Intermittent administration of morphine alters protein expression in rat nucleus accumbens

RESEARCH ARTICLE

Intermittent administration of morphine alters protein

expression in rat nucleus accumbens

Ka Wan Li1, Connie R. Jimenez1, Roel C. van der Schors1, Martin P. Hornshaw2,Anton N. M. Schoffelmeer3 and August B. Smit1

1 Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research,Vrije Universiteit, Amsterdam, The Netherlands

2 Applied Biosystems, Warrington, UK3 Department of Medical Pharmacology, VU Medical Center, Amsterdam, The Netherlands

Repeated exposure to drugs of abuse causes time-dependent neuroadaptive changes in themesocorticolimbic system of the brain that are considered to underlie the expression of majorbehavioral characteristics of drug addiction. We used a 2-D gel-based proteomics approach toexamine morphine-induced temporal changes in protein expression and/or PTM in the nucleusaccumbens (NAc) of morphine-sensitized rats. Rats were pretreated with saline [1 mL/kg sub-cutaneously (s.c.)] or morphine (10 mg/kg, s.c.) once daily for 14 days and the animals weredecapitated 1 day later. The NAc was extracted and proteins resolved by 2-DE. Several proteinfunctional groups were found to be regulated in the morphine-treated group, representing cyto-skeletal proteins, proteins involved in neurotransmission, enzymes involved in energy metabo-lism and protein degradation, and a protein that regulates translation.

Received: January 30, 2005Revised: September 19, 2005

Accepted: September 20, 2005

Keywords:

2-DE / Brain / MALDI-TOF / Rat / TOF MS

Proteomics 2006, 6, 2003–2008 2003

1 Introduction

Unlike sustained exposure to morphine (causing toleranceand physical signs of abstinence), chronic intermittent opiate(or psychostimulant) treatment results in a progressiveincrease in the drug-induced psychomotor activating effectsin rats. This phenomenon, denoted as behavioral sensitiza-tion, is considered to be due to neuroadaptations in themesocorticolimbic system of the brain, and may contributeto the development and maintenance of addictive behavior[1, 2]. Drug-induced behavioral sensitization has been shown

to be associated with enhanced dopamine, glutamate andacetylcholine transmission [3, 4], mediating activation ofdown-stream signaling pathways in the reward-related brainareas, notably the nucleus accumbens (NAc). In turn, thesedrug-induced signal transduction cascades cause progressivechanges in gene expression [5–8], resulting in persistentbiochemical, physiological and morphological alterations ofNAc neurons [2, 9]. These temporal staged adaptive changesare thought to mediate the development and to underlie themaintenance of addictive behavior.

Current studies on the molecular basis of drug addictionemploy high-throughput screening techniques for detectingaltered neuronal gene expression in rat and mouse brainfollowing drug exposure. However, observed changes inmRNA and protein expression levels may not always be cor-related in time, direction, magnitude and space [10, 11].Hence studies at the protein level are of crucial importance.Here, we have used a 2-DE-based proteomics approach as afirst step to examine changes in protein expression in theNAc upon chronic intermittent exposure of rats to the pro-

Correspondence: Dr. Ka Wan Li, Department of Molecular andCellular Neurobiology, Faculty of Earth and Life Sciences, VrijeUniversiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Neth-erlandsE-mail: [email protected]: 131-205989281

Abbreviation: NAc, nucleus accumbens

DOI 10.1002/pmic.200500045

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

2004 K. W. Li et al. Proteomics 2006, 6, 2003–2008

totypical, addictive drug morphine. We used an opiate pre-treatment regimen (14 once-daily drug injections) mediatingrobust neurochemical and behavioral sensitization to mor-phine and psychostimulants [12, 13].

2 Materials and methods

2.1 Animals, drug treatment and dissection

Experiments were approved by the Animal Care Committeeof the Vrije Universiteit, Amsterdam, The Netherlands. MaleWistar rats, weighing 180–200 g (HarlanCPB, Zeist, TheNetherlands) were housed two per cage with food and waterad libitum. Animals were adjusted for 1 week to controlledconditions (lights on from 7 a.m. to 7 p.m.) and were brieflyhandled on the 2 days preceding drug treatment. In theirhome cage, rats (n=5) received a single daily injection witheither saline [1 mL/kg subcutaneously] or with morphine-HCl (10 mg/kg subcutaneously, dissolved in saline) for a pe-riod of 14 days. The rats were killed 24 h after the last saline/morphine treatment. The animals were decapitated and theNAc was dissected from a coronal slice (bregma 11.2 to12.2 mm) and stored at –807C for proteomics analysis.

2.2 2-DE

2-DE was carried out as described [14, 15]. In brief, 10 mg wetweight of NAc was pottered in 400 mL lysis buffer (9 M urea,2% CHAPS, 20 mM Tris pH 7.5, 1% dl-DTT and 0.5% IPGbuffer pH 3–10) using a teflon piston and a glass vial. Aftercentrifugation at 14 0006g for 10 min, 370 mL of the super-natant was used for the rehydration and simultaneouslyloading of the proteins to the IPG strip (Immobiline 18-cmDryStrip 3–10 NL, Amersham Pharmacia Biotech), at 30 Vfor 12 h. The voltage was increased to 8000 V and focused fora total of 65 000 Vh. Immediately after being focused, IPGstrips were wrapped in plastic foil and stored at 2807C. Priorto SDS-PAGE, IPG strips were equilibrated in 6 M urea/2%SDS/1% dl-DTT/50 mM Tris, pH 8.4/30% glycerol for15 min, followed by equilibration in 6 M urea/2% SDS/2.5%iodoacetamide/50 mM Tris, pH 8.4/30% glycerol for 15 min.The second dimension separation was run overnight usingthe Isodalt System (Amersham Pharmacia Biotech) in1.5 mm 11% gels (Duracryl, Genomic Solutions) at 25 mA/gel at 157C. After electrophoresis, gels were fixed and stainedusing silver nitrate.

For the preparative gel, 20 mg wet weight of NAc wasused, and the gel was stained with SYPRORuby and thenfurther stained with CBB G to visualize the protein spots forexcision. Compared to conventional CBB staining, this dou-ble-staining procedure significantly increases the spot inten-sity and facilitates the manual spot picking. The spots weretrypsin digested and subjected to direct MS protein identifi-cation.

2.3 Computer-assisted spot detection and analysis

2-D gel analysis software (PDQuest version 7.1) was used inthis study for spot detection, gel matching and spot quanti-tation. The Mann-Whitney test was applied to compare thespot patterns in gels derived from individual control (n=5)and morphine-treated animals (n=5). Significant spots thatshowed at least 1.5-fold difference in intensity between thegroups were selected for protein identification.

2.4 Digestion of proteins

All of the protein spots of interest were manually excised with around-bottom dermal slicer of 3-mm diameter. The gel pieceswere destained in 60% ACNin 25 mMammonium bicarbonatebuffer, pH 8.5, and then dehydrated with 100% ACN. Theshrunken gel pieces were re-swelled in 25 mM ammoniumbicarbonate buffer, dehydrated again in 100% ACN, and driedin the SpeedVac. For gel pieces that were heavily stained therehydration/dehydration step was repeated once. The gelpieces were rehydrated in 8 mL trypsin solution (20 mg/mL) for1 h, followed by addition of 50 mL 25 mM ammonium bicarbo-nate buffer to completely immerse the gel pieces. After incu-bation overnight at room temperature, 0.5 mL incubation buf-fer was pipetted directly onto the stainless steel sample plate ofthe mass spectrometer, and mixed with 1 mL CHCA. The sam-ples were analyzed by a MALDI-TOF/TOF MS. In cases wherethe MS signals were weak, the incubation buffer was loadedinto a C18 ZipTip (Millipore) according to the manufacturer’sinstructions. The bound peptides were eluted from the ZipTipusing 1.5 mL CHCA, which was directly deposited onto themetal plate. The CHCA matrix concentration was 5 mg/mL in50% ACN/50% water containing 0.1% TFA.

2.5 MS

The mass spectrometer utilized for protein analysis was anApplied Biosystems 4700 Proteomics Analyzer with TOF/TOF Optics as described previously [15]. For PMF each massspectrum was averaged from signals generated from 1500laser shots. For MS/MS 5000–7500 laser shots per samplewere acquired. After data acquisition the mass spectra weresearched against the MASCOTdatabase search engine (MatrixScience). For each peptide mass fingerprint search the masstolerance was set to 25 ppm. One missed tryptic cleavage wasallowed. The mass tolerance for database searching with MS/MS spectra was set to 0.5 Da. All the proteins listed wereidentified with a confidence interval of 95% from the MS and/or MS/MS analysis, unless otherwise stated.

3 Results

To investigate the molecular basis of behavioral sensitization,we have examined the protein constituents of the NAc frommorphine- versus saline-pretreated groups of rats killed 1 day


Proteomics 2006, 6, 2003–2008 Clinical Proteomics 2005

Figure 1. Typical example of a silver-stained 2-D gel of an NAcextract. Spots labeled with numbers showed significant altera-tion in intensities of at least 1.5-fold (n=5 gels) 1 day after 14 dailymorphine pretreatment. The spots were trypsin digested and thepeptides characterized by MS. Spots X, Y and Z did not showchange but were selected for MS analysis to reveal the presenceof multiple spots for single protein. Protein identities are shownin Table 2.

Figure 2. Representative examples of differentially expressedproteins displayed on the 2-D gels. (A) As an example, a portionof the 2-D gels is shown to highlight a spot (arrow, spot 2 fromFig. 1) that shows significant difference between the morphine(M) and the saline (S) groups from five individual runs on fiveseparate 2-D gels. (B) Some proteins exhibit shift in the dimen-sion of pI. For example, spot 13 (enclosed within a square in thesaline-treated group) shows a migration to the left in three (M1–3) of the morphine-treated group.

Table 1. Quantitative change of proteins from NAc in response torepeated morphine administration. Spot number (spotno.) corresponds to the same number shown in Fig. 1.Spot density is the average OD of the same spot fromfive independent samples belonging to the sameexperimental group. CV shows the variation of the OD ofthe spots across these five samples. The CVof spot 8 and13 in the morphine-treated group are exceptionally high.This is underlying in part by the positional shift of thespots resulting in their mismatching in some of the gels(see Fig. 2B)

Spotno.

Saline Morphine p value

Spotdensity

CV Spotdensity

CV

1 902 26.80% 460 40.60% 0.0312 605 27.50% 241 30.10% 0.0083 703 24.40% 438 11.90% 0.0164 382 16.70% 232 17.00% 0.0085 817 57.80% 1815 30.60% 0.0556 464 13.70% 251 6.50% 0.0087 596 23.20% 308 18.40% 0.0088 773 46.40% 124 121.70% 0.0089 1013 23.30% 1607 13.30% 0.016

10 187 5.90% 102 25.40% 0.00811 338 42.70% 585 16.80% 0.03112 162 46.30% 283 6.70% 0.03113 736 8.30% 272 106% 0.01614 540 37.80% 931 16.1% 0.03115 466 13.90% 255 27.90% 0.008

post treatment. For each experimental group five rats wereused. NAc dissected from each rat were separately extracted;the protein constituents fractionated on 2-D gels and stainedwith silver nitrate. The staining detection limit achievedallowed the visualization of faint spots, resulting in theobservation of approximately 1500 spots per gel (Fig. 1).Overall, the 2-D gel protein spot patterns across all the gelswere similar (Fig. 2). To reduce the likelihood of false-posi-tive results in the observation of differences between the gelswe used a filter with p values ,0.05 and a quantitative dif-ference of at least 1.5-fold (Tables 1, 2). To facilitate proteinidentification the differentially regulated spots were matchedto a preparative CBB-stained gel. The protein spots wereseparately cut out from this gel, trypsin digested and thepeptides subjected to MALDI-TOF/TOF MS analysis. Someof the faint silver-stained spots were not detectable on theCBB-stained gel and were not further analyzed.

In total, 22 spots showed significant differences be-tween the morphine- and saline-pretreated groups, and 15of these were successfully characterized (Table 2). Spot 7contained two proteins and thus it was not possible to dis-tinguish which one of the two was specifically regulatedupon morphine pretreatment. The presence of multipleproteins in single spots has frequently been observed [15].



Table 2. Identification of proteins that showed quantitative change in response to morphine administration. Spot no. corresponds to thesame number shown in Fig. 1. As spot 7 contained two distinct proteins, their Mr and pI are not shown. Ratio Mor/Sal representsthe ratio of spot intensity of the morphine- against the saline-treated group. Spots X, Yand Z have similar Mr but differ slightly in pIto that of spot 11 (actin) and were selected for structural characterization to demonstrate the existence of multiple forms of (post-translational modified) actin. Although the MS score for synapsin is low and the CI% is only 77 (data not shown), the observed Mr

and pI agree with the expected values of synapsin and, therefore, this protein is tentatively considered as identified

Spotno.

Protein identity Mr pI MSscore

MS/MSscore

RatioMor/Sal

Accession no.

1 Beta-synuclein 14 495 4.48 180 96 0.51 giu183733262 Ubiquitin C-terminal hydrolase L-1 24 766 5.12 41 128 0.40 giu83945063 Mitochondrial H-ATP synthase subunit d 18 726 6.21 60 51 0.62 giu95064114 NADH-ubiquinone oxidoreductase 26 511 6 73 135 0.61 giu1288675 Triosephosphate isomerase 27 285 6.51 79 40 2.22 giu126210746 Lin-7 20 085 9.32 74 40 0.51 giu597980907 ATP synthase beta/14–3-3 protein gamma – – – – 0.528 Fructose-bisphosphate aldolase 39 691 8.31 190 133 0.16 giu681869 Similar to Atp6v1e1 protein 26 183 8.44 306 170 1.59 giu27712982

10 Enolase 2 gamma 47 111 5.03 55 64 0.54 giu2602394911 Actin beta 41 724 5.29 114 225 1.73 giu7162012 Ribonuclease/angiogenin inhibitor 49 873 4.64 – 43 1.75 giu2080615113 Similar to murine homolog of human ftp-3 53 088 5.81 79 56 0.37 giu3488151314 Neurofilament light polypeptide 61 298 4.63 193 112 1.72 giu1392909815 Synapsin 70 037 9.84 38 – 0.55 giu206933X Actin betaY Actin betaZ Actin beta

The use of narrow pH range IPG strip for 2-DE, in combi-nation with a gel slicer of smaller diameter, might alleviatethis problem.

The major classes of the morphine-regulated proteins arein the categories of energy metabolism (enolase, ATP syn-thase, NADH-ubiquinone oxidoreductase, triosephosphateisomerase and aldolase), neurotransmission (synapsin, Lin-7and b-synuclein), and the cytoskeleton (neurofilament lightpolypeptide and actin b). Several single proteins could not begrouped together, and included ubiquitin C-terminal hydro-lase L1 involved in deubiquitination and protein degradation,H1 transporting V1 subunit E involved in proton transport,heterogeneous nuclear ribonucleoprotein H2 (ftp-3) thatregulates transcription, and ribonuclease/angiogenin inhib-itor.

Previous studies have indicated that individual proteinsmay be resolved as multiple spots on a 2-D gel with a differ-ent pI but similar Mr, probably being caused by a differentialdegree of PTM [15]. Therefore, it is likely that the identifiedproteins from some of the 2-D spots in the present studyrepresent post-translationally modified forms of the proteins.To test this possibility we further analyzed proteins thatformed a chain of spots adjacent to the identified protein ofinterest. Figure 1 indicates that a series of spots, namely X, Yand Z that located next to actin (spot 11) in the 2-D gel werealso characterized as actin. Interestingly, some of the spotsshowed a shift in the dimension of pI (spot 8, fructose-bisphosphate aldolase; and spot 13, ftp-3), causing spot mis-

matching in some of the gels (Fig. 2B). This observationstrongly suggests that morphine pretreatment may causedifferential PTM of proteins.

4 Discussion

Chronic intermittent administration of morphine (as well aspsychostimulants) in rats induces psychomotor sensitiza-tion, which is of particular interest since the neuronal cir-cuits involved overlap with those that mediate the incentivemotivational effects of addictive drugs [16, 17]. In the presentstudy, we examined the nature of molecular changes in NActhat may mediate this morphine-induced behavioral sensiti-zation.

One day after repeated opiate pretreatment, several pro-tein classes in NAc of the morphine group showed signifi-cant alterations as compared to the saline-pretreated group.We observed an increased in neurofilament level, which mayunderlie structural changes of the neuronal dendrites andaxonal caliber [18]. Altered expression level of neurofila-ments have been detected in drug-exposed rats [19, 20], andin human addicts [18, 21]. Furthermore, accumulation ofneurofilaments is characteristic of many human neurode-generative diseases [18]. We detected a subtle change in actinexpression. Among the several spots on the 2-D gel thatcontained actin only one of them showed alteration, indicat-ing that a unique post-translational modified form of actin


Proteomics 2006, 6, 2003–2008 Clinical Proteomics 2007

was affected by repeated morphine administration. In thisrespect, a previous study reported that the expression level ofactin mRNA was not affected by chronic morphine adminis-tration [22]. Actin is the major determinant in the formationand maintenance of the spine head and axonal ending,which is determined by the (de)polymerization of the actinmolecules [23]. Changes in the phosphorylation states ofactin may contribute to this process.

Ubiquitin C-terminal hydrolase L1 was also regulated.This protein deubiquitinates ubiquitinated proteins for recy-cling of free ubiquitin or to remove ubiquitin from incor-rectly tagged proteins. The ubiquitination/proteasome path-way is a major route used to regulate many critical cellularproteins that must be rapidly destroyed. Recently, it wasshown that the ubiquintination/proteasome pathway isinvolved in activity dependent-neuroplasticity [24].

A number of the identified proteins are involved in cel-lular metabolism, some of these are found in the cytoplasmand the others are located within mitochondria. Previousstudies have indicated that even a single injection of mor-phine transiently down-regulated a group of genes thatencode mitochondrial proteins [5]. Interestingly, the pI offructose-bisphosphate aldolase seems to be lower in themorphine-treated group, which was visualized by a shift ofthe spot along the dimension of IEF point. The change in pImay be caused by PTM of the protein; such as phosphoryla-tion. It is a general finding that morphine affects signaltransduction cascades, causing differential phosphorylationof the proteins [25, 26]. As intermittent morphine adminis-tration is well-known to alter NAc neurotransmission [3, 27]and neuronal architecture [16, 17], it is conceivable that therate of energy production [28] is adapted to initiate and sus-tain these changes upon morphine exposure.

Drug-induced neuroplasticity requires new protein syn-thesis. The expression of heterogeneous nuclear ribonucleo-protein, a protein that is involved in translational events [29],was also affected by morphine administration. This mayunderlie the changes in expression of certain proteins in thisbrain region.

Several morphine-regulated proteins are involved insynaptic transmission. The phosphoprotein synapsin Iassociates with synaptic vesicle, and is involved in the reg-ulation of synaptic vesicle exocytosis. The function of synap-sin is modulated by a number of kinases. Interestingly, mul-tiple synapsin spots, representing presumably differentphosphorylation forms of the protein, have been observedusing 2-D gels [15]. In the present study a single spot ofsynapsin was detected, suggesting that intermittent mor-phine pretreatment might have induced specific phospho-rylation of this protein. Repeated morphine administrationhas also been shown to up-regulate the expression of synap-sin mRNA [30]. Lin-7 (also known as veils) is know to interactwith several pre- and post-synaptic proteins, and may couplesynaptic vesicle exocytosis to cell adhesion in the brain [31,32]. Synucleins are the abundant soluble proteins in thenerve terminals and seem to have an important role in the

pathogenesis of Parkinson’s disease [33]. A recent studyindicated that synucleins may have regulatory role on thedopamine levels in the brain, and may contribute to the long-term regulation and/or maintenance of nerve terminalfunctioning [34].

It is generally agreed that addictive drugs may inducesynaptic changes that subserve addictive behavior [16, 17,27]. Indeed, synapsin and Lin-7 have been detected in thesynapse. Nevertheless, it is most likely that the majority ofmorphine-induced changes in the synapse were missed inour analysis. Firstly, 2-D gels displayed fewer than 1500 dis-tinct proteins, which cover only a fraction of the total popu-lation of the brain proteins. Secondly, many synaptic pro-teins, e.g., transmembrane receptors and ion channels, havephysical properties such as high Mr, extreme pI and/or highdegree of overall hydrophobicity that prohibit their resolu-tion on the 2-D gel [15]. Therefore, although our study is inagreement with previous work on altered gene expressionfollowing repeated exposure to drugs of abuse [6, 7, 35, 36],more in depth (organellar) proteomics analyses, focusing on,for example, the synapse proteome and its spatio-temporalcharacteristics, can be expected to cover larger categories ofprotein groups and to further enhance our understanding ofthe role of synaptic plasticity in behavioral sensitization andaddictive behavior.

The authors wish to thank Rob Binnenkade for his contribu-tion to animal care and dissection, Netherlands Research Orga-nization (NWO-MW) for financial support to K.W.L., andCenter of Medical Systems Biology for financial support to K.W.L.and A.B.S.

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Intermittent administration of morphine alters protein expression in rat nucleus accumbens