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Behavioural Brain Research 264 (2014) 1–8

Contents lists available at ScienceDirect

Behavioural Brain Research

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

esearch report

ippocampal monoamine receptor complex levels linked to spatialemory decline in the aging C57BL/6J

ivaprakasam R. Sarojaa, Eun-Jung Kima, Bharanidharan Shanmugasundarama,arald Högerb, Gert Lubeca,∗

Department of Paediatrics, Medical University of Vienna, Währinger Gürtel 18, 1090 Vienna, AustriaCore Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, Brauhausgasse 34,-2325 Himberg, Austria

i g h l i g h t s

Spatial memory deficits in aging are paralleled by monoamine receptor complex changes.In the Morris water maze performance was linked to dopamine receptor complex changes.Declined performance in Morris water maze involves serotonin receptor complexes.Monoamine receptor complex levels are linked to memory retrieval.

r t i c l e i n f o

rticle history:eceived 13 December 2013eceived in revised form 25 January 2014ccepted 28 January 2014vailable online 4 February 2014

eywords:

a b s t r a c t

Although a large series of reports on monoamine receptor (MAR) biochemistry and pharmacology inaging are available, work on MAR complexes rather than subunits is limited. It was the aim of the studyto determine MAR complexes in hippocampi of three different age groups (3–12 and 18 months) in themouse and to link MAR changes to spatial memory retrieval in the Morris water maze (MWM). MAR com-plexes were separated by blue native electrophoresis. Immunohistochemistry was performed in order toshow the pattern of dopamine receptors and its colocalizations. D1R, D2R and 5-HT7R containing receptor

-Hydroxytryptamine receptor 1A5-HT1AR)-Hydroxytryptamine receptor 7 (5-HT7R)opamine receptor D1 (D1R)opamine receptor D2 (D2R)ippocampusging

complex levels were decreasing with age while 5-HT1AR-containing complex levels were increasing withage. D1R, 5-HT7R and 5-HT1AR were significantly correlating with the time spent in the target quadrant,representing retrieval in the MWM. D1R and D2R immunoreactivity was decreasing in an area-dependentpattern and D1R and D2R were colocalized. Individual monoamine receptors are linked to spatial memoryretrieval and are modulated by age. The findings are relevant for interpretation of previous and designof future work on brain receptors, spatial memory and aging.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The hippocampal-dependent cognitive processes during nor-al aging are affected by subtle synaptic alterations rather than

euronal loss [1]. To maintain normal brain functions, monoamineeceptors (MARs) play a vital role but are most vulnerable in aging2]. Although a series of MAR changes have been shown to benvolved in aging of humans and rodents, information on MARsn aging in the mouse is limited and therefore systematic work on

ARs in the aging mouse is needed.Majority of monoamine receptors are G protein-coupled recep-

ors (GPCRs) with seven transmembrane-spanning domains and

∗ Corresponding author. Tel.: +43 1 404003215; fax: +43 1 404006065.E-mail address: gert.lubec@meduniwien.ac.at (G. Lubec).

166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2014.01.042

intracellular loops, capable of inducing intracellular signallingupon receptor activation. GPCRs commonly affected in aging aredopamine receptors (DRs), which can be sub-grouped into D1-likereceptors (D1R and D5R) and D2-like receptors (D2R, D3R and D4R).D1R are expressed on the post synaptic membrane and are cou-pled with G�s/olf to induce cAMP production by activating adenylylcyclase (AC). In contrast to D1R, D2R are expressed both, on pre- andpost-synaptic membranes, interact with G�i/o to suppress cAMPproduction by inhibiting AC. D1R and D2R are acting differentlyon cAMP production, affinity with dopamine and pharmacologicalproperties, but both play important roles in learning and memorywhile D3R, D4R and D5R have less influence on cognitive functions

[3]. So far studies have been focussing on working memory [4,5]and on human, primate or rodent brain and have shown that D1Rand D2R were declining with age [6,7]. There is, however, limitedinformation on spatial memory and D1R or D2R in the mouse and

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o data on D1R or D2R in the aging mouse are available. In D1Rnockout mice spatial learning deficits were described in a Morrisater maze [8], spatial working memory was deficient in mice lack-

ng D2R and D3R [9] and Xing and co-workers [10] demonstratedhat D1R but not the D3R is critical for spatial learning.

A multitude of publications show that 5-hydroxytryptamineeceptor 1A (5-HT1AR) is involved in several forms of learningnd memory mechanisms as mainly shown by 5-HT1A ago-ism/antagonism [11,12] and transgenic studies [13]. The role of-HT1A receptors for learning and memory has been reviewed bygren and co-workers [14]. Age-dependent changes of this recep-

or system have reported and including altered 5-HT1AR bindingites [15,16]. In humans, the number of available 5-HT1AR bind-ng sites declines with age [17]. Age-dependent changes of the-HT1AR were not observed at the transcriptional level [18]. The-hydroxytryptamine receptor 7 (5-HT7R) has been shown to play

significant role for learning and memory [19,20] including spa-ial memory information processing [21] and mice lacking 5-HT7Rshow specific impairments in contextual learning [22]. Duncan andranklin have shown that 5-HT7R transcription is reduced in theging brain of the hamster [23].

In contrast to previous studies the current study was focussingn monoamine receptor complexes which has not been addressedefore but may be of importance as it is the brain recep-ors that are executing key functions in the brain. And indeed,eceptor complexes different between age groups were observednd complement pharmacological and biochemical evidence foronoamine receptor changes with age.

. Materials and methods

.1. Animals

Male C57BL/6J mice, aged 3, 12 and 18 months were obtainedrom JANVIER SAS Laboratories (Le Genest Saint Isle, France) and

aintained in Makrolon cages, supplied with autoclaved wood-hips, standard rodent diet (Altromin, Germany) and water in theore Unit of Biomedical Research, Division of Laboratory Animalcience and Genetics, Medical University of Vienna. Room tem-erature was 22 ± 1 ◦C and relative humidity was 50% ± 10%. The

ight/dark rhythm was 14:10 h. Ventilation with 100% fresh airesulted in an air change rate of 15 times/h. The room was illumi-ated with artificial light at an intensity of about 200 lx in 2 m from 5.m. to 7 p.m. Behavioural tests were performed between 8 a.m. and

p.m. All procedures were carried out according to the guidelines ofhe European Communities Council Directive of 24 November 198686/609/EEC) and were approved by Federal Ministry of Education,cience and Culture, Austria.

.2. Morris water maze

Age-dependent spatial memory abilities were assessed in theWM, a behavioural test that has been used to study cognitive

unctions of rodents, with a standard hidden platform acquisitionrials and retrieval phase without hidden platform to access longerm memory [8]. Mice were trained in four daily acquisition ses-ions (four trials per day with 20 min inter-trial interval) to find

hidden platform in a circular pool (150 cm diameter, wall depth0 cm) filled with water (21 ± 1 ◦C). Mice that failed to locate thelatform within 120 s were manually placed on the platform andllowed to remain on it for 30 s. Memory retrieval was assessed on

ay five (60 s, platform removed). All experiments were recorded byomputerized tracking/image analyzer system (video camcorder:/3′ ′ SSAM HR EX VIEW HAD coupled to the computational trackingystem: TiBeSplit).

in Research 264 (2014) 1–8

2.3. Hippocampal protein preparation

All procedures were carried out at 4 ◦C. Hippocampi werehomogenized in ice-cold homogenization buffer [10 mM HEPES, pH7.5, 300 mM sucrose, one complete protease inhibitor tablet (RocheMolecular Biochemicals, Mannheim, Germany) per 50 mL] by Ultra-Turrax (IKA,Staufen, Germany). The homogenate was centrifugedfor 10 min at 1000 × g and the pellet was discarded. The supernatantwas centrifuged at 50,000 × g for 30 min in an ultracentrifuge (Beck-man Coulter Optima-L-90K). The resulting supernatant was saved(S1), the pellet was resuspended in washing buffer (homogeniza-tion buffer without sucrose), kept on ice for 30 min and centrifugedat 50,000 × g for 30 min to obtain a synaptosomal membrane frac-tion and the resulting supernatant was preserved (S2). Extractionbuffer [1.5 M 6-aminocaproic acid, 300 mM Bis–Tris, pH 7.0 and1% DDM was added to the membrane pellets, incubated for 1 hwith vortexing every 10 min. Following solubilization, sampleswere cleared by centrifugation at 20,000 × g for 60 min. The pro-tein content was estimated using the BCA protein assay kit (Pierce,Rockford, IL, USA). S1 and S2 fractions were pooled together andextracted protein concentrations were measured by the Bradfordmethod (Bio-Rad, Protein assay). Extracted proteins were thenaliquoted and stored at −80 ◦C till use [24].

2.4. Gel electrophoresis and western blotting

Monoamine receptor complexes were separated on 5–18%of BN-PAGE as described previously [24] while protein kinasesCs, PSD-95 (post synaptic density protein 95) and NSF (N-ethylmaleimide-sensitive factor) were separated on 9% ofSDS–PAGE. Separated proteins were transferred to PVDF mem-branes and blocked with 5% of non-fat dry milk in TBST for 1 h.Primary antibodies were added and incubated overnight at 4 ◦C.After membranes were washed for 6 times with TBST, correspond-ing secondary antibodies were added and incubated for 1 hr at21 ◦C (Antibody details are given in Supplementary table). Mem-branes were developed with the ECL solution (Clarity Western ECLSubstrate, Bio-Rad). Ten animals per group were used for quan-tification analysis. Immunoreactive bands were quantified by thesoftware Image J (NIH). Coomassie blue R-350 stained membraneswere used for normalization [25,26].

2.5. Immunoprecipitation (IP)

An antibody against D1R (Sc-14001, Santa Cruz) was used toidentify binding partners of D1R. 10 �g of antibody was usedper 500 �g membrane proteins. The procedures were carried outaccording to the manufacturer’ instructions (The Thermo ScientificPierce Direct IP Kit).

2.6. Immunofluorescence staining and confocal laser scanningmicroscopy

The expression levels of D1R, D2R and colocalization betweenthe D1R and D2R in hippocampus were analyzed on sectionsfrom perfusion-fixed brains processed for single and double-immunofluorescence staining. Five mice per each group wereperformed. In brief, mice were anesthetized and perfused transcar-dially first with saline and then with 4% paraformaldehyde in 0.1 Msodium phosphate buffer, pH 7.4. After perfusion, the brains weretaken and horizontal slices were cut into 100 �m thick sectionsusing a Vibratome 1000 Plus tissue sectioning system (Vibratome

Company, St. Louis, MO, USA). Immuno-histochemistry was per-formed on 2–3 sections of hippocampal regions from each animal.Tissue was processed free-floating as follows: Briefly, slices werepre-incubated in 0.2% Tween in 0.02 M Tris buffered saline for

S.R. Saroja et al. / Behavioural Brain Research 264 (2014) 1–8 3

Table 1One-way ANOVA analysis with post-hoc multiple comparison test (Bonferroni) of receptor complexes, signalling molecules of D1R, hippocampal sub-region and age-dependent expression of D1R and D2R and colocalization of D1R–D2R.

S. no. Contents Mean ± SD P value

3 (m) 12 (m) 18 (m) 3 (m) vs 12 (m) 3 (m) vs 18 (m) 12 (m) vs 18 (m)BN–PAGE from membrane fraction1 D1R complex 0.1533 ± 0.0303 0.08165 ± 0.0304 0.06066 ± 0.0303 <0.0001 <0.0001 0.40172 D2R complex 1.861 ± 0.6914 1.205 ± 0.2698 0.8255 ± 0.3954 0.0177 0.0002 0.25843 5-HT1A R complex

720 kDa 0.3303 ± 0.0649 0.4547 ± 0.0949 0.4929 ± 0.1605 0.0644 0.0108 >0.9999480 kDa 0.8374 ± 0.0699 0.9635 ± 0.1605 1.059 ± 0.0759 0.0490 0.0004 0.1897

4 5-HT7R complex 1.313 ± 0.3201 0.7445 ± 0.2199 0.3435 ± 0.085 <0.0001 <0.0001 0.0017SDS–PAGE from cytosolic fraction5 PKC � 0.09997 ± 0.0095 0.1088 ± 0.0129 0.08227 ± 0.0163 0.4488 0.0177 0.00046 PKC � 0.03756 ± 0.0126 0.02396 ± 0.004 0.03861 ± 0.0094 0.0330 >0.9999 0.00777 PKC � 0.05496 ± 0.0166 0.02856 ± 0.0042 0.02284 ± 0.0021 <0.0001 <0.0001 0.63478 PKC � 0.07903 ± 0.035 0.06592 ± 0.0294 0.07586 ± 0.0656 >0.9999 >0.9999 >0.99999 PSD-95

Membrane fraction 4.027 ± 0.9979 3.244 ± 0.8282 2.917 ± 0.7787 0.1649 0.0252 >0.9999Cytosolic fraction 0.03589 ± 0.0135 0.04938 ± 0.0101 0.05825 ± 0.0109 0.0658 0.0019 0.3918

10 NSF 0.1006 ± 0.0179 0.09424 ± 0.0094 0.07415 ± 0.009 0.8336 0.0003 0.0042Immunohistochemistry11 D1R expression

DG 59.50 ± 29.09 42.99 ± 21.49 0.0129 0.0008 0.5730CA1 73.87 ± 35.08 48.62 ± 22.28 0.2364 0.0063 0.2651CA2 69.38 ± 31.54 49.05 ± 21.29 0.0871 0.0034 0.3896CA3 53.17 ± 23.83 48.67 ± 27.72 0.0048 0.0023 >0.9999

12 D2R expressionDG 53.50 ± 13.26 40.31 ± 21.70 0.0009 <0.0001 0.5434CA1 61.86 ± 16.92 45.60 ± 26.37 0.0179 0.0014 0.5425CA2 67.09 ± 20.51 49.77 ± 20.35 0.0266 0.0014 0.3797CA3 60.87 ± 21.54 44.19 ± 12.17 0.0029 0.0001 0.2687

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—months.

h at 40 ◦C and nonspecific binding was blocked with 10% nor-al donkey serum in 0.1% Tween in 0.02 M Tris buffered saline

or 1 h at room temperature. Slices were incubated overnight in.5% BSA, 0.1% Tween in 0.02 M Tris buffered saline containinghe primary antibody (rabbit anti-Dopamine1 receptor antibody;lomone labs, Jerusalem, Israel, 1:100) and rabbit anti-Dopamine2eceptor antibody (Millipore, Bedford, MA, 1:100) or for colo-alization experiments: goat anti-Dopamine2 receptor antibodyAbcam, Cambridge, UK, 1:100) at 40 ◦C. Slices were then rinsed inlocking solution and were incubated with secondary antibodiesanti-goat IgG labelled with Alexa Fluor 647, 1:1000; Molecu-ar Probes, Invitrogen Corporation, Carslbad, CA and anti-rabbitgG Alexa Fluor 488, 1:1000; Cell Signalling Technology, Boston,

A, USA) for 2 hr at 40 ◦C. Counterstaining of cell nuclei waschieved with DAPI (1.5 �g/ml, 4′,6-diamidino-2-phenylindole,nvitrogen, Carlsbad, CA, USA). After rinsing in 0.1 M phosphateuffered saline, slices were dried and cover slipped with fluo-escence mounting medium (DAKO, Glostrup, Denmark). Slicesere examined by Zeiss LSM 700 confocal laser-scanning micro-

cope (LSM 700, Carl Zeiss GmbH, Jena, Germany) using 20 to3× oil immersion objectives (1.4NA) with the pinhole set at 1iry unit. Images were acquired using LSM 700 software (Carleiss GmbH, Jena, Germany) at 1024 × 1024 pixel resolution. Con-rast and sharpness of the images were adjusted by using theevels and sharpness commands in Adobe Photoshop CS 5 (Adobeystems, San Jose, CA, USA). Specificity of immunostaining wasonfirmed by control experiments with omission of primary anti-ody.

.7. Image analysis

For the analysis of the quantification, the fluorescence intensityf D1R and D2R were quantified using the software ImageJ (NIH).

749 ± 0.1539 0.9673 0.9432 >0.9999066 ± 0.2553 0.0090 0.0092 >0.9999

Briefly, two hippocampal tissue slices containing the CA1, CA2, CA3and DG (dentate gyrus) area were used for each mouse (total 5 miceper group). Within each tissue slice, 10 squares covering each areawere chosen for fluorescence quantification. The mean value from20 squares was adopted and the value of 12 and 18 months oldmice was normalized to the value of the 3 months mice. The anal-ysis of the colocalization of D1R and D2R were performed in single8-bit confocal images using the software ImageJ (NIH) and imagesare acquired from pyramidal cell layer in CA1-3 region and granularcell layer in dentate gyrus in hippocampal slices. Colocalization wasmeasured on single confocal sections using the plugins ‘Colocaliza-tion Finder’ of image J. Pearson coefficients were used to examinethe colocalization. It describes the correlation of the intensity dis-tributions between channels. Its values range between −1, 0 and1, where 0 indicates no significant correlation and 1.0 indicatescomplete positive correlation.

2.8. Statistical analysis

Data from behaviour, immunoblotting, immunofluorescenceand colocalization experiments were analyzed by using ANOVAcombined with post-hoc multiple comparison test (Bonferroni).Pearson correlation coefficients were used to analyze correlationstudies. A probability level of P < 0.05 was considered as statis-tically significant. Calculations were performed using GraphpadPrism 6.

3. Results

3.1. Morris water maze

The animals learned the task as latencies (Fig. 1a) and pathlengths (Fig. 1b) to reach the platform were gradually decreasing,

4 S.R. Saroja et al. / Behavioural Brain Research 264 (2014) 1–8

Fig. 1. Effect of aging on cognition, MA receptor complexes and signalling molecules of D1R. (a) Latency (in s) to reach the platform in acquisition trials. (b) Path length (inm) to reach the platform during acquisition trials. (c) Time spent (in s) in target quadrant in the probe trials. All values are expressed as mean ± s.e.m. n = 15–17 animals pergroup. Two-way ANOVA was used to calculate latency and path length, while time spent in target quadrant was calculated with one-way ANOVA. * P < 0.05, *** P < 0.001, ****

P < 0.0001. (d) Receptor complex analysis from membrane fraction by BN–PAGE. (e) Immunoprecipitation with D1R antibody, and subsequent western blotting with D1Rb analc opreci

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inding partners PKC isoforms-�, -�, and -�, PSD-95 and NSF. (f) SDS–PAGE basedytosolic fraction, except PSD-95# which was from membrane fraction. (g) Immun

ut remained comparable between groups. At the probe trial on day time spent in the target quadrant was significantly decreased inhe 18 months group as compared to the 3 months group of animalsFig. 1c) indicating decreased memory retrieval.

.2. Results from BN–PAGE

Western blot bands are given in Fig. 1d. A single band wasbserved for the D1R-containing complex and as shown in Table 1he receptor complex levels were significantly decreasing withge. A single band was observed for the D2R-containing complexnd Table 1 demonstrates gradually decreasing receptor complexevels resembling the decline D1R-containing complex levels. 5-T1A-containing receptor complexes were presenting with twoands (720 kDa and 480 kDa) and optical density of both bandsere increasing with age (Table 1). 5-HT7-containing receptor

omplexes were represented by a single band and levels wereradually decreasing with age (Table 1). When individual recep-or complex levels were correlated with the time spent in thearget quadrant of the MWM, the key parameter of memoryetrieval, a positive significant correlation was observed for theime spent in the target quadrant and receptor complex levelsontaining D1R and 5-HT7R (Table 2). A negative correlation wasbserved when the time spent in the target quadrant was cor-

elated with 5-HT1A receptor complex levels represented by aand at the apparent molecular weight at 480 kDa (Table 2). Age-ependent expression of D1R was significantly correlated with2R, 5-HT1AR (720 kDa), 5-HT1AR (480 kDa) and 5-HT7R, whereas

ysis of age and memory-dependent alteration of D1R’s signalling molecules frompitation with D1R antibody, and subsequent western blotting with D1R antibody.

D2R correlated significantly with 5-HT7R (Table 2). Moreover, 5-HT7R showed significant correlation with both bands of 5-HT1AR(Table 2).

3.3. Results from immunoprecipitation using an antibody againstD1R

Proteins in the immunoprecipitate were run on SDS–PAGE anddeveloped with antibodies against proteins known to regulateand/or bind to D1R: these proteins included protein kinases C, alpha(�), delta (�), epsilon (�) and gamma (�) isoforms, PSD-95 and NSF[27–30]. As shown in Fig. 1e PKC isoforms-�, -�, and -�, PSD-95 andNSF were observed in the immunoprecipitate. Expression of age-dependent PKCs were analyzed from cytosolic fraction and westernblot bands are shown in Fig. 1f. PKC � was run in addition becausethey are known to phosphorylate D1R. The PKC � isoform wassignificantly reduced at 18 months (Table 1), the � isoform wassignificantly decreased at 12 month and subsequently compara-ble between 3 and 18 months (Table 1). The PKC � was significantlyreduced from 12 months as compared to 3months (Table 1) and the� isoform was comparable between groups (Table 1). Levels of PSD-95 from the membrane fraction were decreasing with age (Table 1)while increasing with age in the cytosolic fraction (Table 1). NSF

levels in the cytosolic fraction were decreasing with age (Table 1).Correlation between D1 and PKC isoforms-�, -� and -�, PSD-95in the membrane and cytosolic fraction and NSF were significant(Table 2).

S.R. Saroja et al. / Behavioural Brain Research 264 (2014) 1–8 5

Fig. 2. Age and hippocampal sub-region dependent levels of D1R and D2R. (a) ExpressionCA3 regions.

Table 2All three groups were combined for Pearson correlations studies by using GraphpadPrism 6.

S. no Pearson correlationcoefficient

P value(two tailed)

Pearson r value

1 D1R vs target quadrant 0.0468 0.43842 D1R vs D2R 0.0030 0.52313 5-HT1A R (480 kDa) vs

target quadrant0.0310 −0.4713

4 5-HT7R vs targetquadrant

0.0162 0.6078

5 5-HT1A R (480 kDa) vs5-HT1A R (720 kDa)

0.0260 0.4060

6 5-HT1A R (720 kDa) vs5-HT7R

0.0040 −0.5097

7 5-HT1A R (480 kDa) vs5-HT7R

<0.0001 −0.7219

8 D1R vs 5-HT1A R(720 kDa)

0.0312 −0.3939

9 D1R vs 5-HT1A R(480 kDa)

0.0004 −0.6035

10 D1R vs 5-HT7R <0.0001 0.813311 D2R vs 5-HT7R 0.0001 0.644112 D1R vs PKC alpha 0.0003 0.801313 D1R vs PKC delta 0.0378 −0.539714 D1R vs PKC epsilon 0.0019 0.733615 D1R vs PSD-95

(membrane fraction)0.0007 0.6558

16 D1R vs PSD-95(cytosolic fraction)

0.0058 −0.5571

17 D1R vs NSF 0.0302 0.4256

of D1R in DG, CA1, CA2 and CA3 regions. (b) Expression of D2R in DG, CA1, CA2 and

3.4. Immunohistochemical findings

Densitometry of D1R immunoreactivity in hippocampal sub-areas DG, CA1, CA2 and CA3 revealed decline of D1R immunore-activity with age (Fig. 2a, Table 1). Densitometry of D2Rimmunoreactivity in hippocampal subareas DG, CA1, CA2 andCA3 revealed decreased D2R immunoreactivity with age (Fig. 2b,Table 1). The presence of D1R and D2R in the same complex isshown in Fig. 1g. Colocalization studies between D1R and D2R inCA subareas (Fig. 3a and b, Table 1) and DG (Table 1, Supplementaryfigure) revealed comparable colocalization in the DG but reducedcolocalization in the CA subareas CA1-CA3.

4. Discussion

Herein we propose the involvement of monoamine receptorcomplexes, rather than receptor subunits in memory retrieval forthe first time, as the subunits may carry out the specific func-tion only when assembled to receptor complexes [26,31]. As to theserotonergic system, two complexes of 5-HT1A receptor containingcomplexes were increased, whereas 5-HT7R-containing complexlevels were decreased towards aging. The 5-HT1AR-containingcomplex at 480 kDa and the 5-HT7R-containing receptor com-plex showed significant correlation with the time spent in targetquadrant, the key parameter of memory retrieval which in turnunambiguously links the serotonergic receptor system to spatial

memory performance. Møller et al. [17] suggested that 5-HT1ARbinding sites were decreased towards aging, it was also demon-strated, however, that 5-HT1AR binding sites were augmentedduring age-associated mild cognitive impairments, but decreased

6 S.R. Saroja et al. / Behavioural Brain Research 264 (2014) 1–8

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Fig. 3. Impact of aging on colocalization of D1R and D

n the Alzheimer diseased patients compared to controls [32]. Ownesults of increased 5-HT1AR containing complex levels are in func-ional agreement with Truchot et al. [32]. Previous studies [33,34]howed that 5-HT7Rs declined with age and we herein showed aositive correlation with age-dependent decline of spatial memoryetrieval.

The functional interaction between serotonin receptors andopamine receptors has been reported [35] and dopamine1 and

receptor-containing complexes were substantially reduced from to 12 and 18 months. Surprisingly there was no reduction from2 to 18 months, proposing that maximum reduction of D1R and2R-containing complex levels occurs within 12 months. The time

pent in the target quadrant representing spatial memory retrieval,owever, was significantly reduced at 18 months only. These dataltogether reveal that even prior to the mild cognitive impair-ent observed at 18 months reduction of D1R and D2R-containing

eceptor complex levels was taking place. The significant corre-ation between D1R and the time spent in the target quadrantndicated a strong link between D1R-containing complex levels andhe age-related memory decline. Addressing the region-specificeceptor complex levels of D1R and D2R the current study shows

ge-dependent hippocampal region-specific levels as DG and CA3egions are involved in spatial tasks, while CA1 is involved inemporal tasks [36,37] and the CA3 region is important for rapidearning of new information and the CA1 region compares the

olocalization pattern in CA3 (a) and CA1 regions (b).

output from the CA3 and the entorhinal cortex [38,39]. Moreover,aged CA3 place cells showed high firing rates and failed to encodenew information compared to young CA3 place cells, whereas agedCA1 cells were similar to young CA1 cells, indicating that agingmay affect individual particular regions [40]. From own immuno-histochemistry studies, decreased D1R levels were observed in allsub-regions. CA1 and CA2 showed significant reduction between 3and 18 months, whereas DG and CA3 regions showed significantreduction from 3 to 12 and 18 months, clearly demonstrating thesub-region specific alterations of D1R in aging. However, D1R lev-els were comparable between 12 and 18 months in all sub-regionsstudied. Gangarossa et al. showed that D1R agonists enhanced Erk-mediated stimulation of Zif268 and Arc in DG, not in CA1 or CA3[41]. In addition, 6-OHDA, a neurotoxin destroying dopaminergicneurons, impaired LTP in DG but not in CA1 [42]. LTP-inducedZif268 and Arc were abolished in the CA1 of D1R knock-out mousehippocampus with memory impairment [43]. Several studiesreported the impact of D1R-mediated LTP and memory forma-tion in the CA1 region [44,45]. Taken together, all hippocampalsub-regions specific D1R may play distinct roles in memory forma-tion and aging. Unlike heterogeneous reduction of D1R, D2R were

substantially reduced from 3 to 12 and 18 months in all sub-regions.Like D1R, no significantly reduced levels were observed between12 and 18 months in any region. Although D1R and D2R levelswere reduced towards aging, both might play different roles as

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onito-Oliva and coworkers showed that 6-OHDA induced memorympairments were rescued by D1R agonists, but not by D2R agonistsn the DG region of a Parkinson disease mouse model [42]. More-ver, D1R and D2R can share a common mechanism [46], probablyhrough forming a heteromeric D1R–D2R complex [47,48]. IPesults from the current study showed that D1R and D2R are form-ng a complex in the hippocampal membrane fraction. Considerablyecreased co-localization of D1R and D2R was observed in the CAegions towards aging, with non-significant reduction in the DGhich suggests that D1R–D2R mediating signalling is impaired dur-

ng aging. However we were unable to find significant differencesither in latency or path length between three groups.

Declined levels of dopamine receptor containing complexesere accompanied by level changes of particularly D1R-associated

ignalling and trafficking molecules Protein kinases C are reporteds important mediators in memory formation [49,50] and particu-arly PKC isoforms-�, -�, -�, and -� were shown to phosphorylate1R [30,51,52]. PKC-� and -� were significantly reduced towardsging and showed positive correlation with D1R and it wasbserved herein that these kinases persist in complex compo-ents of the D1R complex as shown by IP. Although IP resultsevealed that D1R and PKC � were in the same complex, thereere no significant changes in PKC � during aging. PKC � levelsere reduced from 3 to 12 months then increased significantly

nd showed negative correlation with D1R. PSD-95 was showno be a trafficking molecule of D1R in the absence of dopaminegonists [29] and own IP results, together with previous findings,onfirm that D1R interacts with PSD-95 [29]. PSD95 is uncouplingopamine-glutamate interactions in the D1/PSD95/NMDA recep-or complex and knockdown of PSD95 immobilizes D1Rs on theell surface escalating NMDAR-dependent D1 cAMP signalling ineurons [55]. PSD-95 levels in the synaptic membrane fractionere decreased with aging and correlated positively with D1R.

ncreased PSD-95 levels in the cytosolic fraction along with neg-tive correlation with D1R towards aging were demonstrated andhese data suggest that PSD-95 might be an important trafficking

olecule of D1R in aging. In order to understand the differencesetween cytoplasmic and membrane levels of PSD95 levels ofhe guanylate kinase-associated protein (GKAP) that orchestratesctivity-dependent postsynaptic protein remodelling we suggesto carry out further studies on this PSD95 regulating cascade [56].here are several studies on NSF-mediated trafficking of AMPAnd GABA receptors [53,54] but to our knowledge, there is notudy on NSF-mediated trafficking of D1R. It was shown; however,hat NSF had more affinity with D1R than other dopamine recep-or subtypes [28]. Our IP results provided evidence that D1R andSF are in the same complex. As expected considerable reductionf NSF and positive correlation with D1R were observed towardsging.

We herein demonstrate that during mild cognitive impair-ents observed in the MWM, 5-HT1AR-containing complex levels

ncreased while 5-HT7R-containing complex levels decreased inging. Dopamine receptor complexes, their hippocampal region-pecific levels and colocalization studies altogether showed thateduction occurs within 12 months of age. D1R and D2R areeclining almost completely before cognitive impairments start toppear. Furthermore it was demonstrated that along with dimin-shed D1R and D2R-containing complex levels, D1R–D2R mediatingignalling is also declined in aging. The results are in agreementith and extend knowledge of previous work on serotonergic andopaminergic function that decline along with signalling moleculesuring aging and that this may be responsible or paralleling age-

ependent long term spatial memory impairments. Novel evidencet the receptor complex rather than at the receptor subunit level for

strong link between the dopaminergic and serotonergic systemsith memory retrieval in the mouse is provided.

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in Research 264 (2014) 1–8 7

Funding and disclosure

The study was funded by the Medical University of Vienna,Austria.

Conflict of interest statement

There is no conflict of interests in authorship or financial innature.

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.2014.01.042.

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