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ARTICLE IN PRESSG ModelBR 9565 1–12

Behavioural Brain Research xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

rontal cortex and hippocampus neurotransmitter receptor complexevel parallels spatial memory performance in the radial arm maze

haranidharan Shanmugasundarama, Ajinkya Sasea, András G. Miklosia,ernando J. Sialanaa,d, Saraswathi Subramaniyana, Yogesh D. Ahera, Marion Grögerb,arald Högerc, Keiryn L. Bennettd, Gert Lubeca,∗

Department of Pediatrics, Medical University of Vienna, Währinger Gürtel 18, 1090 Vienna, AustriaCore Facility, Medical University of Vienna, Lazarettegasse 14, A-1090 Vienna, AustriaCore Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, Brauhausgasse 34, A-2325 Himberg,ustriaCeMM Research Center for Molecular Medicine of the Austrian Academy of Science, Lazarettgasse 14, AKH BT 25.3, A-1090 Vienna, Austria

i g h l i g h t s

GluA1, GluA2 & GluN2A are modulated in frontal cortex in spatial memory training.GluN2B & DAT-ph (Thr53) of hippocampus are involved in spatial memory training.Whereas, GluN1, D1 & nAChR-�7 are modulated both in hippocampus and frontal cortex.D1 and GluN1 receptors can form complex.

r t i c l e i n f o

rticle history:eceived 10 April 2015eceived in revised form 21 April 2015ccepted 23 April 2015vailable online xxx

eywords:adial arm mazeeurotransmitter receptor complexlue native PAGEpatial memory1-GluN1 complex

a b s t r a c t

Several neurotransmitter receptors have been proposed to be involved in memory formation. However,information on receptor complexes (RCs) in the radial arm maze (RAM) is missing. It was therefore the aimof this study to determine major neurotransmitter RCs levels that are modulated by RAM training becausereceptors are known to work in homo-or heteromeric assemblies. Immediate early gene Arc expressionwas determined by immunohistochemistry to show if prefrontal cortices (PFC) and hippocampi wereactivated following RAM training as these regions are known to be mainly implicated in spatial memory.Twelve rats per group, trained and untrained in the twelve arm RAM were used, frontal cortices andhippocampi were taken, RCs in membrane protein were quantified by blue-native PAGE immunoblot-ting. RCs components were characterised by co-immunoprecipitation followed by mass spectrometricalanalysis and by the use of the proximity ligation assay. Arc expression was significantly higher in PFC oftrained as compared to untrained rats whereas it was comparable in hippocampi. Frontal cortical levelsof RCs containing AMPA receptors GluA1, GluA2, NMDA receptors GluN1 and GluN2A, dopamine receptor

D1, acetylcholine nicotinic receptor alpha 7 (nAChR-�7) and hippocampal levels of RCs containing D1,GluN1, GluN2B and nAChR-�7 were increased in the trained group; phosphorylated dopamine trans-porter levels were decreased in the trained group. D1 and GluN1 receptors were shown to be in the samecomplex. Taken together, distinct RCs were paralleling performance in the RAM which is relevant forinterpretation of previous and design of future work on RCs in memory studies.

© 2015 Published by Elsevier B.V.

Please cite this article in press as: Shanmugasundaram B, et al. Frontlevel parallels spatial memory performance in the radial arm maze. Beh

Abbreviations: AMPA, �-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMD∗ Corresponding author. Tel.: +43 1 40400 3215; fax: +43 1 40400 6065.

E-mail address: gert.lubec@meduniwien.ac.at (G. Lubec).

ttp://dx.doi.org/10.1016/j.bbr.2015.04.043166-4328/© 2015 Published by Elsevier B.V.

al cortex and hippocampus neurotransmitter receptor complexav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

A, N-methyl-D-aspartic acid; PAGE, Polyacrylamide gel electrophoresis.

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. Introduction

Spatial memory is a very essential cognitive component for both,ndividual and species survival. In rodents spatial memory is widelyssessed in the Morris water maze [1] and in the land-based radialrm maze (RAM) [2]. The involvement of neurotransmitter systemsn cognitive functions can be studied using these different spatialearning tasks [3,4]. In RAM the spatial reference memory (identi-ying the position of rewarded arms) and spatial working memoryremembering which arms were visited earlier in the trial) can beimultaneously assessed [5–7]. The gradual decrease in numberf WM errors along the training period signifies that while per-orming the memory task during a session the animal also learns

strategy to perform the task better in the long run that leads toventual improvement in performance during the course of train-ng [8]. Repeated training in the RAM paradigm implies that theearning strategies are consolidated into long term memory afterach training and consolidation encompasses persistent chemicalhanges in the synapse [9,10].

Several neurotransmitter receptors were identified to be mod-lated during memory formation. Ionotropic glutamate receptorsre required and well-studied in the context of learning and mem-ry. Using genetic manipulation studies it was shown that AMPAubunit knockout animals show deficient spatial WM capabilities11–13] and AMPA activation is necessary for the consolida-ion/retention processes [14]. NMDA receptors are important forriggering learning-related plasticity. It has been suggested that thectivation of the NMDA receptor is required for long-term poten-iation (LTP) in the hippocampus [15]. Both, lesion studies andharmacological manipulations in animal models suggest that theMDA-receptor system is important in the induction of memory

ormation [16]. In the RAM task hippocampal NMDA receptors arenvolved in encoding and retrieval processes of spatial WM [14].urthermore, cholinergic receptor systems are important for spatialemory processes. There is significant work on nicotinic receptors

hat can modulate memory and are critical for memory function17–20]. Metabotropic dopamine receptors are also involved inoth, working memory and long term memory processes: Block-

ng receptor activity by pharmacological and knockout approachesave shown that in rodents the D1 receptor in the PFC plays aajor role in spatial memory whereas D2 receptor blockade had

o effect [21,22]. The dopamine transporter (DAT) that pumps theeurotransmitter dopamine back from the synaptic cleft into there-synapse cytosol also plays a major role in WM; inhibition ofAT by a benztropine analog improves WM performance in a PFC-ependent delayed-alteration task [23]. However, its role in longerm spatial memory is not clearly known.

RAM training involves both, spatial reference memory andpatial working memory [24]. Much of the evidence shows themportance of the hippocampus in mediating foraging behavioursing spatial cues [25,26]. Studies also emphasized a role of pre-rontal cortex (PFC) in spatially based foraging behaviours in mazesspecially in the delayed version of RAM [27]. Impairments onelayed spatial tasks after lesions to either the PFC [28] or theippocampal formation [29] suggest that these two brain regionsay interact when an animal is performing spatial memory tasks.part from its role in foraging behaviour of animals in spatial tasks,

he PFC and hippocampus are also involved in long-term memoryrocesses [30,31]. However, information on what major neuro-ransmitter RCs are modulated during spatial memory formationost training in RAM is missing.

Therefore, the aims of the current study were first to find out

Please cite this article in press as: Shanmugasundaram B, et al. Frontlevel parallels spatial memory performance in the radial arm maze. Beh

f PFC and hippocampus are activated following RAM trainingsing Arc expression analysis. Secondly it was tried to answer theuestion which major neurotransmitter RCs and DAT transporteromplexes rather than isolated subunits are paralleling spatial

PRESS Brain Research xxx (2015) xxx–xxx

memory performance of the rat in the RAM using working memoryerrors (for working memory) and latencies (for reference memory)as parameter.

2. Materials and methods

2.1. Animal housing

Male Sprague Dawley rats, aged between 10 and 14 weeks wereused in this study. All rats were purchased and housed in the CoreUnit of Biomedical Research, Division of Laboratory Animal Sci-ence and Genetics, Medical University of Vienna (Himberg, Austria)and maintained in cages made of makrolon and filled with auto-claved woodchips. An autoclaved standard rodent diet (Altromin®,Germany) and water in bottles was available ad libitum. The roomwas illuminated with artificial light from 5:00 h to 19:00 h at anintensity of about 200 lx positioned in 2 m distance. All behaviourexperiments were performed between 8:00 h and 14:00 h.

All procedures were carried out according to the guidelinesof the Ethics committee, Medical University of Vienna, and U.K.Animals (Scientific Procedures) Act, 1986 and associated guide-lines, the European Communities Council Directive of 24 November1986 (86/609/EEC) and were approved by the Federal Ministryof Education, Science and Culture, Austria (BMWF-66.009/0267-II/3b/2012). All efforts were made to minimize animal sufferingand to reduce the number of animals used.

2.2. Radial arm maze

For biochemical analysis twelve rats per group (trained anduntrained) and for Arc expression profiling seven rats per groupwere used. Rats were trained in the twelve arm radial maze. Thesetup is made of black plastic and kept at an elevation of 80 cmabove the floor in a room with visual cues placed distally on allsides. The central platform has a diameter of 50 cm and twelve arms(12 cm × 60 cm) which projects radially outward. A plastic cylinderis used to restrict the movement of rats to the centre before startof the training. The lifting of the cylinder is controlled by a pulleysystem from far end of the room. Food is placed in the arm 1 cmfrom the distal end. Out of twelve arms, eight arms were baitedduring the training and four remained un-baited. The amount offood provided was restricted for five days prior to the experimentto reduce the body weight to 85% to maintain a lean, healthy bodyand also to motivate the rats for foraging behaviour during train-ing. The rats were handled for 30 min/day during these five daysfor adaptation to the experimenter. Water was provided ad libitumduring the whole training.

Before the start of the actual training, rats were given habitu-ation session for two days 5 min each in which some food pelletswere placed scattered all over the maze and rats were allowed toexplore the maze and let consume the food. During the trainingsession, the arms were baited for each rat only once at the begin-ning of each session to assess WM, while the other four arms wereleft un-baited to test reference memory. The pattern of baited andun-baited arms was consistent throughout testing for each rat butdiffered among rats. Each trial began by placing the rat in the cen-tral platform, after 10 s the cylinder was slowly lifted. A sessionlasts eight minutes or until all eight baited arms were entered. Sec-ond time and thereafter entry into a baited arm was counted as aWM error, whereas any entry into an un-baited arm was noted as areference memory error. The rats were given one training sessions

al cortex and hippocampus neurotransmitter receptor complexav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

per day over a period of ten days.Untrained controls were placed in the maze to run the same

amount of time as their trained counterparts. Animals wereexposed to the same spatial cues, but without reward, therefore

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ats did not develop an association between the extra-maze cuesnd the reward.

.3. Histochemical studies

.3.1. Animal perfusion and brain fixationFor Arc immunofluorescence staining, rats were anesthetized

h after the last session of training on day ten with 0.3 ml/kgntraperitoneal injection of sodium penthorbital (Release00 mg/ml, WDT-Wirtschaftsgenossenschaft deutscher Tier-erzte eG) and perfused intracardially with ice-cold PBS (0.1 Mhosphate buffered saline, pH 7.2) containing 0.2% heparin fol-

owed by 4% paraformaldehyde (PFA) at a pH of 7.4 in 0.1 M PBS.rain samples were postfixed in 4% PFA for 24 h at 4 ◦C and thenransferred into 30% sucrose solution (in 0.1 M PBS) for 72 h. Formmunofluorescence fixed brains were embedded with Tissue-Tek

edia (OCT compound, Sakura Finetek Europe, The Netherlands)hen frozen at -20 ◦C and sectioned at 50 �m with a cryostat. Forhe Proximity Ligation Assay fixed brains were embedded withissue-Tek media (OCT compound, Sakura Finetek Europe, Theetherlands) then immersed in isopentane cooled with liquiditrogen and sectioned at 30 �m with a cryostat (Leica CM 3050S,etzlar, Germany). Sections were stored in PBS sodium azide until

urther used.

.3.2. Immunofluorescence stainingFree-floating immunofluorescence staining was performed on

at PFC and hippocampus coronal sections. Samples were washedor 10 min in 0.1 M PBS, then blocked with 10% normal donkeyerum in 0.3% Triton X-100 PBS for 30 min at room temperatureollowed by incubation with anti-Arc rabbit polyclonal antibodyArc/Arg3.1, 1:200, Santa Cruz Biotechnology, Santa Cruz, SA,SA) for 24 h at 4 ◦C. After washing in PBS two times for 5 minach, sections were incubated with secondary antibody (anti-abbit IgG labelled Alexa Fluor 555, 1:1000 dilution, Cell Signalingechnology, Boston, MA, USA) for 1 h in the dark at room tem-erature. Slices were subsequently washed two times 5 min each

n PBS, counterstained by incubation with DAPI (1:1000 dilu-ion, 4′,6-diamidino-2-phenylindole, Invitrogen, Carlsbad, CA, USA)or 10 min followed by washing for 5 min then mounted withuorescence mounting medium (DAKO, Glostrup, Denmark) andoverslipped. Images were acquired with Zeiss Observer.Z1 micro-cope (Carl Zeiss GmbH, Jena, Germany) equipped with TissueFAXSTissue Gnostics GmbH, Vienna, Austria) at 20x magnification keep-ng all acquisition settings even through all samples. Arc expression

as analysed and quantified with TissueQuest software (Tissuenostics GmbH, Vienna, Austria).

.3.3. In-situ proximity ligation assay (PLA)The PLA was performed according to the protocol given by the

anufacturer (O-LINK Bioscience, Uppsala, Sweden) with slightodifications. Free floating brain slices were blocked for 30 min

t room temperature using blocking buffer supplied with the PLAit. After blocking brain slices were incubated with diluted mouseonoclonal antibody against the N-methyl D-aspartate recep-

or 1 (GluN1, 1:100, Abcam, Cambridge, UK) and polyclonal antiopamine D1 receptor primary antibodies (1:100, Alomone Labs,

erusalem, Israel) for 48 h at 4 ◦C on a rocking platform. Follow-ng incubation with primary antibodies slices were washed with

ashing buffer A (O-LINK Bioscience, Uppsala, Sweden) then incu-ated with rabbit PLUS and mouse MINUS probes (1:40, O-LINKioscience, Uppsala, Sweden) for 2 h at 37 ◦C with gentle orbital

Please cite this article in press as: Shanmugasundaram B, et al. Frontlevel parallels spatial memory performance in the radial arm maze. Beh

haking. Ligation was performed according to the manufacturer’srotocol with the exception of a 45 min incubation time instead of0 min mentioned. DNA polymerase was diluted 1:20 and incu-ated for 120 min at 37 ◦C. The rest of the amplification steps

PRESS Brain Research xxx (2015) xxx–xxx 3

remained unchanged. After amplification slices were washed with1x then 0.01x wash buffer B prepared according to the recipe sup-plied in the kit manual. Finally brain slices were transferred toglass slides, mounted with Duolink in Situ Mounting Medium withDAPI (O-LINK Bioscience, Uppsala, Sweden). Images were acquiredwith a Zeiss LSM 700 confocal laser scanning microscope (Carl ZeissGmbH, Jena, Germany) at 20x magnification keeping all acquisitionsettings even through all samples [32,33].

2.4. Biochemical studies

Frontal cortices (FC) and hippocampi was quickly dissected(following a micro dissection procedure described in the book“Neuroproteomics”, chapter “Dissection of Rodent Brain Regions”[34]) from rat brain six hours after the last session of RAM train-ing on day ten by deeply anaesthetizing the animal with CO2 andanimals were killed by neck dislocation. The tissue was stored at−80 ◦C for biochemical analysis.

2.4.1. Crude synaptosome preparationAll procedures were carried out at 4 ◦C. FC and hippocampal tis-

sues were homogenized in ice-cold homogenization buffer [10 mMHEPES, pH 7.5, 300 mM sucrose, one complete protease inhibitortablet (Roche Molecular Biochemicals, Mannheim, Germany) per50 ml] by Ultra-Turrax (IKA, Staufen, Germany). The homogenatewas centrifuged for 10 min at 1000 × g and the pellet was dis-carded. The supernatant was centrifuged at 50,000 × g for 30 minin an ultracentrifuge (Beckman Coulter Optima-L-90 K). The pelletwas re-suspended in washing buffer (homogenization buffer with-out sucrose), kept on ice for 1 h and centrifuged at 50,000 × g for30 min to obtain membrane fraction of crude synaptosome extractas pellet.

2.4.2. Receptor protein extractionAll procedures were carried out at 4 ◦C. An extraction buffer con-

taining 1.5 M 6-aminocaproic acid, 300 mM Bis–Tris, pH 7.0 and1% n-Dodecyl �-d-maltoside (DDM) was added to the membranepellets and incubated for 1 h by gently vortexing every 10 min.Following solubilisation, samples were centrifuged at 15,000 × gfor 60 min. The pellet was discarded. The protein concentrationof the supernatant was estimated using the BCA protein assay kit(Pierce, Rockford, IL, USA). Extracted proteins were then aliquotedand stored at −80 ◦C until used.

2.4.3. Blue native polyacrylamide gel electrophoresis (BN PAGE)and BN PAGE western blot procedure

Equal amount of proteins (30 �g) from trained and untrainedsamples were loaded in the wells and the RCs were separated on5–13% of blue native PAGE gels and the western blot procedurewas carried out using the procedure described previously [35]. Thedetails of antibodies used are given in the supplementary dataTable 1. Immunoreactive bands were quantified by the softwareImage J (NIH). Coomassie blue R-350 stained membranes were usedas loading control and normalized with the western blot densito-metric values [36,37]

2.4.4. Co-immunoprecipitationThe FC crude synaptosome membrane fraction pellet prepared

as described previously was suspended in lysis buffer containing1% DDM, 150 mM NaCl, 1 mM EDTA, 50 mM Tris–HCl (pH 8.0),10 mM NaF, 10 mM Na3VO4, 10 mM Na4O7P2 and protease inhibitorcocktail (Roche, Mannheim, Germany) on a rotation shaker 1 h

al cortex and hippocampus neurotransmitter receptor complexav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

at 4 ◦C. After centrifugation at 15,000 × g, at 4 ◦C for 20 min, thesupernatant was incubated with affinity-purified goat anti-bodyagainst NMDA receptor subunit GluN1 (GluN1; NMDA�1; sc-1467,Santa Cruz Biotechnology) and dopamine receptor subunit D1

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Table 1Showing BN PAGE western blot mean densitometry values (arbitrary units) after normalization and statistical t-test p-values of the individual receptor complexes.

Receptor subunits (apparentmol. wt. in BN PAGE)

FC Hippocampus

mean ± SD p value mean ± SD p value

Trained Untrained Trained Untrained

Glu A1 (∼700 kDa) 1.179 ± 0.186 0.809 ± 0.233 0.0003 1.018 ± 0.239 0.887 ± 0.337 0.2874Glu A2 (∼700 kDa) 0.991 ± 0.101 0.778 ± 0.128 0.0002 1.727 ± 0.299 1.569 ± 0.556 0.3681Glu A2 (∼550 kDa) 0.624 ± 0.492 0.495 ± 0.212 0.4189 – – –Glu A3 (∼700 kDa) 6.059± 1.826 5.242 ± 1.770 0.2783 1.427 ± 0.592 1.424 ± 0.524 0.9886Glu A3 (∼550 kDa) 1.773 ± 0.754 1.383 ± 0.736 0.2136 2.946 ± 1.663 1.968 ± 0.851 0.1008Glu A4 (∼700 kDa) 1.649 ± 0.638 1.697 ± 0.326 0.8219 3.962 ± 1.569 4.800 ± 1.168 0.1532Glu A4 (∼550 kDa) 0.652 ± 0.215 0.786 ± 0.368 0.2926 – – –Glu N1 (∼480 kDa) 0.698 ± 0.159 0.510 ± 0.331 0.0949 4.369 ± 1.508 2.839 ± 0.695 0.0059Glu N1 (∼300 kDa) 0.949 ± 0.595 0.461 ± 0.337 0.0241 – – –Glu N2A (∼700 kDa) 0.910 ± 0.291 0.444 ± 0.137 0.0001 0.736 ± 0.220 0.647 ± 0.244 0.3585Glu N2A (∼500 kDa) – – – 1.141 ± 0.236 1.173 ± 0.363 0.8152Glu N2B (∼700 kDa) – – – 1.237 ± 0.925 0.751 ± 0.303 0.1066Glu N2B (∼500 kDa) 0.682 ± 0.197 0.611 ± 0.132 0.3110 0.930 ± 0.240 0.615 ± 0.143 0.0010nAChR-�7 (∼700 kDa) 1.515 ± 0.331 1.016 ± 0.304 0.0009 0.893 ± 0.272 0.503 ± 0.130 0.0004nAChR-�7 (∼480 kDa) – – – 1.125 ± 0.330 0.554 ± 0.182 0.0001D1 (∼480 kDa) 0.544 ± 0.157 0.431 ± 0.104 0.0510 2.989 ± 0.687 1.893 ± 0.649 0.0006D1 (∼300 kDa) 0.984 ± 0.323 0.647 ± 0.242 0.0090 – – –D2 (∼480 kDa) 0.930 ± 0.287 1.055 ± 0.457 0.4532 – – –D2 (∼300 kDa) 2.541 ± 1.151 2.303 ± 1.106 0.6195 1.460 ± 0.595 1.323 ± 0.323 0.4934DAT (∼500 kDa) 1.344 ± 0.356 1.275 ± 0.473 0.6932 0.530 ± 0.274 0.776 ± 0.413 0.1018

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DAT (∼300 kDa) 1.772 ± 0.555 1.898 ± 0.576

DAT-ph (∼500 kDa) 3.915 ± 1.157 3.561 ± 0.670

DAT-ph (∼300 kDa) 2.098 ± 0.634 2.101 ± 0.894

D1DR Antibody, sc-14001; Santa Cruz Biotechnology) in two sep-rate reactions overnight at 4 ◦C and subsequently incubated withrotein-G agarose beads (GE Healthcare, Uppsala, Sweden) for 4 ht 4 ◦C with gentle rotation. After five times of washing with lysisuffer, proteins bound were denatured with sample buffer con-aining 125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 10% beta-

ercaptoethanol, 0.02% bromophenol blue at 95 ◦C for 3 min. SDS-AGE was performed using 5% stacking and 8% separating gel withn initial current of 50 V for 1 h and then 150 V for 1 h [38]. West-rn blotting was carried out following the procedure as previouslyescribed [39] except the details of the antibodies used in this workre as follows. Membrane blotted with primary antibody anti D1Anti-Dopamine Receptor D1 antibody, ab78021, abcam) 1:1000ilution and anti GluN1 (Anti-NMDAR1 antibody ab134308, abcam):2000 dilution and the secondary antibody used was HRP conju-ated Goat Anti-Mouse IgG H&L (ab97040, abcam) 1:5000 dilution.

.4.5. In-gel digestion of proteinsProtein bands from silver-stained SDS-PAGE gels that were iden-

ified by the corresponding antibodies against the D1 and GluN1ubunits were put into a 1.5 mL tube. Gel pieces were washed with0 mM ammonium bicarbonate and then two times with washinguffer (50% 100 mM ammonium bicarbonate/50% acetonitrile) for0 min each with vortexing. 100 �L of 100% acetonitrile was addedo the tube and the mixture was incubated for 10 min. Gel piecesere dried completely using a SpeedVac concentrator. Reduction

f cysteine residues was carried out with a 10 mM dithiothreitolDTT) solution in 100 mM ammonium bicarbonate pH 8.6 for 60 mint 56 ◦C. After discarding the DTT solution, the same volume of a5 mM iodoacetamide (IAA) solution in 100 mM ammonium bicar-onate buffer pH 8.6 was added and incubated in darkness for5 min at 25 ◦C to alkylate the cysteine residues. The IAA solu-ion was replaced by washing buffer (50% 100 mM ammoniumicarbonate/50% acetonitrile) and washed twice for 15 min each

Please cite this article in press as: Shanmugasundaram B, et al. Frontlevel parallels spatial memory performance in the radial arm maze. Beh

ith vortexing. Gel pieces were washed and dried in 100% ace-onitrile followed by dryness in SpeedVac. Dried gel pieces weree-swollen with 12.5 ng/�L trypsin (Promega, Germany) solutioneconstituted with 25 mM ammonium bicarbonate. Gel pieces were

0.5888 – – –0.3714 0.407 ± 0.201 0.606 ± 0.169 0.01610.9940 0.804 ± 0.376 0.952 ± 0.291 0.2956

incubated for 16 h at 37 ◦C. The supernatant was transferred tonew 0.5 mL tubes, and peptides were extracted with 50 �L of 0.5%formic acid/20% acetonitrile for 20 min in a sonication bath. Thisstep was repeated two times. Samples in extraction buffer werepooled in 0.5 mL tubes and evaporated in a SpeedVac concentrator.The peptides were reconstituted in 5% formic acid and analysed byLC-MS/MS [35].

2.4.6. LC-MS/MS analysisNano-LC-ESI-MS/MS was performed on a linear trap quadrupole

(LTQ) Orbitrap Velos (Thermoscientific, Walthan, MA, USA) cou-pled to an Agilent 1200 HPLC nanoflow system comprised of adual pump with one precolumn and one analytical column (Agi-lent Biotechnologies, Palo Alto, CA, USA) (Bennett et al., 2011).Data were acquired using Xcalibur version 2.1.0. HPLC solventswere as follows: solvent A consisted of 0.4% formic acid in waterand solvent B consisted of 0.4% formic acid in 70% methanol and20% isopropanol. From a thermostated microautosampler, 8 �Lof the peptide mixture were automatically loaded onto a trapcolumn (Zorbax 300SB-C18 5 �m, 5 × 0.3 mm, Agilent Biotechnolo-gies) with a binary pump at a flow rate of 45 �L/min using 0.1%TFA for loading and washing the precolumn. After washing, thepeptides were eluted by back-flushing onto a 16 cm fused silicaanalytical column with an inner diameter of 50 �m packed with aC18 reversed phase material (ReproSil-Pur 120 C18-AQ, 3 �m, Dr.Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides wereeluted from the analytical column with a 27 min gradient rangingfrom 3 to 30% solvent B, followed by a 25 min gradient from 30 to70% solvent B and, finally, a 7 min gradient from 70 to 100% solventB at a constant flow rate of 100 nL/min. The analyses were per-formed in a data-dependent acquisition mode using a top 15 CIDmethod. Dynamic exclusion for selected ions was 60s. A single lockmass at m/z 445.120024 was employed. Maximal ion accumulationtime allowed in MS and MSn mode was 500 and 50ms, respectively.

al cortex and hippocampus neurotransmitter receptor complexav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

Automatic gain control was used to prevent overfilling of the iontrap and was set to 106 ions and 5000 ions for a full Fourier trans-form mass spectrometry scan and MSn, respectively. Peptides weredetected in MS mode at a resolution of 60,000 (at m/z 400).

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ig. 1. a – Working memory error (WME), b – reference memory error (RME) and cay-8, 9 and 10 compared to day-1 (*P < 0.001, F = 4.949). Slightly decreasing trenday-3 onwards compared to day-1 (*P < 0.0001, F = 13.23). Mean and SEM are show

Proteins were identified using Proteome Discoverer version 1.4. spectral peptide peak list was extracted from the raw files andsing both, Mascot and SequestHT, the peak list was matchedgainst a rat protein database (9,595 protein sequence entries fromniProtKB/Swiss-Prot downloaded on March 2013). The MSMS ion

earch parameters were as follows: one missed cleavage site, massolerances of 10 ppm and 0.6 Da for the precursor and fragmentons. Dynamic modifications were: methionine oxidation, serinend threonine phosphorylation. Static modifications were cysteinearbamidomethylation. Matched peptides were filtered as follows:ignificant threshold below 0.05 for Mascot and 0.1 for SEQUEST,nd a 1% false discovery rate cut-off after target decoy search.iltered peptides from the two search engines were merged androtein identifications requiring at least two unique peptides wereeported.

. Results

.1. Radial arm maze

The total number of working memory errors (WME), referenceemory errors (RME) and latency to finish a trial made by the ani-als with respect to the training sessions were shown in Fig. 1.

rained rats showed significant reduction of latency to finish arial and WME over the course of training. Data were analysedsing repeated measurements ANOVA. WME became statistically

Please cite this article in press as: Shanmugasundaram B, et al. Frontlevel parallels spatial memory performance in the radial arm maze. Beh

ifferent from day-8 onwards (F = 4.949, p < 0.0001), on day-10ME were about threefold lower than on day-1. There was a

lightly decreasing trend for RME however, not statistically signif-cant. Latency to finish a trial showed a decreasing trend and was

ency to finish a trial of RAM behaviour test. WMEs were significantly decreased onE curve was not statistically significant. Latency was significantly decreased frome graph. The data were analysed using repeated measurements ANOVA.

significantly different from day-3 onwards compared to day-1(F = 13.23, p < 0.0001).

3.2. Arc expression analysis in PFC and hippocampus

Immunohistochemical analysis (Fig. 2) showed that the imme-diate early gene Arc in PFC was expressed significantly higherin trained animals when compared to the untrained animals. Intrained animals the bright pink spots representing arc expressionin different regions of PFC, ACd (dorsal anterior cingulate area), FR2(frontal cortex area 2), IL (infralimbic area) and PL (prelimbic area)were significantly more abundant, compared with unpaired Stu-dent’s t-test (p < 0.05). The sub-regions of PFC were identified usingthe reference [40]. In hippocampal regions there was no statisticaldifference in Arc expression between trained and untrained groups(data not shown).

3.3. BN PAGE western blot quantification of membrane receptorcomplexes

The RCs were quantified using BN PAGE western blotting. InFC the RCs containing subunits GluA1 at around 700 kDa, GluA2(∼700 kDa), D1 (∼300 kDa), GluN1 (∼300 kDa), GluN2A (∼700 kDa)and nAChR-�7 (∼700 kDa) were significantly increased in thetrained group. In hippocampus RCs containing GluN1 (∼480 kDa),GluN2B (∼500 kDa), D1 (∼480 kDa), and nAChR-�7 (∼700 &

al cortex and hippocampus neurotransmitter receptor complexav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

∼480 kDa) were significantly increased in trained groups whencompared to untrained rats. In hippocampi the phosphorylated,activated form of DAT (DAT-ph) (∼500 kDa) showed lower arbi-trary units of optical density in the trained group as compared by

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Fig. 2. Expression analysis of IEG Arc protein in PFC region of trained and untrained rats by Immunohistochemistry. The bright pink spots in the image represent the expresseda orsal

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rc protein. The image also shows the different regions of PFC. Abbreviations: ACd, drea. Scale-bar in the image represents 500 �m. Arc protein is significantly expresseignificance of difference in unpaired Student’s t-test (*P < 0.05). Mean and SD are s

npaired Student’s t-test. No change in the electrophoretic mobil-ty of the RCs between trained and untrained groups was observed.he mean densitometric values of BN PAGE western blot after nor-alization and statistical t-test p-values of the individual RCs are

isted in the Table 1 (Figs. 3 and 4).

.4. Proximity ligation assay of D1 and GluN1 receptors

Fig. 5 shows the PLA data of D1 and GluN1 receptors in dif-erent regions of rat PFC were presented with red dots indicatingo-localization and proximity of D1 and GluN1 receptors in ACd,R2, IL and PL regions which in turn proposes that D1 and GluN1eceptor subunits may be complex components of the same RCs.

.5. Co-immunoprecipitation of D1 and GluN1 receptor followedy mass spectrometrical identification of receptor complexes

Co-immunoprecipitation was carried out with an immobilizedntibody against D1 as well as an immobilized antibody againstluN1. Western blot analysis shown in Fig. 6 confirmed the pres-nce of both, D1 and GluN1 receptors in both elutions, i.e. thentibody against D1 detected GluN1 and vice versa.

Mass spectrometrical analysis (MS) shows that the immunopre-ipitate using an antibody against D1 contained the GluN1 subunit.otal sequence coverage indicated in Table 2 indicates unambigu-us MS identification of these receptor subunits. The individualeptides obtained from proteolytic cleavage followed by MS are

Please cite this article in press as: Shanmugasundaram B, et al. Frontlevel parallels spatial memory performance in the radial arm maze. Beh

isted in supplementary Table 2. Representative spectra are pro-ided in supplementary Fig. 1.

This finding suggests that the D1 and GluN1 receptors can existn the same complex.

anterior cingulate area; FR2, frontal cortex area 2; IL, infralimbic area; PL, prelimbice in trained group when compared to the untrained rats. Asterisks indicate level of

in the graph.

4. Discussion

Herein, following RAM training in rats, the neuronal activity inPFC and hippocampus were identified using Arc expression analysisand levels of major neurotransmitter RCs in these regions werecompared between trained and untrained animals.

Behavioural results reveal that the animals had learned the taskshowing gradually decreasing latency to finish a session statisti-cally significant from day-3 onwards compared to the first day.Appetite is the motivation for the foraging behaviour of mildlystarving rats in RAM. Animals identify the spatial location of thearm containing food pellets using available distal cues [41]. In orderto retrieve the food efficiently with minimal efforts when a baitedarm is visited for food, the rat has to avoid re-entry. This learn-ing strategy involves working memory and the gradual decreaseof WME over the training days signifies that the learning strate-gies are consolidated to long term memory after each training day[10]. Thus, since the proteomic analysis has been done at the end oftraining, changes in levels of neurotransmitter RCs may be associ-ated with the persistent chemical changes of the neuronal synapsesas a result of RAM training.

Activity regulated cytoskeleton associated protein (Arc) is oneof the Immediate Early Genes (IEG), expressed in the brain regionswhen there is neuronal activation [42–44]. Therefore, IEG expres-sion has been widely used as a marker to identify brain regionsthat are activated in response to learning [45–47]. Own resultsshow that Arc is expressed significantly higher in the trained groupin areas of PFC, ACd, FR2, IL and PL, suggesting that all of these

al cortex and hippocampus neurotransmitter receptor complexav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

areas are activated following RAM training, whereas the hippocam-pal regions do not show any difference in Arc expression betweentrained and untrained groups. The PFC is known for its involve-ment in guiding the animal for goal-directed behaviour and the

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ig. 3. BN PAGE western quantification of receptor complexes in FC tissue. GluA1, Ghen compared to untrained rats. Asterisks indicate level of significance of differenean and SD error bars.

ippocampus is critical for spatial navigation and spatial memoryrocessing. These two structures interact through complex circuitsased on the functional demand and their coordination is neces-ary for optimal performance [48–50]. From the current study its understood that both, hippocampus and PFC are required in theAM paradigm. Inhibition of the activity-dependent expression of

EGs causes impairment of short term memory consolidation [51]nd therefore plays a critical role in the formation of long termemory [52,53]. This implies that Arc is more likely expressed

n brain regions that are involved in memory consolidation. Fromhis perspective, elevated levels of Arc expression in trained ani-

als in PFC signifies that PFC neurons are subjected to memoryonsolidation after the last session, because the learning strate-ies are consolidated as the latency to complete the task decreasesith training. Based upon Arc expression results herein, it may be

uggested that the memory consolidation process in RAM trainingay involve only PFC and not hippocampus. It is known, however,

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hat the hippocampus is essential for acquisition, consolidation andetention of spatial and non-spatial memories [50,54,55]. Own con-rasting result in the hippocampus may propose that after repeatedraining in the RAM, hippocampal neurons do not undergo any

, GluN1, GluN2A, D1, and nAChR-�7 were significantly increased in trained groupsunpaired Student’s t-test (*P < 0.05, **P < 0.01, #P < 0.005). The bar graph shows the

memory consolidation and therefore do not express Arc but proba-bly the hippocampus has been involved in the beginning of training,although the statement has to be reconsidered as significant RCslevel changes were observed.

Previous reports have been already addressing the involvementof neurotransmitter RCs rather than individual receptor subunits inspatial memory formation [37,56–58] but work on the RAM has notbeen published so far to the best of our knowledge. Determinationof RCs is relevant as receptor function is carried out as homo-orheteromeric assemblies of receptors.

Glutamate, the major excitatory neurotransmitter exerts itsaction by binding to glutamate receptors. Ionotropic glutamatereceptors are made up of different types of subunits and basedon the subunit composition the biochemical and electrophysio-logical properties of the RCs vary [59]. Specific AMPA receptorsubunits are delivered to synaptic sites following learning and con-tributes to experience-dependent synaptic strengthening [60,61].

al cortex and hippocampus neurotransmitter receptor complexav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

AMPA receptors play a vital role in WM mechanisms [62]: GluA1subunit deletion in the whole brain results in strong spatial WMdeficits [63]. The molecular mechanism of AMPA receptor subunitfunction in spatial WM is not clearly understood but it has been

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ig. 4. BN PAGE western quantification of receptor complexes in hippocampus. Comrained groups when compared to untrained rats whereas the DAT-ph levels were dtudent’s t-test (*P < 0.05, **P < 0.01, #P < 0.005). The bar graph shows the mean and

ostulated that it includes the activation of AMPA receptors con-aining the GluA1 subunit [63] and consequently during plasticityhe GluA1/GluA2 containing RCs are added to synapses and dur-ng long term memory formation GluA2/GluA3-containing AMPAeceptors constitutively replace synaptic AMPA receptors whileeeping the synaptic strength constant [64,65]. There is evidencehat GluA1-mediated AMPA receptor signalling is essential forpatial memory tasks, particularly in hippocampus [61,66]. It isntriguing that no differences between hippocampal AMPA recep-or containing RCs levels were observed and this finding is of impor-ance to elucidate different memory mechanisms in hippocampus.hese data emphasize the importance of NMDA receptors in acqui-

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ition of spatial memory processes. Since on the last day the rats stillndergo training, NMDA receptor subunits may be modulated afterhe last session. Own results show that in FC GluN2A and GluN1ontaining complexes were increased whereas in hippocampus

containing GluN1, GluN2B, D1, and nAChR-�7 levels were significantly increased ined in trained group. Asterisks indicate level of significance of difference in unpairedror bars.

NR2B and GluN1 containing complexes were elevated in trainedanimals, pointing to different mechanism in hippocampus and FCin NMDA-mediated signalling in performance in the RAM. Pharma-cological manipulation studies in an experimental animal modelsuggest that the NMDA-receptor system may be important in theacquisition of memory, but not for the maintenance [16], hencethe changes in NMDA receptor subunits in the current study couldbe due to the effect of the last training session.nAChR-�7 contain-ing RCs levels were significantly higher in the synaptic membranefraction in trained groups. This result is in agreement with a recentbehavioural study indicating that acetylcholine alpha 7-containingRCs are modulated during memory retrieval in mice tested in the

al cortex and hippocampus neurotransmitter receptor complexav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

multiple-T-maze [67]. Depletion of acetylcholine in dorsolateralPFC markedly impaired WM [17] whereas selective stimulation ofthe neuronal nAChR-�7 receptor improved spatial WM in nonhu-man primates [68] and in rodents [69].

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Please cite this article in press as: Shanmugasundaram B, et al. Frontal cortex and hippocampus neurotransmitter receptor complexlevel parallels spatial memory performance in the radial arm maze. Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.043

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Fig. 5. “In Situ” Proximity ligation assay for detection of co-localized D1 and GluN1 receptors in rat PFC. Representative images showing the D1-GluN1 receptor complex withDAPI counterstained nuclei. Red dots as shown by the arrow indicates the co-localization of D1 and GluN1 receptor. Abbreviations: ACd, dorsal anterior cingulate area; FR2,frontal cortex area 2; IL, infralimbic area; PL, prelimbic area. Negative control is done in parallel with no primary antibody added. Scale-bar in the figure represents 10 �m.

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Table 2Data of mass spectrometrical identification of GluN1 co-immunoprecipitated with D1.

Uniprot accession number Enzyme digestion Gene name �coverage �# Unique Peptides �# Peptides �# PSMs

G3V933D(1A) dopamine receptor

Trypsin Drd1 23.99 8 8 197

P35439-5Glutamate receptor ionotropic,NMDA 1

Trypsin Grin1

Fig. 6. Western blot showing the detection of D1 and GluN1 receptor subunitsimmunoprecipitated with Anti-D1 and Anti-GluN1 antibody. This shows the D1 andGs

wsLaiiicasDraf

atamtbsoso(Dtrp[mUcscuc

ing in the rat. J Neurosci Methods 1984;11:47–60.

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luN1 remains in a complex and are co-immuno precipitated. Cntrl is the total crudeynaptosome membrane fraction protein prepared from rat’s FC tissue.

The involvement of the dopaminergic system in memory isell-studied in hippocampus. The dopamine receptor subtype D1

timulates the onset of protein synthesis-dependent late phase ofTP in the hippocampus [70] and facilitates the persistent stor-ge of hippocampus-dependent memories [71]. Apart from its rolen long-term memory formation, the dopaminergic system is alsonvolved in WM processes. The D1 subtype within the PFC has beenmplicated in normal functioning of WM and its alterations canause WM impairments like schizotypal personality disorder [72]nd schizophrenia [73]. D1 receptor blockade using antagonistshowed dose- and delay-dependent impairments in WM whereas2 receptor blockade had no effect [74]. In the current study it is

evealed that levels of a D1 containing complex in the trained groupre higher and D2 containing RCs levels were unchanged in both,rontal cortex and hippocampus.

Complexes containing D1 and GluN1 were co-migrating atround 300 kDa on BN-PAGE which indicates, that these recep-ors could exist in the same complex. It is well known that D1nd NMDA receptors have profound importance in cognition (likeemory and executive functions) and in synaptic plasticity and

here is evidence that D1 and glutamate receptors can co-exist inrain regions. Sarantis et al. examined the effects of the in-vitrotimulation of D1 and D2 receptors on the phosphorylation statef NMDA and AMPA receptor subunits in rat PFC and hippocampushowing that D1 receptor activation elicits a significant increasef the phosphorylation state of NMDA (GluN1, GluN2B) and AMPAGluA1) receptor subunits [75]. In another study it was shown that1 and NR1 receptors interact through protein-protein interac-

ions in single pyramidal neurons and inter-neurons in the adultat PFC [76]. D1 and NMDA (GluN1, GluN2B) receptors form a com-lex through the carboxyl tails and can be co-immunoprecipitated77]. In electrophysiological studies it was demonstrated that D1

odulates NMDA currents in hippocampal neuron cultures [77].sing PLA herein it was observed that all regions in PFC showedo-localisation of D1 and GluN1 receptor subunits. Since the PLA

Please cite this article in press as: Shanmugasundaram B, et al. Frontlevel parallels spatial memory performance in the radial arm maze. Beh

hows proximity and does not confirm physical interactions, theomplex formation between D1 and GluN1 was further examinedsing co-immunoprecipitation experiments that revealed that D1o-eluted with an antibody against GluN1 and vice versa, moreover,

5.76 5 5 5

mass spectrometrical analysis of the immunoprecipitate using anantibody against D1 identified the presence of GluN1. Nai andco-workers explored the effects of the D1–GluN1 interaction onNMDA receptor-dependent LTP and WM reporting that uncou-pling the D1–GluN1 interaction led to impaired WM and decreasedGluN1–CaMKII association and CaMKII activity in rat hippocampus[78].

The DAT is a presynaptic membrane-spanning protein knownto mediate dopamine re-uptake. Reuptake inhibition has beenalready used for cognitive enhancement in the treatment of ADHD.Schmeichel et al. observed improved performance in rats using aPFC-dependent delayed-alternation task of spatial working mem-ory by the administration of a well-characterized benztropineanalog, AHN 2-005 [23]. However, not much is known about itsinvolvement in long term memory formation. Own results showthat levels of DAT containing complexes were comparable betweentrained and untrained animals in frontal cortex and hippocampuswhereas levels of complexes containing the phosphorylated activeform of DAT (phosphorylated at amino acid position Thr53) werelower in trained animals in hippocampus but not in FC.

The presence and modulation of a high molecular weight com-plex containing the activated form of DAT in spatial memoryperformance in the current work points to the probable involve-ment of a large transporter complex that remains to be furthercharacterised by subsequent studies.

Taken together, RCs changes paralleling spatial memory withreference and working memory components in the PFC as well asin hippocampus at the end of the RAM testing were revealed, con-firming the participation of both regions in spatial and workingmemory mechanisms. Moreover, a complex was shown to containD1 and GluN1 and a complex containing phosphorylated, i.e. acti-vated DAT in FC was decreased in rats trained in the RAM probablyindicating a role for dopamine reuptake in memory formation. Thiswork may be relevant for the interpretation of previous work on sig-naling and design of future studies on neurotransmitter receptorsand dopamine transport in spatial memory.

Acknowledgements

The skilful technical assistance of Sabine Rauscher from theimaging core facility is highly appreciated. We acknowledge com-ments from Prof. Dr. Volker Korz, Univ. Vienna. We appreciate thefinancial assistance from the Medical University of Vienna to carryout this work.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bbr.2015.04.043

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