One 8 (2013) e66024. R.E. Ulloa, H. Nicolini, A. Fernández-Guasti, Sex differences on spontaneous alternation in prepubertal rats: implications for an animal model of obsessive-compulsive disorder, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 28 (2004) 687–692. R.N. Hughes, Sex differences in spontaneous alternation and open-field behavior of hamsters: habituation differences, Curr. Psychol. 8 (1989) 144–150. R.N. Hughes, The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory, Neurosci. Biobehav. Rev. 28 (2004) 497–505. C.L. Richman, W.N. Dember, P. Kim, Spontaneous alternation behavior in animals: a review, Curr. Psychol. Res. Rev. 5 (1987) 358–391. S.A. Williams, E. Jasarevic, G.M. Vandas, D.A. Warzak, D.C. Geary, M.R. Ellersieck, R.M. Robert, C.S. Rosenfeld, Effects of developmental bisphenol A exposure on reproductive-related behaviors in California mice (Peromyscus californicus): a monogamous animal model, PLoS One 8 (2013) e55698. A.A. Walf, C. Koonce, K. Manley, C.A. Frye, Proestrous compared to diestrous wildtype, but not estrogen receptor beta knockout, mice have better performance in the spontaneous alternation and object recognition tasks and reduced anxiety-like behavior in the elevated plus and mirror maze, Behav. Brain Res. 196 (2009) 254–260. K.S. Ervin, J.M. Lymer, R. Matta, A.E. Clipperton, M. Kavaliers, E. Choleris, Estrogen involvement in social behavior in rodents: rapid and long-term actions, Horm. Behav. 74 (2015) 53–76. B. Li, L. Wang, Y. Liu, Y. Chen, Z. Zhang, J. Zhang, Jujube promotes learning and memory in a rat model by increasing estrogen levels in the blood and nitric oxide and acetylcholine levels in the brain, Exp. Ther. Med. 5 (2013) 1755–1759. M. Daniel, Effects of oestrogen on cognition: what have we learned from basic research? J. Neuroendocrinol. 18 (2006) 787–795. L.S. Chen, C.M. Stellrecht, V. Gandhi, RNA-directed agent, cordycepin, induces cell death in multiple myeloma cells, Br. J. Haematol. 140 (2008) 682–691. X. Zhou, C.U. Meyer, P. Schmidtke, F. Zepp, Effect of cordycepin on interleukin-10 production of human peripheral blood mononuclear cells, Eur. J. Pharmacol. 453 (2002) 309–317. T. Ramesh, S.K. Yoo, S.W. Kim, S.Y. Hwang, S.H. Sohn, I.W. Kim, S.K. Kim, Cordycepin (3′-deoxyadenosine) attenuates age-related oxidative stress and ameliorates antioxidant capacity in rats, Exp. Gerontol. 47 (2012) 979–987. Y.J. Tsai, L.C. Lin, T.H. Tsai, Pharmacokinetics of adenosine and cordycepin, a bioactive constituent of Cordyceps sinensis in rat, J. Agric. Food Chem. 58 (2010) 4638–4643. S. Latini, F. Pedata, Adenosine in the central nervous system: release mechanisms and extracellular concentrations, J. Neurchem. 79 (2001) 463–484.
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