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igma Receptor Agonists: Receptor Binding and Effectsn Mesolimbic Dopamine Neurotransmission Assessedy Microdialysis

inda Garcés-Ramírez, Jennifer L. Green, Takato Hiranita, Theresa A. Kopajtic, Maddalena Mereu,lexandra M. Thomas, Christophe Mesangeau, Sanju Narayanan, Christopher R. McCurdy,

onathan L. Katz, and Gianluigi Tanda

ackground: Subtypes of sigma (�) receptors, �1 and �2, can be pharmacologically distinguished, and each may be involved in substance-buse disorders. �-Receptor antagonists block cocaine place conditioning and �-receptor agonists are self-administered in rats thatreviously self-administered cocaine. Self-administration of abused drugs has been related to increased dopamine (DA) neurotransmission,owever, �-receptor agonist effects on mesolimbic DA are not fully characterized.

ethods: Receptor-binding studies assessed affinities of �-receptor ligands for �-receptor subtypes and the DA transporter; effects on DAransmission in the rat nucleus accumbens shell were assessed using in vivo microdialysis.

esults: Cocaine (.1–1.0 mg/kg intravenous [IV]), the nonselective �1/2-receptor agonist DTG (1.0 –5.6 mg/kg IV), and the selective

1-receptor agonist PRE-084 (.32–10 mg/kg IV) dose-dependently increased DA to �275%, �150%, and �160% maxima, respectively.TG-induced stimulation of DA was antagonized by the nonselective �1/2-receptor antagonist BD 1008 (10 mg/kg intraperitoneal [IP]) and

he preferential �2-receptor antagonist SN 79 (1–3 mg/kg IP), but not by the preferential �1-receptor antagonist, BD 1063 (10 –30 mg/kg IP).either PRE-084 nor cocaine was antagonized by BD 1063 or BD 1008.

onclusions: �-Receptor agonists stimulated DA in a brain area critical for reinforcing effects of cocaine. DTG effects on DA appear to beediated by �2-receptors rather than �1-receptors. However, DA stimulation by cocaine or PRE-084 does not likely involve �-receptors. The

elatively low potency on DA transmission of the selective �1-receptor agonist, PRE-084, and its previously reported potent reinforcingffects, suggest a dopamine-independent reinforcing pathway that may contribute to substance-abuse disorders.

ey Words: Abuse liability, addiction, cocaine, dopamine microdi-lysis, nucleus accumbens, reinforcing effects, sigma receptors

igma (�) receptors, initially proposed as opioid (1) and laterphencyclidine receptors (2), were demonstrated to representa unique binding site in the mammalian brain and peripheral

rgans (3–5) and are expressed throughout the central nervousystem. The �-receptor system has been implicated in a variety ofhysiological functions and disease states (6).

Pharmacological and structural studies distinguished two re-eptor subtypes: sigma 1 (�1) and sigma 2 (�2) (5,7). The �1-receptoras cloned and characterized as a 29 kDa single polypeptide havingo homology with any other known mammalian proteins (8). Inontrast, the �2-receptor is an 18 to 21 kDa protein that has not yeteen cloned (9). The �1-receptors possess two transmembraneomains and have been localized subcellularly at the endoplasmic

rom the Psychobiology Section (LG-R, JLG, TH, TAK, MM, AMT, JLK, GT),Medication Discovery Research Branch, Intramural Research Program,National Institute on Drug Abuse, National Institutes of Health, Depart-ment of Health and Human Services; and Japanese Society for the Pro-motion of Science (TH), Research Fellowship for Japanese Biomedicaland Behavioral Researchers at National Institutes of Health, Baltimore,Maryland; Escuela Nacional de Ciencias Biológicas (LG-R), InstitutoPolitécnico Nacional, Mexico City, México; and Department of MedicinalChemistry (CM, SN, CRM), University of Mississippi, Oxford, Mississippi.

ddress correspondence to Gianluigi Tanda, Ph.D., National Institute on DrugAbuse, National Institutes of Health, Department of Health and HumanServices, Intramural Research Program, Psychobiology Section, Medica-tions Discovery Research Branch, Building C, Room 321, 5500 Nathan ShockDrive, Baltimore, MD 21224; E-mail: gtanda@intra.nida.nih.gov.

eceived Mar 24, 2010; revised Jul 26, 2010; accepted Jul 28, 2010.

006-3223/$36.00oi:10.1016/j.biopsych.2010.07.026 Publish

reticulum. It has been suggested that, after stimulation by � li-gands, �1-receptors translocate to the plasma membrane (10).

Several classes of compounds, including neurosteroids, neuro-leptics, dextrobenzomorphans, and psychostimulants, such asmethamphetamine and cocaine, have been shown to bind to �-re-ceptors (11,12). The psychomotor stimulant and reinforcing effectsof cocaine are primarily mediated through stimulation of dopamine(DA) neurotransmission by inhibiting DA reuptake. However, itsmoderate affinity for �-receptors (13–15) suggests that cocaine-induced effects could involve actions at �1-receptor and/or �2-receptor subtypes (16). Consequently, studies have examined theeffects of �-receptor ligands on various effects of cocaine related toits abuse (see reviews [11,17,18]). For example, �-receptor antago-nists blocked the locomotor-stimulant effects of cocaine (19 –22)and the development of cocaine-induced locomotor sensitization(22,23). Further, �-receptor antagonists attenuated cocaine-in-duced place conditioning (24), though these compounds failed tosubstantially alter cocaine self-administration (25,26), indicatingthat �-receptors are not directly involved in the reinforcing effectsof cocaine. However, the preferential �1-receptor antagonistN-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethyl-amine (BD 1047) attenuated the reinstatement of cocaine-rein-forced behavior induced by cocaine-related stimuli, suggestingthat �-receptors might be involved in brain mechanisms and be-havioral activities that trigger cocaine use. Finally, several �-recep-tor antagonists can block or attenuate acute cocaine-induced tox-icities, such as convulsions and lethality (13). Together, thesestudies suggest that �1-receptors participate in the mechanisms ofaction of cocaine and that �-receptor antagonists might be poten-tially useful as medications for treating cocaine abuse, dependence,

and overdose (24,27).

BIOL PSYCHIATRY 2011;69:208–217ed by Elsevier Inc on behalf of Society of Biological Psychiatry

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Recently, Hiranita et al. (26) showed reinforcing effects of �-re-eptor agonists in rats trained to self-administer cocaine. Reinforc-

ng effects of most drugs of abuse appear to be mediated by stim-lation of DA transmission, and it has been repeatedly shown that

he acute effects of drugs abused by humans at doses able toaintain self-administration in rodents preferentially or selectively

ncrease DA transmission in the nucleus accumbens (NAc) shellompared with the NAc core or dorsal striatum (28 –30). Increases inA transmission induced by nonselective �-receptor agonists haveeen reported (31,32), though contrasting results and sometimesiphasic effects have been reported after systemic or local intrastri-tal administration (33–36). Thus, the goal of the present study waso test the hypothesis that intravenous (IV) administration of �-re-eptor agonists would increase DA transmission in the NAc shell. Tohis end, rats were implanted with microdialysis probes in the NAchell and effects of various IV doses of 1,3-di(2tolyl)guanidine (DTG),

nonselective �-receptor agonist, and 2-(4-morpholinoethyl)-1-henylcyclohexane-1-carboxylate (PRE-084), a selective �1-recep-

or agonist, on extracellular DA levels were compared with theffects of IV cocaine. In addition, the effects of the �-receptor ago-ists as well as cocaine were studied in combination with �-recep-

or antagonists to confirm the pharmacological specificity of theffect. The �-receptor ligands were also studied in binding experi-ents to test their affinities for �1-receptor and �2-receptor sub-

ypes, as well as the DA transporter (DAT), the main pharmacologi-al target of cocaine.

ethods and Materials

1 and �2 Receptor BindingFrozen whole guinea pig brains (minus cerebellum) were used

nd processed as already published (37) (see also Supplement 1 foromplete details). The guinea pig brains were preferred over ratrains due to their use as a standard for �-receptor binding becausef the relatively higher density of those receptors in that tissueompared with the rat (38). Ligand binding experiments were con-ucted for �1-receptor studies with 3 nmol/L [3H](�)-pentazocine

specific activity 28 Ci/mmol) and 8.0 mg tissue. Nonspecific bind-ng was determined using 10 �mol/L haloperidol. For �2-receptortudies, each tube contained 3 nmol/L [3H]1,3-di(2tolyl)guanidine[3H]DTG) (specific activity 48 Ci/mmol), 200 nmol/L (�)-pentazo-ine, and 8.0 mg tissue. Nonspecific binding was determined using00 �mol/L haloperidol.

AT BindingBrains from male Sprague-Dawley rats weighing 200 g to 225 g

Taconic Laboratories, Germantown, New York) were used, and ex-

able 1. Affinities of Various �-Receptor Ligands and Cocaine on Binding t3H](�)-Pentazocine, [3H]DTG, and [3H]WIN 35428, Respectively

ompound �1 Ki Value (nmol/L) �2 Ki Va

TGa 57.4 (49.3–66.7) 21.9 (13520 (2

RE-084 53.2 (44.8 – 63.2) 32,100 (2D 1008 2.13 (1.77–2.56) 16.6 (1

20,500 (9D 1047 3.13 (2.68 –3.65) 47.5 (3

55,300 (2D 1063 8.81 (7.15–10.9) 625 (4

53,700 (1ocaine 5190 (3800 –7060) 19,300 (1

�1, sigma 1; �2, sigma 2.

aValues for �2-receptors are Kd values obtained by homologous competition

periments were carried out as published previously (39). Bindingwas assessed with .5 nmol/L [3H](-)-2�-Carbomethoxy-3�-(4-flu-orophenyl)tropane, [3H]WIN 35,428 (specific activity 84 Ci/mmol)and 1.0 mg striatal tissue. Nonspecific binding was determinedusing .1 mmol/L (-)-cocaine hydrochloride.

In Vivo Microdialysis StudiesMale Sprague-Dawley rats (Charles River Laboratories, Wilming-

ton, Massachusetts) weighing 275 g to 350 g served as subjects, andall methods were as described previously (30,40 – 42) (see details inSupplement 1). Rats were doubly housed in a temperature- andhumidity-controlled room maintained on a 12-hour light/dark cycle(lights on: 07:00 –19:00 hours) and had free access to food andwater. Experiments were conducted during the light phase.

Surgical procedures were conducted under a mixture of ket-amine and xylazine (60.0 and 12.0 mg/kg intraperitoneal [IP], re-spectively) anesthesia. Rats were first implanted with a silastic cath-eter into the external jugular vein, with the catheter exiting the skinat the back between the shoulders. Rats were then placed in astereotaxic apparatus and implanted with concentric dialysisprobes aimed at the NAc shell (uncorrected coordinates from therat brain atlas of Paxinos and Watson [43]: anterior � �2.0 mm frombregma, lateral � �1.0 mm from bregma, vertical � �7.9 mm fromdura). The histological confirmations of probe placement are in-cluded in Supplement 1.

Experiments were performed on freely moving rats, about 22to 24 hours after probe implant. Dialysate was sampled every 10minutes and immediately analyzed. After stable DA values (lessthan 10% variability) were obtained for at least three consecu-tive samples (after about 1–2 hours), rats were treated with onedose of one of the test drugs (cocaine, DTG, PRE-084, or saline) orwith an antagonist (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide [BD 1008], N-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride [BD1063], or 6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one di-hydrochloride [SN 79]) followed 30minutes (or 10 minutes for SN 79) later by saline or one of the testdrugs. Different groups of naive rats were used for each differenttreatment. In another series of experiments in different groups ofnaive rats, after obtaining stable DA levels, microdialysis probeswere perfused with calcium-free Ringer’s solution (147.0 mmol/LNaCl, and 4.0 mmol/L KCl) for 60 minutes before IV injections of DTGor PRE-084 (44,45).

DA was detected in dialysate samples by high-performance liq-uid chromatography coupled with a coulometric detector (5200aCoulochem II, or Coulochem III, ESA, Chelmsford, Massachusetts).

eceptor and �2-Receptor Subtypes and to the DAT as Labeled with

mol/L) �2/�1 DAT Ki Value (nmol/L)

2.4),200)

.382 93,500 (80,000 –109,000)

– 44,700) 603 19,600 (17,600 –21,900)1.1)43,500)

7.79 2510 (2250 –2790)

1.4)–122,000)

15.2 3220 (2820 –3670)

7)–174,000)

70.9 8020 (7100 –9060)

–23,300) 3.72 76.6 (72.6 – 80.5)

o �1-R

lue (n

4.8 –357– 483,1003.0 –2640 –6.7– 65,00047– 876,5006,000

experiments.

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he average basal DA values in dialysates from the NAc shell in theresent experiments were 42.3 � 1.0 fmoles (�SEM) in a 10 �Lample, n � 171. No significant differences were found in basal DAoncentrations from the different experimental groups [analysis ofariance (ANOVA), F (30,140) � .516, p � .98].

rugsThe following were used: (-)-cocaine hydrochloride (.1–1.0 mg/kg

V); the mixed �1/2-receptor agonist, DTG (1.0–5.6 mg/kg IV); the selec-ive �1-receptor agonist, PRE-084 (.32–10 mg/kg IV); the preferential

1-receptor antagonist, BD 1063 (10–30 mg/kg IP); the mixed �1/2-eceptor antagonist, BD 1008 (10–30 mg/kg IP); and the preferential

2-receptor antagonist, SN 79 (1–3 mg/kg IP) (synthesized at the De-artment of Medicinal Chemistry at the University of Mississippi)

18,46). The preferential �1-receptor antagonist BD 1047 was used ininding studies. Sources of the compounds, their preparation, andose selection rationale are provided in Supplement 1.

ata AnalysisData from the in vitro binding studies were fit by nonlinear

egression using GraphPad prism 5.03 (GraphPad Software, Saniego, California). The fits of data for DTG displacement of [3H]DTGere globally fit to one- or two-site homologous competition mod-

ls. Because those data were better fit (F test) to a two-site model,ompetition studies of all drugs tested for displacement of [3H]DTGere compared for global fits to both one- and two-site models foreterologous competition, with data for the best fit reported. Thets of displacement data against the other ligands were consistentith a single site model, and therefore single affinities are reported.

Results of in vivo microdialysis studies were expressed as a per-entage of basal DA values, which were calculated as means of thehree consecutive samples preceding the test drug or saline injec-ion, with results presented as group means (�SEM). Statisticalnalysis (Statistica software, Stat Soft, Tulsa, Oklahoma) was carriedut using one-, two-, or three-way ANOVA for repeated measuresver time, with drug dose, time, and antagonist pretreatments as

actors. Significant results were followed by post hoc Tukey’s tests.hanges were considered significant when p .05.

esults

n Vitro Binding ExperimentsAs has been previously reported (47,48), DTG and PRE-084 had

igh affinity for �-receptors. The homologous competition of DTGodeled better for two than one binding site (Table 1). The high

ffinity binding Kd value of 21.9 nmol/L is comparable with thateported previously for �2-receptors in the papers cited above andndicates a twofold to threefold selectivity for �2 over �1-receptors,s also reported previously. In contrast, PRE-084 had a high affinityor �1-receptors (53.2 nmol/L) and was approximately 600-fold se-ective for this site over �2-receptors. Among the �-receptor antag-nists tested, BD 1008 had the highest affinity for both �1-receptorsnd �2-receptors; however, this compound was the least selectivef the antagonists tested. The �-receptor antagonist BD 1047 hadpproximately the same affinity for �1-receptors as BD 1008 butad lower affinity at �2-receptors. BD 1063 had lower affinity atoth �1-receptors and �2-receptors but was the most selective of

he three for the �1-receptor. None of the compounds except co-aine had high affinity for the DAT, and that affinity was approxi-ately 70-fold greater than its affinity for the �1-receptor (Table 1).

n Vivo Microdialysis ExperimentsAdministration of cocaine (.1–1.0 mg/kg IV) significantly in-

reased extracellular levels of DA within the first 10 minutes after

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injection to values approximating 275% of baseline levels at thehighest dose tested [two-way ANOVA gave main effects of cocainedose: F (2,16) � 3.73, p .05; time: F (6,96) � 17.8, p .001; and doseby time interaction: F (12,96) � 3.53, p .001]. Stimulation of DAlevels was transient, and levels of DA approached basal levelswithin 50 minutes after injection of the highest dose (Figure 1A).The effects of cocaine obtained in the first 30 minutes after injectionwere significant and directly related to dose (Figure 1B) [two-wayANOVA showed significant main effects of dose: F (2,16) � 4.58, p .05; time: F (3,48) � 22.7, p .001; and dose by time interaction:F (6,48) � 4.14, p .001].

Figure 1. Effects of intravenous (i.v.) administration of cocaine (.1–1.0 mg/kg) on dopamine (DA) levels in dialysates from the nucleus accumbens shellin rats. (A) shows the time course of effects of different cocaine doses; (B)shows the average change in DA levels, related to cocaine doses, obtainedin the first 30 minutes (min) after cocaine injection. Results are means, withvertical bars representing SEM, of the amount of DA in 10-min dialysatesamples, expressed as percentage of basal values (A) or of the average of DAvalues during the first 30 min after cocaine injection calculated as percent-age of basal values (B). Basal DA values (fmoles/sample) and group size (n)were 36.9 � 6.4 (4), 45.5 � 6.5 (8), and 44.8 � 6.8 (7) for cocaine .1, .32, and1.0 mg/kg, respectively. *p .05 compared with corresponding basal DA

values.

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L. Garcés-Ramírez et al. BIOL PSYCHIATRY 2011;69:208–217 211

Administration of the nonselective �-receptor agonist DTG1.0 –5.6 mg/kg IV) transiently and significantly increased DA levels,

ith values returning to the nonstimulated baseline levels withinpproximately 45 minutes after administration (Figure 2A) [two-ay ANOVA gave significant main effects of dose: F (2,18) � 6.76, .01; time: F (6,108) � 5.58, p .001; and dose by time interac-

ion: F (12,108) � 4.15, p .001]. Maximal effects of the highest (5.6g/kg) dose were observed within 10 minutes after injection,hereas the maximal effects of 3.2 mg/kg were observed at the

0-minute sampling time. The effects of DTG in the first 30 minutes,s with cocaine, show a linear increase over the dose range studiedFigure 2B). Maximal increases in DA concentration were obtainedt 5.6 mg/kg, which was approximately 50% of the effect obtainedith the highest dose of cocaine studied. At higher doses, DTGroduced signs of acute toxicity. At the doses tested, DTG was lessotent than cocaine. The selective �1-receptor agonist, PRE-084

.32–10 mg/kg IV), also increased DA levels and the effect wasignificant at the highest dose tested (Figure 2C) [two-way ANOVAave significant main effects of dose: F (4,17) � 3.94, p .02; time:(9,153) � 5.96, p .001; and dose by time interaction: F (36,153) �.32, p .001]. The maximal increase in DA levels obtained with the0 mg/kg dose of PRE-084 was about 170% of basal values at 10inutes after administration (Figure 2D).

The preferential �1-receptor antagonist, BD 1063 (10 –30 mg/kgP), administered alone induced a small, transient ( 30 minutes)onsignificant increase in DA levels of about 30% and a later smallecrease in DA levels to about 80% of baseline (Figure 3A,B; openquares). A 30-minute pretreatment with the preferential �1-recep-or antagonist BD 1063 did not significantly alter the time course of.6 mg/kg of DTG (Figures 3A,B) or its maximal effects (Figure 3C)

three-way ANOVA gave significant main effects of DTG treatment:(1,30) � 7.07, p .05; time: F (6,180) � 9.40, p .001; and the DTG

reatment by time interaction: F (6,180) � 11.4, p .001). In addi-

ion, nonsignificant effects of BD 1063 pretreatment: F (2,30) � 1.39;

and the interactions of DTG treatment by BD 1063 pretreatment:F (2,30) � .035; BD 1063 pretreatment by time: F (12,180) � 1.05; andDTG treatment by BD 1063 pretreatment by time: F (12,180) � .420were obtained].

The nonselective �-receptor antagonist, BD 1008 (10 mg/kg IP),had no immediate effects of its own (Figure 4A,B; open squares),though modest decreases in DA levels were obtained over the2-hour observation period to levels approximating 75% of basalcontrol values at 2 hours after injection. The BD 1008 pretreatmentsignificantly attenuated the effects of 5.6 mg/kg of DTG (Figure 4A)and completely blocked the effects of 3.2 mg/kg of DTG (Figure4B,C) [three-way ANOVA gave main effects of DTG treatment:F (2,31) � 3.39, p .05; BD 1008 pretreatment: F (1,31) � 15.5, p .001; time: F (6,186) � 10.0, p .001; and the interactions of DTGtreatment by time: F (12,186) � 2.97, p .001; BD 1008 pretreat-ment by time: F (6,186) � 4.20, p .001; and DTG by BD 1008 bytime: F (12,186) � 1.90, p .05; though a nonsignificant interactionof DTG treatment by BD 1008 pretreatment: F (2,31) � .103].

The preferential �2-receptor antagonist, SN 79 (18,46), adminis-tered alone (1–3 mg/kg IP) did not significantly modify extracellularlevels of DA up to 100 minutes after its administration (Figure 5A,B).However, SN 79 pretreatment dose-dependently and significantlyantagonized the effects of 3.2 mg/kg and 5.6 mg/kg of DTG (Figure5A,B). [Three-way ANOVA gave significant main effects of DTG:F (2,32) � 3.72, p .05; SN 79 pretreatment: F (1,32) � 12.4, p .005;time: F (3,96) � 10.9, p .001; and the interactions of DTG treat-ment by time: F (6,96) � 5.93, p .001; SN 79 pretreatment by time:F (3,96) � 2.80, p .05; and DTG treatment by SN 79 pretreatmentby time: F (6,96) � 2.50, p .05. The interaction of DTG by SN 79pretreatment was not significant: F (2,32) � 2.810, p � .07]. Over therange of DTG doses studied, 1.0 mg/kg of SN 79 significantly antag-onized the effects of DTG (Figure 5C).

A 30-minute pretreatment with the preferential � -receptor an-

Figure 2. Effects of intravenous (i.v.) administration of thenonselective �1/2-receptor agonist DTG (1–5.6 mg/kg) andthe selective �1-receptor agonist PRE-084 (.32–10.0 mg/kg)on dopamine (DA) levels in dialysates from the nucleus ac-cumbens shell in rats. (A) and (C) show the time course of theadministration of different doses of DTG and PRE-084, respec-tively; (B) and (D) show the changes in DA levels, related todrug doses, obtained during the first 30 minutes after DTG orPRE-084 injections, respectively. Basal DA values (fmoles/sample) and group size (n) were 41.3 � 5.4 (8), 42.2 � 6.4 (7),and 52.5 � 3.0 (6) for DTG 1.0, 3.2, and 5.6 mg/kg, respectively,and were 51.9 � 7.5 (4), 45.5 � 5.1 (5), 43.4 � 9.8 (5), 40.0 � 3.6(4), and 35.8 � 6.9 (4) for PRE-084 .32, 1.0, 3.2, 5.6, and 10mg/kg, respectively. *p .05 compared with correspondingbasal DA values. See Figure 1 legend for other details.

1

tagonist, BD 1063 (10 mg/kg IP), had no significant effects of its own

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212 BIOL PSYCHIATRY 2011;69:208–217 L. Garcés-Ramírez et al.

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igure 3. Effects of pretreatments with BD 1063 (10 –30 mg/kg intraperito-eal), a preferential �1-receptor antagonist, on DTG-induced stimulation ofopamine (DA) levels in dialysates from the nucleus accumbens shell in rats.

A) and (B) show the time course of effects of BD 1063 injected 30 minutesmin) before the most effective dose of DTG, 5.6 mg/kg intravenous (i.v.);C) shows the same data expressed as the average change in DA levelsbtained in the first 30 min after DTG injection. Basal DA values (fmoles/ample) and group size (n) were 44.6 � 7.9 (8) and 39.5 � 2.5 (6) for BD063 10 mg/kg � saline (Sal) or � DTG 5.6 mg/kg, respectively, and 47.9 �.6 (7) and 47.3 � 1.6 (5) for BD 1063 30 mg/kg � saline or � DTG 5.6 mg/kg,

espectively. *p .05 compared with corresponding basal DA values. Seeigure 1 legend for other details.

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Figure 4. Effects of pretreatment with BD 1008, a nonselective �1/2-receptor an-tagonist(10mg/kgintraperitoneal),onDTG-inducedstimulationofdopamine(DA)levels in dialysates from the nucleus accumbens shell in rats. (A) and (B) show thetime course of 10 mg/kg of BD 1008 effects on DA levels stimulated by differentdosesofDTG,5.6and3.2mg/kgintravenous(i.v.), respectively.BD1008wasadmin-istered 30 minutes (min) before DTG; (C) shows the same data expressed as theaverage change in DA levels, related to DTG doses, obtained during the first 30 minafter DTG administration. Basal DA values (fmoles/sample) and group size (n) were32.3 � 7.0 (6), 49.5 � 7.9 (6), and 49.0 � 8.1 (7) for BD 1008 10 mg/kg � saline, BD1008 10 mg/kg � DTG 5.6 mg/kg, and BD 1008 10 mg/kg � DTG 3.2 mg/kg,respectively. #p .05 compared with the corresponding time point of administra-

tion of BD 1008 10 mg/kg � DTG 5.6 mg/kg (A,C) or BD 1008 10 mg/kg � DTG 3.2mg/kg (B). See Figure 1 legend for other details.

aDaemtiteot

L. Garcés-Ramírez et al. BIOL PSYCHIATRY 2011;69:208–217 213

nd did not significantly modify PRE-084 –induced stimulation ofA transmission in the NAc shell (Figure 6A) [two-way ANOVA gavemain effect of time: F (9,54) � 10.181, p .001; but no significantffects of BD 1063 pretreatment: F (1,6) � .025; or BD 1063 pretreat-ent by time interaction: F (9,54) � .517]. Similarly, the nonselec-

ive �-receptor antagonist BD 1008 (10 –30 mg/kg IP) did not signif-cantly modify PRE-084 –induced stimulation of DA transmission inhe NAc shell (Figure 6B) [two-way ANOVA gave a significant mainffect of time: F (9,81) � 26.775, p .001; but no significant effectsf BD 1008 pretreatment: F (2,9) � .971; or BD 1008 pretreatment by

ime interaction: F (18,81) � .498].

As with PRE-084, the effects of cocaine on stimulation of DAtransmission were not significantly modified by pretreatments witheither 10 mg/kg of BD 1063 or BD 1008 administered 30 minutesbefore (Figure 7A,B) [two-way ANOVAs conducted individually foreach drug interaction gave significant main effects of time F (9,99) �19.609, p .001 (BD 1063 and cocaine) or F (9,99) � 11.357, p .001(BD 1008 and cocaine) but nonsignificant effects of BD 1063 pre-treatment: F (1,11) � .028 and its interaction with time: F (9,99) �.595; or BD 1008 pretreatment: F (1,11) � .001 and its interactionwith time: F (98,99) � .568].

Local perfusion of the NAc shell with a calcium-free Ringer’ssolution delivered by the microdialysis probe markedly decreasedextracellular DA levels compared with basal levels collected withnormal Ringer’s solution (Figure 8A,B). Decreases were apparentfrom the first sample after treatment, and the maximal decreasewas to about 15% to 20% of basal control values obtained withnormal Ringer’s solution (Figure 8A,B). Administration of vehicle(not shown), DTG (5.6 mg/kg), or PRE-084 (10 mg/kg) 60 minutesafter the start of local perfusion with calcium-free Ringer’s solutiondid not significantly modify the low extracellular levels of DA (Fig-ure 8A,B) [two-way ANOVAs conducted individually for eachdrug gave significant main effects of time F (14,84) � 75.0, p .001 (DTG) or F (14,84) � 106, p .001 (PRE-084) but nonsignifi-cant effects of DTG treatment: F (1,6) � .525 and its interactionwith time: F (14,84) � .760; or PRE-084 treatment: F (1,6) � .655and its interaction with time: F (14,84) � 1.73].

Discussion

A recent study with rodents on self-administration of the �-re-ceptor agonists DTG and PRE-084 (26) prompted the present studyof the effects of these drugs on DA levels in the NAc shell. The goalof the present study was to assess whether the effectiveness of DTGand PRE-084 in maintaining self-administration behavior wouldcorrespond with an increase in DA neurotransmission in the NAcshell after acute administration. This correspondence has beenfound with most drugs abused by humans (28 –30) and is consid-ered a biological substrate for drug abuse psychiatric disorders(49 –51). We observed that acute IV administration of DTG, a nonse-lective �-receptor agonist, as well as the selective �1-receptor ago-nist, PRE-084, dose-dependently increased DA levels in the NAcshell. We found no previously published reports on the effects ofselective �1-receptor agonists, like PRE-084, on DA transmission inlimbic areas. Contrasting effects of nonselective �-receptor ago-nists, like DTG, on DA neurotransmission in striatal areas have beenreported, and inconsistency of the results shown in those studiesmay be related to procedural differences (for example, to local

Figure 5. Effects of pretreatment with SN 79, a preferential �2-receptorantagonist (1–3 mg/kg intraperitoneal), on DTG-induced stimulation of do-pamine (DA) levels in dialysates from the nucleus accumbens shell in rats.(A) shows the time course of 1 to 3 mg/kg of SN 79 effects on DA levelsstimulated by DTG, 5.6 mg/kg intravenous (i.v.), injected 10 minutes (min)after SN 79, while (B) shows the effects of SN 79 pretreatment on DA levelsstimulated by DTG, 3.2 mg/kg i.v., under the same experimental conditions.(C) shows the same data expressed as the average change in DA levels,related to DTG doses, obtained during the first 30 min after DTG administra-tion. Basal DA values (fmoles/sample) and group size (n) were 37.5 � 4.1 (5),28.5 � 7.0 (6), 51.9 � 9.9 (6), 50.0 � 4.6 (6), and 42.3 � 4.2 (6) for SN 79 3mg/kg � saline (Sal), SN 79 3 mg/kg � DTG 5.6 mg/kg, SN 79 1 mg/kg �saline, SN 79 1 mg/kg � DTG 5.6 mg/kg, and SN 79 1 mg/kg � DTG 3.2mg/kg, respectively. #p .05 compared with the corresponding time pointof administration of SN 79 � DTG 5.6 mg/kg (A,C) or SN 79 � DTG 3.2 mg/kg

(B). See Figure 1 legend for other details.

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ersus systemic administration of drugs or anesthetized versuswake animals) (33–36).

The present study extends the validity of the correlation be-ween stimulation of DA levels and maintenance of self-administra-ion behavior to �-receptor agonists like DTG and PRE-084. How-ver, although the effects of the nonselective agonist DTG on DAransmission were obtained within a range of doses that approxi-

ated the doses per injection that maintained self-administrationn rats (26), the doses of the selective �1-receptor agonist PRE-084hat significantly increased DA levels were about 10 to 30 timesigher than the doses per injection in the self-administration study.

igure 6. Effects of pretreatment with BD 1063 (10 mg/kg intraperitonealIP]), a preferential �1-receptor antagonist, and BD 1008 (10 –30 mg/kg IP), aonselective �1/2-receptor antagonist, on PRE-084 –induced stimulation ofopamine (DA) levels in dialysates from the nucleus accumbens shell in rats.

A) shows the time course of effects of a 30-minute (min) pretreatment dose,0 mg/kg IP, of BD 1063 on DA levels stimulated by PRE-084, 10 mg/kg

ntravenous (i.v.); (B) shows the time course of the effects of 10 and 30g/kg IP doses of BD 1008 on DA levels stimulated by PRE-084, 10 mg/kg i.v.

asal DA values (fmoles/sample) and group size (n) were 38.8 � 2.2 (4),8.5 � 9.2 (4), and 33.1 � 6.4 (4) for BD 1063 10 mg/kg � PRE-084 10 mg/kg,D 1008 10 mg/kg � PRE-084 10 mg/kg, and BD 1008 30 mg/kg � PRE-0840 mg/kg, respectively. *p .05 compared with corresponding basal DAalues. See Figure 1 legend for other details.

n addition, PRE-084 was about threefold more potent than DTG in

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the self-administration study (26), whereas it was less potent thanDTG in increasing DA levels in the present study, suggesting thatdifferent mechanisms might underlie the dopaminergic and rein-forcing effects of these �-receptor agonists. To better understandthe mechanisms underlying these differences, studies of antago-nism were conducted with several �-receptor antagonists.

The previously reported preferential �1-receptor antagonist ef-fects of BD 1063 (13) were confirmed in the present binding studies.When tested in microdialysis studies, BD 1063, up to 30 mg/kg,failed to antagonize the effects of any dose tested of DTG or PRE-084 and cocaine. The lack of antagonism by the preferential �1-receptor antagonist BD 1063 suggests that �1-receptors are notinvolved in the acute effects of cocaine, PRE-084, and DTG on DAlevels in the NAc shell. In contrast, the relatively nonselective � -

Figure 7. Effects of pretreatment with BD 1063, 10 mg/kg intraperitoneal(A), a preferential �1-receptor antagonist, and BD 1008, 10 mg/kg intra-peritoneal (B), a nonselective �1/2-receptor antagonist, on cocaine-inducedstimulation of dopamine (DA) levels in dialysates from the nucleus accum-bens shell in rats. Both compounds were administered 30 minutes (min)before the intravenous (i.v.) injection of a .32 mg/kg dose of cocaine. BasalDA values (fmoles/sample) and group size (n) were 39.1 � 4.0 (5) and 39.2 �5.8 (6) for BD 1063 10 mg/kg � cocaine .32 mg/kg and BD 1008 10 mg/kg �cocaine .32 mg/kg, respectively. *p .05 compared with correspondingbasal DA values. See Figure 1 legend for other details.

1/2

receptor antagonist BD 1008 (20) significantly antagonized the

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L. Garcés-Ramírez et al. BIOL PSYCHIATRY 2011;69:208–217 215

cute effects of the nonselective �1/2-receptor agonist DTG on DAransmission in the NAc shell.

In the present binding studies, BD 1008 was characterized as theeast selective of the compounds assessed for affinity at �-receptorubtypes but nonetheless had higher affinity for �1-receptors than

2-receptors. We therefore examined the antagonism of the effectsf DTG by the preferential �2-receptor antagonist SN 79 (18,20,46).s with BD 1008, the DTG effects were antagonized by this novel

igure 8. Effects of removal of calcium (Ca��) from the perfusion Ringer’solution on DTG-induced and PRE-084 –induced stimulation of dopamineDA) levels in dialysates from the nucleus accumbens shell in rats. (A) and (B)how the time course of the different effects of administration of DTG orRE-084, respectively, on stimulation of DA levels during perfusion of theicrodialysis probes with normal Ringer’s solution or with a Ca��-free

inger’s solution. Microdialysis probes were perfused with Ca��-free Ring-r’s solution from 60 minutes before injection (�60 minutes) until the end ofhe experiment (solid line above the x axis). Solid circles show the lack offfects of DTG or PRE-084 on DA levels under these conditions, comparedith their significant effects on DA levels obtained with perfusion of regularinger’s solution (open circles). Basal DA values (fmoles/sample) and groupize (n) were 27.7 � 5.8 (4) and 42.3 � 2.8 (4) for Ca��-free Ringer’s solution

PRE-084 10 mg/kg and Ca��-free Ringer’s solution � DTG 5.6 mg/kg,espectively. See Figure 1 legend for other details. i.v., intravenous; min,

inutes.

referential �2-receptor antagonist. Thus, the results with SN 79

confirm the results with BD 1008, suggesting again that the effectsof DTG on DA levels in the NAc shell are due to its effects on�2-receptors.

In contrast to the effects obtained with DTG, the effects of theselective �1-receptor agonist PRE-084 were antagonized by neitherof the �-receptor antagonists examined (BD 1008 and BD 1063).Because of the high affinity and selectivity for �1-receptors and thevery low affinity for the DAT that we found for PRE-084 in bindingstudies, we therefore tested the possibility that its effects on DAlevels were the result of a nonspecific DA-releasing action of thedrug that was not related to a physiological activation of the DAsystem. When the NAc shell was perfused with a calcium-free Ring-er’s solution through the microdialysis probes, neither �-receptoragonist PRE-084 nor DTG effectively increased DA levels, suggest-ing that the increase in DA was the result of a physiological synapticactivity resulting in a vesicular, calcium-dependent DA release (45).Thus, the mechanism for the high-dose effects of PRE-084 on DAlevels is not known at this time but appears to be independent of itsactions at �-receptors and unlike the DAT-mediated actions of co-caine. Further, the high selectivity of PRE-084 for �1-receptors,which has not previously been reported, is consistent with a con-clusion that �2-receptors mediate the effects of the nonselective�-receptor agonist DTG on DA and suggests that �1-receptors areminimally involved in this effect, if at all.

Although the effects of DTG on DA levels appear mediated by�-receptors, the effects of cocaine do not, as neither of the �-recep-tor antagonists tested (BD 1008 and BD 1063) altered the acuteeffects of cocaine on extracellular DA levels. Cocaine has approxi-mately 70-fold higher affinity for the DAT than for �-receptors andamong �-receptors has selectivity for �1- over �2-receptors. As theeffects of the selective �1-receptor agonist PRE-084 indicate littleinvolvement of �1-receptors in its effects on DA, the present resultssuggest that the effects of cocaine on extracellular DA are minimallyinfluenced, if at all, by its affinity for �-receptors. Thus, although theacute effects of DTG and PRE-084 on DA transmission appear onfirst blush to be in agreement with their reinforcing effects, theireffects on DA are not mediated by similar mechanisms. Further,both �-receptor antagonists BD 1008 and BD 1063 blocked theself-administration of �-receptor agonists (26), indicating that theirreinforcing effects are mediated by �-receptors. Thus, previous andpresent results raise questions about the role of DA transmission inthe reinforcing effects of �-receptor agonists.

In conclusion, the present study shows that �-receptor agonistscan stimulate DA transmission in the NAc shell, a brain area that hasbeen involved in the mediation of reinforcing effects of drugsabused by humans. The �2-receptors appear to be primarily in-volved in the DA-stimulating effects of DTG, which may underlie thepreviously reported reinforcing effects of �-receptor agonists (26).Further, the present experiments excluded a direct involvement of�-receptors in the acute DA-stimulating effects of cocaine and PRE-084. Finally, the present results, along with previous findings thatboth DTG and PRE-084 have reinforcing effects (26), suggest adopamine-independent reinforcing pathway that may contributeto substance abuse disorders.

This work was supported by the Intramural Research Program ofthe National Institute on Drug Abuse (NIDA), National Institutes ofHealth, Department of Health and Human Services, and in part byNIDA Grant (DA023205 [CRM]). LG-R was recipient of a scholarshipfrom Consejo Nacional de Ciencia y Tecnología, México.

Animals used in the study were experimentally naive at the start ofexperiments. The animal housing facilities were fully accredited by the

Association for Assessment and Accreditation of Laboratory Animal

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are International and all procedures were conducted in accordanceith the guidelines of the Institutional Care and Use Committee of theIDA Intramural Research Program and the 1996 National Researchouncil and Guide for Care and Use of Laboratory Animals.

We thank Ms. Patty Ballerstadt for administrative support.CRM, CM, and SN are co-inventors of SN 79, one of the compounds

escribed in this manuscript. The patent for this invention is pendingnd belongs to the University of Mississippi; it has not been licensed tonyone. All other authors declare no biomedical financial interests orotential conflicts of interest.

Supplementary material cited in this article is available online.

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