Deletion of Go2alpha abolishes cocaine-induced behavioral sensitization by disturbing the striatal...

The FASEB Journal • Research Communication

Deletion of Go2� abolishes cocaine-induced behavioralsensitization by disturbing the striatal dopaminesystem

Irene Brunk,*,1 Christian Blex*,1 Carles Sanchis-Segura,†,2 Jan Sternberg,*Stephanie Perreau-Lenz,† Ainhoa Bilbao,† Heide Hortnagl,‡ Jens Baron,*Judyta Juranek*,3, Gregor Laube,* Lutz Birnbaumer,§ Rainer Spanagel,†,4 andGudrun Ahnert-Hilger*,4

*Institute for Integrative Neuroanatomy, Center for Anatomy, Charite-Universitatsmedizin Berlin,Berlin, Germany; †Department of Psychopharmacology, Central Institute of Mental Health,Mannheim, Germany; ‡Institute for Pharmacology, Charite-Universitatsmedizin Berlin, Berlin,Germany; and §National Institute of Environmental Health Sciences, Research Triangle Park,North Carolina, USA

ABSTRACT The �-subunits of the trimeric Go classof GTPases, comprising the splice variants Go1� andGo2�, are abundantly expressed in brain and reside onboth plasma membrane and synaptic vesicles. Go2� isinvolved in the vesicular storage of monoamines but itsphysiological relevance is still obscure. We now showthat genetic depletion of Go2� reduces motor activityinduced by dopamine-enhancing drugs like cocaine, asrepeated injections of cocaine fail to provoke behav-ioral sensitization in Go2��/� mice. In Go2��/� mice,D1 receptor signaling in the striatum is attenuated dueto a reduced expression of Golf� and Gs�. Followingcocaine treatment, Go2��/� mice have lower D1 andhigher D2 receptor amounts compared to wild-typemice. The lack of behavioral sensitization correlateswith reduced dopamine levels in the striatum anddecreased expression of tyrosine hydroxylase. Onereason for the neurochemical changes may be a re-duced uptake of monoamines by synaptic vesicles fromGo2��/� mice as a consequence of a lowered set pointfor filling. We conclude that Go2� optimizes vesicularfilling which is instrumental for normal dopaminefunctioning and for the development of drug-inducedbehavioral sensitization.—Brunk, I., Blex, C., Sanchis-Segura, C., Sternberg, J., Perreau-Lenz, S., Bilbao, A.,Hortnagl, H., Baron, J., Juranek, J., Laube, G., Birn-baumer, L., Spanagel, R., Ahnert-Hilger, G. Deletion ofGo2� abolishes cocaine-induced behavioral sensitiza-tion by disturbing the striatal dopamine system. FASEBJ. 22, 3736–3746 (2008)

Key Words: vesicular regulation � VMAT2 � locomotor activity� psychostimulants

A great variety of brain functions, including motorcontrol, cognition, and emotions, as well as drug-induced reinforcement and sensitization, is linked todopaminergic neurotransmission. Dopamine is synthe-

sized by tyrosine hydroxylase (TH) and DOPA decar-boxylase, and cytosolic dopamine is concentrated intosynaptic vesicles (SVs) of dopaminergic neurons by theaction of the vesicular monoamine transporter 2(VMAT2). Cytosolic dopamine can be degraded bymonoaminooxidases (MAOs). Released dopamine ex-erts its physiological response on dopaminergic recep-tors of the dopamine 1 (D1) -like or D2-like subfamilies.Dopamine is regained by the plasma membrane dopa-mine transporter (DAT). In contrast to DAT, VMAT2 isnot selective for dopamine but also transports seroto-nin, noradrenaline, and adrenaline with comparableefficiency (1, 2). A perfect balance between synthesis,degradation, and reuse guarantees adequate amountsof monoamines for their diverse physiological func-tions. An alteration in any of the components involvedin dopamine synthesis, transport, and degradation usu-ally results in a disturbance at the systemic level, asobserved by pharmacological interventions or knock-out models. The DAT knockout, for example, mostprominently shows a hyperactive phenotype (3). Dele-tion of VMAT2 is fatal, and even mice heterozygous forVMAT2 display altered drug-induced locomotor activityand MPTP toxicity (4). Indeed, compensatory mecha-nisms exist that counteract imbalances of dopaminehomeostasis, particularly with respect to vesicular stor-age. Thus, TH expression is increased in VMAT2

1 These authors contributed equally to this work.2 Current address: Laboratorio de Psicofamacologia del

Etanol, Universitat Jaume I, Castello (12071), Spain.3 Current address: Department of Surgery, Columbia Uni-

versity Medical Center, BB 1708, New York, NY 10032, USA.4 Correspondence: G.A.H., AG Functional Cell Biology,

Center for Anatomy, Charite-Universitatsmedizin Berlin, Phil-ippstr. 12, 10115 Berlin, Germany. E-mail: [email protected]; or R.S., Department of Psychopharmacology, Cen-tral Institute of Mental Health, J5, 68159 Mannheim, Ger-many. E-mail: [email protected]

doi: 10.1096/fj.08-111245

3736 0892-6638/08/0022-3736 © FASEB

knockout animals (5), and enhanced monoamine syn-thesis is observed when VMAT2 activity is blocked byreserpine (6).

We have recently shown that vesicular storage ofdopamine, as well as of other monoamines, is con-trolled by Go2� (7). Go1� and Go2� are splice variantsof Go�, which when associated with G�� dimers con-stitute 1 to 2% of total brain membrane protein (8).Besides their occurrence at the plasma membrane, Goproteins have been found on endomembranes, includ-ing SVs (9, 10) suggesting an effect on vesicular func-tion. Indeed monoamine storage mediated by VMAT2is regulated by SV-associated Go2� but not by Go1�,thus confirming that the two splice variants exertdifferent functions. In this signaling pathway, VMAT2assumes the role of a G-protein-coupled receptor thatsenses intravesicular transmitter concentrations andprovides a direct feedback loop between the degree ofvesicle filling and uptake activity (7, 11).

Recently, mouse strains have been characterized thatare deficient in either Go1� or Go2�. Although Go1�knockouts are severely impaired, Go2��/� mice do notpresent obvious phenotypical changes (12–14). Consid-ering the role of Go2� in regulating vesicular mono-amine storage discussed above, there are two possibleexplanations for the lack of gross phenotypical andbehavioral changes in these mice. First, compensatorymechanisms may exist that substitute for Go2�, thusensuring that vesicular monoamine storage is main-tained within a normal range. Alternatively, storage isaltered, but the resulting effects are too subtle to beobvious without closer analyses.

In the present study, we examined whether in Go2�-deficient mice dopaminergic dysfunctions become ap-parent when dopamine homeostasis is pharmacologi-cally challenged. We found that Go2��/� mutants lackbehavioral sensitization following repeated injectionsof cocaine, probably caused by an imbalance in D1receptor signaling within the striatum and impaireddopamine storage dynamics. Our results provide evi-dence that fine-tuning of vesicular dopamine content isimportant in modulating the effects of psychostimu-lants in the striatal dopaminergic system.

MATERIALS AND METHODS

Antibodies

Mouse monoclonal antibodies against Go� proteins werepreviously described (15). A monoclonal antibody againstsynaptophysin (16) and polyclonal antibodies against VMAT2(17) and against the 116-kDa subunit of the vacuolar protonpump were obtained from Synaptic Systems (Gottingen,Germany). Antibodies against TH (rabbit), DAT (rabbit),dopamine D1 (mouse) and D2 (rabbit) receptors were fromChemicon International (Temecula CA, USA). Antibodiesagainst Gq� (rabbit) were purchased from Calbiochem (LaJolla, CA, USA), against Golf� and Gs� (both rabbit) and Go�(sc13532) from Santa Cruz Biotechnology (Santa Cruz, CA,USA), respectively. A polyclonal antiserum against G-protein

�-subunits 1–4 (18) was a generous gift from B. Nurnberg(Heinrich-Heine-Universitat, Dusseldorf, Germany).

Secondary antibodies, horse anti-mouse and goat anti-rabbit conjugated with horseradish peroxidase, were pur-chased from Vector Laboratories (Burlingame, CA, USA).Fluorescent dye-labeled secondary antibodies (goat anti-mouse IgG Cy2 or Cy3 and goat anti-rabbit IgG Cy2 or Cy3)were obtained from Dianova (Hamburg, Germany).

Chemicals

Guanolyl 5�-imidodiphosphate [GMP-P(NH)P], dopamine,serotonin, reserpine, nigericine, and bafilomycine were pur-chased from Sigma-Aldrich (St. Louis, MO, USA). 5-Hydroxy-[3H]tryptamine trifluoroacetate (serotonin; specific activity4.33 TBq/mmol) and 7,8-[3H]dopamine (specific activity1.78 TBq/mmol) were obtained from GE Healthcare (NewYork, NY, USA). GBR12909 was purchased from Tocris Bio-science (Bristol, UK). Cocaine was purchased from Sigma-Aldrich. Streptolysin O (SLO) from �-hemolytic streptococciwas purified as described previously (19).

G-protein deletion mutants

Go1� and Go2� splice variant-specific deletion mutants aswell as Go� deletion mutants were generated and bred asdescribed previously (12, 14). The respective wild-type litter-mates (129/SvxC57/BL6) were derived from heterozygousbreeding. All experimental procedures were approved by theCommittee on Animal Care and Use and were carried out inaccordance with the local Animal Welfare Act and the Euro-pean Communities Council Directive of 24 November 1986(86/609/EEC).

Synaptosomes and SVs

Synaptosomes were either prepared from whole mouse brainor from the striatal area, as described previously (20). SVswere prepared from whole mouse brain, as described previ-ously (15).

Monoamine uptake

Uptake was performed into either SVs or permeabilizedsynaptosomes. SVs were resuspended in potassium glutamate(KG) buffer (150 mM KG; 20 mM 1,4-piperazinediethanesul-fonic acid; 4 mM EGTA; 2.9 mM MgCl2, equivalent to 1 mMfree Mg2�; and 2 mM ATP, adjusted to pH 7.0 with KOH) bypassing 10 times through a 27-gauge needle, before they weredivided into individual reaction cups. Uptake was started byadding KG-ATP buffer, supplemented with 1 mM ascorbicacid and 40 nM [3H]serotonin or [3H]dopamine. Additivessuch as GMP-P(NH)P (100 �M), cocaine (5 �M), or reser-pine (6 �M, to determine nonspecific uptake), were appliedduring this step. Incubation was performed for 10 min at25°C and stopped by addition of 500 �l of ice-cold KG bufferfollowed by rapid centrifugation at 440,000 g for 10 min at4°C. The pellets were lysed in 0.4% Triton X-100 to deter-mine radioactivity by liquid scintillation counting and proteincontent using the bicinchoninic acid method.

For permeabilization, synaptosomes were resuspended inKG buffer supplemented with 5000 hemolytic units (HE)/mlSLO and incubated for 10 min at 4°C. Under these condi-tions, SLO monomers bind to the cholesterol of the plasmamembrane. After removal of unbound SLO by centrifugation(16,000 g, 2 min, 4°C), oligomerization and pore formationcan be initiated by elevating the temperature, thereby restrict-

3737COCAINE RESPONSE AND VESICULAR DOPAMINE IN GO2�–/– MICE

ing permeability to the plasma membrane. The pellet con-taining permeabilized synaptosomes was resuspended in KGbuffer, and monoamine uptake was performed for 10 min at37°C as described for SVs.

Dopamine uptake by intact synaptosomes was performedin sodium buffer (10 mM glucose; 5 mM KCl; 140 mM NaCl;5 mM NaHCO3;1 mM MgCl2; 1.2 mM Na2HPO4; 20 mMHEPES, adjusted to pH 7.4 with NaOH), and nonspecificuptake was determined in the presence of 20 �M GBR 12909.

HPLC analysis

Wild-type and Go2��/� mice of different ages [adult, post-natal day (P) 2, 4, 8, or 12] were decapitated; after removal,brains were immediately frozen on dry ice and kept at –80°Cuntil use. From frozen P12 and adult brains, different areas(bulbus olfactorius, frontal cortex, striatum, hypothalamus,hippocampus, and cerebellum) were dissected on a cold plate(–15°C). Samples were weighed and stored at –80°C untilhomogenization. After adding of 10–20 vol of deionizedwater, frozen samples were homogenized by ultrasonicationat 4°C. Homogenates were added to an equal volume of 0.2 Nperchloric acid and centrifuged for 10 min at 25,000 g and4°C. The supernatant was used for determination of mono-amines by HPLC with electrochemical detection, as describedpreviously (21).

Immunoblot analysis

Subcellular fractions from brain homogenates were resus-pended in sucrose buffer, analyzed for protein content, andsubjected to SDS-PAGE. After transfer to nitrocellulose mem-branes, proteins were analyzed using the indicated antibodiesand the enhanced chemiluminescence (ECL) detection sys-tem (GE Healthcare). ECL-processed films were scanned, andprotein bands were densitometrically quantified using theLabimage 1D 2006 program (Kapelan GmbH, Halle, Ger-many). It was ensured that signals were in the linear range ofthe ECL detection system. Quantification of proteins betweenwild-type and knockout samples was performed from one gelusing either synaptophysin or synaptobrevin as internal stan-dards.

Measurement of MAO activities

Activities of MAO A and B were determined with the AmplexRed MAO assay kit from Molecular Probes, Invitrogen (Tu-bingen, Germany).

Immunofluorescence microscopy

Anesthesized mice were transcardially perfused by a fixativecontaining 4% formaldehyde, 0.05% glutaraldehyde, and0.2% picric acid and dissolved in 0.1 M phosphate buffer (pH7.4) for 30 min. The fixative was removed by a subsequentperfusion with 0.15 M sucrose in 0.1 M phosphate buffer (pH7.4). Immunocytochemistry was performed on cryostat sec-tions (20 or 30 �m), as described previously (17).

Cocaine-induced conditioned place preference andsensitization

The procedure of conditioning and testing of cocaine-in-duced conditioned place preference (CPP) was conducted asdescribed previously (22). During the course of this experi-ment, the effects of cocaine on acute locomotion and onlocomotor sensitization were also measured. CPP was con-

ducted in six white acrylic chambers (32 cm long, 16 cm wide,22 cm high) using two different types of floors, spaced metalbars (rod) or holed metal plate (hole), as conditioningstimulus (CS). The whole procedure consisted of 6 phases: 1preconditioning session and 8 conditioning sessions of 30min, and 1 session of 15 min for the preference test. Allsessions were monitored by a video-tracking system (Ethovi-sion 2.0; Noldus, Wageningen, The Netherlands) to deter-mine locomotion and spatial placement of each mouse each0.2 s across the whole session. On the first day (precondition-ing session), all subjects were placed inside the conditioningchamber with a distinctive floor. The conditioning phasestarted the next day and followed an unbiased pavlovianconditioning procedure in which mice belonging to thedifferent genotype-based groups were randomly assigned toone of two experimental conditions (CS�: rod� or hole�; orCS�: rod� or hole�). During the conditioning phase, micehad access to the entire apparatus, but only one type of floorwas presented. On CS� trials, each mouse received aninjection of cocaine (10 mg/kg i.p.), whereas in the CS�trials, animals were injected with equivalent volumes of saline.Immediately after injection, mice were placed in the condi-tioning apparatus containing the corresponding floor. Theeffects of an acute drug challenge on mice locomotion wereassessed by comparing the distance traveled (cm/30 min)during the first CS� and the first CS� trial. Furthermore,drug-induced behavioral sensitization was assessed by analyz-ing the changes in locomotion across the four CS� trials. Thepreference test took place 24 h after the last conditioningtrial. To conduct this test, both types of floor were presentedin each chamber, and mice were placed in the center of thechamber without any previous injection. The relative position(left vs. right) of each floor was counterbalanced. During the15 min of the preference test session, the time spent on CS�and CS� floor was assessed. The mice were killed immedi-ately after the end of the CPP test. Their brains werecollected, frozen in an isopentane solution for 2 min, andstored at –80°C for further analyses.

Data analysis

All biochemical and in vivo experiments were repeated atleast 3 times, and samples were run in triplicate. Data arepresented as means � sd (biochemical experiments) ormeans � se (HPLC and behavioral experiments), and asignificance level of P � 0.05 was applied throughout thisstudy. The behavioral data were analyzed by 1- or 2-wayANOVA, with repeated measures when necessary, followed byDuncan’s post hoc tests, when appropriate. CPP was analyzedby means of a paired Student’s t test for independent samples;for biochemical data, a Student’s t test for independentsamples was applied.

RESULTS

Lack of cocaine-induced behavioral sensitization andreduced dopamine D1 receptor signaling in Go2��/�

mice

Go2��/� mice show normal basal motor activity. Alter-ations in motor activity might become apparent follow-ing a pharmacological challenge of the dopaminergicsystem. An acute injection of cocaine increased activity,but no significant changes could be observed betweenthe genotypes (Fig. 1A). Repeated injections of cocaine

3738 Vol. 22 October 2008 BRUNK ET AL.The FASEB Journal

led to the development of behavioral sensitization inwild-type mice (Fig. 1A). In contrast, Go2��/� mice didnot develop cocaine-induced behavioral sensitization(Fig. 1A). The observed phenotypical changes werespecific for locomotor-stimulating effects because rein-forcing effects of cocaine measured by a conditionedplace preference test (23) revealed no differencesbetween the genotypes (Fig. 1B).

Cocaine mainly exerts its effect by inhibition of theplasma membrane transporter DAT, thereby increasingthe extracellular amount of dopamine. To exclude analtered effect of cocaine on plasma membrane andvesicular transporters in Go2��/� compared to wild-type mice, we examined the effects of cocaine ondopamine uptake, mediated either by DAT or VMAT2,into striatal synaptosomes. As expected, cocaine com-pletely blocked DAT-mediated dopamine uptake intointact synaptosomes but had no effect on VMAT2-mediated uptake following SLO permeabilization, withno difference between synaptosomes derived from thetwo genotypes (Fig. 2).

Reduced motor activity in Go2��/� mice suggestschanges at the level of G proteins or their upstreamdopamine receptors. Go� has been linked to dopaminereceptors; however, the effect of the splice variantsremained unclear (24). Motor activity mainly dependson functional D1 receptors. In the striatum, coupling ofdopamine D1 receptors to adenylyl cyclase is mediatedby Golf� and Gs�, and alterations in these G proteinscan affect locomotor responses of psychostimulants(25, 26). Therefore, we asked whether the levels ofGo1�, Golf�, and Gs� were altered in Go2��/� micecompared to wild-type animals.

First, the amounts of Go� subunits were analyzed byWestern blot analysis from synaptosomal preparations ofwild-type, Go� �/�, Go1��/�, and Go2��/� mice using amonoclonal antibody that recognizes both splice variants

(101.1), a Go2�-specific antibody (101.4) (15), and acommercially available Go� antibody (sc13532) that pref-erentially recognizes Go1�. Go2� represents on averageone-third of total Go� in mouse brain (Fig. 3A). Nosignificant up-regulation of Go1� was observed inGo2��/� mice (according to the Go1� antibody). Paral-lel quantification of actin, synaptophysin, and synaptobre-vin showed no changes and served as internal controls. Incerebellar slices, the immunosignal for Go� (using the101.1 antibody) was reduced by 30 to 40% in Go2��/�

mice compared to their wild-type littermates (Fig. 3A anddata not shown). Next, we compared the amounts ofGolf� and Gs� in striatal membrane preparations fromwild-type and Go2��/� mice. A significant decrease inboth G-protein subunits was observed in the knockoutanimals, whereas Gq� or G� subunits were not affected(Fig. 3B).

These data indicate a possible reduction in D1-receptor signaling in the knockout animals as a resultof lowered levels of Golf and Gs. The amounts of D1receptors showed a tendency toward being reducedthat was not significant, whereas D2 receptors did notdiffer between genotypes (Fig. 3C). These initial find-ings were changed when analyzing striatal membranepreparations of the two genotypes following cocainetreatment. Now the amount of D1 receptor was signif-icantly reduced, and the amount of D2 receptor wasincreased in Go2��/� mice (Fig. 4A), whereas thedifferences in the protein levels of Golf� and Gs� wereleveled out between the genotypes (Fig. 4B).

Reduced striatal dopamine, decreased amounts of TH,and increased amounts of VMAT2 in Go2��/� mice

The data described so far suggest that lack of cocaine-induced sensitization in Go2��/� mice is caused by an

Figure 1. Cocaine-induced behavior in wild-type (wt) and Go2��/� mice. A) Effects of acute and repeated intermittentinjections of cocaine on locomotor activity of wt (n16) and Go2��/� (n15) littermates. Single administration of cocaineincreased locomotor activity in both genotypes (cocaine F1, 2744.77, P0.0001); however, there were only slight, but notsignificant, differences between the genotypes (genotype F1, 271.43, P0.24). Subsequently, daily injections of cocaine led toan increase in locomotor activity (behavioral sensitization) in wild-type mice (Trial F3, 810.04, P0.0001). In contrast,Go2��/� mice showed no development of sensitization (genotype F1, 277.79, P0.01). *P 0.05. B) Both genotypes did notdiffer in the conditioned place preference induced by cocaine (T260.059, P0.95). The dashed line indicates the time spentin the drug-paired side before drug administration.


impairment of D1-receptor-mediated signaling and ashift toward D2-receptor-mediated effects. In additionto dopamine receptor and G-protein expression, wealso analyzed the overall amounts of dopamine, nor-adrenaline, and serotonin, including their metabolitesduring postnatal development in Go2��/� mice andtheir wild-type littermates. Analysis of whole brainsfrom days P2, 4, and 8 revealed no differences betweenwild-type and knockout littermates (Fig. 5A). However,on P12 and in adult Go2��/� mice, striatal dopaminelevels were significantly reduced, while levels of seroto-

nin and noradrenaline remained unchanged (Fig. 5B).Analysis of dopamine, noradrenaline, and serotonin inall other regions, including forebrain, hippocampus,bulbus olfactorius, hypothalamus, and cerebellum, re-vealed no differences between the genotypes (Supple-mental Table 1).

To explore reasons for the reduced dopamine levelsin the striatum, we examined whether synthesizing ordegrading enzymes or an alteration of the respectivetransporters for dopamine are changed in Go2��/�

mice. We found reduced amounts of TH in Go2��/�

mice (Fig. 6A), but no changes in MOA A or B activities(Fig. 6B). The reduction in TH expression was notaffected by cocaine (data not shown). As discussed inthe introduction, TH is up-regulated in VMAT-defi-cient mice, suggesting that compensatory regulatorymechanisms exist to rescue an impairment of vesicularstorage. The fact that TH is reduced in Go2�-deficientmice raised the question whether the reverse is alsotrue, i.e., whether VMAT2 is up-regulated when THlevels are low. Indeed, VMAT2 amounts were increasedin SV preparations from Go2��/� mice (Fig. 6C),which may compensate for the reduced dopaminesynthesis and allow for sufficient vesicular loading (seeabove). Comparable to the reduction of TH, up-regu-lation of VMAT2 was not affected by cocaine treatment(data not shown).

The data so far show that Go2��/� mice havediminished dopamine levels in the striatum. This re-duction disturbs dopaminergic signaling and leads toalterations in the development of cocaine-induced sen-sitization. We next asked whether the down-regulationof TH and the resulting reduction of striatal dopaminelevels are primarily caused by a dysregulation of vesic-ular dopamine storage.

Reduced vesicular monoamine uptake in Go2��/�

mice

We have previously shown that persistent G-proteinactivation by the GTP-analog GMP-P(NH)P diminishesvesicular uptake of monoamines, as seen both in per-meabilized synaptosomes and in isolated SVs. Thiseffect of GMP-P(NH)P was absent in preparations fromknockout animals and unchanged in preparations de-rived from heterozygous mice (Fig. 7A–C). These datashow that Go2� is the neuronal regulator for vesicularmonoamine storage, thereby confirming previous find-ings (17). However, the overall amount of monoamineuptake was slightly (�10%) but significantly reduced inGo2��/� mice, regardless of whether SLO-permeabil-ized synaptosomes or isolated SVs were analyzed (Fig.7D).

Next, we tested whether uptake was reduced becauseof a perturbation of the electrochemical proton gradi-ent ��H� that drives monoamine uptake by VMAT2.In addition to providing the driving force for uptake,the low intravesicular pH also traps monoamines in thevesicles and thus may contribute to dopamine storageby an independent mechanism (27). No differences

α α

α α

Figure 2. In vitro effects of cocaine on synaptosomes fromwild-type (wt) and Go2��/� mice. A) Synaptosomes from wtor Go2��/� mouse brains were loaded with [3H]dopaminefor 10 min at 37°C, either in the absence or presence of 5 �Mcocaine. Nonspecific uptake using 20 �M GBR 12909, aspecific inhibitor of DAT, was subtracted. As expected, co-caine completely blocked uptake into synaptosomes with nodifference between the genotypes. *P 0.05. B) Synapto-somes from both wild-type and Go2��/� mice were perme-abilized by SLO, and vesicular uptake was determined in theabsence or presence of 5 �M cocaine. Nonspecific uptake inthe presence of reserpine was subtracted. Vesicular uptakewas not affected by cocaine, and there was no differencebetween the genotypes. Data represent means � sd of 3determinations.


between wild-type and knockout animals were detectedwhen the pH dependence of monoamine uptake wasmeasured. Furthermore, the level of the vacuolar pro-ton ATPase was unchanged (Fig. 7E and data notshown).

Taken together, loss of Go2� diminishes dopamineuptake by SVs by an as yet unknown mechanism.

DISCUSSION

The �-subunit of Go2 is an important regulator ofdopamine homeostasis. Deletion of the protein results

in an imbalance of dopamine pools due to alteredlevels of TH and VMAT2 and reduced uptake by SVs. Asa consequence, cocaine-induced behavioral sensitiza-tion is abolished. These behavioral changes can beattributed to a diminished dopamine D1-receptor-me-diated signal transduction via Golf� and Gs�.

Mechanisms underlying behavioral consequences

Motor activity is regulated by neuronal circuits involv-ing the basal ganglia. Dopamine promotes motor activ-ity by activating the D1-receptor-mediated direct path-way and by blocking the D2-receptor-mediated indirect

Figure 3. Expression of trimeric G-protein subunits and D1- and D2-receptor proteins in wild-type (wt) and Go2��/� mice.A) Ratio of Go1� and Go2� expression. Blots: synaptosomal fractions (P2) from whole brains of Go1��/� (P20), Go��/� (P30),or Go2��/� (adult) and of the corresponding wt littermates were analyzed using the monoclonal antibody clone 101.1(recognizing both splice variants), the Go2�-specific clone 101.4 (only applicable for Western blot analysis), and the sc13532Go� antibody preferentially recognizing Go1�. Note that in Go��/� preparations, none of the antibodies gives a signal, whereassignals for clone 101.4 or sc13532 antibodies are almost or completely absent in Go2��/� or Go1��/� mice, respectively. Go2�represents one-third of Go� in P20 (left bars) or adult (right bars) brains, as revealed by quantification using synaptobrevin(Syb) as an internal standard. Go1� is not up-regulated in Go2��/� mice compared to wt littermates. Immunofluorescence:cerebellar sections were labeled by an antibody (clone 101.1), recognizing both Go� subunits (15). The mean immunofluo-rescence/100 �m2 of Go� subunits checked was significantly reduced in the molecular layer from 105.2 � 6.8 in wt to 72.8 �9.4 in Go2��/� mice and in the granular cell layer from 41.7 � 9.0 (wt) to 21.9 � 1.8 (knockout) (means � sd).B) Quantification was performed with synaptosomal preparations from striata of four pairs of wt and Go2��/� mice. Theamounts of Golf� and Gs�, but not of Gq� or G� subunits, in general (G�1–4), were reduced in Go2��/� mice. C) There wasno significant difference in the amounts of D1- and D2-receptor protein between wt and Go2��/� mice. For each quantificationin B and C, 5, 10, and 15 �g of protein of the respective sample was loaded. Quantification is given as relative optical density(OD) and was performed using synaptophysin (Syp) or synaptobrevin (Syb) as an internal control. Graphs show thequantification from the 10 �g protein load. Values are expressed as means � sd. *P 0.05.


pathway (Fig. 8). Actually, the D1 receptor has a lowaffinity, thus requiring high levels of dopamine to getactivated. Because of the high affinity of D2 receptors,the indirect pathway is blocked, even under restingconditions when striatal dopamine concentration is low(Fig. 8).

Expression of D1 receptor is decreased in Go2��/�

mice compared to wild-type animals, especially aftercocaine treatment, indicating a reduction of activitywithin the D1-receptor pathway. As a consequence, thedevelopment of cocaine-induced behavioral sensitiza-tion is absent. This is the first phenotypical change thatcan be directly linked to Go2 at the systemic level.Drug-induced behavioral sensitization is a dopamine-dependent, very robust phenomenon that has beenobserved across several species and that is frequentlyused as a test for the initiation of a compulsive, drug-seeking behavior (28). The observed phenotypicalchanges in Go2��/� mice were specific for locomotor-stimulating effects, because reinforcing effects of co-caine measured by a conditioned place preference test(23) revealed no differences in genotypes. In thiscontext, it is important to note that cocaine reinforce-ment is not strictly dependent on dopamine. For exam-ple, DAT knockout animals develop normal condi-tioned place preference following cocaine application.Cocaine-induced place preference is only abolished indopamine/serotonin transporter double knockouts(29). Furthermore, two recent meta-analyses showedthat cocaine-induced dopamine overflow is linearlyincreased over a dose range of 5–30 mg/kg (30).However, cocaine-induced place preference does notparallel this dose-response relationship (31), demon-strating that dopamine levels do not correlate well withdrug reinforcement measures.

Coupling of dopamine D1 receptors to adenylylcyclase is mediated by Golf� and Gs�, and reducedstriatal levels of these stimulatory G proteins havepronounced effects on locomotor response to psycho-stimulants (25, 26, 32). Psychostimulant-mediated ef-fects appear to rely more on the presence of Golf�,because genetic down-regulation of Golf�, but not ofD1 receptor, severely affects behavioral responses to

Figure 4. Expression of dopamine (D1, D2) receptor proteinsand G-protein �-subunits in wild-type (wt) and Go2��/� micefollowing cocaine treatment. Western blot analyses wereperformed from postnuclear membrane preparation fromfrozen striata of both genotypes using cocaine-treated animalspresented in Fig. 1. A) After treatment with cocaine, amountsof D1 receptor were lower and of D2 receptor were higher instriata of Go2��/� mice compared to wt littermates. B) Theexpression of Golf� and Gs� leveled out between the geno-types following cocaine treatment. For each quantification, 5,10, and 15 �g of protein of the respective sample was loaded;the graphs show the quantification from the 10-�g proteinload. Quantification is given as relative OD and was per-formed using either Syp or Syb as an internal control. Valuesare expressed as means � sd. *P 0.05.

Figure 5. Monoamine levels in brain areas of wild-type (wt) and Go2��/� mice. Serotonin, noradrenaline, and dopamine wereanalyzed in extracts from frozen brains or brain areas using HPLC. A) Until day P8, there are no significant differences in wholebrain monoamine concentrations between wt and Go2��/� mice. B) In the striatum of P12 and adult Go2��/� animals,dopamine amounts are decreased compared to wt littermates. Values represent means � se; n 6. *P 0.05.


cocaine (26). In line with this observation, Go2� �/�

mice exhibit reduced Golf� levels before cocaine treat-ment and show a lack in the development of behavioralsensitization following repeated intermittent injectionsof cocaine. However, cocaine treatment offsets thereduced Golf� and Gs� levels in Go2��/� mice, whileD1 receptors are decreased, suggesting that these Gproteins may also couple to other receptors. Indeed,following cocaine treatment, D2 receptors are higher inGo2��/� mice compared to wild type. The differencesin the amount of key molecules of dopamine signalingbetween wild-type and knockout animals under restingcondition and following cocaine treatment are summa-rized in Table 1.

Following cocaine treatment, the ratio between D1and D2 receptors is lower in Go2��/� compared towild-type mice. This finding may be explained in twoways. An increase in functional D2 receptors couldenhance the inhibition of the indirect pathway. Thismay provide a compensatory mechanism for a reducedstimulation of the direct pathway due to a lack of D1receptor signaling. An increase of the interaction of D2receptor with the NMDA receptor subunit NR2B afteracute cocaine administration has been described re-cently (33). By complexing NR2B, the D2 receptorprevents the interaction of NR2B with the Ca2�/cal-modulin-dependent protein kinase II and suppressesthe inhibitory indirect pathway (33). This mechanism

may amplify D2-receptor-mediated effects in Go2��/�

mice.Alternatively, increased amounts of D2 receptor in

Go2��/� mice may reflect accumulated nonfunctionalreceptors, which lack G-protein coupling. In this sce-nario, an increase in D2-receptor expression representsan unsuccessful compensating mechanism in Go2��/�

mice. In addition, D2 receptors may not be able tointeract with the NR2B receptor in the absence of Go2,thereby potentiating negative effects on motor activityfollowing cocaine stimulation.

In summary, the absence of cocaine-induced sensitiza-tion is due to an imbalance in the dopamine system,caused by changes in both D1- and D2-receptor signaling.

Mechanisms involved in Go2�-mediated regulation ofmonoamine storage

Go2 heterotrimers are localized to SVs (10, 34), includ-ing monoaminergic vesicles (17). We could show in thepast that Go2� or Gq� specifically modulate vesicularmonoamine uptake mediated by VMAT2 in neurons orplatelets, respectively (9, 11). In these in vitro ap-proaches, both G� subunits proved to be negativeregulators of monoamine uptake. The loss of a GMP-P(NH)P-mediated effect in Go2��/� mice clearlyproves that Go2� regulates vesicular monoamine up-

Figure 6. Dopamine synthesizing and metabolizing enzymes, as wellas transporters in wild-type (wt) and Go2��/� mice. A) Amounts ofTH and DAT were analyzed in synaptosomal preparations (wholebrain) from wt and Go2��/� mice. Amount of TH is reduced inGo2��/� mice. Values represent means � se; n 4. B) Determi-nation of MAO A � B activity in synaptosomes from wt andGo2��/� mice revealed no difference between the genotypes.Values represent the means � se; n 3. C) VMAT2 expression inwt and Go2��/� mice. Subcellular fractionation [H, homogenate;P2, synaptosomes; LP1, lysed synaptosomal pellet 1 at 29,000 g; LP2,lysed synaptosomal pellet 2 (SVs) at 360,000 g] of wt and Go2��/�

mice revealed that VMAT2 is enriched in the SV fraction (LP2) inboth genotypes. Surprisingly, more VMAT2 is expressed in the SVfraction of Go2��/� mice compared to wt littermates. For eachquantification, 5, 10, and 15 �g of protein of the respective samplewas loaded. Quantification is given as relative optical density (OD)and was performed using Syp as a reference. Graphs show thequantification from 10-�g protein load. Values represent means �se; n 4 animals/genotype. *P 0.05.


take. The absence of Go2 regulation decreases theoverall monoamine uptake and probably increases theamount of cytosolic dopamine. The permanently in-creased concentration of dopamine in the cytosol maylead to a reduction of the expression of TH andincrease the expression of VMAT2 in order to keep

cytosolic dopamine concentrations tolerable, while suf-ficient vesicular loading is ensured. A correlation be-tween VMAT2 and TH expression is also seen inVMAT2 deletion mutants that show an increase in THexpression (5). Thus, the ratio between cytosolic andvesicular dopamine appears to be a sensitive regulatorfor VMAT2 and TH expression and is altered inGo2��/� mice.

Surprisingly, enhanced VMAT2 expression in Go2�deletion mutants is accompanied by reduced mono-amine uptake, as well as striatal dopamine content invivo (35). This discrepancy may be explained by two, sofar, hypothetical and controversial models.

Figure 7. Go2� modulates [3H] monoamine uptake into VMAT2-containing SVs. A, B) SLO-permeabilized synaptosomes (A) or iso-lated SVs (B) of either wt or Go2��/� mice were subjected to [3H]serotonin uptake in the absence or presence of 5 or 100 �M ofGMP-P(NH)P, respectively. GMP-P(NH)P failed to modulate vesicu-lar uptake in knockout animals. C) SVs from either wt, heterozygous,or knockout animals were subjected to serotonin uptake in theabsence or presence of 100 �M GMP-P(NH)P. Values are expressedas the GMP-P(NH)P-mediated inhibition (%). Note that there is noinhibition in the knockout-derived SVs. D) [3H]serotonin uptake intopermeabilized synaptosomes or isolated SVs is moderately decreasedin Go2��/� animals compared to wt littermates. Values were takenfrom 10 different experiments, each run in triplicate. The reserpine-sensitive uptake of wt animals was set at 100%. Bars indicate means �sd. E) The amount of the vacuolar ATPase in various subcellularfractions analyzed using an antibody against the 116-kDa subunitrevealed an enrichment in the SV fraction (LP2), with no difference

between wt- and Go2��/�-derived membranes. Graphs show the quantification from 10-�g protein load. Data werenormalized to Syp; means � sd; n 4 animals/genotype. *P 0.05.

Figure 8. Effects of D1- and D2-receptor activation on motoractivity. Dopamine enhances motor activity both by promot-ing the direct pathway via the stimulatory D1 receptor signal-ing and by slowing down the indirect pathway via the inhib-itory D2-receptor signaling.

TABLE 1. Differences in the amount of key molecules of thedopaminergic system between wild-type and Go2��/� (knockout)mice

Molecule Untreated Following cocaine treatment

D1 receptor wtko wt koD2 receptor wtko wtkoGs� wt ko wtkoGolf� wt ko wtkoTH wt ko wt koVMAT2 wtko wtko

wt, wild type; ko, knockout.


As one possibility, VMAT2 in the presence of nonac-tivated Go2 may be constitutively more active, whereasthe activation of Go2� (mediated by the vesicularfilling) reduces the activity of the transporter. In thisscenario, the reduced uptake in Go2��/� mice wouldreflect the dissociation of an activated Go2� fromVMAT2 or from other vesicular compounds in wild-typemice. So far, we have not found an indication for adirect interaction between VMAT2 and Go2�, whenperforming immunoprecipitation using the respectiveantibodies. Besides VMAT2, other vesicular or vesicle-associated proteins may be bound by Go2 and releasedby an activation of the G protein subsequently reducingvesicular storage.

As a second possibility, Go2� regulates the set pointof vesicular filling. Monoaminergic vesicles exhibit non-specific leakage of transmitters not mediated byVMAT2. A high �pH provided by the proton pump andchloride transporters counteracts this leakage by trap-ping monoamines in their protonated form in thevesicular lumen. Increased amounts of VMAT2 increasequantal size, provided the driving force for uptake, thatis, the H� electrochemical gradient, is comparable. Aslight decrease in the H� electrochemical gradientcaused by loss of Go2� could increase the efflux ofmonoamines, which may be counteracted by an in-creased expression of VMAT2 (27). So the decreasedmonoamine uptake observed in SVs of Go2��/� micemay indicate a disturbance of the H� electrochemicalgradient. At the moment, we can only speculate on howand by which steps Go2� interferes with vesicularstorage, but its main target appears to reside on thevesicle.

The present findings show the effects of subtlechanges at the level of SVs for systemic functions. Inconclusion, Go2� is responsible for the fine-tuning ofvesicular dopamine content and is important for mod-ulating the effects of psychostimulants like cocaine andprobably also of other pharmacological compoundsthat do affect the striatal dopamine system.

This work was supported by the Deutsche Forshungsge-meinschaft (AH 67/3–3), the Forschungsforderung derCharite to G.A.H., by the German Federal Ministry of Educa-tion and Research (BMBF; Nationales Genomforschung-snetz) to R.S., and by the Intramural Research Program of theNational Institutes of Health to L.B. (Z01ES01643). Techni-cal assistance of Marion Mobes and Birgit Metze (bothCharite) is gratefully acknowledged.

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Received for publication April 3, 2008.Accepted for publication June 12, 2008.


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