Requirement for the EndocannabinoidSystem in Social Interaction ImpairmentInduced by Coactivation of Dopamine D1and D2 Receptors in the Piriform Cortex

Michelle Zenko,1 Yongyong Zhu,2 Eliyahu Dremencov,1 Wei Ren,3

Lin Xu,2 and Xia Zhang1*1Institute of Mental Health Research and Departments of Psychiatry and Cellular and Molecular Medicine,University of Ottawa, Ottawa, Ontario, Canada2Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology,Chinese Academy of Science, Kunming, People’s Republic of China3School of Life Sciences, Shaanxi Normal University, Xian, Shaanxi Province, People’s Republic of China

The dopamine receptor family consists of D1–D5receptors (D1R–D5R), and we explored the contri-butions of each dopamine receptor subtype in the piri-form cortex (PirC) to social interaction impairment (SII).Rats received behavioral tests or electrophysiologicalrecording of PirC neuronal activity after injection of theD1R/D5R agonist SKF38393, the D2R/D3R/D4R agonistquinpirole, or both, with or without pretreatment withdopamine receptor antagonists, D1R or D5R antisenseoligonucleotides, the cannabinoid CB1 receptor anta-gonist AM281, or the endocannabinoid transporterinhibitor VDM11. Systemic injection of SKF38393 andquinpirole together, but not each one alone, induced SIIand increased PirC firing rate, which were blocked byD1R or D2R antagonist. Intra-PirC microinfusion ofSKF38393 and quinpirole together, but not each onealone, also induced SII, which was blocked by D1Rantisense oligonucleotides or D2R antagonist but notby D3R or D4R antagonist or D5R antisense oligo-nucleotides. SII induced by intra-PirC SKF38393/quin-pirole was blocked by AM281 and enhanced byVDM11, whereas neither AM281 nor VDM11 aloneaffected social interaction behavior. Coadministration ofSKF38393 and quinpirole produced anxiolytic effectswithout significant effects on locomotor activity, olfac-tion, and acquisition of olfactory short-term memory.These findings suggest that SII induced by coactivationof PirC D1R and D2R requires the endocannabinoidsystem. VVC 2011 Wiley-Liss, Inc.

Key words: cannabinoid receptor; dopamine receptor;social interaction

The dopamine receptor family consists of D1-likereceptors, including both D1 receptor (D1R) and D5receptor (D5R), and D2-like receptors, including D2receptor (D2R), D3 receptor (D3R), and D4 receptor(D4R). Activation of dopamine receptors is known to

influence a variety of behaviors, including social beha-viors. Thus, it has been demonstrated that low and highdoses of dopamine receptor agonists enhance andsuppress aggression, respectively (for review see Korzanet al., 2006). While dopamine transporter terminatesdopamine transmission by retaking up released dopamineback into the presynaptic neuron, knockout of the dopa-mine transporter gene enhances aggressive behavior(Rodriguiz et al., 2004). Social withdrawal andgambling-like behavior occur after increasing synapticdopamine concentrations in the nucleus accumbens byreduced expression of dopamine transporter (Adrianiet al., 2010). Loss of prefrontal cortical dopamine axonsby intracerebral injection of 6-hydroxydopamine reducessocial interaction, i.e., the sum duration of sniffingelicited by the experimental rat toward a stimulating rat(Espejo, 2003). Amphetamine pretreatment impairedmating-induced partner preferences through activation ofD1R, but not D2R, in the nucleus accumbens (Liuet al., 2010). Systemic administration of D2-like receptoragonists has a more selective effect of disrupting maternal

M. Zenko and Y. Zhu contributed equally to this work.

Contract grant sponsor: Canadian Institutes of Health Research (to

X.Z.); Contract grant sponsor: Chinese Education Ministry (to X.Z.);

Contract grant sponsor: National Natural Science Foundation of China

(to L.X.).

Eliyahu Dremencov’s current address is Brains On-Line, BV, and the Uni-

versity of Groningen Institute of Pharmacy, Groningen, The Netherlands.

*Correspondence to: Xia Zhang, Institute of Mental Health Research

and Departments of Psychiatry and Cellular and Molecular Medicine,

University of Ottawa, 1145 Carling Ave, Ottawa, Ontario, K1Z 7K4,

Canada. E-mail: xia.zhang@rohcg.on.ca

Received 5 October 2010; Revised 19 November 2010; Accepted 19

November 2010

Published online 6 May 2011 in Wiley Online Library

(wileyonlinelibrary.com). DOI: 10.1002/jnr.22580

Journal of Neuroscience Research 89:1245–1258 (2011)

' 2011 Wiley-Liss, Inc.

potentiation than systemic administration of D1-likereceptor agonists (Muller et al., 2009). Contact quietingto the dam, but not to littermates, was disrupted byeither blockade or exogenous stimulation of D1-likereceptors. In contrast, the D2-like receptor agonist quin-pirole permitted or enhanced contact quieting. Activa-tion of striatal D2 receptors can enhance, but is not nec-essary for, contact quieting to the dam (Shair et al.,2009). Human studies also suggest that social status andsocial support are positively correlated with D2R/D3Rbinding in the striatum (Martinez et al., 2010), whereassocial desirability is positively correlated with D2R bind-ing in the hippocampus-amygdala complex (Cervenkaet al., 2010). The potent D2R/5-HT2 receptor antago-nist risperidone has also been shown to be effective intreating social interaction impairment (SII) in autisticchildren (Troost et al., 2005).

These lines of evidence suggest that dopaminereceptors are involved in social behavior. Because thepiriform cortex (PirC) not only contains a high densityof dopaminergic axons (Datiche and Cattarelli, 1996;Wilson et al., 2006) and D1R, D2R (Maltais et al.,2000), D3R (Ariano and Sibley, 1994), D4R (Wedzonyet al., 2000), and D5R (Khan et al., 2000) but also isinvolved in social behavior (Barton, 2006) and cognition(Sevelinges et al., 2008), we hypothesized that dopaminereceptors in the PirC make contributions to SII, i.e.,reduce the duration of sniffing elicited by the experi-mental rat toward a stimulating rat. In addition to dopa-mine receptors, the PirC also contains a moderatedensity of the cannabinoid CB1 receptor (CB1R)-containing immunoreactive neurons and nerve fibers,and the hippocampus and the cerebellum contain a highdensity (Tsou et al., 1997; Moldrich and Wenger, 2000).Therefore, considering that social play behavior isdecreased by systemic administration of the CB1Ragonist WIN55,212-2 (Trezza and Vanderschuren,2008a,b, 2009), we further hypothesized that the dopa-mine receptor system participates in social interactionbehavior by interaction with the PirC endocannabinoidsystem in adult rats. To test these hypothesis critically,we have conducted a series of behavioral tests and elec-trophysiological recording of PirC neuronal activity aftersystemic or intra-PirC injection of the D1R/D5R ago-nist SKF38393, the D2R/D3R/D4R agonist quinpirole,or both, with or without pretreatment with dopaminereceptor antagonists, D1R or D5R antisense oligo-nucleotides, the CB1R antagonist AM281, or the endo-cannabinoid transporter inhibitor VDM11.

MATERIALS AND METHODS

Animals

Adult male Fisher-344 rats or Sprague-Dawley ratsweighing between 225 and 250 g (Charles River) were usedfor all behavioral tests. Adult male Long-Evans rats weighingbetween 225 and 250 g (Charles River) were used as thestimulus rats in the social interaction test, because our pilotexperiment had shown that the testing rat spent longer time

in exploring a different strain of stimulus rat than the same strainof stimulus rat. Animals were individually housed upon arrivaland given at least 5 days to habituate to the vivarium beforetesting or surgery. Standard conditions, including a 12-hr light/dark cycle (lights on at 7:00 AM and off at 7:00 PM), a tempera-ture between 228C and 238C, and humidity levels of 35–50%,were maintained in the vivarium at all times. Cages were linedwith wood chips, and free access to food and water was granted,unless otherwise noted. All procedures were performed inaccordance with the guidelines established by the CanadianCouncil on Animal Care as approved by Animal Care Commi-ttees of the University of Ottawa Institute of Mental HealthResearch and the Chinese Academy of Sciences.

Drugs

The D1R/D5R agonist SKF38393, the D2R/D3R/D4R agonist quinpirole, the D1R/D5R antagonist SCH23390,the D2R/D3R antagonist raclopride, and the D1R/D2Rantagonist LEE300 were purchased from Sigma-Aldrich (St.Louis, MO). The selective D2R antagonist L-741,626, theselective D3R antagonist GR 103691, the selective D4R anta-gonist L-745,870, the CB1R antagonist AM281, and the endo-cannabinoid (anandamide) transporter inhibitor VDM11 werepurchased from Tocris Bioscience (Ellisville, MO).

Behavioral Tests

The social interaction test was conducted as describedelsewhere (Shah and Treit, 2003; Kwon et al., 2006). Briefly,on the first testing day, adult male Fisher-344 rats or Sprague-Dawley rats were placed in their home cages in a behavioraltesting room for a 5-min habituation period. Then, in thetwo-trial protocol, a stimulus rat was introduced into thehome cage for a 5-min trial (trial 1), followed 24 hr later by asecond trial (trial 2) with the same stimulus rat. In the three-trial protocol, rats received a 5-min trial (trial 1), followed2 hr later by another 5-min trial (trial 2) or 24 hr later byanother 5-min trial (trial 3; trials 2 and 3 were independentgroups). Animal behavior in each trial was recorded by videocamera and analyzed by raters blind to experimental treatment.In accordance with previous studies (Espejo, 2003; Shah andTreit, 2003; Kwon et al., 2006), the time spent sniffing thestimulus rat, from head to tip of tail, was recorded. Sniffingwas characterized by the proximity of the nose of the testingrat to the stimulus rat (touching or very near touching), and afaint but visible wiggle of the nose of the testing rat.

To validate the results obtained with the social inter-action test, another behavioral test, i.e., nest formation test,was conducted as described previously (Lijam et al., 1997;Moretti et al., 2005). Briefly, nesting material, a 10 3 10 cm2

piece of cotton, was introduced in the rat home cage. After90 min, the quality and height of the nest were recorded.Nest quality was measured on the following scale: 0, nestingmaterial unmodified; 1, flat nest with partially shredded nest-ing material; 2, shallow nest with shredded material but lack-ing fully formed walls; 3, nest with well-developed walls; and4, nest in the shape of a cocoon with partial or complete roof.

The olfactory discrimination test was performed asdescribed elsewhere (Prediger et al., 2005). Briefly, a modified

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home cage was built to allow the separation of used and newbedding. A clear plexiglass insert (1.5 inches in height) was slidinto the center of a cage and held into place by a plexiglasstrack mounted on either side of the cage, thus separating thecage into two areas. One side of the modified home cage wasfilled with new bedding up to the level of the insert, and theother side was filled with the bedding from the rat home cagethat had not been changed for at least 4 days prior to testing,and thus was soiled. A 5-min trial was recorded with a videocamera, during which rats had unrestricted access to either sideof the modified home cage. The percentage of time spentsniffing the new beddings was the measure of interest. Thestarting position for each rat was randomly assigned.

The locomotor activity test and open-field test wereconducted as described by Ji et al. (2006). Briefly, the appara-tus, made of opaque plexiglass, had a floor of 18 inches 3 18inches (divided by black lines into 36 squares of 3 inches 3 3inches), and walls were 6 inches high. Each rat was randomlyplaced in a corner of the open field, and the number of totalsquares crossed by the hind legs and the total time spent rear-ing were evaluated over a 6-min trial. Also evaluated was theproportion of time spent in center squares vs. the time spentat the perimeter as a measure of anxiety.

The elevated plus maze test was performed as describedelsewhere (Ji et al., 2006). Briefly, the maze, constructed ofhard wood, consists of two opposite open arms (50 cm 3 10cm) and two opposite enclosed arms (50 cm 3 10 cm 3 40cm), with the open arms surrounded by a 1-cm-high Plexiglasledge, set up 50 cm above the floor. The junction area of thefour arms (central platform) measures 10 cm 3 10 cm.Immediately after the locomotor activity test, the rat wasremoved from the activity box and placed onto the centralplatform of the plus maze, facing an open arm, and allowed toexplore the apparatus for 5 min. Times spent in and entriesinto open arms and enclosed arms were recorded. An armentry was recorded when all four paws of the rat were in thearm. Measures of anxiety included entry ratio, the ratio ofclosed arm to open arm entries, and dwell ratio, the ratio ofdwell time in the closed arms to the dwell time in all fourarms, with higher values indicating higher levels of anxiety.

Animal Treatment

Eight experiments were conducted (Table I). In experi-ment 1, adult rats were subjected to the social interactiontest and nest formation test 25 min after receiving vehicle(control), SKF38393 (3 mg/kg, i.p.), quinpirole (2 mg/kg,i.p.), or both SKF38393 and quinpirole. The SKF38393 andquinpirole dosage was chosen based on the previous findingsthat SKF38393 at a dosage of 3 mg/kg (i.p. or s.c.) blockedbehavioral sensitization of rats (Shuto et al., 2006) andincreased the behavioral responsiveness in unilaterally dopa-mine-depleted rats (Van De Witte et al., 2002) and that Fosexpression in the rat prefrontal cortex, nucleus accumbens,and septal nucleus induced by the D2R antagonist sulpiridewas reversed by quinpirole at a dosage of 2 mg/kg (i.p.; Moet al., 2005).

In experiment 2 (Table I), adult rats were injected withthe D1R/D5R antagonist SCH23390 (0.5 mg/kg, i.p.), the

D2R/D3R antagonist raclopride (1.0 mg/kg, i.p.), or vehicle.Thirty minutes later, the same rats were given SKF38393 (3mg/kg, i.p.) and quinpirole (2 mg/kg, i.p.), followed 25 minlater by the social interaction test with the two-trial protocol.We determined these doses of SCH23390 and racloprideaccording to previous reports: SCH23390 pretreatment (0.5mg/kg, i.p.) antagonized rotation and c-Fos expression in thestriatum induced by the D1R/D5R agonist SKF83822(Wirtshafter, 2007), and raclopride at the dosage of 1.0 mg/kg(i.p.) significantly reduced rotation of ci2 mutant rats(Schirmer et al., 2007).

In experiment 3, all compounds were administeredintravenously (i.v.) during recording of individual PirCneurons. SKF38393 and quipirole were administered, alone orin combination, at cumulative doses of 0.1–1.0 or individualdoses of 1.0–3.0 mg/kg, to test their dose-response effects onthe firing activity of PirC pyramidal neurons. The D1R/D2Rantagonist LEE300 and the D2R antagonist L-741,626 (1 mg/kg each) were administered after the agonists in an attempt toreverse the effect of SKF38393 and quinpirole on the firingactivity of PirC pyramidal neurons.

Although a coadministration of SKF38393 (5 lg/side)and quinpirole (10 lg/side) into the rat nucleus accumbensinduced turning behavior (Saigusa et al., 1995; Moribe et al.,2005), in experiment 4 we tested two doses of SKF38393 (30lg/1.0 ll/side or 5 lg/0.5 ll/side) and quinpirole (20 lg/1.0ll/side or 10 lg/0.5 ll/side). Adult rats were given a bilateralintra-PirC microinjection of SKF38393 (30 or 5 lg/side),quinpirole (20 or 10 lg/side), both SKF38393 and quinpirole,or vehicle, followed 25 min later by the social interaction testwith the two-trial protocol.

In experiment 5, both D1R and D5R share the sameagonists and antagonists, so we employed the antisense oligo-deoxynucleotide strategy for a selective suppression of D1Rand D5R mRNA expression. The antisense (30 to 50)-oligo-deoxynucleotides (D1R: 50-CCG TTC AGT GGA TCCATT GG-30; D5R: 50-CAG CAT GTC GCG CTG AGT-30;Apostolakis et al., 1996; Filip et al., 2000) designed to coversymmetrically the translation initiation sites of the targetsequences have been shown to suppress D1R and D5Rexpression effectively (Apostolakis et al., 1996), respectively,to produce behavioral changes (Apostolakis et al., 1996; Filipet al., 2000). A scrambled oligonucleotide (50-ATA CTTCAC GCC GAT GG-30) was designed that had thermo-dynamic properties similar to those of antisense oligonucleo-tides (Filip et al., 2000). Phosphorothiolated oligonucleotideswere synthesized, lyophilized, and redissolved in sterile dis-tilled water. With naıve rats as control, experimental ratsreceived a bilateral intra-PirC microinjection of D1R or D5Rantisense oligonucleotides or control oligonucleotides (2.5nmol/0.5 ll/side; Filip et al., 2000), the selective D2R antag-onist L-741,626 (5 lg/0.5 ll/side; Wei et al., 2009), theselective D3R antagonist GR 103691 (100 nmol/0.5 ll/side;Clemens and Hochman, 2004), the selective D4R antagonistL-745,870 (1 lg/0.5 ll/side; Succu et al., 2007), or vehicle.One day later (for antisense oligonucleotide injection) or10 min later (for antagonist or vehicle injection), the samerats were given a bilateral intra-PirC microinjection of bothSKF38393 (5 lg/0.5 ll/side) and quinpirole (10 lg/0.5 ll/side),

Dopamine Receptor and Social Interaction 1247

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followed 25 min later by the social interaction test with thetwo-trial protocol.

Experiment 6 used the selective cannabinoid CB1Rantagonist AM281 to block the action of endocannabinoids(the CB1R antagonist AM251 was not used because it is alsoa GPR55 agonist); VDM11 was employed as an inhibitor ofthe cellular uptake of the endocannabinoid anandamide,thereby potentiating its actions (Trezza and Vanderschuren,2009). Six groups of adult rats underwent the social inter-action test (with a two trial protocol) at 25 min after an intra-PirC injection of AM281 (5 pmol/0.5 ll/side; Padley et al.2003), VDM11 (10 pmol/0.5 ll/side; de Lago et al., 2004;D’Argenio et al., 2006; Clarke et al., 2008), or vehicle, withor without both SKF38393 (5 lg/0.5 ll/side) and quinpirole(10 lg/0.5 ll/side) or vehicle (Table I).

Experiment 7 examined the effects of systemic andintra-PirC coadministration of SKF38393 and quinpirole onolfaction and olfactory learning and memory (Table I). The

olfactory discrimination test was conducted in adult rats25 min after receiving either an i.p. injection of SKF38393 (3mg/kg), quinpirole (2 mg/kg), both SKF38393 and quin-pirole, or vehicle (control) or a bilateral intra-PirC injectionof both SKF38393 (5 lg/0.5 ll/side) and quinpirole (10 lg/0.5 ll/side) or vehicle. To study the acquisition of olfactorymemory, adult rats received either an i.p. injection ofSKF38393 (3 mg/kg) and quinpirole (2 mg/kg) together orvehicle or a bilateral intra-PirC injection of SKF38393 (5 lg/0.5 ll/side) and quinpirole (10 lg/0.5 ll/side) together orvehicle 25 min before the first or third trial of the social inter-action test.

Finally, experiment 8 used the locomotor activity test,open-field test, and elevated-plus maze test 25 min afterSKF38393 (3 mg/kg, i.p.), quinpirole (2 mg/kg, i.p.), bothSKF38393 and quinpirole, or vehicle (control). An additionalsix groups of rats were subjected to locomotor activity test 1)25 min after an i.p. injection of vehicle, the D1R/D5R

TABLE I. Diagram of Animal Treatment*

Exp Treatment 1 Treatment 2 Behavioral tests

1 i.p.

SKF38393 Social interaction test

Qinpirole ?SKF38393 1 qinpirole Nest formation test

Vehicle

2 i.p. i.p.

D1R/D5R antagonist

D2R/D3R antagonist ? SKF38393 1 qinpirole ? Social interaction test

Vehicle

3 i.v. i.v.

D1R/D2R antagonist SKF38393 Record the firing rate of

D2R antagonist ? Qinpirole ? PirC pyramidal neuron

SKF38393 1 qinpirole

4 intra-PirC

SKF38393

Qinpirole ? Social interaction test

SKF38393 1 qinpirole

Vehicle

5 intra-PirC intra-PirC

D1R antisense SKF38393

D2R antagonist ? Qinpirole ? Social interaction test

D3R antagonist SKF38393 1 qinpirole

D4R antagonist Vehicle

D5R antisense

6 intra-PirC

Vehicle 1 SKF38393 1 qinpirole or vehicle 1 vehicle

AM281 or VDM11 1 SKF38393 1 qinpirole,

AM281, or VDM11

? Social interaction test

7 i.p. SKF38393, qinpirole, SKF38393 1 qinpirole Olfactory discrimination

or vehicle ? test

intra-PirC SKF38393 1 qinpirole Social interaction test

8 i.p. SKF38393, qinpirole, SKF38393 1 qinpirole ? Locomotor,open filed or

or vehicle elevated plus maze test

i.p. Vehicle 1 SKF38393 1 qinpirole

D1R/D5R antagonist 1 SKF38393 1 qinpirole

D2R/D3R antagonist 1 SKF38393 1 qinpirole ? Locomotor test

intra-PirC D1R antisense 1 SKF38393 1 qinpirole

Vehicle 1 SKF38393 1 qinpirole

D2R antagonist 1 SKF38393 1 qinpirole

*Exp, experiments (n 5 6 to 10 per group); intra-PirC, injection into the piriform cortex; i.p., intraperitoneal injection.

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antagonist SCH23390, or the D2R/D3R antagonist raclopridetogether with SKF38393 and quinpirole; 2) 24 hr after anintra-PirC injection of D1R antisense oligonucleotidetogether with SKF38393 and quinpirole; or 3) 10 min after anintra-PirC injection of vehicle or the D2R antagonist L-741,626 together with SKF38393 and quinpirole.

Intra-PirC Cannulation

Intra-PirC cannulation was conducted with animalsunder isoflurance anesthesia. After placing rats onto the stereo-taxic frame, the skull was exposed and two holes were made,followed by implantation of bilateral cannulae (26 Ga) intothe PirC (11.7 AP, 13.9 ML, 27.6 DV). Then, cannulaewere secured to the skull with three jewelers’ screws and den-tal acrylic. Rats were allowed a 10-day recovery before testingbegan. All drugs were freshly prepared. A total of 1 or 0.5 llsolution was administered through each cannula within10 min, and the cannulae were held in place for 30 sec afteragonist administration.

After behavioral tests, rats receiving cannulation wereanesthetized with 40% chloral hydrate and perfused intra-cardially with 200 ml of 0.1 M phosphate-buffered saline,followed by 400 ml of 4% buffered paraformaldehyde asdescribed elsewhere (Zhang et al., 2001; Ji et al., 2006). Afterdecapitation, brains were removed and immersed in 4%buffered paraformaldehyde for 1 day and then in 40% sucrosesolution for 2 days. Brains were cut on a sliding microtome(Leica) at a thickness of 40 lm. The sections were mountedonto glass slides and then stained with cresyl violet for theidentification of cannula location in the brain. Only those ratswith the bilateral location of cannula tips within the PirC(11.0 22.2 AP) were used for further data analysis.

Single-Unit Electrophysiological Recording

Electrophysiological recording of PirC neuronal activitywas conducted as described by Ji et al. (2006). Briefly, ratswere anesthetized with chloral hydrate (400 mg/kg, i.p.) andmounted in a stereotaxic apparatus (David Kopf Instuments,Tujunga, CA). Supplementary doses of chloral hydrate weregiven to maintain constant level of anesthesia. Body tempe-rature was maintained at 378C throughout the experimentsutilizing a water heating pad. A 2-mm burr hole was drilledin the skull, and a recording electrode was placed into thePirC (11.7 AP, 13.9 ML, 27.6 DV) for recordings of thePirC pyramidal neuronal activity. Posterior PirC was chosenbecause the firing activity of its neurons is independent of thebreathing cycle (Litaudon et al., 2003). Extracellular unitaryrecordings were conducted with single-barreled glass electro-des filled with a 2 M NaCl solution. Their impedance rangewas between 4 and 6 MX. Single-unit recordings were carriedout using amplitude discrimination. Data were acquitted withSpike-2 software (CED, Cambridge, United Kingdom). Crite-ria for identifying a PirC neuron are both a breathing-relatedrhythmic activity and an odor-evoked change of activity(Bouret and Sara, 2002; Litaudon et al., 2003; Illig, 2007).Specifically, spontaneous activity was synchronized withbreathing, and odor stimuli (ethyl alcohol) evoked a synchron-ized excitatory response. After identification of a PirC neuron,

its firing activity was recorded for at least 2 min to establish abaseline measure of firing rate. Changes in firing rate werecalculated by averaging the effects of the drugs for a 2-minperiod when the effects of drug were visible and comparingthem with the mean of the predrug baseline. The effects ofthe compound on PirC neuronal firing activity were expressedas percentage 6 SEM of baseline. Rats were perfused withsaline, followed by buffered formalin. Brain sections (40 lm)were cut with a sliding microtome. The sections were stainedwith cresyl violet for the examination of the placement ofrecording electrodes.

Data Analysis

Results are reported as mean 6 SEM. Statistical analysisof the data was performed by using a paired-samples t-test (tocompare the effects of the same treatment on trials 1 and 2),one-way ANOVA (to compare the effects within trial 1 or 2),or one-way ANOVA for repeated measures (to compare theeffects between trials 1 and 2 or multiple effects in electro-physiological study), followed by LSD post hoc test. Statisticalsignificance was set at P < 0.05.

RESULTS

Coactivation of Dopamine Receptors ImpairsSocial Behavior

An i.p. injection of the D1R/D5R agonistSKF38393, the D2R/D3R/D4R agonist quinpirole,both SKF38393 and quinpirole, or vehicle produced asignificant group effect in the first trial of the socialinteraction test (F3,28 5 7.929, P < 0.01, n 5 8/group).LSD post hoc test further showed that both control andSKF38393-treated rats exposed to a novel conspecific ratexhibited similar behavior of approaching and sniffing(P 5 0.281), but such initial social interaction was pro-foundly increased and decreased in rats receiving a sys-temic injection of quinpirole alone and both SKF38393and quinpirole, respectively (P < 0.05; Fig. 1A). Whenboth the first and the second trials were assessedtogether, there was a significant group effect (F7,56 515.405, P < 0.001, n 5 8/group), suggesting a signifi-cant change in social interaction in the second trial thatwas conducted 1 day after the first trial. Specifically,when reexposed to the same stimulus rat after 1 day,vehicle-, SKF38393-, and quinpirole-treated rats exhib-ited a typical decrease in social interaction comparedwith the initial interaction (P < 0.001; Fig. 1A), indica-ting recognition of the familiar stimulus rat and normalsocial learning. However, rats receiving a systemic injec-tion of SKF38393 and quinpirole together did notdecrease their interaction in the second trial (P 5 0.398;Fig. 1A), indicating impaired social learning or inabilityto identify the stimulus rat because of the low level ofinitial interaction.

Then, we examined nest formation, a test for homecage behavior (Lijam et al., 1997; Moretti et al., 2005). Asignificant group effect was also observed after a systemicinjection of vehicle, SKF38393, quinpirole, or bothSKF38393 and quinpirole (F3,28 5 12.215, P < 0.01,

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n 5 8/group). Similarly to the case in the social inter-action test, SKF38393-treated rats showed typical nestformation similar to that of the vehicle-treated rats (LSD

post hoc test, P 5 0.371), whereas rats receiving eitherquinpirole alone or SKF38393 and quinpirole togetherexhibited increased or decreased nest-forming activity (P <0.05; Fig. 1B). Because both social interaction test and nestformation test showed consistent results, we conductedsocial interaction test only in the following experiments.

Next, we explored the effects of pretreatment withdopamine receptor antagonists on social interactionimpairment induced by coadministration of SKF38393and quinpirole. There was a significant group effect inrats receiving a systemic injection of the D1R/D5Rantagonist SCH23390, the D2R/D3R antagonistraclopride, or vehicle before coadministration ofSKF38393 and quinpirole (F2,15 5 12.220, P < 0.01, n5 8/group). LSD post hoc test showed that, relative tovehicle pretreatment, SCH23390 or raclopride pretreat-ment blocked the SII induced by a systemic coadminis-tration of SKF38393 and quinpirole together (LSD posthoc test, P < 0.05; Fig. 1C).

Coactivation of D1R and D2R ActivatesPirC Neurons

Because a systemic injection of the D1R/D5Ragonist SKF38393 and the D2R/D3R/D4R agonistquinpirole together impaired social behavior as shownabove, we further examined whether coadministration ofSKS38393 and quinpirole was able to activate the PirCneurons with extracellular recording of the firing rate ofpyramidal neurons in the PirC layer II. When cumula-tive administration of 0.1–1 mg/kg or individual injec-tion of 1, 2, or 3 mg/kg of SKF38393 or quinpirole wasconducted, neither SKF38393 nor quinpirole signifi-cantly altered the firing activity of PirC pyramidal neu-rons (P > 0.05, n 5 3/group; Fig. 2A–C). Cumulativeadministration of 0.1–0.9 mg/kg of SKF38393 and quin-pirole together did not significantly change the firingrate either (P > 0.05, n 5 5/group; Fig. 2A). However,when 1 mg/kg SKF38393 and quinpirole was cumula-tively administered, the firing activity was significantlyincreased (P < 0.05, n 5 3–5/group; Fig. 2A). The ani-mals receiving a coadministration of SKF38393 (3 mg/kg, i.v.) and quinpirole (1 mg/kg, i.v.) also showed asignificant increase of the firing rate of PirC pyramidalneurons (P < 0.05, n 5 5/group), which was reversedby the D1R/D2R antagonist LEE300 (Fig. 2B).Similarly, the animals receiving coadministration ofSKF38393 (3 mg/kg, i.v.) and quinpirole (2 mg/kg, i.v.)exhibited a significant increase of the firing rate (P <0.05, n 5 5/group), which was reversed by the D2Rantagonist L-741,626 (Fig. 2C).

Coactivation of PirC D1R and D2R ImpairsSocial Interaction

The above-mentioned results demonstrate that asystemic injection of SKF38393 and quinpirole impairssocial interaction, likely through activation of PirCpyramidal neurons. To provide further evidence sup-porting this idea, we examined whether an intra-PirC

Fig. 1. Effects of dopamine receptor agonists and antagonists onsocial interaction behavior (n 5 8/group). A: Sniffing time for ratsreceiving vehicle, SKF38393 (D1-like), quinpirole (D2-like), or bothSKF38393 and quinpirole, with a 24-hr interval between trial 1 andtrial 2 in the social interaction test. B: Nest thickness in rats receivingvehicle, SKF38393 (D1-like), quinpirole (D2-like), or bothSKF38393 and quinpirole. C: Sniffing time for SKF38393- and quin-pirole-treated rats receiving pretreatment with vehicle, the D1R/D5R antagonist SCH23390, or the D2R/D3R antagonist raclopride,with a 24-hr interval between trial 1 and trial 2 in the social interac-tion test. *P < 0.05, **P < 0.01.

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coadministration of SKF38393 and quinpirole was ableto induce SII. Similarly to systemic injection, a bilateralintra-PirC microinjection (Fig. 3A) of SKF38393 (30lg/1.0 ll/side) and quinpirole (20 lg/1.0 ll/side) alsoproduced significant group effects (F3,24 5 3.235, P <0.05, n 5 6–8/group). LSD post hoc test furthershowed that the combined intra-PirC treatment withSKF38393 and quinpirole prior to trial 1 significantlydecreased the time spent sniffing the stimulus rat com-pared with the vehicle group or the groups that receivedeither agonist alone (P < 0.05; Fig. 3B). Similar resultswere obtained after a bilateral intra-PirC microinjectionof a smaller dose, i.e., SKF38393 (5 lg/0.5 ll/side) andquinpirole (10 lg/0.5 ll/side; F3,24 5 10.051, P <0.05, n 5 7/group). These findings indicate that coacti-vation of D1R, D2R, D3R, D4R, and D5R in thePirC is able to induce SII.

Next, we explored contributions of each dopaminereceptor subtype in the PirC to SII induced by intra-PirC infusion of the D1R/D5R agonist SKF38393 andthe D2R/D3R/D4R agonist quinpirole. Naıve controlrats and experimental rats receiving an intra-PirC micro-injection of D1R antisense oligonucleotides, the D2Rantagonist L-741,626, the D3R antagonist GR 103691,the D4R antagonist L-745,870, D5R antisense oligonu-cleotides, control oligonucleotides or vehicle prior to anintra-PirC infusion of SKF38393 and quinpirole togetherexhibited significant group effects (D1R and D5R anti-sense oligonucleotides: F3,28 5 9.408, P < 0.01, n 5 8/group; D2R, D3R and D4R antagonists: F3,28 516.658, P < 0.01, n 5 8/group). Specifically, in com-parison with naıve rats, rats receiving D1R antisenseoligonucleotide pretreatment showed normal socialinteraction, whereas rats pretreated with either D5Rantisense oligonucleotides or scrambled oligonucleotidesexhibited a significant SII (Fig. 3C). Relative to vehiclepretreatment, whichy exerted no significant effects onsocial interaction impairment, D2R antagonist pretreat-ment, but not D3R or D4R antagonist, reversed the SIIinduced by SKF38393 and quinpirole together (Fig.3D). The sniffing times during trial 2 were generallysimilar between rats receiving either systemic or intra-PirC injection of SKF38393, quinpirole, or both with orwithout dopamine receptor antagonists. Therefore, co-activation of PirC D1R and D2R, but not D3R, D4R,or D5R, likely is able to induce SII.

Requirement for PirC EndocannabinoidSystem in SII

To determine whether the PirC endocannabinoidsystem is involved in the SII induced by coactivation ofPirC D1R and D2R, we tested the effects of bilateralintra-PirC microinfusion of the specific CB1R antago-nist AM281. AM281 produced significant group effectswhen both trial 1 and trial 2 were compared (F7,48 57.370, P < 0.01, n 5 7/group). Relative to rats receiv-ing vehicle alone, rats receiving SKF38393, quinpirole,and the vehicle for AM281 exhibited a significant

Fig. 2. Effects of dopamine receptor agonists and antagonists on neu-ronal firing rate of the piriform cortex (n 5 3–5/group). A: Relativefiring rate after cumulative doses of SKF38393 (D1-like), quinpirole(D2-like), or both SKF38393 and quinpirole. B: Relative firing rateafter injection of SKF38393 (D1) at 1, 2, or 3 mg/kg, both SKF38393(D1, 3 mg/kg) and quinpirole (D2, 1 mg/kg), or SKF38393 (D1, 3mg/kg), quinpirole (D2, 1 mg/kg), and LE300 (1 mg/kg) together.C: Relative firing rate after injection of quinpirole (D2) at 1, 2, or 3mg/kg, both quinpirole (D2, 2 mg/kg) and SKF38393 (D1, 3 mg/kg),or quinpirole (D2, 2 mg/kg), SKF38393 (D1, 3 mg/kg), and L741626(1 mg/kg) together. *P < 0.05, **P < 0.01.

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decrease in sniffing time (P < 0.01; Fig. 4A), which wascomparable to that of rats receiving intra-PirC injectionof SKF38393 and quinpirole (Fig. 3B). These resultsconfirm that PirC coadministration of SKF38393 andquinpirole induced SII. A simultaneous intra-PirC infu-sion of AM218 completely blocked the SII induced byintra-PirC SKF38393/quinpirole, whereas AM281 alonedid not significantly affect social interaction behavior incomparison with vehicle (Fig. 4A). When reexposed tothe same stimulus rat after 1 day, rats receiving vehicleand AM281 or SKF38393, quinpirole, and AM281exhibited a typical decrease in social interactioncompared with the initial interaction (LSD post hoc test,P < 0.001; Fig. 4A), indicating recognition of the fami-liar stimulus rat and normal social learning. However,rats receiving SKF38393, quinpirole, and vehicle did notsignificantly decrease their interaction in the second trial(Fig. 4A), indicating impaired social learning or inabilityto identify the stimulus rat because of the low level ofinitial interaction.

Because intra-PirC infusion of AM281 completelyblocked the SII produced by intra-PirC coadministration

of SKF38393 and quinpirole, we hypothesized thatincreased PirC concentrations of endocannabinoidsmight enhance the SII induced by intra-PirCSKF38393/quinpirole. To test this hypothesis, weinfused the endocannabinoid transporter inhibitorVDM11 into the PirC with or without a simultaneousintra-PirC microinjection of both SKF38393 and quin-pirole. These treatments produced a significant groupeffect when trial 1 and trial 2 were compared (F7,48 519.446, P < 0.01, n 5 7/group). As expected, relativeto rats receiving SKF38393, quinpirole, and vehicle, ratsreceiving SKF38393, quinpirole, and VDM11 exhibiteda significant reduction in sniffing time (P < 0.05),although VDM11 alone did not significantly affect socialinteraction behavior (Fig. 4B).

Effects of Coactivation of Dopamine Receptorson Olfactory Function

Our results suggest that both systemic and intra-PirC injection of SKF38393 and quinpirole togetherproduced SII by exhibiting decreased sniffing time.However, it is also possible that the decreased sniffing

Fig. 3. Effects of intrapiriform cortical injection of dopamine recep-tor agonists and antagonists on social interaction behavior (n 5 6–8/group). A: Nissl-stained section showing the location of the micro-pipette tip in the piriform cortex (PirC) at the level of Bregama 1.7mm. aca, Anterior part of the anterior commissura. B: Sniffing timefor rats receiving intra-PirC injection of vehicle (control), SKF38393(D1-like), quinpirole (D2-like), or both SKF38393 and quinpirole,with a 24-hr interval between trial 1 and trial 2 in the social interac-tion test. C: Sniffing time spent in naıve rats (control) and experi-

mental rats receiving intra-PirC injection of both SKF38393 andquinpirole, together with scrambled oligonucleotides (scrambledON), D1 receptor antisense oligonucleotides (D1R AON), or D5receptor antisense oligonucleotides (D5R AON). D: Sniffing timefor rats receiving intrapiriform cortical injection of both SKF38393and quinpirole, together with vehicle (Vehicle), the D2 receptorantagonist L-741,626 (D2R antagonist), the D3 receptor antagonistGR 103691 (D3R antagonist), or the D4 receptor antagonist L-745,870 (D4R antagonist). *P < 0.05, **P < 0.01.

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time with coadministration of both SKF38393 and quin-pirole resulted from the suppressive effects of SKF38393and quinpirole on olfaction (i.e., olfactory sensation). Toexamine this possibility, we conducted the modifiedhome cage version of an olfactory discrimination task(i.e., percentage of time spent sniffing the new bedding)following an i.p. injection of vehicle, SKF38393 (3 mg/kg), quinpirole (2 mg/kg), or SKF38393 and quinpiroletogether. As shown in Figure 5A, one-way ANOVA didnot show significant group effects (F3,27 5 1.671, P 50.197, n 5 7 or 8/group). Similar results were observedin rats receiving an intra-PirC infusion of SKF38393 andquinpirole together (F3,24 5 9.702, P 5 0.206, n 5 7/group; Fig. 5B).

Another possibility is that the decreased sniffingtime with coadministration of SKF38393 and quinpirole

resulted from the suppressive effects of SKF38393 andquinpirole on olfactory learning. To examine this possi-bility, we conducted the social interaction test with athree-trial protocol as described in detail in Materialsand Methods. Vehicle or both SKF38393 and quinpirolewere injected 25 min prior to the trial 1, which allowedus to examine their effects on the acquisition of short-term social interaction memory when comparison wasmade between trial 1 and trial 2. Significant groupeffects were observed in rats receiving either systemic orintra-PirC injection of SKF38393 and quinpiroletogether or vehicle (systemic injection: F5,36 5 12.301,P < 0.01, n 5 7/group; intra-PirC injection: F5,36 57.021, P < 0.01, n 5 7/group). Rats receiving a sys-temic injection of SKF38393 and quinpirole togetherspent significantly less time exploring the stimulus ratthan vehicle-treated rats during the trial 1 (P < 0.01;Fig. 5C), indicating that social interaction was reducedby coadministration of SKF38393 and quinpirole. Thissignificant difference was maintained in trial 2 at 2 hr(Fig. 5C). The difference between trial 1 and trial 2 wassignificant for both groups (P < 0.01), and the percent-age reductions in time spent exploring the familiar sti-mulus rat were quite similar (vehicle, 257.1% 6 3.1%;both SKF38393 and quinpirole, 257.2% 6 2.6%).Similar results were observed in rats receiving an intra-PirC microinjection of SKF38393 and quinpiroletogether or vehicle (Fig. 5D). These data indicate thatneither systemic nor intra-PirC injection of SKF38393and quinpirole together significantly affected the acquisi-tion of short-term social interaction memory. In the trial3, however, the time spent on social interaction eitherwas increased in rats receiving SKF38393 and quinpiroletogether or remained low in vehicle-treated rats (Fig.5C,D), indicating that short-term social interactionmemory had worn off in rats receiving SKF38393 andquinpirole together but not in vehicle-treated rats.

Effects of Coactivation of Dopamine Receptorson Locomotor Activity and Anxiety

Decreased sniffing time with coadministration ofboth SKF38393 and quinpirole may be produced eitherby the suppressive effects of SKF38393 and quinpiroleon motor activity or by their anxiogenic effects insteadof actual SII. To investigate the first possibility, weexamined the effects of various treatments on locomotoractivity. One-way ANOVA revealed that a systemicinjection of vehicle, SKF38393 (3 mg/kg), quinpirole(2 mg/kg), or both SKF38393 and quinpirole did notproduce significant group effects (F3,28 5 0.473, P 50.704, n 5 8/group; Fig. 6A). Similar results wereobtained either after a systemic injection of vehicle, theD1R/D5R antagonist SCH23390, or the D2R/D3Rantagonist raclopride together with both SKF38393 andquinpirole (F2,21 5 0.501, P 5 0.824, n 5 8/group;Fig. 6B) or after a bilateral intra-PirC injection ofD1R antisense oligonucleotides, vehicle, or the D2Rantagonist L-741,626 together with both SKF38393 and

Fig. 4. Effects of intrapiriform cortical injection of dopamine recep-tor agonists and endocannabinoid interference on social interactionbehavior (n 5 7/group). A: Sniffing time for rats receiving intra-PirC injection of vehicle, SKF38393 (D1-like), quinpirole (D2-like),and/or the CB1 receptor antagonist AM281, with a 24-hr intervalbetween trial 1 and trial 2 in the social interaction test. B: Sniffingtime for rats receiving intra-PirC injection of vehicle, SKF38393(D1-like), quinpirole (D2-like), and/or the endocannabinoid trans-porter VDM11. *P < 0.05, **P < 0.01.

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quinpirole (F2,21 5 0.454, P 5 0.730, n 5 8/group;Fig. 6C).

To explore the second possibility, we examinedthe effects of systemic injection of vehicle, SKF38393 (3mg/kg), quinpirole (2 mg/kg), or both SKF38393 andquinpirole on anxiety with the open field test and ele-vated plus maze test. A significant group effect wasrevealed in the open field test by one-way ANOVA(F3,28 5 4.077, P < 0.05, n 5 8/group). Post hocanalysis illustrated that quinpirole- and the combinedSKF38393 and quinpirole-treated groups displayed sig-nificantly less anxiety than control by spending moretime at the center of the open field (P < 0.05; Fig. 6D).However, because they were not different from eachother, it is assumed that the D2R agonist administrationis mediating this effect and not the coadministration ofagonists. The elevated plus maze also showed significantgroup effects (entry ratio: F3,24 5 12.104, P < 0.05,n 5 7/group; dwell ratio: F3,24 5 11.046, P < 0.05, n5 7/group). Specifically, quinpirole- and the combinedSKF38393- and quinpirole-treated groups exhibitedsignificantly decreased entry ratio (Fig. 6E) and dwell ra-tio (Fig. 6F) compared with vehicle-treated control rats(P < 0.05), suggesting less anxiety in rats treated withquinpirole or with combined SKF38393 and quinpirole.

DISCUSSION

The main measure in the present study to establishSII was the time spent sniffing the unfamiliar rats in thesocial interaction test. Both the social interaction test andthe nest formation test showed consistent results regard-ing SII following administration of SKF38393 andquinpirole together but not each alone, suggesting thatcoadministration of SKF38393 and quinpirole impairssocial interaction but not the ability of rats to sniff. Thisidea is further supported by our control experimentshowing that the proportion of time spent sniffing thenovel bedding in the olfactory discrimination test wasnot significantly different among rats receivingSKF38393, quinpirole, or both. Because neither systemicnor intra-PirC injection of SKF38393 and quinpiroletogether significantly affected olfactory sensation assessedvia olfactory discrimination test, we do not attributeSKF38393/quinpirole-induced social SII to deficienciesin olfactory sensation. Along the same lines, we do notattribute SKF38393/quinpirole-induced SII to deficien-cies in olfactory learning, because neither systemic norintra-PirC injection of SKF38393 and quinpiroletogether significantly affected the acquisition of short-term social interaction memory. Furthermore, we donot attribute SKF38393/quinpirole-induced SII to the

Fig. 5. Effects of dopamine receptor agonists on olfactory sensationand olfactory memory (n 5 7 or 8/group). A: With the olfactorydiscrimination test, time spent sniffing the new bedding in ratsreceiving systemic injection of vehicle, SKF38393 (D1-like), quinpir-ole (D2-like), or both SKF38393 and quinpirole. B: With the olfac-tory discrimination test, time spent sniffing the new bedding in ratsreceiving intra-PirC injection of vehicle, SKF38393 (D1-like), quin-pirole (D2-like), or both SKF38393 and quinpirole. C: Sniffing time

for rats receiving systemic injection of vehicle or both SKF38393(D1-like) and quinpirole (D2-like), with a 2-hr interval between trial1 and trial 2 and a 24-hr interval between trial 2 and trial 3 in thesocial interaction test. D: Sniffing time for in rats receiving intrapiri-form cortical injection of vehicle or both SKF38393 (D1-like) andquinpirole (D2-like), with a 2-hr interval between trial 1 and trial 2and a 24-hr interval between trial 2 and trial 3 in the social inter-action test. *P < 0.05.

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suppressive effects of SKF38393/quinpirole on motoractivity or to anxiogenic effects of SKF38393/quinpirole,because we observed that coadministration of SKF38393and quinpirole produced anxiolytic effects withoutsignificant effects on locomotor activity. The blockadeeffects of D1R or D2R antagonism on the SII inducedby systemic or intra-PirC injection of SKF38393 andquinpirole together likely are not produced by thesuppressive effects of D1R or D2R antagonism onmotor activity, because these treatments did not signifi-cantly alter locomotor activity.

Accumulating lines of evidence suggest a role ofdopamine receptor activation in SII (Espejo, 2003;Troost et al., 2005; Adriani et al., 2010), a key clinicalfeature of autism (Rapin, 1997; American PsychiatricAssociation, 2000). The exact mechanism remainsunknown, however. In the present study, we providethe first evidence that systemic administration of theD1R/D5R agonist SKF38393 and the D2R/D3R/D4Ragonist quinpirole together, but not either alone,induced significant SII. We also show that SII inducedby SKF38393/quinpirole was blocked by systemic injec-tion of D1R/D5R antagonist or D2R/D3R antagonist.These results suggest a role of global coactivation ofD1R, D2R, D3R, D4R, and D5R in SII.

We then tried to explore the brain region responsi-ble for the SII following dopamine receptor coactivation.Te dopamine system in many brain regions such as theprefrontal cortex (Espejo, 2003) has been shown to beinvolved in social interaction behavior. Although it isunknown whether the PirC plays a role in SII, we areinterested in this brain region because of its intensivedopaminergic innervation (Datiche and Cattarelli, 1996;Wilson et al., 2006) and the existence of all subtypes ofdopamine receptors (Ariano and Sible, 1994; Khanet al., 2000; Maltais et al., 2000; Wedzony et al., 2000).We found that systemic SKF38393 and quinpirole to-gether, but not either alone, significantly increased PirCneuronal firing rate, which was disrupted by the D1R/D2R antagonist LEE300 and the D2R antagonist L-741,626. These data indicate that coadministration ofSKF38393 and quinpirole impairs social interaction likelythrough activation of D1R and D2R in the PirC. Weobtained further evidence in support of this hypothesis.Intra-PirC microinfusion of SKF38393 and quinpiroletogether, but not alone, induced SII, which was blockedby intra-PirC microinfusion of D1R antisense oligo-nucleotides or D2R antagonist but not by D3R orD4R antagonist or D5R antisense oligonucleotides.Thus, coactivation of D1R and D2R, but not D3R,

Fig. 6. Effects of dopamine receptor agonists on locomotor activityand anxiety (n 5 8/group). A–C: Number of squares crossed in anopen field in rats receiving systemic injection of vehicle, SKF38393(D1-like), quinpirole (D2-like), or both SKF38393 and quinpirole(A); in rats receiving a systemic injection of vehicle, the D1R/D5Rantagonist SCH23390, or the D2R/D3R antagonist raclopridetogether with both SKF38393 and quinpirole (B); or in rats receivinga bilateral intra-PirC injection of D1R antisense oligonucleotides,vehicle, or the D2R antagonist L-741,626 together with bothSKF38393 and quinpirole (C). D: Time spent within the cetral area

of an open field in rats receiving systemic injection of vehicle,SKF38393 (D1-like), quinpirole (D2-like), or both SKF38393 andquinpirole. E: The entry ratio (entries into the enclosed arms/entriesinto the open arms) during the elevated plus maze test in rats receiv-ing systemic injection of vehicle, SKF38393 (D1-like), quinpirole(D2-like), or both SKF38393 and quinpirole. F: The dwell ratio(time spent in the enclosed arms/total time spent in both the openand enclosed arms) during the elevated plus maze test in rats receiv-ing systemic injection of vehicle, SKF38393 (D1-like), quinpirole(D2-like), or both SKF38393 and quinpirole. *P < 0.05.

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D4R, or D5R, in the PirC is able to impair social inter-action behavior.

Next, we determined the possible involvement ofthe endocannabinoid system in the SII induced by co-activation of D1R and D2R in the PirC. Cannabis ormarijuana, its primary psychoactive ingredient D9-tetra-hydrocannabinol, and a variety of synthetic analogs(collectively known as cannabinoids) act on CB1R andCB2R, which are distributed mainly in the brain andimmune system, respectively, and also targeted byendogenous cannabinoids (or endocannabinoids) such asanandamide and 2-arachidonylglycerol (Fride andMechoulam, 2003; Iversen, 2003; Piomelli, 2003; Meyerand Quenzer, 2004). The fatty acid amide hydrolase,which catalyzes the intracellular hydrolysis of anand-amide, can be inhibited by URB597 (Fride andMechoulam, 2003; Piomelli, 2003). Before intracellularhydrolysis, anandamide must be transported into the cell,which can be inhibited by AM404 and VDM11 (Frideand Mechoulam, 2003; Piomelli, 2003). AlthoughAM404 has both cannabinoid and noncannabinoidtargets (Lambert and Fowler, 2005), VDM is more selec-tive for the cannabinoid system (De Petrocellis et al.,2000). It has recently been shown that social play behav-ior was increased by systemic administration of URB597and VDM11 and decreased by systemic administration ofthe CB1R agonist WIN55,212-2 (Trezza and Vander-schuren, 2008a,b, 2009). The effects of URB597 andVDM11 on social play were mediated by CB1 anddopamine receptors, whereas WIN55,212-2 reducedsocial play behavior via a nondopaminergic, CB1R-mediated mechanism (Trezza and Vanderschuren,2008a,b, 2009). Because endocannabinoids are releasedon demand (Piomelli, 2003), these results suggest thatanandamide release within the neural circuits mediatingsocial behavior facilitates social interaction, whereasstimulating cannabinoid neurotransmission outside thiscircuitry may interfere with the normal execution ofcomplex social acts (Trezza and Vanderschuren, 2008a,b,2009). In the present study, we found that the SIIinduced by intra-PirC microinfusion of both SKF38393and quinpirole was blocked by the selective CB1Rantagonist AM281 and enhanced by VDM11, althoughneither AM281 nor VDM11 alone affected social inter-action behavior. These results suggest that the SIIinduced by coactivation of PirC D1R and D2R requiresthe endocannabinoid system. Specifically, coactivation ofPirC D1R and D2R may activate the PirC endocanna-binoid system to induce SII. Our findings of enhancedSII by intra-PirC VDM11 might not be in conflict withthe recent findings of enhanced social play with systemicVDM11 (Trezza and Vanderschuren, 2008a,b, 2009) ifwe consider the possibility that the endocannabinoidsystem in different brain regions may make differentcontributions to social interaction behavior.

We show here that coactivation of D1R and D2Rleads to SII, and similar synergistic effects have recentlybeen reported repeatedly. Thus, systemic or intracerebraladministration of SKF38393 and quipirole together, but

not either alone, is required for a significant increase inFos expression in the striatum (LaHoste et al., 1993,1996; Svennningsson et al., 2000); in neuronal firing rateof globus pallidus (Walters et al., 1987); in repetitive oralmovements, tongue protrusions, and chewing (Waszczaket al., 2002; Presti et al., 2004); or in repetitive andstereotyped behavior (Capper-Loup et al., 2002).Although it is not clear why coactivation of D1R andD2R leads to these functional synergistic effects, theseeffects may be produced by a coactivation of D1R:D2Rheterodimers, because heterooligomers have been foundto have ligand-binding and signaling properties thatdiffer from those associated with their monomeric con-stituents (George et al., 2002). It has recently beenshown that coactivation of both D1R and D2R whencoexpressed in vitro resulted in an increase in intra-cellular calcium via the phospolipase C pathway (Leeet al., 2004). This signal was not observed when eitherreceptor was expressed alone or when the coexpressedreceptors were stimulated by just one agonist. Moreinterestingly, experiments with coimmunoprecipitationassay and Forster resonance energy transfer techniquewere able to suggest that D1R and D2R form acomplex in cultured cells (Lee et al., 2004; Dziedzicka-Wasylewska et al., 2006). The possible existence of theheterodimer D1R:D2R in the PirC is further supportedby our observation that numerous PirC neurons contain-ing D1R mRNA are doubly stained with D2R antibody(Maltais et al., 2000). Future studies are required toexamine whether the heterodimer D1R:D2R exists inthe PirC, which is responsible, upon activation, for theSII that requires activation of the endocannabinoidsystem in the PirC. In conclusion, our novel findings sug-gest that the PirC endocannabinoid system is required forthe SII induced by coactivation of PirC D1R and D2R.

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