Frontal cortex and hippocampus neurotransmitter receptor complex level parallels spatial memory performance in the radial arm maze.

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Contents lists available at ScienceDirect

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Research report

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Frontal cortex and hippocampus neurotransmitter receptor complex level parallels spatial memory performance in the radial arm maze

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Bharanidharan Shanmugasundaram a , Ajinkya Sase a , András G. Miklosi a , Fernando J. Sialana a,d , Saraswathi Subramaniyan a , Yogesh D. Aher a , Marion Gröger b , Harald Höger c , Keiryn L. Bennett d , Gert Lubec a,∗ a

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

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h 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.

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a r t i c l e

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a b s t r a c t

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Article history: Received 10 April 2015 Received in revised form 21 April 2015 Accepted 23 April 2015 Available online xxx

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Keywords: Radial arm maze Neurotransmitter receptor complex Blue native PAGE Spatial memory D1-GluN1 complex

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 aim of this study to determine major neurotransmitter RCs levels that are modulated by RAM training because receptors are known to work in homo-or heteromeric assemblies. Immediate early gene Arc expression was determined by immunohistochemistry to show if prefrontal cortices (PFC) and hippocampi were activated 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 and hippocampi were taken, RCs in membrane protein were quantified by blue-native PAGE immunoblotting. RCs components were characterised by co-immunoprecipitation followed by mass spectrometrical analysis and by the use of the proximity ligation assay. Arc expression was significantly higher in PFC of trained as compared to untrained rats whereas it was comparable in hippocampi. Frontal cortical levels of 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 transporter levels were decreased in the trained group. D1 and GluN1 receptors were shown to be in the same complex. Taken together, distinct RCs were paralleling performance in the RAM which is relevant for interpretation of previous and design of future work on RCs in memory studies. © 2015 Published by Elsevier B.V.

Abbreviations: AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartic acid; PAGE, Polyacrylamide gel electrophoresis. ∗ Corresponding author. Tel.: +43 1 40400 3215; fax: +43 1 40400 6065. E-mail address: [email protected] (G. Lubec). http://dx.doi.org/10.1016/j.bbr.2015.04.043 0166-4328/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Shanmugasundaram B, et al. Frontal cortex and hippocampus neurotransmitter receptor complex level 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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1. Introduction

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Spatial memory is a very essential cognitive component for both, individual and species survival. In rodents spatial memory is widely assessed in the Morris water maze [1] and in the land-based radial arm maze (RAM) [2]. The involvement of neurotransmitter systems in cognitive functions can be studied using these different spatial learning tasks [3,4]. In RAM the spatial reference memory (identifying the position of rewarded arms) and spatial working memory (remembering which arms were visited earlier in the trial) can be simultaneously assessed [5–7]. The gradual decrease in number of WM errors along the training period signifies that while performing the memory task during a session the animal also learns a strategy to perform the task better in the long run that leads to eventual improvement in performance during the course of training [8]. Repeated training in the RAM paradigm implies that the learning strategies are consolidated into long term memory after each training and consolidation encompasses persistent chemical changes in the synapse [9,10]. Several neurotransmitter receptors were identified to be modulated during memory formation. Ionotropic glutamate receptors are required and well-studied in the context of learning and memory. Using genetic manipulation studies it was shown that AMPA subunit knockout animals show deficient spatial WM capabilities [11–13] and AMPA activation is necessary for the consolidation/retention processes [14]. NMDA receptors are important for triggering learning-related plasticity. It has been suggested that the activation of the NMDA receptor is required for long-term potentiation (LTP) in the hippocampus [15]. Both, lesion studies and pharmacological manipulations in animal models suggest that the NMDA-receptor system is important in the induction of memory formation [16]. In the RAM task hippocampal NMDA receptors are involved in encoding and retrieval processes of spatial WM [14]. Furthermore, cholinergic receptor systems are important for spatial memory processes. There is significant work on nicotinic receptors that can modulate memory and are critical for memory function [17–20]. Metabotropic dopamine receptors are also involved in both, working memory and long term memory processes: Blocking receptor activity by pharmacological and knockout approaches have shown that in rodents the D1 receptor in the PFC plays a major role in spatial memory whereas D2 receptor blockade had no effect [21,22]. The dopamine transporter (DAT) that pumps the neurotransmitter dopamine back from the synaptic cleft into the pre-synapse cytosol also plays a major role in WM; inhibition of DAT by a benztropine analog improves WM performance in a PFCdependent delayed-alteration task [23]. However, its role in long term spatial memory is not clearly known. RAM training involves both, spatial reference memory and spatial working memory [24]. Much of the evidence shows the importance of the hippocampus in mediating foraging behaviour using spatial cues [25,26]. Studies also emphasized a role of prefrontal cortex (PFC) in spatially based foraging behaviours in mazes especially in the delayed version of RAM [27]. Impairments on delayed spatial tasks after lesions to either the PFC [28] or the hippocampal formation [29] suggest that these two brain regions may interact when an animal is performing spatial memory tasks. Apart from its role in foraging behaviour of animals in spatial tasks, the PFC and hippocampus are also involved in long-term memory processes [30,31]. However, information on what major neurotransmitter RCs are modulated during spatial memory formation post training in RAM is missing. Therefore, the aims of the current study were first to find out if PFC and hippocampus are activated following RAM training using Arc expression analysis. Secondly it was tried to answer the question which major neurotransmitter RCs and DAT transporter complexes rather than isolated subunits are paralleling spatial

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

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2. Materials and methods

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2.1. Animal housing

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Male Sprague Dawley rats, aged between 10 and 14 weeks were used in this study. All rats were purchased and housed in the Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna (Himberg, Austria) and maintained in cages made of makrolon and filled with autoclaved woodchips. An autoclaved standard rodent diet (Altromin® , Germany) and water in bottles was available ad libitum. The room was illuminated with artificial light from 5:00 h to 19:00 h at an intensity of about 200 lx positioned in 2 m distance. All behaviour experiments were performed between 8:00 h and 14:00 h. All procedures were carried out according to the guidelines of the Ethics committee, Medical University of Vienna, and U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the Federal Ministry of Education, Science and Culture, Austria (BMWF-66.009/0267II/3b/2012). All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Radial arm maze For biochemical analysis twelve rats per group (trained and untrained) and for Arc expression profiling seven rats per group were used. Rats were trained in the twelve arm radial maze. The setup is made of black plastic and kept at an elevation of 80 cm above the floor in a room with visual cues placed distally on all sides. The central platform has a diameter of 50 cm and twelve arms (12 cm × 60 cm) which projects radially outward. A plastic cylinder is used to restrict the movement of rats to the centre before start of the training. The lifting of the cylinder is controlled by a pulley system from far end of the room. Food is placed in the arm 1 cm from the distal end. Out of twelve arms, eight arms were baited during the training and four remained un-baited. The amount of food provided was restricted for five days prior to the experiment to reduce the body weight to 85% to maintain a lean, healthy body and also to motivate the rats for foraging behaviour during training. The rats were handled for 30 min/day during these five days for adaptation to the experimenter. Water was provided ad libitum during the whole training. Before the start of the actual training, rats were given habituation session for two days 5 min each in which some food pellets were placed scattered all over the maze and rats were allowed to explore the maze and let consume the food. During the training session, the arms were baited for each rat only once at the beginning of each session to assess WM, while the other four arms were left un-baited to test reference memory. The pattern of baited and un-baited arms was consistent throughout testing for each rat but differed among rats. Each trial began by placing the rat in the central platform, after 10 s the cylinder was slowly lifted. A session lasts eight minutes or until all eight baited arms were entered. Second time and thereafter entry into a baited arm was counted as a WM error, whereas any entry into an un-baited arm was noted as a reference memory error. The rats were given one training sessions 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 were exposed to the same spatial cues, but without reward, therefore


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rats did not develop an association between the extra-maze cues and the reward.

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2.3. Histochemical studies

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2.3.1. Animal perfusion and brain fixation For Arc immunofluorescence staining, rats were anesthetized 2 h after the last session of training on day ten with 0.3 ml/kg intraperitoneal injection of sodium penthorbital (Release 300 mg/ml, WDT-Wirtschaftsgenossenschaft deutscher Tieraerzte eG) and perfused intracardially with ice-cold PBS (0.1 M phosphate buffered saline, pH 7.2) containing 0.2% heparin followed by 4% paraformaldehyde (PFA) at a pH of 7.4 in 0.1 M PBS. Brain samples were postfixed in 4% PFA for 24 h at 4 ◦ C and then transferred into 30% sucrose solution (in 0.1 M PBS) for 72 h. For immunofluorescence fixed brains were embedded with Tissue-Tek media (OCT compound, Sakura Finetek Europe, The Netherlands) then frozen at -20 ◦ C and sectioned at 50 ␮m with a cryostat. For the Proximity Ligation Assay fixed brains were embedded with Tissue-Tek media (OCT compound, Sakura Finetek Europe, The Netherlands) then immersed in isopentane cooled with liquid nitrogen and sectioned at 30 ␮m with a cryostat (Leica CM 3050S, Wetzlar, Germany). Sections were stored in PBS sodium azide until further used. 2.3.2. Immunofluorescence staining Free-floating immunofluorescence staining was performed on rat PFC and hippocampus coronal sections. Samples were washed for 10 min in 0.1 M PBS, then blocked with 10% normal donkey serum in 0.3% Triton X-100 PBS for 30 min at room temperature followed by incubation with anti-Arc rabbit polyclonal antibody (Arc/Arg3.1, 1:200, Santa Cruz Biotechnology, Santa Cruz, SA, USA) for 24 h at 4 ◦ C. After washing in PBS two times for 5 min each, sections were incubated with secondary antibody (antirabbit IgG labelled Alexa Fluor 555, 1:1000 dilution, Cell Signaling Technology, Boston, MA, USA) for 1 h in the dark at room temperature. Slices were subsequently washed two times 5 min each in PBS, counterstained by incubation with DAPI (1:1000 dilution, 4 ,6-diamidino-2-phenylindole, Invitrogen, Carlsbad, CA, USA) for 10 min followed by washing for 5 min then mounted with fluorescence mounting medium (DAKO, Glostrup, Denmark) and coverslipped. Images were acquired with Zeiss Observer.Z1 microscope (Carl Zeiss GmbH, Jena, Germany) equipped with TissueFAXS (Tissue Gnostics GmbH, Vienna, Austria) at 20x magnification keeping all acquisition settings even through all samples. Arc expression was analysed and quantified with TissueQuest software (Tissue Gnostics GmbH, Vienna, Austria). 2.3.3. In-situ proximity ligation assay (PLA) The PLA was performed according to the protocol given by the manufacturer (O-LINK Bioscience, Uppsala, Sweden) with slight modifications. Free floating brain slices were blocked for 30 min at room temperature using blocking buffer supplied with the PLA kit. After blocking brain slices were incubated with diluted mouse monoclonal antibody against the N-methyl D-aspartate receptor 1 (GluN1, 1:100, Abcam, Cambridge, UK) and polyclonal anti dopamine D1 receptor primary antibodies (1:100, Alomone Labs, Jerusalem, Israel) for 48 h at 4 ◦ C on a rocking platform. Following incubation with primary antibodies slices were washed with washing buffer A (O-LINK Bioscience, Uppsala, Sweden) then incubated with rabbit PLUS and mouse MINUS probes (1:40, O-LINK Bioscience, Uppsala, Sweden) for 2 h at 37 ◦ C with gentle orbital shaking. Ligation was performed according to the manufacturer’s protocol with the exception of a 45 min incubation time instead of 30 min mentioned. DNA polymerase was diluted 1:20 and incubated for 120 min at 37 ◦ C. The rest of the amplification steps

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remained unchanged. After amplification slices were washed with 1x then 0.01x wash buffer B prepared according to the recipe supplied in the kit manual. Finally brain slices were transferred to glass slides, mounted with Duolink in Situ Mounting Medium with DAPI (O-LINK Bioscience, Uppsala, Sweden). Images were acquired with a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss GmbH, Jena, Germany) at 20x magnification keeping all acquisition settings 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 training on day ten by deeply anaesthetizing the animal with CO2 and animals were killed by neck dislocation. The tissue was stored at −80 ◦ C for biochemical analysis. 2.4.1. Crude synaptosome preparation All procedures were carried out at 4 ◦ C. FC and hippocampal tissues were homogenized in ice-cold homogenization buffer [10 mM HEPES, pH 7.5, 300 mM sucrose, one complete protease inhibitor tablet (Roche Molecular Biochemicals, Mannheim, Germany) per 50 ml] by Ultra-Turrax (IKA, Staufen, Germany). The homogenate was centrifuged for 10 min at 1000 × g and the pellet was discarded. The supernatant was centrifuged at 50,000 × g for 30 min in an ultracentrifuge (Beckman Coulter Optima-L-90 K). The pellet was re-suspended in washing buffer (homogenization buffer without sucrose), kept on ice for 1 h and centrifuged at 50,000 × g for 30 min to obtain membrane fraction of crude synaptosome extract as pellet. 2.4.2. Receptor protein extraction All procedures were carried out at 4 ◦ C. An extraction buffer containing 1.5 M 6-aminocaproic acid, 300 mM Bis–Tris, pH 7.0 and 1% n-Dodecyl ␤-d-maltoside (DDM) was added to the membrane pellets and incubated for 1 h by gently vortexing every 10 min. Following solubilisation, samples were centrifuged at 15,000 × g for 60 min. The pellet was discarded. The protein concentration of the supernatant was estimated using the BCA protein assay kit (Pierce, Rockford, IL, USA). Extracted proteins were then aliquoted and 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 untrained samples were loaded in the wells and the RCs were separated on 5–13% of blue native PAGE gels and the western blot procedure was carried out using the procedure described previously [35]. The details of antibodies used are given in the supplementary data Table 1. Immunoreactive bands were quantified by the software Image J (NIH). Coomassie blue R-350 stained membranes were used as loading control and normalized with the western blot densitometric values [36,37] 2.4.4. Co-immunoprecipitation The FC crude synaptosome membrane fraction pellet prepared as described previously was suspended in lysis buffer containing 1% DDM, 150 mM NaCl, 1 mM EDTA, 50 mM Tris–HCl (pH 8.0), 10 mM NaF, 10 mM Na3 VO4 , 10 mM Na4 O7 P2 and protease inhibitor cocktail (Roche, Mannheim, Germany) on a rotation shaker 1 h at 4 ◦ C. After centrifugation at 15,000 × g, at 4 ◦ C for 20 min, the supernatant was incubated with affinity-purified goat anti-body against NMDA receptor subunit GluN1 (GluN1; NMDA␨1; sc-1467, Santa Cruz Biotechnology) and dopamine receptor subunit D1


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

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Hippocampus

mean ± SD

Glu A1 (∼700 kDa) Glu A2 (∼700 kDa) Glu A2 (∼550 kDa) Glu A3 (∼700 kDa) Glu A3 (∼550 kDa) Glu A4 (∼700 kDa) Glu A4 (∼550 kDa) Glu N1 (∼480 kDa) Glu N1 (∼300 kDa) Glu N2A (∼700 kDa) Glu N2A (∼500 kDa) Glu N2B (∼700 kDa) Glu N2B (∼500 kDa) nAChR-␣7 (∼700 kDa) nAChR-␣7 (∼480 kDa) D1 (∼480 kDa) D1 (∼300 kDa) D2 (∼480 kDa) D2 (∼300 kDa) DAT (∼500 kDa) DAT (∼300 kDa) DAT-ph (∼500 kDa) DAT-ph (∼300 kDa)

282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297

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p value

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Untrained

1.179 ± 0.186 0.991 ± 0.101 0.624 ± 0.492 6.059± 1.826 1.773 ± 0.754 1.649 ± 0.638 0.652 ± 0.215 0.698 ± 0.159 0.949 ± 0.595 0.910 ± 0.291 – – 0.682 ± 0.197 1.515 ± 0.331 – 0.544 ± 0.157 0.984 ± 0.323 0.930 ± 0.287 2.541 ± 1.151 1.344 ± 0.356 1.772 ± 0.555 3.915 ± 1.157 2.098 ± 0.634

0.809 ± 0.233 0.778 ± 0.128 0.495 ± 0.212 5.242 ± 1.770 1.383 ± 0.736 1.697 ± 0.326 0.786 ± 0.368 0.510 ± 0.331 0.461 ± 0.337 0.444 ± 0.137 – – 0.611 ± 0.132 1.016 ± 0.304 – 0.431 ± 0.104 0.647 ± 0.242 1.055 ± 0.457 2.303 ± 1.106 1.275 ± 0.473 1.898 ± 0.576 3.561 ± 0.670 2.101 ± 0.894

(D1DR Antibody, sc-14001; Santa Cruz Biotechnology) in two separate reactions overnight at 4 ◦ C and subsequently incubated with protein-G agarose beads (GE Healthcare, Uppsala, Sweden) for 4 h at 4 ◦ C with gentle rotation. After five times of washing with lysis buffer, proteins bound were denatured with sample buffer containing 125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 10% betamercaptoethanol, 0.02% bromophenol blue at 95 ◦ C for 3 min. SDSPAGE was performed using 5% stacking and 8% separating gel with an initial current of 50 V for 1 h and then 150 V for 1 h [38]. Western blotting was carried out following the procedure as previously described [39] except the details of the antibodies used in this work are as follows. Membrane blotted with primary antibody anti D1 (Anti-Dopamine Receptor D1 antibody, ab78021, abcam) 1:1000 dilution and anti GluN1 (Anti-NMDAR1 antibody ab134308, abcam) 1:2000 dilution and the secondary antibody used was HRP conjugated Goat Anti-Mouse IgG H&L (ab97040, abcam) 1:5000 dilution. 2.4.5. In-gel digestion of proteins Protein bands from silver-stained SDS-PAGE gels that were identified by the corresponding antibodies against the D1 and GluN1 subunits were put into a 1.5 mL tube. Gel pieces were washed with 50 mM ammonium bicarbonate and then two times with washing buffer (50% 100 mM ammonium bicarbonate/50% acetonitrile) for 30 min each with vortexing. 100 ␮L of 100% acetonitrile was added to the tube and the mixture was incubated for 10 min. Gel pieces were dried completely using a SpeedVac concentrator. Reduction of cysteine residues was carried out with a 10 mM dithiothreitol (DTT) solution in 100 mM ammonium bicarbonate pH 8.6 for 60 min at 56 ◦ C. After discarding the DTT solution, the same volume of a 55 mM iodoacetamide (IAA) solution in 100 mM ammonium bicarbonate buffer pH 8.6 was added and incubated in darkness for 45 min at 25 ◦ C to alkylate the cysteine residues. The IAA solution was replaced by washing buffer (50% 100 mM ammonium bicarbonate/50% acetonitrile) and washed twice for 15 min each with vortexing. Gel pieces were washed and dried in 100% acetonitrile followed by dryness in SpeedVac. Dried gel pieces were re-swollen with 12.5 ng/␮L trypsin (Promega, Germany) solution reconstituted with 25 mM ammonium bicarbonate. Gel pieces were

0.0003 0.0002 0.4189 0.2783 0.2136 0.8219 0.2926 0.0949 0.0241 0.0001 – – 0.3110 0.0009 – 0.0510 0.0090 0.4532 0.6195 0.6932 0.5888 0.3714 0.9940

mean ± SD

p value

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Untrained

1.018 ± 0.239 1.727 ± 0.299 – 1.427 ± 0.592 2.946 ± 1.663 3.962 ± 1.569 – 4.369 ± 1.508 – 0.736 ± 0.220 1.141 ± 0.236 1.237 ± 0.925 0.930 ± 0.240 0.893 ± 0.272 1.125 ± 0.330 2.989 ± 0.687 – – 1.460 ± 0.595 0.530 ± 0.274 – 0.407 ± 0.201 0.804 ± 0.376

0.887 ± 0.337 1.569 ± 0.556 – 1.424 ± 0.524 1.968 ± 0.851 4.800 ± 1.168 – 2.839 ± 0.695 – 0.647 ± 0.244 1.173 ± 0.363 0.751 ± 0.303 0.615 ± 0.143 0.503 ± 0.130 0.554 ± 0.182 1.893 ± 0.649 – – 1.323 ± 0.323 0.776 ± 0.413 – 0.606 ± 0.169 0.952 ± 0.291

0.2874 0.3681 – 0.9886 0.1008 0.1532 – 0.0059 – 0.3585 0.8152 0.1066 0.0010 0.0004 0.0001 0.0006 – – 0.4934 0.1018 – 0.0161 0.2956

incubated for 16 h at 37 ◦ C. The supernatant was transferred to new 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. This step was repeated two times. Samples in extraction buffer were pooled in 0.5 mL tubes and evaporated in a SpeedVac concentrator. The peptides were reconstituted in 5% formic acid and analysed by LC-MS/MS [35]. 2.4.6. LC-MS/MS analysis Nano-LC-ESI-MS/MS was performed on a linear trap quadrupole (LTQ) Orbitrap Velos (Thermoscientific, Walthan, MA, USA) coupled to an Agilent 1200 HPLC nanoflow system comprised of a dual pump with one precolumn and one analytical column (Agilent Biotechnologies, Palo Alto, CA, USA) (Bennett et al., 2011). Data were acquired using Xcalibur version 2.1.0. HPLC solvents were as follows: solvent A consisted of 0.4% formic acid in water and solvent B consisted of 0.4% formic acid in 70% methanol and 20% isopropanol. From a thermostated microautosampler, 8 ␮L of the peptide mixture were automatically loaded onto a trap column (Zorbax 300SB-C18 5 ␮m, 5 × 0.3 mm, Agilent Biotechnologies) with a binary pump at a flow rate of 45 ␮L/min using 0.1% TFA for loading and washing the precolumn. After washing, the peptides were eluted by back-flushing onto a 16 cm fused silica analytical column with an inner diameter of 50 ␮m packed with a C18 reversed phase material (ReproSil-Pur 120 C18-AQ, 3 ␮m, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides were eluted from the analytical column with a 27 min gradient ranging from 3 to 30% solvent B, followed by a 25 min gradient from 30 to 70% solvent B and, finally, a 7 min gradient from 70 to 100% solvent B at a constant flow rate of 100 nL/min. The analyses were performed in a data-dependent acquisition mode using a top 15 CID method. Dynamic exclusion for selected ions was 60s. A single lock mass at m/z 445.120024 was employed. Maximal ion accumulation time allowed in MS and MSn mode was 500 and 50ms, respectively. Automatic gain control was used to prevent overfilling of the ion trap and was set to 106 ions and 5000 ions for a full Fourier transform mass spectrometry scan and MSn , respectively. Peptides were detected in MS mode at a resolution of 60,000 (at m/z 400).


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training sessions (days) Fig. 1. a – Working memory error (WME), b – reference memory error (RME) and c – latency to finish a trial of RAM behaviour test. WMEs were significantly decreased on day-8, 9 and 10 compared to day-1 (*P < 0.001, F = 4.949). Slightly decreasing trend in RME curve was not statistically significant. Latency was significantly decreased from day-3 onwards compared to day-1 (*P < 0.0001, F = 13.23). Mean and SEM are shown in the graph. The data were analysed using repeated measurements ANOVA.

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Proteins were identified using Proteome Discoverer version 1.4. A spectral peptide peak list was extracted from the raw files and using both, Mascot and SequestHT, the peak list was matched against a rat protein database (9,595 protein sequence entries from UniProtKB/Swiss-Prot downloaded on March 2013). The MSMS ion search parameters were as follows: one missed cleavage site, mass tolerances of 10 ppm and 0.6 Da for the precursor and fragment ions. Dynamic modifications were: methionine oxidation, serine and threonine phosphorylation. Static modifications were cysteine carbamidomethylation. Matched peptides were filtered as follows: significant threshold below 0.05 for Mascot and 0.1 for SEQUEST, and a 1% false discovery rate cut-off after target decoy search. Filtered peptides from the two search engines were merged and protein identifications requiring at least two unique peptides were reported.

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3. Results

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3.1. Radial arm maze

3.3. BN PAGE western blot quantification of membrane receptor complexes

The total number of working memory errors (WME), reference memory errors (RME) and latency to finish a trial made by the animals with respect to the training sessions were shown in Fig. 1. Trained rats showed significant reduction of latency to finish a trial and WME over the course of training. Data were analysed using repeated measurements ANOVA. WME became statistically different from day-8 onwards (F = 4.949, p < 0.0001), on day-10 WME were about threefold lower than on day-1. There was a slightly decreasing trend for RME however, not statistically significant. Latency to finish a trial showed a decreasing trend and was

The RCs were quantified using BN PAGE western blotting. In FC 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 the trained group. In hippocampus RCs containing GluN1 (∼480 kDa), GluN2B (∼500 kDa), D1 (∼480 kDa), and nAChR-␣7 (∼700 & ∼480 kDa) were significantly increased in trained groups when compared to untrained rats. In hippocampi the phosphorylated, activated form of DAT (DAT-ph) (∼500 kDa) showed lower arbitrary units of optical density in the trained group as compared by

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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 immediate early gene Arc in PFC was expressed significantly higher in trained animals when compared to the untrained animals. In trained animals the bright pink spots representing arc expression in 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 Student’s t-test (p < 0.05). The sub-regions of PFC were identified using the reference [40]. In hippocampal regions there was no statistical difference in Arc expression between trained and untrained groups (data not shown).


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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 expressed arc protein. The image also shows the different regions of PFC. Abbreviations: ACd, dorsal anterior cingulate area; FR2, frontal cortex area 2; IL, infralimbic area; PL, prelimbic area. Scale-bar in the image represents 500 ␮m. Arc protein is significantly expressed more in trained group when compared to the untrained rats. Asterisks indicate level of significance of difference in unpaired Student’s t-test (*P < 0.05). Mean and SD are shown in the graph.

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unpaired Student’s t-test. No change in the electrophoretic mobility of the RCs between trained and untrained groups was observed. The mean densitometric values of BN PAGE western blot after normalization and statistical t-test p-values of the individual RCs are listed in the Table 1 (Figs. 3 and 4). 3.4. Proximity ligation assay of D1 and GluN1 receptors Fig. 5 shows the PLA data of D1 and GluN1 receptors in different regions of rat PFC were presented with red dots indicating co-localization and proximity of D1 and GluN1 receptors in ACd, FR2, IL and PL regions which in turn proposes that D1 and GluN1 receptor subunits may be complex components of the same RCs. 3.5. Co-immunoprecipitation of D1 and GluN1 receptor followed by mass spectrometrical identification of receptor complexes Co-immunoprecipitation was carried out with an immobilized antibody against D1 as well as an immobilized antibody against GluN1. Western blot analysis shown in Fig. 6 confirmed the presence of both, D1 and GluN1 receptors in both elutions, i.e. the antibody against D1 detected GluN1 and vice versa. Mass spectrometrical analysis (MS) shows that the immunoprecipitate using an antibody against D1 contained the GluN1 subunit. Total sequence coverage indicated in Table 2 indicates unambiguous MS identification of these receptor subunits. The individual peptides obtained from proteolytic cleavage followed by MS are listed in supplementary Table 2. Representative spectra are provided in supplementary Fig. 1. This finding suggests that the D1 and GluN1 receptors can exist in the same complex.

4. Discussion Herein, following RAM training in rats, the neuronal activity in PFC and hippocampus were identified using Arc expression analysis and levels of major neurotransmitter RCs in these regions were compared between trained and untrained animals. Behavioural results reveal that the animals had learned the task showing gradually decreasing latency to finish a session statistically significant from day-3 onwards compared to the first day. Appetite is the motivation for the foraging behaviour of mildly starving rats in RAM. Animals identify the spatial location of the arm containing food pellets using available distal cues [41]. In order to retrieve the food efficiently with minimal efforts when a baited arm is visited for food, the rat has to avoid re-entry. This learning strategy involves working memory and the gradual decrease of WME over the training days signifies that the learning strategies are consolidated to long term memory after each training day [10]. Thus, since the proteomic analysis has been done at the end of training, changes in levels of neurotransmitter RCs may be associated with the persistent chemical changes of the neuronal synapses as a result of RAM training. Activity regulated cytoskeleton associated protein (Arc) is one of the Immediate Early Genes (IEG), expressed in the brain regions when there is neuronal activation [42–44]. Therefore, IEG expression has been widely used as a marker to identify brain regions that are activated in response to learning [45–47]. Own results show that Arc is expressed significantly higher in the trained group in areas of PFC, ACd, FR2, IL and PL, suggesting that all of these areas are activated following RAM training, whereas the hippocampal regions do not show any difference in Arc expression between trained and untrained groups. The PFC is known for its involvement in guiding the animal for goal-directed behaviour and the


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Fig. 3. BN PAGE western quantification of receptor complexes in FC tissue. GluA1, GluA2, GluN1, GluN2A, D1, and nAChR-␣7 were significantly increased in trained groups when compared to untrained rats. Asterisks indicate level of significance of difference in unpaired Student’s t-test (*P < 0.05, **P < 0.01, #P < 0.005). The bar graph shows the mean and SD error bars.

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hippocampus is critical for spatial navigation and spatial memory processing. These two structures interact through complex circuits based on the functional demand and their coordination is necessary for optimal performance [48–50]. From the current study it is understood that both, hippocampus and PFC are required in the RAM paradigm. Inhibition of the activity-dependent expression of IEGs causes impairment of short term memory consolidation [51] and therefore plays a critical role in the formation of long term memory [52,53]. This implies that Arc is more likely expressed in brain regions that are involved in memory consolidation. From this perspective, elevated levels of Arc expression in trained animals in PFC signifies that PFC neurons are subjected to memory consolidation after the last session, because the learning strategies are consolidated as the latency to complete the task decreases with training. Based upon Arc expression results herein, it may be suggested that the memory consolidation process in RAM training may involve only PFC and not hippocampus. It is known, however, that the hippocampus is essential for acquisition, consolidation and retention of spatial and non-spatial memories [50,54,55]. Own contrasting result in the hippocampus may propose that after repeated training in the RAM, hippocampal neurons do not undergo any

memory consolidation and therefore do not express Arc but probably the hippocampus has been involved in the beginning of training, although the statement has to be reconsidered as significant RCs level changes were observed. Previous reports have been already addressing the involvement of neurotransmitter RCs rather than individual receptor subunits in spatial memory formation [37,56–58] but work on the RAM has not been published so far to the best of our knowledge. Determination of RCs is relevant as receptor function is carried out as homo-or heteromeric assemblies of receptors. Glutamate, the major excitatory neurotransmitter exerts its action by binding to glutamate receptors. Ionotropic glutamate receptors are made up of different types of subunits and based on the subunit composition the biochemical and electrophysiological properties of the RCs vary [59]. Specific AMPA receptor subunits are delivered to synaptic sites following learning and contributes to experience-dependent synaptic strengthening [60,61]. AMPA receptors play a vital role in WM mechanisms [62]: GluA1 subunit deletion in the whole brain results in strong spatial WM deficits [63]. The molecular mechanism of AMPA receptor subunit function in spatial WM is not clearly understood but it has been


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Fig. 4. BN PAGE western quantification of receptor complexes in hippocampus. Complex containing GluN1, GluN2B, D1, and nAChR-␣7 levels were significantly increased in trained groups when compared to untrained rats whereas the DAT-ph levels were decreased in trained group. Asterisks indicate level of significance of difference in unpaired Student’s t-test (*P < 0.05, **P < 0.01, #P < 0.005). The bar graph shows the mean and SD error bars.

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postulated that it includes the activation of AMPA receptors containing the GluA1 subunit [63] and consequently during plasticity the GluA1/GluA2 containing RCs are added to synapses and during long term memory formation GluA2/GluA3-containing AMPA receptors constitutively replace synaptic AMPA receptors while keeping the synaptic strength constant [64,65]. There is evidence that GluA1-mediated AMPA receptor signalling is essential for spatial memory tasks, particularly in hippocampus [61,66]. It is intriguing that no differences between hippocampal AMPA receptor containing RCs levels were observed and this finding is of importance to elucidate different memory mechanisms in hippocampus. These data emphasize the importance of NMDA receptors in acquisition of spatial memory processes. Since on the last day the rats still undergo training, NMDA receptor subunits may be modulated after the last session. Own results show that in FC GluN2A and GluN1 containing complexes were increased whereas in hippocampus

NR2B and GluN1 containing complexes were elevated in trained animals, pointing to different mechanism in hippocampus and FC in NMDA-mediated signalling in performance in the RAM. Pharmacological manipulation studies in an experimental animal model suggest that the NMDA-receptor system may be important in the acquisition of memory, but not for the maintenance [16], hence the changes in NMDA receptor subunits in the current study could be due to the effect of the last training session.nAChR-␣7 containing RCs levels were significantly higher in the synaptic membrane fraction in trained groups. This result is in agreement with a recent behavioural study indicating that acetylcholine alpha 7-containing RCs are modulated during memory retrieval in mice tested in the multiple-T-maze [67]. Depletion of acetylcholine in dorsolateral PFC markedly impaired WM [17] whereas selective stimulation of the neuronal nAChR-␣7 receptor improved spatial WM in nonhuman primates [68] and in rodents [69].


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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 with DAPI 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 2 Data of mass spectrometrical identification of GluN1 co-immunoprecipitated with D1. Uniprot accession number

Enzyme digestion

Gene name

coverage

# Unique Peptides

# Peptides

# PSMs

G3V933 D(1A) dopamine receptor P35439-5 Glutamate receptor ionotropic, NMDA 1

Trypsin

Drd1

23.99

8

8

197

Trypsin

Grin1

5.76

5

5

5

Fig. 6. Western blot showing the detection of D1 and GluN1 receptor subunits immunoprecipitated with Anti-D1 and Anti-GluN1 antibody. This shows the D1 and GluN1 remains in a complex and are co-immuno precipitated. Cntrl is the total crude synaptosome membrane fraction protein prepared from rat’s FC tissue.

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The involvement of the dopaminergic system in memory is well-studied in hippocampus. The dopamine receptor subtype D1 stimulates the onset of protein synthesis-dependent late phase of LTP in the hippocampus [70] and facilitates the persistent storage of hippocampus-dependent memories [71]. Apart from its role in long-term memory formation, the dopaminergic system is also involved in WM processes. The D1 subtype within the PFC has been implicated in normal functioning of WM and its alterations can cause WM impairments like schizotypal personality disorder [72] and schizophrenia [73]. D1 receptor blockade using antagonists showed dose- and delay-dependent impairments in WM whereas D2 receptor blockade had no effect [74]. In the current study it is revealed that levels of a D1 containing complex in the trained group are higher and D2 containing RCs levels were unchanged in both, frontal cortex and hippocampus. Complexes containing D1 and GluN1 were co-migrating at around 300 kDa on BN-PAGE which indicates, that these receptors could exist in the same complex. It is well known that D1 and NMDA receptors have profound importance in cognition (like memory and executive functions) and in synaptic plasticity and there is evidence that D1 and glutamate receptors can co-exist in brain regions. Sarantis et al. examined the effects of the in-vitro stimulation of D1 and D2 receptors on the phosphorylation state of NMDA and AMPA receptor subunits in rat PFC and hippocampus showing that D1 receptor activation elicits a significant increase of the phosphorylation state of NMDA (GluN1, GluN2B) and AMPA (GluA1) receptor subunits [75]. In another study it was shown that D1 and NR1 receptors interact through protein-protein interactions in single pyramidal neurons and inter-neurons in the adult rat PFC [76]. D1 and NMDA (GluN1, GluN2B) receptors form a complex through the carboxyl tails and can be co-immunoprecipitated [77]. In electrophysiological studies it was demonstrated that D1 modulates NMDA currents in hippocampal neuron cultures [77]. Using PLA herein it was observed that all regions in PFC showed co-localisation of D1 and GluN1 receptor subunits. Since the PLA shows proximity and does not confirm physical interactions, the complex formation between D1 and GluN1 was further examined using co-immunoprecipitation experiments that revealed that D1 co-eluted with an antibody against GluN1 and vice versa, moreover,

mass spectrometrical analysis of the immunoprecipitate using an antibody against D1 identified the presence of GluN1. Nai and co-workers explored the effects of the D1–GluN1 interaction on NMDA receptor-dependent LTP and WM reporting that uncoupling the D1–GluN1 interaction led to impaired WM and decreased GluN1–CaMKII association and CaMKII activity in rat hippocampus [78]. The DAT is a presynaptic membrane-spanning protein known to mediate dopamine re-uptake. Reuptake inhibition has been already used for cognitive enhancement in the treatment of ADHD. Schmeichel et al. observed improved performance in rats using a PFC-dependent delayed-alternation task of spatial working memory by the administration of a well-characterized benztropine analog, AHN 2-005 [23]. However, not much is known about its involvement in long term memory formation. Own results show that levels of DAT containing complexes were comparable between trained and untrained animals in frontal cortex and hippocampus whereas levels of complexes containing the phosphorylated active form of DAT (phosphorylated at amino acid position Thr53 ) were lower in trained animals in hippocampus but not in FC. The presence and modulation of a high molecular weight complex containing the activated form of DAT in spatial memory performance in the current work points to the probable involvement of a large transporter complex that remains to be further characterised by subsequent studies. Taken together, RCs changes paralleling spatial memory with reference and working memory components in the PFC as well as in hippocampus at the end of the RAM testing were revealed, confirming the participation of both regions in spatial and working memory mechanisms. Moreover, a complex was shown to contain D1 and GluN1 and a complex containing phosphorylated, i.e. activated DAT in FC was decreased in rats trained in the RAM probably indicating a role for dopamine reuptake in memory formation. This work may be relevant for the interpretation of previous work on signaling and design of future studies on neurotransmitter receptors and dopamine transport in spatial memory. Acknowledgements The skilful technical assistance of Sabine Rauscher from the imaging core facility is highly appreciated. We acknowledge comments from Prof. Dr. Volker Korz, Univ. Vienna. We appreciate the financial assistance from the Medical University of Vienna to carry out this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2015.04.043 References [1] Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984;11:47–60. [2] David S, Olton, Robert J, Samuelson. Remembrance of places passed: spatial memory in rats. J Exp Psychol Anim Behav Process 1976;2:97–116. [3] Levin ED. Psychopharmacological effects in the radial-arm maze. Neurosci Biobehav Rev 1988;12:169–75.


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[4] Butelman ER. The effect of NMDA antagonists in the radial arm maze task with an interposed delay. Pharmacol Biochem Behav 1990;35:533–6. [5] Buresová O, Bures J. Radial maze as a tool for assessing the effect of drugs on the working memory of rats. Psychopharmacology (Berl) 1982;77:268–71. [6] Levin ED, Timofeeva OA, Yang L, Petro A, Ryde IT, Wrench N, et al. Early postnatal parathion exposure in rats causes sex-selective cognitive impairment and neurotransmitter defects which emerge in aging. Behav Brain Res 2010;208:319–27, http://dx.doi.org/10.1016/j.bbr.2009.11.007. [7] Spritzer MD, Daviau ED, Coneeny MK, Engelman SM, Prince WT, Rodriguez-Wisdom KN. Effects of testosterone on spatial learning and memory in adult male rats. Horm Behav 2011;59:484–96, http://dx.doi.org/10.1016/j.yhbeh.2011.01.009. [8] McNamara DS, Scott JL. Working memory capacity and strategy use. Mem Cognit 2001;29:10–7. [9] Mizumori SJ, Rosenzweig MR, Bennett EL. Long-term working memory in the rat: effects of hippocampally applied anisomycin. Behav Neurosci 1985;99:220–32. [10] Mizumori SJ, Channon V, Rosenzweig MR, Bennett EL. Short- and long-term components of working memory in the rat. Behav Neurosci 1987;101:782–9. [11] Zamanillo D, Sprengel R, Hvalby O, Jensen V, Burnashev N, Rozov A, et al. Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning. Science 1999;284:1805–11. [12] Reisel D, Bannerman DM, Schmitt WB, Deacon RMJ, Flint J, Borchardt T, et al. Spatial memory dissociations in mice lacking GluR1. Nat Neurosci 2002;5:868–73, http://dx.doi.org/10.1038/nn910. [13] Sanderson DJ, Good MA, Seeburg PH, Sprengel R, Rawlins JNP, Bannerman DM. The role of the GluR-A (GluR1) AMPA receptor subunit in learning and memory. Prog Brain Res 2008;169:159–78, http://dx.doi.org/10.1016/S0079-6123(07)00009-X. [14] Yoshihara T, Ichitani Y. Hippocampal N-methyl-d-aspartatereceptor-mediated encoding and retrieval processes in spatial working memory: Delayinterposed radial maze performance in rats. Neuroscience 2004;129:1–10, http://dx.doi.org/10.1016/j.neuroscience.2004.07.030. [15] Izquierdo I. Pharmacological evidence for a role of long-term potentiation in memory. FASEB J Off Publ Fed Am Soc Exp Biol 1994;8:1139–45. [16] Izquierdo I. Role of NMDA receptors in memory. Trends Pharmacol Sci 1991;12:128–9. [17] Croxson PL, Kyriazis DA, Baxter MG. Cholinergic modulation of a specific memory function of prefrontal cortex. Nat Neurosci 2011;14:1510–2, http://dx.doi.org/10.1038/nn.2971. [18] Hidaka N, Suemaru K, Kato Y, Araki H. Involvement of ␣4␤2 nicotinic acetylcholine receptors in working memory impairment induced by repeated electroconvulsive seizures in rats. Epilepsy Res 2013;104:181–5, http://dx.doi.org/10.1016/j.eplepsyres.2012.09.017. [19] Yang Y, Paspalas CD, Jin LE, Picciotto MR, Arnsten AFT, Wang M. Nicotinic ␣7 receptors enhance NMDA cognitive circuits in dorsolateral prefrontal cortex. Proc Natl Acad Sci USA 2013;110:12078–83, http://dx.doi.org/10.1073/pnas.1307849110. [20] Deiana S, Platt B, Riedel G. The cholinergic system and spatial learning. Behav Brain Res 2011;221:389–411, http://dx.doi.org/10.1016/j.bbr.2010.11.036. [21] Xing B, Guo J, Meng X, Wei S, Li S. The dopamine D1 but not D3 receptor plays a fundamental role in spatial working memory and BDNF expression in prefrontal cortex of mice. Behav Brain Res 2012;235:36–41, http://dx.doi.org/10.1016/j.bbr.2012.06.035. [22] Wan P, Wang S, Zhang Y, Lv J, Jin QH. Involvement of dopamine D1 receptors of the hippocampal dentate gyrus in spatial learning and memory deficits in a rat model of vascular dementia. Pharmazie 2014;69:709–10. [23] Schmeichel BE, Zemlan FP, Berridge CW. A selective dopamine reuptake inhibitor improves prefrontal cortex-dependent cognitive function: potential relevance to attention deficit hyperactivity disorder. Neuropharmacology 2013;64:321–8, http://dx.doi.org/10.1016/j.neuropharm.2012.07.005. [24] Olton DS, Collison C, Werz MA. Spatial memory and radial arm maze performance of rats. Learn Motiv 1977;8:289–314, http://dx.doi.org/10.1016/ 0023-9690(77)90054-6. [25] Olton DS, Paras BC. Spatial memory and hippocampal function. Neuropsychologia 1979;17:669–82, http://dx.doi.org/10.1016/0028-3932(79)90042-3. [26] Jarrard LE. On the role of the hippocampus in learning and memory in the rat. Behav Neural Biol 1993;60:9–26. [27] Seamans JK, Floresco SB, Phillips AG. Functional differences between the prelimbic and anterior cingulate regions of the rat prefrontal cortex. Behav Neurosci 1995;109:1063–73. [28] Kesner RP. Retrospective and prospective coding of information: role of the medial prefrontal cortex. Exp Brain Res 1989;74:163–7. [29] Dunnett SB. Role of prefrontal cortex and striatal output systems in shortterm memory deficits associated with ageing, basal forebrain lesions, and cholinergic-rich grafts. Can J Psychol 1990;44:210–32. [30] Blumenfeld RS, Ranganath C. Prefrontal cortex and long-term memory encoding: an integrative review of findings from neuropsychology and neuroimaging. Neuroscientist 2007;13:280–91, http://dx.doi.org/10.1177/ 1073858407299290. [31] Ramírez-Amaya V, Balderas I, Sandoval J, Escobar ML, Bermúdez-Rattoni F. Spatial long-term memory is related to mossy fiber synaptogenesis. J Neurosci Off J Soc Neurosci 2001;21:7340–8.

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[32] Trifilieff P, Rives M-L, Urizar E, Piskorowski RA, Vishwasrao HD, Castrillon J, et al. Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum. BioTechniques 2011;51:111–8, http://dx.doi.org/10.2144/000113719. [33] Balasuriya D, Stewart AP, Edwardson JM. The ␴-1 receptor interacts directly with GluN1 but not GluN2A in the GluN1/GluN2A NMDA receptor. J Neurosci 2013;33:18219–24, http://dx.doi.org/10.1523/JNEUROSCI. 3360-13.2013. [34] Sabine S. Dissection of rodent brain regions. In: Neuroproteomics. Humana Press; 2011. p. 13–26. [35] Kang S-U, Fuchs K, Sieghart W, Pollak A, Csaszar E, Lubec G. Gel-based mass spectrometric analysis of a strongly hydrophobic GABAA-receptor subunit containing four transmembrane domains. Nat Protoc 2009;4:1093–102, http://dx.doi.org/10.1038/nprot.2009.92. [36] Welinder C, Ekblad L. Coomassie staining as loading control in Western blot analysis. J Proteome Res 2011;10:1416–9, http://dx.doi.org/10.1021/ pr1011476. [37] Sase S, Khan D, Sialana F, Höger H, Russo-Schlaff N, Lubec G. Modafinil improves performance in the multiple T-Maze and modifies GluR1, GluR2, D2 and NR1 receptor complex levels in the C57BL/6J mouse. Amino Acids 2012;43:2285–92, http://dx.doi.org/10.1007/s00726-012-1306-y. [38] Falsafi SK, Deli A, Höger H, Pollak A, Lubec G. Scopolamine administration modulates muscarinic, nicotinic and NMDA receptor systems. PloS One 2012;7:e32082, http://dx.doi.org/10.1371/journal.pone.0032082. [39] Ganesan MK, Jovanovic M, Secerov B, Ignjatovic M, Bilban M, Andjus P, et al. Radiation protection from whole-body gamma irradiation (6.7 Gy): behavioural effects and brain protein-level changes by an aminothiol compound GL2011 in the Wistar rat. Amino Acids 2014;46:1681–96, http://dx.doi.org/10.1007/s00726-014-1728-9. [40] Heidbreder CA, Groenewegen HJ. The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based upon functional and anatomical characteristics. Neurosci Biobehav Rev 2003;27:555–79, http://dx.doi.org/10. 1016/j.neubiorev.2003.09.003. [41] Mazmanian DS, Roberts WA. Spatial memory in rats under restricted viewing conditions. Learn Motiv 1983;14:123–39, http://dx.doi.org/10.1016/ 0023-9690(83)90001-2. [42] Abraham WC, Mason SE, Demmer J, Williams JM, Richardson CL, Tate WP, et al. Correlations between immediate early gene induction and the persistence of long-term potentiation. Neuroscience 1993;56:717–27. [43] Guzowski JF, McNaughton BL, Barnes CA, Worley PF. Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles. Nat Neurosci 1999;2:1120–4, http://dx.doi.org/10.1038/16046. [44] Montag-Sallaz M, Welzl H, Kuhl D, Montag D, Schachner M. Novelty-induced increased expression of immediate-early genes c-fos and arg 3.1 in the mouse brain. J Neurobiol 1999;38:234–46. [45] Zhang Y, Fukushima H, Kida S. Induction and requirement of gene expression in the anterior cingulate cortex and medial prefrontal cortex for the consolidation of inhibitory avoidance memory. Mol Brain 2011;4:4, http://dx.doi.org/10.1186/1756-6606-4-4. [46] Link W, Konietzko U, Kauselmann G, Krug M, Schwanke B, Frey U, et al. Somatodendritic expression of an immediate early gene is regulated by synaptic activity. Proc Natl Acad Sci USA 1995;92:5734–8. [47] Lyford GL, Yamagata K, Kaufmann WE, Barnes CA, Sanders LK, Copeland NG, et al. Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites. Neuron 1995;14:433–45. [48] Hasselmo ME. A model of prefrontal cortical mechanisms for goal-directed behavior. J Cogn Neurosci 2005;17:1115–29, http://dx.doi.org/10.1162/ 0898929054475190. [49] Miller EK, Cohen JD. An integrative theory of prefrontal cortex function. Annu Rev Neurosci 2001;24:167–202, http://dx.doi.org/10.1146/annurev. neuro.24.1.167. [50] Vertes RP. Interactions among the medial prefrontal cortex, hippocampus and midline thalamus in emotional and cognitive processing in the rat. Neuroscience 2006;142:1–20, http://dx.doi.org/10.1016/j.neuroscience. 2006.06.027. [51] Guzowski JF, Lyford GL, Stevenson GD, Houston FP, McGaugh JL, Worley PF, et al. Inhibition of activity-dependent arc protein expression in the rat hippocampus impairs the maintenance of long-term potentiation and the consolidation of long-term memory. J Neurosci Off J Soc Neurosci 2000;20: 3993–4001. [52] Robertson HA. Immediate-early genes, neuronal plasticity, and memory. Biochem Cell Biol Biochim Biol Cell 1992;70:729–37. [53] Kaczmarek L. Molecular biology of vertebrate learning: is c-fos a new beginning? J Neurosci Res 1993;34:377–81, http://dx.doi.org/10.1002/ jnr.490340402. [54] Vnek N, Rothblat LA. The hippocampus and long-term object memory in the rat. J Neurosci Off J Soc Neurosci 1996;16:2780–7. [55] Jarrard LE. Selective hippocampal lesions and behavior: effects of kainic acid lesions on performance of place and cue tasks. Behav Neurosci 1983;97:873–89. [56] Keihan Falsafi S, Ghafari M, Pollak A, Höger H, Lubec G. Hippocampal AMPAtype receptor complexes containing GluR3 and GluR4 are paralleling training


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in the Multiple T-Maze. Neurochem Int 2012;60:425–30, http://dx.doi.org/ 10.1016/j.neuint.2012.01.005. Ghafari M, Falsafi SK, Hoeger H, Lubec G. Hippocampal levels of GluR1 and GluR2 complexes are modulated by training in the multiple T-Maze in C57BL/6J mice. Brain Struct Funct 2012;217:353–62, http://dx.doi.org/ 10.1007/s00429-011-0335-8. Saroja SR, Kim E-J, Shanmugasundaram B, Höger H, Lubec G. Hippocampal monoamine receptor complex levels linked to spatial memory decline in the aging C57BL/6J. Behav Brain Res 2014;264:1–8, http://dx.doi.org/10.1016/j.bbr.2014.01.042. Collingridge GL, Olsen RW, Peters J, Spedding M. A nomenclature for ligandgated ion channels. Neuropharmacology 2009;56:2–5, http://dx.doi.org/ 10.1016/j.neuropharm.2008.06.063. Mitsushima D, Ishihara K, Sano A, Kessels HW, Takahashi T. Contextual learning requires synaptic AMPA receptor delivery in the hippocampus. Proc Natl Acad Sci USA 2011;108:12503–8, http://dx.doi.org/10.1073/ pnas.1104558108. Rumpel S, LeDoux J, Zador A, Malinow R. Postsynaptic receptor trafficking underlying a form of associative learning. Science 2005;308:83–8, http://dx.doi.org/10.1126/science.1103944. Bannerman DM. Fractionating spatial memory with glutamate receptor subunit-knockout mice. Biochem Soc Trans 2009;37:1323–7, http://dx.doi.org/10.1042/BST0371323. Freudenberg F, Marx V, Seeburg PH, Sprengel R, Celikel T. Circuit mechanisms of GluA1-dependent spatial working memory. Hippocampus 2013;23:1359–66, http://dx.doi.org/10.1002/hipo.22184. Joels G, Lamprecht R. Interaction between N-ethylmaleimide-sensitive factor and GluR2 is essential for fear memory formation in lateral amygdala. J Neurosci 2010;30:15981–6, http://dx.doi.org/10.1523/JNEUROSCI. 1872-10.2010. Shi S, Hayashi Y, Esteban JA, Malinow R. Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 2001;105:331–43. Malinow R, Malenka RC. AMPA receptor trafficking and synaptic plasticity. Annu Rev Neurosci 2002;25:103–26, http://dx.doi.org/10.1146/annurev. neuro.25.112701.142758. Subramaniyan S, Heo S, Patil S, Li L, Hoger H, Pollak A, et al. A hippocampal nicotinic acetylcholine alpha 7-containing receptor complex is linked to memory retrieval in the multiple-T-maze in C57BL/6j mice. Behav Brain Res 2014;270:137–45, http://dx.doi.org/10.1016/j.bbr.2014.05.012.

[68] Castner SA, Smagin GN, Piser TM, Wang Y, Smith JS, Christian EP, et al. Immediate and sustained improvements in working memory after selective stimulation of ␣7 nicotinic acetylcholine receptors. Biol Psychiatry 2011;69:12–8, http://dx.doi.org/10.1016/j.biopsych.2010.08.006. [69] Boess FG, De Vry J, Erb C, Flessner T, Hendrix M, Luithle J, et al. The novel alpha7 nicotinic acetylcholine receptor agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide improves working and recognition memory in rodents. J Pharmacol Exp Ther 2007;321:716–25, http://dx.doi.org/10.1124/jpet.106.118976. [70] Huang Y-Y, Kandel ER. Age-related enhancement of a protein synthesisdependent late phase of LTP induced by low frequency paired-pulse stimulation in hippocampus. Learn Mem Cold Spring Harb N 2006;13:298–306, http://dx.doi.org/10.1101/lm.166906. [71] Rossato JI, Bevilaqua LRM, Izquierdo I, Medina JH, Cammarota M. Dopamine controls persistence of long-term memory storage. Science 2009;325:1017–20, http://dx.doi.org/10.1126/science.1172545. [72] Thompson JL, Rosell DR, Slifstein M, Girgis RR, Xu X, Ehrlich Y, et al. Prefrontal dopamine D1 receptors and working memory in schizotypal personality disorder: a PET study with [(11)C]NNC112. Psychopharmacology (Berl) 2014, http://dx.doi.org/10.1007/s00213-014-3566-6.

835

[73] Kebir O, Tabbane K. Working memory in schizophrenia: a review. L’Encéphale 2008;34:289–98, http://dx.doi.org/10.1016/j.encep.2006.12.008. [74] Aultman JM, Moghaddam B. Distinct contributions of glutamate and dopamine receptors to temporal aspects of rodent working memory using a clinically relevant task. Psychopharmacology (Berl) 2001;153:353–64. [75] Sarantis K, Matsokis N, Angelatou F. Synergistic interactions of dopamine D1 and glutamate NMDA receptors in rat hippocampus and prefrontal cortex: Involvement of ERK1/2 signaling. Neuroscience 2009;163:1135–45, http://dx.doi.org/10.1016/j.neuroscience.2009.07.056.

855

[76] Kruse MS, Prémont J, Krebs M-O, Jay TM. Interaction of dopamine D1 with NMDA NR1 receptors in rat prefrontal cortex. Eur Neuropsychopharmacol 2009;19:296–304, http://dx.doi.org/10.1016/j.euroneuro.2008.12.006.

864

[77] Lee FJS, Xue S, Pei L, Vukusic B, Chéry N, Wang Y, et al. Dual regulation of NMDA receptor functions by direct protein-protein interactions with the dopamine D1 receptor. Cell 2002;111:219–30, http://dx.doi.org/10.1016/S0092-8674(02)00962-5. [78] Nai Q, Li S, Wang S-H, Liu J, Lee FJS, Frankland PW, et al. Uncoupling the D1N-methyl-D-aspartate (NMDA) receptor complex promotes NMDA-dependent long-term potentiation and working memory. Biol Psychiatry 2010;67:246–54, http://dx.doi.org/10.1016/j.biopsych.2009.08.011.

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Nigella sativa Oil Enhances the Spatial Working Memory Performance of Rats on a Radial Arm Maze.

Spatial memory impairments in amnestic mild cognitive impairment in a virtual radial arm maze.

Post-training Inactivation of the Anterior Thalamic Nuclei Impairs Spatial Performance on the Radial Arm Maze.

Effects of intraventricular infusion of the N-methyl-D-aspartate (NMDA) receptor antagonist AP5 on spatial memory of rats in a radial arm maze.

The effects of prenatal nicotine on radial-arm maze performance in rats.

Chronic nicotine and withdrawal effects on radial-arm maze performance in rats.

7-NI and ODQ Disturbs Memory in the Elevated Plus Maze, Morris Water Maze, and Radial Arm Maze Tests in Mice.

Integrating Spatial Working Memory and Remote Memory: Interactions between the Medial Prefrontal Cortex and Hippocampus.

Interactive effects of D1 and D2 agonists with scopolamine on radial-arm maze performance.

Long-term effects of developmental halothane exposure on radial arm maze performance in rats.

Radial maze performance following hippocampal kindling.

Septal serotonin depletion in rats facilitates working memory in the radial arm maze and increases hippocampal high-frequency theta activity.

Effects of rolipram and zaprinast on learning and memory in the Morris water maze and radial arm maze tests in naive mice.

Delayed-non-match-to-sample performance in the radial arm maze: effects of dopaminergic and gabaergic agents.

A Virtual Radial Arm Maze for the Study of Multiple Memory Systems in a Functional Magnetic Resonance Imaging Environment.

Western Diet Chow Consumption in Rats Induces Striatal Neuronal Activation While Reducing Dopamine Levels without Affecting Spatial Memory in the Radial Arm Maze.

Capacity of working memory in rats as determined by performance on a radial maze.

Hippocampal mossy fibers and radial-maze learning in the mouse: a correlation with spatial working memory but not with non-spatial reference memory.

The effects of chronic alcohol consumption or thiamin deficiency on radial-arm maze performance in the rat.

Nicotinic and muscarinic interactions and choice accuracy in the radial-arm maze.

Early postnatal hyperthyroidism improves both working and reference memory in a spatial radial-maze task in adult mice.

Dopaminergic drugs reverse the impairment of radial-arm maze performance caused by lesions involving the cholinergic medial pathway.

Learning about cognition risk with the radial-arm maze in the developmental neurotoxicology battery.

Functions of the primate hippocampus in spatial and nonspatial memory.