Peptides 62 (2014) 155–158
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
The role of apelin-13 in novel object recognition memory Ren-wen Han a , Hong-jiao Xu b , Rui-san Zhang b , Rui Wang b,∗ a
Institute of Translational Medicine, Nanchang University, Nanchang 330008, China Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China b
a r t i c l e
i n f o
Article history: Received 4 September 2014 Received in revised form 7 October 2014 Accepted 7 October 2014 Available online 17 October 2014 Keywords: Apelin-13 APJ Object recognition Short-term memory Long-term memory
a b s t r a c t Apelin and its receptor APJ (apelin receptor) are prominently expressed in brain regions involved in learning and memory. However, the role of apelin in cognition was largely unclear. Here, the role of apelin-13 in memory processes was investigated in mice novel object recognition task. Post-training injection of apelin-13 (0.3 and 1 nmol) dose-dependently impaired short-term memory (STM), however, pre-training infusion of apelin-13 (1 nmol) did not affect STM, suggesting apelin-13 blocks formation but not acquisition of STM. Apelin-13 (1 nmol) administered immediately, 30, 60 or 120 min post-training impaired long-term memory (LTM) in a time-dependent manner (30 min), however, both pre-training and pre-test infusion of apelin-13 (1 nmol) did not affect LTM, suggesting apelin-13 impaired consolidation but not acquisition and recall of LTM. Taken together, for the first time, our results indicate that apelin-13 blocks STM formation and LTM consolidation in novel object recognition task. © 2014 Elsevier Inc. All rights reserved.
Introduction Neuropeptide apelin, the endogenous ligand of the G-proteincoupled receptor APJ (apelin receptor) [19,25], exhibits several biologically active forms, including apelin-36, apelin-17 and apelin13 [15]. Apelin is extremely conserved among different species, and the peptide apelin-13, studied in our present study, is completely conserved across all species investigated [13]. Both apelin and its receptor are widely expressed in both the CNS and peripheral tissues [5,10,13,16,23,24]. Therefore, lots of physiological and pathophysiological roles for apelin/APJ system have been reported, including regulation of cardiovascular system, immune system, fluid homeostasis, and adiposity [7,12,22]. Apelin and APJ are strongly expressed in the learning- and memory-associated brain regions, including hippocampus, amygdala and cerebral cortex [10,13,16], suggesting apelin/APJ system may take part in the regulation of memory processes. This possibility is also raised by the following evidences. First, apelin/APJ system could negatively regulate the cAMP pathway [25], which is crucial for learning and memory [6]. Second, apelin has been reported to attenuate N-methyl-d-aspartate (NMDA) receptor-induced intracellular Ca2+ accumulation and excitotoxicity in the cortical and
∗ Corresponding author at: School of Basic Medical Sciences, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, PR China. Tel.:+86 931 8912567/85234003755; fax: +86 931 8911255/852 23649932. E-mail addresses: [email protected], [email protected] (R. Wang). http://dx.doi.org/10.1016/j.peptides.2014.10.003 0196-9781/© 2014 Elsevier Inc. All rights reserved.
hippocampal neurons [4,18,28], suggesting that apelin could inhibit the NMDA receptor pathway, which plays an essential role in learning and memory [14]. However, the role of apelin/APJ system in cognition was still unclear; except that, most recently, Telegdy et al. reported that intracerebroventricular (icv) injection of apelin-13 facilitated passive avoidance memory consolidation [26]. Novel object recognition (NOR) task is a non-aversive learning paradigm which is based on animals’ spontaneous preference for the novel object, thus it has the advantage of avoiding the potential confounds of using differential rewards or punishments, and is widely used to evaluate the effects of various drugs on learning and memory processes (see review [2]). The present study was undertaken to investigate whether apelin-13 could regulate short-term memory (STM) and long-term memory (LTM) in mice NOR task.
Materials and methods Animals Male Kunming strains of Swiss mice were obtained from the Experimental Animal Center of Lanzhou University, China. Animals were housed in an animal room that was maintained at 22 ± 2 ◦ C with a 12-h light: 12-h dark cycle. Food and water were available ad libitum. All the protocols in this study were approved by the Ethics Committee of Lanzhou University, China.
R.-w. Han et al. / Peptides 62 (2014) 155–158
Each mouse (20–24 g) was anesthetized with sodium pentobarbital (75 mg/kg; Sigma–Aldrich, USA) and placed in a stereotaxic frame (Leica, Germany). According to the atlas of Paxinos and Franklin [21], a 9 mm 26-gauge stainless-steel guide cannula was implanted over the right lateral ventricle (0.5 mm posterior to bregma, 1.0 mm lateral to midline, 2.0 mm ventral to skull surface). After surgery, mice were housed individually and were allowed to recover 5–7 days.
A 100
B 100
##
Discrimination index (%)
Surgery
Discrimination index (%)
156
80
** 60
**
40 20 0 Vehicle
NOR task
0.3
1.0
80 60
***
**
40 20 0 Vehicle Apelin-13
Apelin-13 (nmol)
The procedure of NOR task was based on our previous reports [8,9], and that described by Okamura et al. [20]. Briefly, each mouse was tested in its home cage in a sound-attenuated room with somber lighting. The general procedure consisted of a training phase and a retention phase, separated by a delay. Each mouse was handed 3 min per day for three consecutive days prior to training. During the training phase, two identical objects were placed in the opposite sides of the home cage. The sample phase ended when mouse had explored two identical objects for a total of 20 s. Mouse showing a total exploratory time (TET) < 20 s within 10 min was excluded. In the test phase, a familiar object from the training phase and a novel object were placed in the same locations as in the training phase. The test phase was ended when mouse had explored two objects for a total of 25 s, or after 5 min had passed, whichever came first. All objects were made of plastic or glass, similar in size (4–5 cm high) but different in color and shape. There were several copies of each object for use interchangeably. Throughout the experiments, the objects used as novel or familiar were counterbalanced to exclude possible preference for special object. Moreover, the locations of the novel and familiar objects were also counterbalanced in the test to exclude possible spatial bias. Objects were cleaned thoroughly between trials to ensure absence of olfactory cue. Exploration was defined as sniffing or touching object with nose and/or forepaws. Resting against or turning around object was not considered exploratory behavior. The time spent exploring each object was recorded by an observer blind to the treatments. A discrimination index (DI) in the test phase was calculated as a percentage of the time spent exploring the novel object over the total time spent exploring both objects. A DI of 50% corresponds to the chance level and a higher DI reflects intact recognition memory.
Fig. 1. The effect of apelin-13 on short-term memory (STM). (A) Apelin-13 (0.3 and 1 nmol) injected immediately after training dose-dependently impaired STM formation. Vehicle, n = 10; apelin-13 0.3 nmol, n = 8; apelin-13 1 nmol, n = 10. (B) Apelin-13 (1 nmol) infused 5 min before training did not affect STM acquisition. N = 8 for both group. The dashed line indicates 50% chance level. ## p < 0.01 compared with vehicle; **p < 0.01 and ***p < 0.001 compared with chance level. Vertical lines represent SEM.
Drug infusion
Results
Apelin-13 (Glu-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-ProPhe) was synthesized and purified as described in our previous report [3]. Apelin-13 was dissolved in artificial CSF containing (in mM) 126.6 NaCl, 27.4 NaHCO3 , 2.4 KCl, 0.5 KH2 PO4 , 0.89 CaCl2 , 0.8 MgCl2 , 0.48 Na2 HPO4 , and 7.1 glucose, pH 7.4. For icv infusion, the infusion cannula extended 0.5 mm beyond the tip of the guide cannula. Apelin-13 (0.3 or 1 nmol) or vehicle (2 l) was infused over a period of 2 min via a 25 l Hamilton syringe (Hamilton) mounted on a microdrive pump (KD Scientific). The infusion cannula remained in place for 1 min after infusion to allow for drug diffusion. After completion of experiment, the placement of cannula was verified by histological examination and the animal with misplaced icv injection was excluded.
The role of apelin-13 in STM
Experiment design STM and LTM were determined at a delay of 30 min and 24 h, respectively. Initially, three groups of mice (vehicle, 0.3 and 1 nmol apelin-13) were used to determine whether apelin-13 injected immediately post-training could regulate the formation of STM.
Then, two groups of mice (vehicle and 1 nmol apelin-13) were utilized to detect whether apelin-13 could influence STM acquisition when it was infused 5 min before training. Furthermore, four vehicle groups and four apelin groups of mice were adopted to investigate whether apelin-13 (1 nmol) could modulate LTM consolidation when it was delivered immediately, 30, 60 or 120 min after training. Finally, two vehicle groups and two apelin groups of mice were used to determine the role of apelin-13 in LTM acquisition and recall by infusion of apelin-13 (1 nmol) 5 min before training and test, respectively.
Statistical analysis Data were expressed as mean ± SEM. Statistical analysis was conducted using SPSS 17.0. One-sample t-test was used to determine whether the DI differed from the chance level (50%) for each group. Differences between two groups were determined by unpaired Student’s t-test. Differences among more than two groups were determined by one-way ANOVA followed by Bonferroni post hoc test. P < 0.05 was considered significance.
When tested 30 min after training, the mice treated with vehicle or 0.3 nmol apelin-13 immediately post-training showed significant preference for the novel objects, as indicated by the DI of both groups was significantly higher than 50% chance level (p < 0.01 for both groups; Fig. 1A). However, when a higher dose of apelin-13 (1 nmol) was infused immediately after training, the DI of the mice was almost equal to the chance level (Fig. 1A). One-way ANOVA analysis indicated that post-training injection of apelin-13 (0.3 and 1 nmol) dose-dependently impaired STM expression (F2,25 = 0.007; P < 0.01; Fig. 1A). Further post-hoc analysis revealed that the DI of 1 nmol apelin-13 group was significantly lower than that of the control mice (p < 0.01; Fig. 1A). However, when apelin-13 (1 nmol) was delivered 5 min before training, the DI of the mice was significantly higher than the chance level (p < 0.01; Fig. 1B), and no significant difference was detected between the DI of vehicle and apelin-13 groups (Fig. 1B). There is no significant difference between treatments in the training phase duration, as well as in the duration and TET of the test phase (Table 1).
R.-w. Han et al. / Peptides 62 (2014) 155–158
157
Table 1 Duration of sample phase (s), duration of test phase (s) and total exploration time (TET) in test phase (s) for each group. Fig.
Group
Duration of sample phase
Duration of test phase
TET in test phase
1A
Vehicle Apelin-13 0.3 nmol Apelin-13 1.0 nmol Vehicle Apelin-13 Vehicle (immediately) Apelin-13 (immediately) Vehicle (30 min) Apelin-13 (30 min) Vehicle (60 min) Apelin-13 (60 min) Vehicle (120 min) Apelin-13 (120 min) Vehicle (Pre-training) Apelin-13 (Pre-training) Vehicle (Pre-test) Apelin-13 (Pre-test)
190.0 ± 26.8 170.6 ± 30.8 185.7 ± 26.6 160.6 ± 22.8 186.3 ± 29.7 163.9 ± 39.5 171.3 ± 27.1 203.7 ± 27.1 194.6 ± 16.7 200.4 ± 34.9 238.9 ± 28.3 197.8 ± 26.4 224.3 ± 32.9 181.9 ± 22.2 170.6 ± 23.1 171.3 ± 24.2 183.5 ± 30.5
237.0 ± 15.5 250.0 ± 27.8 204.0 ± 25.3 195.0 ± 26.3 255.0 ± 22.0 229.3 ± 16.6 227.5 ± 27.6 243.0 ± 18.5 228.8 ± 23.6 240.1 ± 14.7 261.7 ± 19.2 227.5 ± 21.4 231.3 ± 19.6 216.3 ± 16.8 205 ± 18.6 207.5 ± 28.0 237.5 ± 26.1
22.0 ± 1.2 22.6 ± 2.3 23.3 ± 1.0 23.0 ± 1.3 21.2 ± 1.4 23.8 ± 0.7 24.1 ± 0.6 20.9 ± 1.3 22.9 ± 0.8 24.6 ± 0.3 24.0 ± 0.5 22.4 ± 1.8 24.2 ± 0.6 24.6 ± 0.4 23.2 ± 1.7 23.0 ± 1.0 24.4 ± 0.3
2
3
The role of apelin-13 in LTM The mice treated with apelin-13 (1 nmol) immediately, 60 min or 120 min after training showed good memory performance at a 24-h delay (immediately: p < 0.01; 60 min: p < 0.05; 120 min: p < 0.01; Fig. 2), and the DI of these groups was not significantly different from their corresponding controls, respectively (Fig. 2). However, the mice infused with apelin-13 30 min post-training could not discriminate between the novel and familiar objects, and the DI of these mice was significantly lower than that of the control mice (p < 0.01; Fig. 2). No significant difference was detected between treatments in the duration of the training phase, as well as in the duration and TET of the test phase (Table 1). Both pre-training and pre-test 1 nmol apelin-13-treated groups showed good memory performance at a delay of 24 h (p < 0.01 for both groups; Fig. 3), and the DI of both groups was not significantly different from their corresponding control groups, respectively (Fig. 3). There is no significant difference between treatments in the duration of the training phase as well as in the duration and TET of the test phase (Table 1). Discussion
Discrimination index (%)
The present study, for the first time, demonstrates that icv injection of apelin-13 impairs both STM and LTM in mice novel object recognition task. Our result is consistent with the predominant
100
Vehicle ##
80 60
Apelin-13
distribution of apelin and APJ in brain tissues correlated with learning and memory [10,13,16]. The role of apelin-13 in STM formation was firstly investigated. Our data showed that apelin-13 injected immediately post-training at the dose of 1 nmol but not 0.3 nmol significantly impaired memory performance at a delay of 30 min, suggesting apelin13 inhibited STM formation. Apelin-13 treated immediately after training did not affect the duration and TET of the test phase given 30 min after training, suggesting the inhibitory effect of apelin-13 on STM was not secondary to its effect on exploratory behavior. Then, the effect of apelin-13 on STM acquisition was determined by infusion of apelin-13 5 min before training. Pre-training injection of 1 nmol apelin-13 did not affect memory expression when the testing was given 30 min after training, suggesting apelin-13 had no influence on STM acquisition. Taken together, our results indicate that icv injection of apelin-13 disrupts STM formation but not acquisition. Then, we further determined the role of apelin-13 in LTM. Inconsistent with the effect of apelin-13 on STM, infusion of apelin-13 immediately after training did not affect memory performance at a delay of 24 h. The different effects of apelin-13 on STM and LTM suggest that STM may be separated from LTM, and successful performance of LTM is not dependent on normal STM expression. In support of our results, many previous reports have demonstrated that some treatments can differently regulate STM and LTM [11]. The consolidation phase of LTM is well demonstrated to persist for at least several hours after training, during which LTM can be interfered by pharmacological manipulations [1]. Thus, apelin-13 was injected 30, 60 or 120 min after training to further
**
** **
** *
*** **
40 20 0 immediately
30 min
60 min
120 min
Post-training injection time
Discrimination index (%)
1B
100
Vehicle
80 60
**
**
Apelin-13
***
**
40 20 0
Fig. 2. The effect of apelin-13 on long-term memory (LTM) consolidation. Apelin-13 (1 nmol) injected 30 min but not immediately, 60 or 120 min after training blocks LTM consolidation. Vehicle, n = 7, 10, 10 and 8 for immediately, 30, 60 and 120 min, respectively; Apelin-13, n = 8, 12, 9, and 8 for immediately, 30, 60 and 120 min, respectively. The dashed line indicates 50% chance level. ## p < 0.01 compared with corresponding vehicle control; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with chance level. Vertical lines represent SEM.
Pre-training
Pre-test
Fig. 3. The effect of apelin-13 on long-term memory (LTM) acquisition and recall. Injection of apelin-13 (1 nmol) 5 min before training and test did not affect LTM acquisition and recall, respectively. N = 8 for each group. The dashed line indicates 50% chance level. **p < 0.01 and ***p < 0.001 compared with chance level. Vertical lines represent SEM.
158
R.-w. Han et al. / Peptides 62 (2014) 155–158
determine its possible role in memory consolidation. Interestingly, our results showed that LTM was significantly impaired by apelin-13 injected 30 min post-training. However, no significant effect was observed when apelin-13 was given 60 or 120 min posttraining. These results indicated that apelin-13 disrupted LTM in a time-dependent manner. A similar phenomenon is observed with H1-receptor antagonist, pyrilamine, which also blocks LTM consolidation in a time dependent manner. From previous report [17], we speculated that apelin-13 only inhibited LTM consolidation at a single time point might due to its relatively short half-life in vivo. In support of this opinion, our unpublished data indicated that icv injection of apelin-13 (3 nmol) produced an analgesic effect, which reached a maximum at 5 min and terminated at 30 min after icv infusion. In addition, Xu et al. also reported that the analgesic effect of apelin-13 (icv, 3 g) was almost terminated at 30 min post-injection [27]. Finally, we found that both pre-training and pre-test infusion of apelin-13 did not affect LTM expression, suggesting apelin-13 did not regulate both acquisition and retrieval of LTM. Taken together, our behavioral data suggests that apelin-13 negatively regulates STM formation and LTM consolidation. However, the underlying mechanisms are not studied here. From previous reports, it can be found that apelin inhibits cAMP pathway and NMDA receptor-mediated functions [4,18,25,28]. In addition, blocking cAMP pathway or NMDA receptor results in memory impairment [6,14], implying that apelin-13 might hinder memory processes. Thus, our results are consistent with these reports. However, it should be mentioned that, recently, apelin-13 is indicated to promote memory consolidation in mice passive avoidance task [26]. Hence, different influences of apelin on memory may be obtained due to different paradigms, experimental conditions or animals used. Form this respect, it is important to further investigate the role of apelin in memory processes by using various paradigms and animal species. Overall, our present data and previous reports suggest that apelin/APJ system may be a potential target for the regulation of memory. Acknowledgements We are grateful for grants from the National Natural Science Foundation of China (nos. 91213302, 21272102, 21432003, 81473095, 81460546), the Key National Science and Technology Program “Major New Drug Development” of the Ministry of Science and Technology of China (2012ZX09504001–003), the Natural Science Foundation of Jiangxi Province (20142BAB215018), and the Foundation of Jiangxi Provincial Education (GJJ14216). References [1] Abel T, Lattal KM. Molecular mechanisms of memory acquisition, consolidation and retrieval. Curr Opin Neurobiol 2001;11:180–7. [2] Antunes M, Biala G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 2012;13:93–110. [3] Chang M, Peng YL, Dong SL, Han RW, Li W, Yang DJ, et al. Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor. Regul Pept 2005;130:116–22.
[4] Cook DR, Gleichman AJ, Cross SA, Doshi S, Ho W, Jordan-Sciutto KL, et al. NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury. J Neurochem 2011;118:1113–23. [5] De Mota N, Lenkei Z, Llorens-Cortes C. Cloning, pharmacological characterization and brain distribution of the rat apelin receptor. Neuroendocrinology 2000;72:400–7. [6] Ferguson GD, Storm DR. Why calcium-stimulated adenylyl cyclases? Physiology (Bethesda) 2004;19:271–6. [7] Galanth C, Hus-Citharel A, Li B, Llorens-Cortes C. Apelin in the control of body fluid homeostasis and cardiovascular functions. Curr Pharm Des 2012;18:789–98. [8] Han RW, Xu HJ, Zhang RS, Wang P, Chang M, Peng YL, et al. Neuropeptide S interacts with the basolateral amygdala noradrenergic system in facilitating object recognition memory consolidation. Neurobiol Learn Mem 2014;107: 32–6. [9] Han RW, Zhang RS, Xu HJ, Chang M, Peng YL, Wang R, Neuropeptide S. enhances memory and mitigates memory impairment induced by MK801, scopolamine or A1-42 in mice novel object and object location recognition tasks. Neuropharmacology 2013;70:261–7. [10] Hosoya M, Kawamata Y, Fukusumi S, Fujii R, Habata Y, Hinuma S, et al. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J Biol Chem 2000;275: 21061–7. [11] Izquierdo LA, Barros DM, Vianna MR, Coitinho A, deDavid e Silva T, Choi H, et al. Molecular pharmacological dissection of short- and long-term memory. Cell Mol Neurobiol 2002;22:269–87. [12] Kleinz MJ, Davenport AP. Emerging roles of apelin in biology and medicine. Pharmacol Ther 2005;107:198–211. [13] Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, et al. Characterization of apelin, the ligand for the APJ receptor. J Neurochem 2000;74:34–41. [14] Lynch MA. Long-term potentiation and memory. Physiol Rev 2004;84:87–136. [15] Masri B, Knibiehler B, Audigier Y. Apelin signalling: a promising pathway from cloning to pharmacology. Cell Signal 2005;17:415–26. [16] Medhurst AD, Jennings CA, Robbins MJ, Davis RP, Ellis C, Winborn KY, et al. Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin. J Neurochem 2003;84:1162–72. [17] Mesmin C, Dubois M, Becher F, Fenaille F, Ezan E. Liquid chromatography/tandem mass spectrometry assay for the absolute quantification of the expected circulating apelin peptides in human plasma. Rapid Commun Mass Spectrom 2010;24:2875–84. [18] O‘Donnell LA, Agrawal A, Sabnekar P, Dichter MA, Lynch DR, Kolson DL. Apelin an endogenous neuronal peptide, protects hippocampal neurons against excitotoxic injury. J Neurochem 2007;102:1905–17. [19] O‘Dowd BF, Heiber M, Chan A, Heng HH, Tsui LC, Kennedy JL, et al. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene 1993;136:355–60. [20] Okamura N, Garau C, Duangdao DM, Clark SD, Jungling K, Pape HC, et al. Neuropeptide S enhances memory during the consolidation phase and interacts with noradrenergic systems in the brain. Neuropsychopharmacology 2011;36:744–52. [21] Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates. 2nd Ed. San Diego: Academic Press; 2001. [22] Pitkin SL, Maguire JJ, Bonner TI, Davenport AP. International Union of Basic and Clinical Pharmacology LXXIV. Apelin receptor nomenclature, distribution, pharmacology, and function. Pharmacol Rev 2010;62:331–42. [23] Reaux A, De Mota N, Skultetyova I, Lenkei Z, El Messari S, Gallatz K, et al. Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain. J Neurochem 2001;77:1085–96. [24] Reaux A, Gallatz K, Palkovits M, Llorens-Cortes C. Distribution of apelinsynthesizing neurons in the adult rat brain. Neuroscience 2002;113:653–62. [25] Tatemoto K, Hosoya M, Habata Y, Fujii R, Kakegawa T, Zou MX, et al. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Biophys Res Commun 1998;251:471–6. [26] Telegdy G, Adamik A, Jaszberenyi M. Involvement of neurotransmitters in the action of apelin-13 on passive avoidance learning in mice. Peptides 2013;39:171–4. [27] Xu N, Wang H, Fan L, Chen Q. Supraspinal administration of apelin-13 induces antinociception via the opioid receptor in mice. Peptides 2009;30:1153–7. [28] Zeng XJ, Yu SP, Zhang L, Wei L. Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons. Exp Cell Res 2010;316:1773–83.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
The role of apelin-13 in novel object recognition memory Ren-wen Han a , Hong-jiao Xu b , Rui-san Zhang b , Rui Wang b,∗ a
Institute of Translational Medicine, Nanchang University, Nanchang 330008, China Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China b
a r t i c l e
i n f o
Article history: Received 4 September 2014 Received in revised form 7 October 2014 Accepted 7 October 2014 Available online 17 October 2014 Keywords: Apelin-13 APJ Object recognition Short-term memory Long-term memory
a b s t r a c t Apelin and its receptor APJ (apelin receptor) are prominently expressed in brain regions involved in learning and memory. However, the role of apelin in cognition was largely unclear. Here, the role of apelin-13 in memory processes was investigated in mice novel object recognition task. Post-training injection of apelin-13 (0.3 and 1 nmol) dose-dependently impaired short-term memory (STM), however, pre-training infusion of apelin-13 (1 nmol) did not affect STM, suggesting apelin-13 blocks formation but not acquisition of STM. Apelin-13 (1 nmol) administered immediately, 30, 60 or 120 min post-training impaired long-term memory (LTM) in a time-dependent manner (30 min), however, both pre-training and pre-test infusion of apelin-13 (1 nmol) did not affect LTM, suggesting apelin-13 impaired consolidation but not acquisition and recall of LTM. Taken together, for the first time, our results indicate that apelin-13 blocks STM formation and LTM consolidation in novel object recognition task. © 2014 Elsevier Inc. All rights reserved.
Introduction Neuropeptide apelin, the endogenous ligand of the G-proteincoupled receptor APJ (apelin receptor) [19,25], exhibits several biologically active forms, including apelin-36, apelin-17 and apelin13 [15]. Apelin is extremely conserved among different species, and the peptide apelin-13, studied in our present study, is completely conserved across all species investigated [13]. Both apelin and its receptor are widely expressed in both the CNS and peripheral tissues [5,10,13,16,23,24]. Therefore, lots of physiological and pathophysiological roles for apelin/APJ system have been reported, including regulation of cardiovascular system, immune system, fluid homeostasis, and adiposity [7,12,22]. Apelin and APJ are strongly expressed in the learning- and memory-associated brain regions, including hippocampus, amygdala and cerebral cortex [10,13,16], suggesting apelin/APJ system may take part in the regulation of memory processes. This possibility is also raised by the following evidences. First, apelin/APJ system could negatively regulate the cAMP pathway [25], which is crucial for learning and memory [6]. Second, apelin has been reported to attenuate N-methyl-d-aspartate (NMDA) receptor-induced intracellular Ca2+ accumulation and excitotoxicity in the cortical and
∗ Corresponding author at: School of Basic Medical Sciences, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, PR China. Tel.:+86 931 8912567/85234003755; fax: +86 931 8911255/852 23649932. E-mail addresses: [email protected], [email protected] (R. Wang). http://dx.doi.org/10.1016/j.peptides.2014.10.003 0196-9781/© 2014 Elsevier Inc. All rights reserved.
hippocampal neurons [4,18,28], suggesting that apelin could inhibit the NMDA receptor pathway, which plays an essential role in learning and memory [14]. However, the role of apelin/APJ system in cognition was still unclear; except that, most recently, Telegdy et al. reported that intracerebroventricular (icv) injection of apelin-13 facilitated passive avoidance memory consolidation [26]. Novel object recognition (NOR) task is a non-aversive learning paradigm which is based on animals’ spontaneous preference for the novel object, thus it has the advantage of avoiding the potential confounds of using differential rewards or punishments, and is widely used to evaluate the effects of various drugs on learning and memory processes (see review [2]). The present study was undertaken to investigate whether apelin-13 could regulate short-term memory (STM) and long-term memory (LTM) in mice NOR task.
Materials and methods Animals Male Kunming strains of Swiss mice were obtained from the Experimental Animal Center of Lanzhou University, China. Animals were housed in an animal room that was maintained at 22 ± 2 ◦ C with a 12-h light: 12-h dark cycle. Food and water were available ad libitum. All the protocols in this study were approved by the Ethics Committee of Lanzhou University, China.
R.-w. Han et al. / Peptides 62 (2014) 155–158
Each mouse (20–24 g) was anesthetized with sodium pentobarbital (75 mg/kg; Sigma–Aldrich, USA) and placed in a stereotaxic frame (Leica, Germany). According to the atlas of Paxinos and Franklin [21], a 9 mm 26-gauge stainless-steel guide cannula was implanted over the right lateral ventricle (0.5 mm posterior to bregma, 1.0 mm lateral to midline, 2.0 mm ventral to skull surface). After surgery, mice were housed individually and were allowed to recover 5–7 days.
A 100
B 100
##
Discrimination index (%)
Surgery
Discrimination index (%)
156
80
** 60
**
40 20 0 Vehicle
NOR task
0.3
1.0
80 60
***
**
40 20 0 Vehicle Apelin-13
Apelin-13 (nmol)
The procedure of NOR task was based on our previous reports [8,9], and that described by Okamura et al. [20]. Briefly, each mouse was tested in its home cage in a sound-attenuated room with somber lighting. The general procedure consisted of a training phase and a retention phase, separated by a delay. Each mouse was handed 3 min per day for three consecutive days prior to training. During the training phase, two identical objects were placed in the opposite sides of the home cage. The sample phase ended when mouse had explored two identical objects for a total of 20 s. Mouse showing a total exploratory time (TET) < 20 s within 10 min was excluded. In the test phase, a familiar object from the training phase and a novel object were placed in the same locations as in the training phase. The test phase was ended when mouse had explored two objects for a total of 25 s, or after 5 min had passed, whichever came first. All objects were made of plastic or glass, similar in size (4–5 cm high) but different in color and shape. There were several copies of each object for use interchangeably. Throughout the experiments, the objects used as novel or familiar were counterbalanced to exclude possible preference for special object. Moreover, the locations of the novel and familiar objects were also counterbalanced in the test to exclude possible spatial bias. Objects were cleaned thoroughly between trials to ensure absence of olfactory cue. Exploration was defined as sniffing or touching object with nose and/or forepaws. Resting against or turning around object was not considered exploratory behavior. The time spent exploring each object was recorded by an observer blind to the treatments. A discrimination index (DI) in the test phase was calculated as a percentage of the time spent exploring the novel object over the total time spent exploring both objects. A DI of 50% corresponds to the chance level and a higher DI reflects intact recognition memory.
Fig. 1. The effect of apelin-13 on short-term memory (STM). (A) Apelin-13 (0.3 and 1 nmol) injected immediately after training dose-dependently impaired STM formation. Vehicle, n = 10; apelin-13 0.3 nmol, n = 8; apelin-13 1 nmol, n = 10. (B) Apelin-13 (1 nmol) infused 5 min before training did not affect STM acquisition. N = 8 for both group. The dashed line indicates 50% chance level. ## p < 0.01 compared with vehicle; **p < 0.01 and ***p < 0.001 compared with chance level. Vertical lines represent SEM.
Drug infusion
Results
Apelin-13 (Glu-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-ProPhe) was synthesized and purified as described in our previous report [3]. Apelin-13 was dissolved in artificial CSF containing (in mM) 126.6 NaCl, 27.4 NaHCO3 , 2.4 KCl, 0.5 KH2 PO4 , 0.89 CaCl2 , 0.8 MgCl2 , 0.48 Na2 HPO4 , and 7.1 glucose, pH 7.4. For icv infusion, the infusion cannula extended 0.5 mm beyond the tip of the guide cannula. Apelin-13 (0.3 or 1 nmol) or vehicle (2 l) was infused over a period of 2 min via a 25 l Hamilton syringe (Hamilton) mounted on a microdrive pump (KD Scientific). The infusion cannula remained in place for 1 min after infusion to allow for drug diffusion. After completion of experiment, the placement of cannula was verified by histological examination and the animal with misplaced icv injection was excluded.
The role of apelin-13 in STM
Experiment design STM and LTM were determined at a delay of 30 min and 24 h, respectively. Initially, three groups of mice (vehicle, 0.3 and 1 nmol apelin-13) were used to determine whether apelin-13 injected immediately post-training could regulate the formation of STM.
Then, two groups of mice (vehicle and 1 nmol apelin-13) were utilized to detect whether apelin-13 could influence STM acquisition when it was infused 5 min before training. Furthermore, four vehicle groups and four apelin groups of mice were adopted to investigate whether apelin-13 (1 nmol) could modulate LTM consolidation when it was delivered immediately, 30, 60 or 120 min after training. Finally, two vehicle groups and two apelin groups of mice were used to determine the role of apelin-13 in LTM acquisition and recall by infusion of apelin-13 (1 nmol) 5 min before training and test, respectively.
Statistical analysis Data were expressed as mean ± SEM. Statistical analysis was conducted using SPSS 17.0. One-sample t-test was used to determine whether the DI differed from the chance level (50%) for each group. Differences between two groups were determined by unpaired Student’s t-test. Differences among more than two groups were determined by one-way ANOVA followed by Bonferroni post hoc test. P < 0.05 was considered significance.
When tested 30 min after training, the mice treated with vehicle or 0.3 nmol apelin-13 immediately post-training showed significant preference for the novel objects, as indicated by the DI of both groups was significantly higher than 50% chance level (p < 0.01 for both groups; Fig. 1A). However, when a higher dose of apelin-13 (1 nmol) was infused immediately after training, the DI of the mice was almost equal to the chance level (Fig. 1A). One-way ANOVA analysis indicated that post-training injection of apelin-13 (0.3 and 1 nmol) dose-dependently impaired STM expression (F2,25 = 0.007; P < 0.01; Fig. 1A). Further post-hoc analysis revealed that the DI of 1 nmol apelin-13 group was significantly lower than that of the control mice (p < 0.01; Fig. 1A). However, when apelin-13 (1 nmol) was delivered 5 min before training, the DI of the mice was significantly higher than the chance level (p < 0.01; Fig. 1B), and no significant difference was detected between the DI of vehicle and apelin-13 groups (Fig. 1B). There is no significant difference between treatments in the training phase duration, as well as in the duration and TET of the test phase (Table 1).
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Table 1 Duration of sample phase (s), duration of test phase (s) and total exploration time (TET) in test phase (s) for each group. Fig.
Group
Duration of sample phase
Duration of test phase
TET in test phase
1A
Vehicle Apelin-13 0.3 nmol Apelin-13 1.0 nmol Vehicle Apelin-13 Vehicle (immediately) Apelin-13 (immediately) Vehicle (30 min) Apelin-13 (30 min) Vehicle (60 min) Apelin-13 (60 min) Vehicle (120 min) Apelin-13 (120 min) Vehicle (Pre-training) Apelin-13 (Pre-training) Vehicle (Pre-test) Apelin-13 (Pre-test)
190.0 ± 26.8 170.6 ± 30.8 185.7 ± 26.6 160.6 ± 22.8 186.3 ± 29.7 163.9 ± 39.5 171.3 ± 27.1 203.7 ± 27.1 194.6 ± 16.7 200.4 ± 34.9 238.9 ± 28.3 197.8 ± 26.4 224.3 ± 32.9 181.9 ± 22.2 170.6 ± 23.1 171.3 ± 24.2 183.5 ± 30.5
237.0 ± 15.5 250.0 ± 27.8 204.0 ± 25.3 195.0 ± 26.3 255.0 ± 22.0 229.3 ± 16.6 227.5 ± 27.6 243.0 ± 18.5 228.8 ± 23.6 240.1 ± 14.7 261.7 ± 19.2 227.5 ± 21.4 231.3 ± 19.6 216.3 ± 16.8 205 ± 18.6 207.5 ± 28.0 237.5 ± 26.1
22.0 ± 1.2 22.6 ± 2.3 23.3 ± 1.0 23.0 ± 1.3 21.2 ± 1.4 23.8 ± 0.7 24.1 ± 0.6 20.9 ± 1.3 22.9 ± 0.8 24.6 ± 0.3 24.0 ± 0.5 22.4 ± 1.8 24.2 ± 0.6 24.6 ± 0.4 23.2 ± 1.7 23.0 ± 1.0 24.4 ± 0.3
2
3
The role of apelin-13 in LTM The mice treated with apelin-13 (1 nmol) immediately, 60 min or 120 min after training showed good memory performance at a 24-h delay (immediately: p < 0.01; 60 min: p < 0.05; 120 min: p < 0.01; Fig. 2), and the DI of these groups was not significantly different from their corresponding controls, respectively (Fig. 2). However, the mice infused with apelin-13 30 min post-training could not discriminate between the novel and familiar objects, and the DI of these mice was significantly lower than that of the control mice (p < 0.01; Fig. 2). No significant difference was detected between treatments in the duration of the training phase, as well as in the duration and TET of the test phase (Table 1). Both pre-training and pre-test 1 nmol apelin-13-treated groups showed good memory performance at a delay of 24 h (p < 0.01 for both groups; Fig. 3), and the DI of both groups was not significantly different from their corresponding control groups, respectively (Fig. 3). There is no significant difference between treatments in the duration of the training phase as well as in the duration and TET of the test phase (Table 1). Discussion
Discrimination index (%)
The present study, for the first time, demonstrates that icv injection of apelin-13 impairs both STM and LTM in mice novel object recognition task. Our result is consistent with the predominant
100
Vehicle ##
80 60
Apelin-13
distribution of apelin and APJ in brain tissues correlated with learning and memory [10,13,16]. The role of apelin-13 in STM formation was firstly investigated. Our data showed that apelin-13 injected immediately post-training at the dose of 1 nmol but not 0.3 nmol significantly impaired memory performance at a delay of 30 min, suggesting apelin13 inhibited STM formation. Apelin-13 treated immediately after training did not affect the duration and TET of the test phase given 30 min after training, suggesting the inhibitory effect of apelin-13 on STM was not secondary to its effect on exploratory behavior. Then, the effect of apelin-13 on STM acquisition was determined by infusion of apelin-13 5 min before training. Pre-training injection of 1 nmol apelin-13 did not affect memory expression when the testing was given 30 min after training, suggesting apelin-13 had no influence on STM acquisition. Taken together, our results indicate that icv injection of apelin-13 disrupts STM formation but not acquisition. Then, we further determined the role of apelin-13 in LTM. Inconsistent with the effect of apelin-13 on STM, infusion of apelin-13 immediately after training did not affect memory performance at a delay of 24 h. The different effects of apelin-13 on STM and LTM suggest that STM may be separated from LTM, and successful performance of LTM is not dependent on normal STM expression. In support of our results, many previous reports have demonstrated that some treatments can differently regulate STM and LTM [11]. The consolidation phase of LTM is well demonstrated to persist for at least several hours after training, during which LTM can be interfered by pharmacological manipulations [1]. Thus, apelin-13 was injected 30, 60 or 120 min after training to further
**
** **
** *
*** **
40 20 0 immediately
30 min
60 min
120 min
Post-training injection time
Discrimination index (%)
1B
100
Vehicle
80 60
**
**
Apelin-13
***
**
40 20 0
Fig. 2. The effect of apelin-13 on long-term memory (LTM) consolidation. Apelin-13 (1 nmol) injected 30 min but not immediately, 60 or 120 min after training blocks LTM consolidation. Vehicle, n = 7, 10, 10 and 8 for immediately, 30, 60 and 120 min, respectively; Apelin-13, n = 8, 12, 9, and 8 for immediately, 30, 60 and 120 min, respectively. The dashed line indicates 50% chance level. ## p < 0.01 compared with corresponding vehicle control; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with chance level. Vertical lines represent SEM.
Pre-training
Pre-test
Fig. 3. The effect of apelin-13 on long-term memory (LTM) acquisition and recall. Injection of apelin-13 (1 nmol) 5 min before training and test did not affect LTM acquisition and recall, respectively. N = 8 for each group. The dashed line indicates 50% chance level. **p < 0.01 and ***p < 0.001 compared with chance level. Vertical lines represent SEM.
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determine its possible role in memory consolidation. Interestingly, our results showed that LTM was significantly impaired by apelin-13 injected 30 min post-training. However, no significant effect was observed when apelin-13 was given 60 or 120 min posttraining. These results indicated that apelin-13 disrupted LTM in a time-dependent manner. A similar phenomenon is observed with H1-receptor antagonist, pyrilamine, which also blocks LTM consolidation in a time dependent manner. From previous report [17], we speculated that apelin-13 only inhibited LTM consolidation at a single time point might due to its relatively short half-life in vivo. In support of this opinion, our unpublished data indicated that icv injection of apelin-13 (3 nmol) produced an analgesic effect, which reached a maximum at 5 min and terminated at 30 min after icv infusion. In addition, Xu et al. also reported that the analgesic effect of apelin-13 (icv, 3 g) was almost terminated at 30 min post-injection [27]. Finally, we found that both pre-training and pre-test infusion of apelin-13 did not affect LTM expression, suggesting apelin-13 did not regulate both acquisition and retrieval of LTM. Taken together, our behavioral data suggests that apelin-13 negatively regulates STM formation and LTM consolidation. However, the underlying mechanisms are not studied here. From previous reports, it can be found that apelin inhibits cAMP pathway and NMDA receptor-mediated functions [4,18,25,28]. In addition, blocking cAMP pathway or NMDA receptor results in memory impairment [6,14], implying that apelin-13 might hinder memory processes. Thus, our results are consistent with these reports. However, it should be mentioned that, recently, apelin-13 is indicated to promote memory consolidation in mice passive avoidance task [26]. Hence, different influences of apelin on memory may be obtained due to different paradigms, experimental conditions or animals used. Form this respect, it is important to further investigate the role of apelin in memory processes by using various paradigms and animal species. Overall, our present data and previous reports suggest that apelin/APJ system may be a potential target for the regulation of memory. Acknowledgements We are grateful for grants from the National Natural Science Foundation of China (nos. 91213302, 21272102, 21432003, 81473095, 81460546), the Key National Science and Technology Program “Major New Drug Development” of the Ministry of Science and Technology of China (2012ZX09504001–003), the Natural Science Foundation of Jiangxi Province (20142BAB215018), and the Foundation of Jiangxi Provincial Education (GJJ14216). References [1] Abel T, Lattal KM. Molecular mechanisms of memory acquisition, consolidation and retrieval. Curr Opin Neurobiol 2001;11:180–7. [2] Antunes M, Biala G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 2012;13:93–110. [3] Chang M, Peng YL, Dong SL, Han RW, Li W, Yang DJ, et al. Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor. Regul Pept 2005;130:116–22.
[4] Cook DR, Gleichman AJ, Cross SA, Doshi S, Ho W, Jordan-Sciutto KL, et al. NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury. J Neurochem 2011;118:1113–23. [5] De Mota N, Lenkei Z, Llorens-Cortes C. Cloning, pharmacological characterization and brain distribution of the rat apelin receptor. Neuroendocrinology 2000;72:400–7. [6] Ferguson GD, Storm DR. Why calcium-stimulated adenylyl cyclases? Physiology (Bethesda) 2004;19:271–6. [7] Galanth C, Hus-Citharel A, Li B, Llorens-Cortes C. Apelin in the control of body fluid homeostasis and cardiovascular functions. Curr Pharm Des 2012;18:789–98. [8] Han RW, Xu HJ, Zhang RS, Wang P, Chang M, Peng YL, et al. Neuropeptide S interacts with the basolateral amygdala noradrenergic system in facilitating object recognition memory consolidation. Neurobiol Learn Mem 2014;107: 32–6. [9] Han RW, Zhang RS, Xu HJ, Chang M, Peng YL, Wang R, Neuropeptide S. enhances memory and mitigates memory impairment induced by MK801, scopolamine or A1-42 in mice novel object and object location recognition tasks. Neuropharmacology 2013;70:261–7. [10] Hosoya M, Kawamata Y, Fukusumi S, Fujii R, Habata Y, Hinuma S, et al. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J Biol Chem 2000;275: 21061–7. [11] Izquierdo LA, Barros DM, Vianna MR, Coitinho A, deDavid e Silva T, Choi H, et al. Molecular pharmacological dissection of short- and long-term memory. Cell Mol Neurobiol 2002;22:269–87. [12] Kleinz MJ, Davenport AP. Emerging roles of apelin in biology and medicine. Pharmacol Ther 2005;107:198–211. [13] Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, et al. Characterization of apelin, the ligand for the APJ receptor. J Neurochem 2000;74:34–41. [14] Lynch MA. Long-term potentiation and memory. Physiol Rev 2004;84:87–136. [15] Masri B, Knibiehler B, Audigier Y. Apelin signalling: a promising pathway from cloning to pharmacology. Cell Signal 2005;17:415–26. [16] Medhurst AD, Jennings CA, Robbins MJ, Davis RP, Ellis C, Winborn KY, et al. Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin. J Neurochem 2003;84:1162–72. [17] Mesmin C, Dubois M, Becher F, Fenaille F, Ezan E. Liquid chromatography/tandem mass spectrometry assay for the absolute quantification of the expected circulating apelin peptides in human plasma. Rapid Commun Mass Spectrom 2010;24:2875–84. [18] O‘Donnell LA, Agrawal A, Sabnekar P, Dichter MA, Lynch DR, Kolson DL. Apelin an endogenous neuronal peptide, protects hippocampal neurons against excitotoxic injury. J Neurochem 2007;102:1905–17. [19] O‘Dowd BF, Heiber M, Chan A, Heng HH, Tsui LC, Kennedy JL, et al. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene 1993;136:355–60. [20] Okamura N, Garau C, Duangdao DM, Clark SD, Jungling K, Pape HC, et al. Neuropeptide S enhances memory during the consolidation phase and interacts with noradrenergic systems in the brain. Neuropsychopharmacology 2011;36:744–52. [21] Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates. 2nd Ed. San Diego: Academic Press; 2001. [22] Pitkin SL, Maguire JJ, Bonner TI, Davenport AP. International Union of Basic and Clinical Pharmacology LXXIV. Apelin receptor nomenclature, distribution, pharmacology, and function. Pharmacol Rev 2010;62:331–42. [23] Reaux A, De Mota N, Skultetyova I, Lenkei Z, El Messari S, Gallatz K, et al. Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain. J Neurochem 2001;77:1085–96. [24] Reaux A, Gallatz K, Palkovits M, Llorens-Cortes C. Distribution of apelinsynthesizing neurons in the adult rat brain. Neuroscience 2002;113:653–62. [25] Tatemoto K, Hosoya M, Habata Y, Fujii R, Kakegawa T, Zou MX, et al. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Biophys Res Commun 1998;251:471–6. [26] Telegdy G, Adamik A, Jaszberenyi M. Involvement of neurotransmitters in the action of apelin-13 on passive avoidance learning in mice. Peptides 2013;39:171–4. [27] Xu N, Wang H, Fan L, Chen Q. Supraspinal administration of apelin-13 induces antinociception via the opioid receptor in mice. Peptides 2009;30:1153–7. [28] Zeng XJ, Yu SP, Zhang L, Wei L. Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons. Exp Cell Res 2010;316:1773–83.
