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Peptides 44 (2013) 60–65

Contents lists available at SciVerse ScienceDirect

Peptides

j ourna l ho me pa g e: www.elsev ier .com/ locate /pept ides

he effect of ghrelin on MK-801 induced memory impairment in rats

atemeh Goshadroua,b,∗, Mojtaba Kermanib, Abdolaziz Ronaghia,b, Samad Sajjadi c

Physiology Department, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, IranNeuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, IranEnglish Language Department, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

r t i c l e i n f o

rticle history:eceived 8 July 2012eceived in revised form 18 March 2013ccepted 18 March 2013vailable online 26 March 2013

eywords:hrelinMDA receptor antagonistK-801emory

assive avoidance task

a b s t r a c t

Accumulating evidence indicates that the brain-gut peptide ghrelin which is expressed in hippocampusimproves memory and learning processes. The MK-801, a noncompetitive NMDA receptor antagonist,has also shown amnesic properties in animal model. The current study was to find out whether intracere-broventricular administration of ghrelin can prevent amnesia induced by MK-801 in rats. A week after thesurgery, during which cannuals were implanted in the lateral ventricular, the animals were trained andtested in a step-through type passive avoidance task. Memory retrieval was measured by step-throughlatency (STL) and total time in dark compartments (TDC). In the first series of experiments, we estab-lished a dose–response relationship for ghrelin on the passive avoidance paradigm. In the second set ofexperiments, animals were divided to two groups. In the first group, MK-801 (0.075, 0.15 and 0.3 mg/kg)was injected intraperitoneally (i.p.) immediately after the acquisition session and in the second groupMK-801 (same doses) was injected (i.p.) 30 min before the retention session. Analysis of data showedthat in both groups, MK-801 impaired learning and memory. In the third set of experiments, adminis-

tration of ghrelin (200 ng/rat) right after the acquisition session (i.e. before MK-801 injection) improvedthe MK-801 induced memory impairment, but administration of ghrelin before retrieval session did notaffect the MK-801 induced memory impairment.

These results show an interaction between ghrelin and glutamatergic system. A novel finding in thisstudy is that ghrelin can prevent amnesia produced by NMDA antagonist in rats when injected in post-training phase.

. Introduction

Ghrelin, a unique 28 amino acid peptide hormone, has recentlyeen identified as an endogenous ligand of the growth hormoneecretagogue receptor and is characterized by a novel posttrans-ational acylation that ensures bioactivity [24,42]. It is primarilyeleased from X/A like the endocrine cells of oxyntic glands andirculates in the blood in the fasting process [10,24]. The centralervous system (CNS) might produce miniscule amounts of ghrelin21,27]. Ghrelin receptors are expressed throughout the brain,ncluding the hippocampus, a structure related to learning and

emory [8,17,43]. In the hippocampus, the circulating ghrelin waseported to cross the blood–brain barrier and bind to specific recep-

ors located on the hippocampus that cause an increase in synapticlasticity and the creation of new synaptic connections betweeneurons [11]. Long-term potentiation (LTP) is a molecular and

∗ Corresponding author at: Physiology Department, College of Paramedical Sci-nces, Darband Street, Ghods Square (Tajrish), Tehran, Iran. Tel.: +98 21 22718530;obile: +98 912 2115747.

E-mail address: fgoshadrou@yahoo.com (F. Goshadrou).

196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.peptides.2013.03.022

© 2013 Elsevier Inc. All rights reserved.

cellular model for synaptic plasticity in the hippocampus [3] and asubstrate for learning and memory processes [4]. Extensive studieshave shown that N-methyl-d-aspartate receptor (NMDA) systemplays a crucial role in the process of learning and memory forma-tion. It has also been suggested that the activation of the NMDAreceptor is required for LTP in the hippocampus, amygdale andmedial septum [22,32,33]. This mechanism has been implicated inmemory formation, with involvement of glutamate-receptor sys-tem and LTP is strongly linked to new learning and memory inanimal models [34,41]. The NMDA receptor is the predominantmolecular device for controlling synaptic plasticity and memoryfunction. Thus, an understanding of the control and action of theNMDA receptors at central synapses may provide clues to thera-peutic strategies in treating memory disorders. The NMDA receptorconstitutes the principal cellular machinery responsible for initiat-ing many forms of synaptic plasticity in different areas of the brain.It is a heterotetramer consisting of two obligatory NR1 subunitsand two regionally localized NR2 subunits [26]. A well-accepted

key molecule in the induction and maintenance of hippocamal LTPis CREB (cAMP response-element binding protein). According to arecent study, ghrelin in the hippocampus stimulated CREB throughthe activation of cAMP, protein kinase A (PKA) and PKA-dependent

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hosphorylation of NR1 subunit of the NMDA receptor [9]. The up-egulation of CREB by ghrelin, reported in the hippocampus, mayesult in the enhancement of LTP which then improves memoryormation.

The aim of the present set of experiments was to exam-ne whether intracerebroventricularly administered ghrelin couldmprove learning and memory deficits induced by MK-801 usingtep-through passive avoidance task in rats. It is well accepted thatK-801, a non-competitive NMDA receptor antagonist, impairs

he acquisition memory for passive avoidance learning and hasmnesic properties in the animal model [2,39]. Nonetheless, theres no report to date about the effect of ghrelin on MK-801 inducedmpairment of memory in rats. Just recently, however, it was sug-ested that there could be an interaction between ghrelin andlutamatergic system (through NMDA receptor) on feeding intaken broiler cockerels [36].

. Materials and methods

.1. Animals

Adult male Wistar rats (200 + 20 g) were used in this study. Thenimals were kept under standard laboratory conditions, with tapater and regular rat chow ad libitum. Upon arrival, the rats were

llowed to habituate in groups of four in standard cages, for at leastne week before initiation of the experiment. They were kept atemperature- and humidity-controlled vivarium on 12 h light/darkycle. All the experiments were conducted in accordance with theuide for the Care and Use of Laboratory Animals (National Institutef Health Publication No. 80-23, revised 1996). The experimentsere approved by the Research and Ethics Committee of Shahideheshti University of Medical Sciences.

.2. Surgery

Experimental animals were prepared with guide cannulamplantation (23 gauge needle) at least 8–10 days before their use.he rats were anesthetized with intraperitoneal (i.p.) injection ofetamine 10% (100 mg/kg) and xylazine 2% (10 mg/kg) and the can-ulae were stereotaxically (Stoelting, stereotaxic apparatus, USA)

mplanted in their lateral ventricles. The necessary coordinationas made based on Paxinos and Watson atlas [29] such as AP:0.8 mm caudal to bregma, Lat: 1.6 mm, DV: 3 mm ventral from

he skull surface (the guide cannulae were 1 mm above the appro-riate injection place). The guide cannulae were secured in placesing two stainless steel screws anchored to the skull with den-al acrylic cement. They were sealed with occluding stylette in theecovery period (8–10 days).

.3. Passive avoidance task (PA)

Training was performed in a conditioned apparatus with twoarts of the same size (30 × 20 × 20 cm) separated by guillotine typeoor. The floor of both compartments was a grid made of stain-

ess steel (1 mm diameter) with a distance of 1 cm apart. Subjectsere tested on the passive avoidance task (PA) after recovery period

f the surgery and all behavioral testing was performed between:00 am to 12:00 am. There were two habituations (10 min each)ith 30-min interval. In each session, the animal was placed in the

ight compartment and the sliding door was raised following a 10-selay while it was allowed to move freely for 10 min. In the trainingession which was performed 30 min after the second habituation,

imilar steps were applied. Once the rat fully crossed into the darkompartment (with all four paws on the shock grid floor), the slideoor was lowered, and after a 10-s delay, a 0.3-mA shock (50 Hz)as applied to the grid floor for 3 s. Animals that did not cross into

es 44 (2013) 60–65 61

the dark compartment during the training time, despite the prac-tices carried out in habituation sessions, were eliminated from thestudy. A 3-s shock was delivered after 2 min only if the same ratentered the dark compartment in spite of the first foot shock. Therats were immediately removed from the dark compartment andreturned back to their home cage.

A retention test was given to assess these mentioned trained ratsafter 24, 48 and 72 h in the same process as the training session;however, there was no shock applied to the grid floor when the ratsentered the dark compartment [15,37]. During the retention test,the rats were given access to the dark compartment for a maximumof 600 s. In this session, the latency of entering the dark compart-ment or step-through latency (STL) and the time spent in the darkcompartment (TDC) in 10 min were recorded.1

2.4. Drug administration

Ghrelin (Tocris) and MK-801 (Sigma–Aldrich) were dissolvedin normal saline to get a final intended concentration of 100, 200,400 ng/rat, and 0.075, 0.15 and 0.3 mg/kg, respectively, immedi-ately before use. Ghrelin injection was performed by a stainlesssteel needle (30 gauges) which was directly inserted into the guidecannula, with 1 mm beyond the tip of the cannula. The injectorcannula was connected to a 1-�l Hamilton syringe by polyethyl-ene tubing (PE-20) and 5 �l of ghrelin or the vehicle was infusedin over 1 min [25]. The injector was left in situ for 30 s after drugadministration, followed by replacement of the occluding stylette.Administration of MK-801 was performed intraperitoneally.

2.5. Experimental design

The researchers performed three sets of studies as follows.

2.5.1. Experiment 1: dose–response effect of ghrelin on thepassive avoidance response

In this set of experiment, we established a dose–response rela-tionship for ghrelin on the passive avoidance paradigm. Rats wereadministered 100, 200 and 400 ng/rat of ghrelin intracerebroven-tricularly immediately after foot shock in the passive avoidancetask (one series of animals; n = 8/group; total n = 24). The step-through latency and the time spent in the dark compartment duringthe retrieval test were recorded.

2.5.2. Experiment 2: dose–response effect of MK-801 on thepassive avoidance response

In this set of experiment the animals were randomly assignedto two groups designated by whether they received saline or MK-801 after the training session (group I) or prior to the testing phase(group II). In both groups the rats were administered 0.075, 0.15and 0.3 mg/kg of MK-801 intraperitoneally in the passive avoidancetask (two series of animals; n = 8/group; total n = 48).

2.5.3. Experiment 3: effect of ghrelin on MK-801-induced amnesiaIn this set of experiment, to evaluate the possible effect of

ghrelin on MK801-induced memory impairment, the animalsreceived ghrelin (200 ng/5 �l; icv) immediately after training and15 min prior to administration of MK-801. In the second group, theanimals received ghrelin 15 min prior to administration of MK-801(0.15 mg/kg; ip). MK-801 was injected 30 min before the retentiontest.

2.6. Histology

After commencement of all behavioral testing, the rats wereanaesthetized with a lethal dose of sodium pentobarbital, perfused

62 F. Goshadrou et al. / Peptides 44 (2013) 60–65

Fig. 1. The effect of post-training intracerebroventricular (i.c.v.) injections of salineand different doses of ghrelin on passive avoidance task. The treatments were givenimmediately after training. Step-through latency (A) and time in the dark compart-ment (B) during the retrieval test were given 24, 48 and 72 h after training. Thecd*

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Fig. 2. The effect of post-training intraperitoneal (i.p.) injections of saline and dif-ferent doses of MK-801 on the passive avoidance task. The treatments were givenimmediately after training. Step-through latency (A) and time in the dark compart-ment (B) during the retrieval test were given 24, 48 and 72 h after training. The

ontrol group (CTRL) received saline while the test groups (Ghr) received differentoses of ghrelin. Data are expressed as mean + SEM. n = 8 for each group. **P < 0.01,**P < 0.001, different from saline-treated group.

ranscardially with saline solution, followed by a 10% formalin solu-ion. The brain was removed and stored in buffered Formalin 10%efore sectioning it by a vibratome (Campden Instruments Ltd,K). The data reported here are limited to animals in which thelacement of cannula was histologically verified.

.7. Statistics

The latencies in passive avoidance tasks were analyzed using Two-way Analysis of Variance (ANOVA) for repeated measuresver time, followed by Bonferroni post hoc test for multiple com-arisons. The results of the behavioral studies are presented asean + SEM. A probability value of P < 0.05 was considered as sta-

istically significant.

. Results

.1. Dose–response effect of ghrelin on passive avoidanceesponse

The latency time and time in the dark compartment (Fig. 1)re regarded as an index of memory retention after i.c.v.hrelin administration. Compared with control animals receiv-ng saline, the animals receiving ghrelin with doses of 100, 200nd 400 ng/5 �l manifested significant dose-dependent increasen memory retention. Unlike the saline-treated group, the ani-

als receiving different doses of ghrelin and post-injection timesevealed significantly different responses as revealed by a two-wayNOVA, followed by Bonferroni’s test [factor ghrelin vs. saline: F(3,4) = 14.42, P < 0.0001; factor post injection time: F(2, 54) = 0.2588,

= 0.7729; treatment × time interaction effect: F(6, 54) = 1.522,

= 0.1886]. According to Bonferroni post test, the STL significantlyncreased in groups that received 200 ng/rat and 400 ng/rat ofhrelin (P < 0.01), compared with the control group. The statisti-al analyses also indicated a significant difference between the

control group (CTRL) received saline and test groups (Ghr) received different dosesof MK-801. Data are expressed as mean + SEM. n = 8 for each group. ***P < 0.001,different from saline-treated group.

experimental group, receiving ghrelin, and control group in relationto TDC, [factor ghrelin vs. saline: F(3, 54) = 41.01, P < 0.0001; factorpost injection time F(2, 54) = 0.8532, P = 0.4317; treatment × timeinteraction effect: F(6, 54) = 1.099, P = 0.3750]. As illustrated inFig. 1B, depicting Bonferroni post test results, different doses ofghrelin (200 ng/rat and 400 ng/rat) resulted in significant decreasein TDC, P < 0.001. The maximal recuperative response of ghrelinappeared at the doses of 200 ng/rat which lasted for three days; theresearchers accordingly chose this dose for further experiment.

3.2. Dose–response effect of MK-801 administration aftertraining and before the retention test

In this study we used two protocols for inducing the MK-801amnesia. In the first protocol, MK-801 was administered after thetraining while in the second it was administered before the retrievaltest. Fig. 2A shows the dose–response effects of different doses ofMK-801 (0.075, 0.15 and 0.3 mg/kg; i.p.) injected after training, onthe step-through latency. Two-way ANOVA for repeated measuresover time, followed by Bonferroni’s test, showed significant differ-ence between the control and various administrations of MK-801doses [treatment main effect: F(3, 56) = 24.69.25, P < 0.0001; timemain effect F(2, 56) = 2.169, P = 0.1238; treatment × time interac-tion effect: F(6, 56) = 0.5977, P = 0.9817]. Based on the data, all thethree doses of MK801 could decrease STL time at 24 h, 48 h, and72 h retrieval tests.

Also, the dose–response effects of different levels of MK-801(0.075, 0.15 and 0.3 mg/kg; i.p.) on TDC in 24 h, 48 h and 72 h afterthe training are shown in Fig. 2B. Two-way ANOVA for repeatedmeasures over time followed by Bonferroni’s test showed sig-nificant difference between the control and experimental groupsusing various doses of MK-801 each day [treatment main effect:

F(3, 56) = 16.93.38, P < 0.0001; time main effect F(2, 56) = 10.17,P = 0.0002; treatment × time interaction effect: F(6, 56) = 1.247,P = 0.2969]. Data showed that the three doses of MK801 increasedTDC times at 24 h, 48 h, and 72 h retrieval tests.

F. Goshadrou et al. / Peptides 44 (2013) 60–65 63

Fig. 3. The effect of pre-test intraperitoneal (i.p.) injections of saline or differentdoses of MK-801 on the passive avoidance task. The treatments were given 30 minbefore the retrieval test. Step-through latency (A) and time in the dark compart-ment (B) during the retrieval test were given 24, 48 and 72 h after training. Thecdd

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Fig. 4. The effect of post-training administration of ghrelin on the MK-801 inducedreduction of step-through latency (A) and time in the dark compartment (B) duringthe retrieval test given 24, 48 and 72 h after training. Ghrelin (200 ng/rat, i.c.v.), wasadministered 15 min before MK-801 injection (0.075 mg/kg, i.p.) and immediately

ontrol group (CTRL) received saline but the test groups (Ghr) received differentoses of MK-801. Data are expressed as mean + SEM. n = 8 for each group. ***P < 0.001,ifferent from saline-treated group.

In the second protocol, animals received various doses of MK-01 (0.075, 0.15 and 0.3 mg/kg; i.p.) 30 min before the retrievalest. Two-way ANOVA for repeated measures over time (Fig. 3A)hows a significant difference between the groups [treatment mainffect: F(3, 56) = 59.41, P < 0.0001; time main effect F(2, 56) = 12.99,

< 0.0001; treatment × time interaction effect: F(6, 56) = 3.387, = 0.0064]. Further analysis with Bonferroni post test showed sig-ificant difference between the control group and experimentalroups using 0.15 and 0.3 mg/kg doses of MK-801; P < 0.001.

On the other hand, different doses of MK-801 used in thistudy significantly affected TDC [treatment main effect: F(3,6) = 58.80, P < 0.0001; time main effect F(2, 56) = 11.18, P = 0.0001;reatment × time interaction effect: F(6, 56) = 1.596, P = 0.1655].onferroni post test showed significant difference between theontrol group and experimental groups using various doses of MK-01 (0.15 and 0.3 mg/kg), P < 0.001 (Fig. 3B).

The i.p. administration of MK-801 significantly decreased theatency time but increased TDC time, as memory impairment, atoses of 0.075, 0.015 and 0.3 at 24, 48 and 72 h after training sessionsee Fig. 2). However, as can be seen in Fig. 3, the decrease wasignificant only at the last two doses (0.015 and 0.03 mg/kg) of preetrieval test therapy.

.3. Effect of central ghrelin administration on demolisher effectf MK-801 in memory retention

In this set of experiments, ghrelin 200 ng/rat and MK 801.075 mg/kg were selected as effective doses. To assess the protec-ive effect of ghrelin on MK-801 memory impairment, ghrelin and

K-801 were co-administrated. Fig. 4A shows the effect of pos-raining administration of ghrelin on the MK801-induced memory

eduction. The data indicated significant differences between thetudy groups [treatment main effect: F(3, 56) = 180.9, P < 0.0001;ime main effect F(2, 56) = 0.9294, P = 0.4008; treatment × timenteraction effect: F(6, 56) = 0.6033, P = 0.7256]. Further analysis

after training. Data are expressed as mean + SEM. n = 8 for each group. **P < 0.01 and***P < 0.001, different from saline–treated group. †P < 0.05, ††P < 0.01 and †††P < 0.001different from MK-801-treated group.

using Bonferroni’s post test revealed that ghrelin attenuated theMK-801 effect on STL when MK-801 injected after training, P < 0.05.Also there was significant difference between the treated groupsin TDC criteria [treatment main effect: F(3, 56) = 159.8, P < 0.0001;time main effect F(2, 56) = 4.667, P = 0.0133; treatment × time inter-action effect: F(6, 56) = 1.954, P = 0.0879]. In this regard, two-wayANOVA results appear in Fig. 4B. According to Bonferroni’s test,there was significant difference between animals that received MK-801, P < 0001, compared with the groups receiving ghrelin beforeMK-801. This data shows that ghrelin has decreased MK-801 effectwhile the animals were in the dark compartment. In other words,as can be seen in Fig. 4, in comparison with the rats exposed to MK-801, the rats subjected to co-administration of ghrelin and MK-801manifested more latency time which lasted for the 3-day retrievaltest.

In another section of this experiment, the effect of ghrelin onmemory retrieval was evaluated. In this set of experiment all drugswere administrated before the retrieval session. Two-way repeatedmeasure ANOVA indicated the significant effect of each partic-ular treatment on STL [treatment main effect: F(3, 56) = 210.3,P < 0.0001; time main effect F(2, 56) = 0.2590, P = 0.7728; treat-ment × time interaction effect: F(6, 56) = 0.6805, P = 0.6659]. Asshown in Fig. 5A, Bonferroni’s test didn’t indicate any significantdifference between MK-801 on the one hand and co-administrationof ghrelin and MK-801on the other. The TDC score showed a sig-nificant difference between the groups in this set of experiment[treatment main effect: F(3, 56) = 61.44, P < 0.0001; time main effectF(2, 56) = 1.4, P = 0.2551; treatment × time interaction effect: F(6,56) = 1.350, P = 0.2509]. After the post test, however, there was nosignificant difference between animals that received MK-801 onlyand those that received co-administration of ghrelin and MK-801together, Fig. 5B.

4. Discussion

This seems to be the first study investigating the possibileinvolvement of glutamatergic circuits in ghrelin controlling of

64 F. Goshadrou et al. / Peptid

Fig. 5. The effect of administration of ghrelin on the MK-801 induced reduction ofStep-through latency (A) and time in the dark compartment (B) during the retrievaltest given 24, 48 and 72 h after training. Ghrelin (200 ng/rat, i.c.v.), was administered15 min before MK-801 injection (0.15 mg/kg, i.p.) and 30 min before retrieval test.Dd

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ata are expressed as mean + SEM. n = 8 for each group. **P < 0.01 and ***P < 0.001,ifferent from saline-treated group.

earning and memory behavior in rats. The main finding of the studys that ghrelin could reverse MK-801-induced impaired memory inats’ passive avoidance task, if administrated after training. Never-heless, if ghrelin is administrated before the test session, it cannotrevent amnesia produced by NMDA antagonist.

However, according to the result obtained from experiment, the intracerebroventricular injection of ghrelin could increase

atency time in step-through task while improving memory reten-ion. The results in this experiment are in line with those of previoustudies according to which, in a dose-dependent manner, ghrelinould increase the latency time in the step-down test [11]. Theaximum response to ghrelin happened at dose 200 ng/rat, i.c.v.hich was accordingly chosen for further experiments. The results

rom experiment 2 showed that intraperitoneal administration ofK-801 could significantly decrease the latency time while induc-

ng dose-dependent impairment of memory consolidation whennjected after the training session. This result agrees with thendings showing the significance of NMDA receptor’s activity for

earning in a variety of procedures [13,16,20]. Because the NMDAeceptors are required at the time of training and a few secondsfterwards [23], our results are in line with the idea that hip-ocampal NMDA receptors participate in the consolidation phase ofemory processing [35]. Also our results showed that i.p. injection

f MK-801 before the test session can decrease latency time duringhe retention session. In this set of experiments, 0.075 mg/kg of MK-01, that could be effective if injected immediately after training,emained ineffective. Only doses greater than 0.1 mg, e.g. 0.15 and.3 mg/kg of MK-801 could induce amnesia. This result agrees withhat of previous studies done by Hlinak and Krejci [18,19]; nonethe-ess, the noticeable point is that, in addition to memory impairment,

K-801 could induce changes in motor activity [14]. Therefore, its important to know whether MK-801 effects on memory are sec-ndary to its effects on motor disturbance. In an attempt to addresshis important issue, Carey et al. [5] examined the effects of MK-

01 on retention of habituation in a novel environment and motorctivity. Based on their results, a low dose of 0.1 mg/kg MK-801id not affect locomotor activity.The result of experiment 3 (first

es 44 (2013) 60–65

group) showed that administration of ghrelin before MK-801 ame-liorates memory impairment induced by MK-801 if it is injectedafter training. Although the proper mechanism by which ghrelinprevents the MK-801 induced amnesia cannot be explained on thebasis of the present data, there is recently some evidence indicatingcellular signaling mechanism of ghrelin in the hippocampus [9]. Itis well known that the glutamatergic system plays a crucial rolein learning and memory [12,30]. The activation of NMDA recep-tors increases intracellular calcium levels and triggers a cascadeof events leading to enhanced synaptic activity in the hippocam-pus, a phenomenon known as long-term potentiation (LTP) that isbelieved to be important for learning and memory [28,38]. In thehippocampus, ghrelin stimulated CREB signaling by ghrelin recep-tor and cAMP/PKA signaling pathway and the cAMP-dependentamplification of PKA resulted in the enhancement of NMDA recep-tor function by increasing the number of phosphorylated NR1subunits [9]. Also in Diano et al.’s report, ghrelin induced new den-dritic spine with synapses providing a structural mechanism bywhich ghrelin exerted its effect on hippocampal physiology [11].To the authors, such a prolonged response could be attributable toenhanced NMDA receptor activation. The possibility that the newspine synapses induced by ghrelin are enriched in NMDA receptorsis consistent with the electrophysiological finding [7].

The result of experiment 3 (second group) showed that co-administration of ghrelin and MK-801, before retrieval session,cannot affect the memory deficit induced by MK-801. The lowesteffective dose of MK-801 used in this experiment was 0.15 mg/kg.Abel and Lattal have demonstrated that during memory acqui-sition the animal associates the context and the shock. Duringconsolidation, which can last from minutes to days, this mem-ory moves from a labile to a more persistent state. During theretrieval phase, the animal was returned to a new context toassess the memory for the context-shock association [1]. It canbe quite difficult to isolate experimentally the different stagesof memory because experimental techniques potentially affecttwo or more stages of memory, depending on the time course ofthe manipulations. In this study we used two time courses forexperimental manipulation of different stages of memory. Manip-ulations immediately after acquisition could affect early stagesof consolidation, and manipulations before retrieval may affectlate stages of consolidation or retention. According to Carliniet al., ghrelin is more effective in the modulation of the mem-ory consolidation but cannot affect the retrieval; they accordinglyhypothesized that ghrelin modulates specific molecular interme-diates involved in the memory acquisition/consolidation processesbut not those related to retrieval [6]. By using a pharmacologicalapproach to inactivate the hippocampus with an AMPA receptorantagonist, Riedel et al. found that long-term inactivation dur-ing consolidation disrupted performance in a retention test [31].Thus, the hippocampus is important in the consolidation of spa-tial memories, independent of its role in acquisition or retrieval.A similar technique has been used to investigate the role ofthe lateral amygdale in acquisition and consolidation. Wilenskyet al. found that temporary inactivation of the lateral amygdaleby muscimol, a GABAA agonist, immediately before condition-ing, blocked auditory fear conditioning, but inactivation happeningimmediately after conditioning did not [40]. This may indicatethat the lateral amygdale has a specific role in memory acqui-sition but not in memory consolidation owing to Pavlovian fearof conditioning.To sum up, in this study, ghrelin amelioratedMK-801-induced memory impairment in the passive avoidancetask using rats. The results suggest that the beneficial effects of

Although the finding of this study may not provide clinicallyuseful outcomes for patients or healthy individuals, they do sug-gest the beneficial effects of ghrelin in treating diseases, such as

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lzheimer and schizophrenia that could cause cognitive impair-ents.

cknowledgment

The authors would like to express their deep gratitude to theuthorities at Neuroscience Research Center of Shahid Beheshtiniversity, Tehran, for offering a grant to cover the expenses of

he research.

eferences

[1] Abel T, Lattal KM. Molecular mechanisms of memory acquisition, consolidationand retrieval. Curr Opin Neurobiol 2001;11:180–7.

[2] Benvenga MJ, Spaulding TC. Amnesic effect of the novel anticonvulsant MK-801.Pharmacol Biochem Behav 1988;30:205–7.

[3] Bliss TVP, Collingridge GL. A synaptic model of memory: long-term potentiationin the hippocampus. Nature 1993;361:31–9.

[4] Böhme GA, Bon C, Lemaire M, Reibaud M, Piot O, Stutzmann JM, et al. Alteredsynaptic plasticity and memory formation in nitric oxide synthase inhibitor-treated rats. Proc Natl Acad Sci USA 1993;90:9191.

[5] Carey RJ, Dai H, Gui J. Effects of dizocilpine (MK-801) on motor activity andmemory. Psychopharmacology 1998;137:241–6.

[6] Carlini VP, Ghersi M, Schiöth HB, de Barioglio SR. Ghrelin and memory: differ-ential effects on acquisition and retrieval. Peptides 2010;31:1190–3.

[7] Chen L, Xing T, Wang M, Miao Y, Tang M, Chen J, et al. Local infusion of ghrelinenhanced hippocampal synaptic plasticity and spatial memory through acti-vation of phosphoinositide 3-kinase in the dentate gyrus of adult rats. Eur JNeurosci 2011;33:266–75.

[8] Cowley MA, Smith RG, Diano S, Tschöp M, Pronchuk N, Grove KL, et al. The dis-tribution and mechanism of action of Ghrelin in the CNS demonstrates a novelhypothalamic circuit regulating energy homeostasis. Neuron 2003;37:649–61.

[9] Cuellar JN, Isokawa M. Ghrelin-induced activation of cAMP signal transduc-tion and its negative regulation by endocannabinoids in the hippocampus.Neuropharmacology 2011;60:842–51.

10] Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, et al.Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized ina distinct endocrine cell type in the gastrointestinal tracts of rats and humans.Endocrinology 2000;141:4255–61.

11] Diano S, Farr SA, Benoit SC, McNay EC, da Silva I, Horvath B, et al. Ghrelin controlshippocampal spine synapse density and memory performance. Nat Neurosci2006;9:381–8.

12] Dingledine R, Borges K, Bowie D, Traynelis SF. The glutamate receptor ion chan-nels. Pharmacoll Rev 1999;51:7–62.

13] Escobar ML, Alcocer I, Chao V. The NMDA receptor antagonist CPP impairs con-ditioned taste aversion and insular cortex long-term potentiation in vivo. BrainRes 1998;812:246–51.

14] Ford LM, Norman AB, Sanberg PR. The topography of MK-801-induced locomo-tor patterns in rats. Physiol Behav 1989;46:755–8.

15] Ghavipanjeh GR, Alaei H, Khazaei M, Pourshanazari AA, Hoveida R. Effect ofacute and chronic hypertension on short- and long-term spatial and avoidancememory in male rats. 2010; 17:39–44.

16] Golden GJ, Houpt TA. NMDA receptor in conditioned flavor-taste preferencelearning: blockade by MK-801 and enhancement by d-cycloserine. PharmacolBiochem Behav 2007;86:587–96.

17] Guan X-M, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJS, et al.Distribution of mRNA encoding the growth hormone secretagogue receptor in

brain and peripheral tissues. Mol Brain Res 1997;48:23–9.

18] Hlinák Z, Krejcí I. MK-801 induced amnesia for the elevated plus-maze in mice.Behav Brain Res 2002;131:221–5.

19] Hlinák Z, Krejcí I. Kynurenic acid prevented social recognition deficits inducedby MK-801 in rats. Physiol Res 2003;52:805–8.

[

[

es 44 (2013) 60–65 65

20] Homayoun H, Stefani MR, Adams BW, Tamagan GD, Moghaddam B. Func-tional interaction between NMDA and mGluS receptors: effects on workingmemory, instrumental learning motor behaviors, and dopamine release. Neu-ropsychopharmacology 2004.

21] Horvath TL, Diano S, Sotonyi P, Heiman M, Tschöp M. Minireview: ghrelin andthe regulation of energy balance – a hypothalamic perspective. Endocrinology2001;142:4163–9.

22] Izquierdo I. Pharmacological evidence for a role of long-term potentiation inmemory. FASEB J 1994;8:1139–45.

23] Izquierdo I, Medina JH. Memory formation: the sequence of biochemical eventsin the hippocampus and its connection to activity in other brain structures.Neurobiol Learn Mem 1997;68:285–316.

24] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelinis a growth-hormone-releasing acylated peptide from stomach. Nature1999;402:656–60.

25] Lee CK, Choi YJ, Park SY, Kim JY, Won KC, Kim YW. Intracerebroventricu-lar injection of metformin induces anorexia in rats. Diab Metab J 2012;36:293–9.

26] Li F, Tsien JZ. Memory and the NMDA receptors. N Engl J Med 2009;361:302–3.

27] Lu S, Guan JL, Wang QP, Uehara K, Yamada S, Goto N, et al. Immunocytochemicalobservation of ghrelin-containing neurons in the rat arcuate nucleus. NeurosciLett 2001;321:157–60.

28] Lynch G, Kessler M, Arai A, Larson J. The nature and causes of hippocampallong-term potentiation. Prog Brain Res 1990;83:233.

29] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Hard Cover Edi-tion Academic Press; 2007.

30] Rao VR, Finkbeiner S. NMDA and AMPA receptors: old channels, new tricks.Trends Neurosci 2007;30:284–91.

31] Riedel G, Micheau J, Lam A, Roloff E, Martin S, Bridge H, et al. Reversible neuralinactivation reveals hippocampal participation in several memory processes.Nat Neurosci 1999;2:898–905.

32] Rockstroh S, Emre M, Pokorny R, Tarral A. Effects of the novel NMDA-receptorantagonist SDZ EAA 494 on memory and attention in humans. Psychopharma-cology (Berl) 1996;124:261–6.

33] Scatton B, Carter C, Benavides J, Giroux C. N-methyl d-aspartate receptor antag-onists: a novel therapeutic perspective for the treatment of ischemic braininjury. Cerebrovasc Dis 1991;1:121–35.

34] Scheetz A, Constantine-Paton M. Modulation of NMDA receptor function:implications for vertebrate neural development. FASEB J 1994;8:745–52.

35] Steele R, Morris R. Delay-dependent impairment of a matching-to-place taskwith chronic and intrahippocampal infusion of the NMDA-antagonist D-AP5.Hippocampus 1999;9:118–36.

36] Taati M, Nayebzadeh H, Zendehdel M. The effects of DL-AP5 and glutamateon ghrelin-induced feeding behavior in 3-h food-deprived broiler cockerels. JPhysiol Biochem 2011;67:217–23.

37] Tamburella A, Micale V, Mazzola C, Salomone S, Drago F. The selec-tive norepinephrine reuptake inhibitor atomoxetine counteracts behavioralimpairments in trimethyltin-intoxicated rats.

38] Tsien JZ, Huerta PT, Tonegawa S. The essential role of hippocampalCA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell1996;87:1327–38.

39] Venable N, Kelly PH. Effects of NMDA receptor antagonists on passive avoid-ance learning and retrieval in rats and mice. Psychopharmacology (Berl)1990;100:215–21.

40] Wilensky AE, Schafe GE, LeDoux JE. The amygdala modulates memory consol-idation of fear-motivated inhibitory avoidance learning but not classical fearconditioning. J Neurosci 2000;20:7059–66.

41] Wong RWC, Setou M, Teng J, Takei Y, Hirokawa N. Overexpression of motorprotein KIF17 enhances spatial and working memory in transgenic mice. ProcNatl Acad Sci USA 2002;99:14500.

42] Wren A, Small C, Ward H, Murphy K, Dakin C, Taheri S, et al. The novel hypo-thalamic peptide ghrelin stimulates food intake and growth hormone secretion.Endocrinology 2000;141:4325–8.

43] Zigman JM, Jones JE, Lee CE, Saper CB, Elmquist JK. Expression of ghrelin recep-tor mRNA in the rat and the mouse brain. J Comp Neurol 2006;494:528.