Enhanced latent inhibition in dopamine receptor-deficient miceis sex-specific for the D1 but not D2 receptor subtype:implications for antipsychotic drug action

Cecilie Bay-Richter1, Colm M. P. O’Tuathaigh2, Gerard O’Sullivan2, David M. Heery3, JohnL. Waddington2, and Paula M. Moran1

1School of Psychology, University of Nottingham, Nottingham, UK 2Molecular and CellularTherapeutics, Royal College of Surgeons in Ireland, St Stephen’s Green, Dublin, Ireland 3Schoolof Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK

AbstractLatent inhibition (LI) is reduced learning to a stimulus that has previously been experiencedwithout consequence. It is an important model of abnormal allocation of salience to irrelevantinformation in patients with schizophrenia. In rodents LI is abolished by psychotomimetic drugsand in experimental conditions where LI is low in controls, its expression is enhanced byantipsychotic drugs with activity at dopamine (DA) receptors. It is however unclear what theindependent contributions of DA receptor subtypes are to these effects. This study thereforeexamined LI in congenic DA D1 and D2 receptor knockout (D1 KO and D2 KO) mice.Conditioned suppression of drinking was used as the measure of learning in the LI procedure.Both male and female DA D2 KO mice showed clear enhancement of LI reproducingantipsychotic drug effects in the model. Unexpectedly, enhancement was also seen in D1 KOfemale mice but not in D1 KO male mice. This sex-specific pattern was not replicated inlocomotor or motor coordination tasks nor in the effect of DA KOs on baseline learning in controlgroups indicating some specificity of the effect to LI. These data suggest that the dopaminergicmechanism underlying LI potentiation and possibly antipsychotic action may differ between thesexes, being mediated by D2 receptors in males but by both D1 and D2 receptors in females. Thesedata suggest that the DA D1 receptor may prove an important target for understanding sexdifferences in the mechanisms of action of antipsychotic drugs and in the aetiology of aberrantsalience allocation in schizophrenia.

KeywordsAntipsychotic; dopamine D1 receptor; dopamine D2 receptor; dopamine knockout mice; latentinhibition; sex difference

Copyright © 2008 CINP

Address for correspondence: P. M. Moran, Ph.D., School of Psychology, University of Nottingham, Nottingham NG7 2RD, UK. Tel.:+44 115 9515312 Fax : +44 115 9515324, paula.moran@nottingham.ac.uk.

Statement of InterestNone.

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Published in final edited form as:Int J Neuropsychopharmacol. 2009 April ; 12(3): 403–414. doi:10.1017/S1461145708009656.

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IntroductionLatent inhibition (LI) is a process of learning to ignore irrelevant stimuli and is manifestedas slower learning about a stimulus that has been pre-exposed without consequence (PE),compared to learning about the same stimulus without pre-exposure (NPE) (Lubow andMoore, 1959). LI is used to model abnormalities in information filtering in schizophrenia,where patients have difficulty selecting between relevant and irrelevant information (Barakand Weiner, 2007; Gray et al., 1997; Kapur et al., 2005; Lubow, 2005; Weiner, 2003). LI isdisrupted in acute schizophrenia patients, their relatives and high schizotypal individuals,and normalized by antipsychotic drug treatment in patients (Baruch et al., 1988; Cohen etal., 2004; Gray et al., 1991; Lubow, 2005; Rascle et al., 2001). LI shows parallelpharmacological profiles in animals and humans (Moser et al., 2000; Weiner, 2003). Inrodents, psychotomimetic drugs such as amphetamine impair LI, while antipsychotic drugsenhance low levels of LI produced by decreasing the amount of pre-exposure (Moran et al.,1996; Moser et al., 2000; Palsson et al., 2005; Weiner, 2003; Weiner et al., 2003). Thesefeatures make it a powerful preclinical animal model of relevance to schizophrenia.

Pharmacology, microdialysis and lesion studies in rats have implicated the importance of theneurotransmitter dopamine (DA) in the regulation of LI (Gray et al., 1997; Joseph et al.,2000; Weiner, 2003; Young et al., 2005). In the brain the effects of DA are mediatedthrough activation of differently coupled receptor subtypes referred to as either DA D1-like(D1, D5) or DA D2-like (D2, D3, D4) (Missale et al., 1998). There is increasing evidence fora differential contribution of DA D1 and DA D2 receptors (hereafter designated D1 and D2respectively) to a variety of individual behaviours such as locomotor activity, memory andreward as well as responses to drugs of abuse and neuroprotection (Bozzi and Borrelli, 2006;Clifford et al., 2000; Doherty et al., 2008; Drago et al., 1994; Fowler et al., 2002; Kruzichand Grandy, 2004; Welter et al., 2007). The assignment of specific roles for DA receptorsubtypes in behaviour has been facilitated by the availability of knockout (KO) mice foreach DA receptor subtype (El-Ghundi et al., 2007; Holmes et al., 2004; Tan et al., 2003;Waddington et al., 2005). Given the importance of the DA hypothesis of schizophreniawhich suggests an overactivity in mesolimbic DA (Carlsson, 1988; Guilin et al., 2007;Kapur and Mamo, 2003; Kapur et al., 2005; Toda and Abi-Dargham, 2007) and the affinityof antipsychotic drugs for both D1 and D2 receptors (Missale et al., 1998; Miyamoto et al.,2005), it is of particular importance to establish the role of these receptor subtypes inbehaviours relevant to schizophrenia. Furthermore, understanding the potential separation ofside-effect and antipsychotic consequences of D1 and D2 blockade is important for thedevelopment of novel antipsychotic drugs with fewer extra-pyramidal side-effects (Farde etal., 1992; Horacek et al., 2006; Reynolds, 2007; Strange, 2001; Zai et al., 2007).

A number of studies have shown that D2 but not D1 antagonists enhance low levels of LIinduced by reducing the amount of stimulus pre-exposure, suggesting an action of DAthrough D2 but not D1 receptors in LI. D2 antagonists also reverse LI disruption byamphetamine while D1 antagonists do not (Moran et al., 1996; Moser et al., 2000; Trimbleet al., 2002; Warburton et al., 1994). However, nicotine-induced disruption of LI(considered to be mediated by nucleus accumbens DA release), is reversed by the D1antagonist SCH 23390 (Young et al., 2005). None of the drugs used in these studies is fullyselective for either the D1 or D2 receptor with many of the atypical antipsychotic drugshaving similar affinity for both receptors as well as for D3 and D4 (Missale et al., 1998;Miyamoto et al., 2005).

In this study congenic D1 or D2 receptor KO mice were used to determine whether theenhancing effects of D2 antagonists and the lack of effect of D1 antagonists on low LI arereproduced in mice that have genes for D1 or D2 receptors deleted independently and

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whether D1 or D2 receptors are independently essential for the normal expression of (high)LI. LI was investigated using two levels of pre-exposure, one producing LI in wild-type(WT) animals (60 PE) and one not (40 PE). This facilitates the observation of potentialenhancement or reduction of LI. To assess consistency with prior behavioural studies andrelevance to motor side-effects of antipsychotic drugs, locomotor activity and motorcoordination on the rotarod were also assessed.

Materials and methodsAnimals

The original F2 hybrid strain (129/Sv×C57BL/6J) containing the mutated D1 or D2 receptoralleles were generated as reported previously (Drago et al., 1994; Kelly et al., 1997). Thetargeted gene deletion was constructed in 129/Sv embryonic stem cells and male chimaerasmated with C57BL/6 females to produce heterozygous mutants (D1

+/- or D2+/-). Incipient

congenic D1 and D2 lines were established by back-crossing heterozygous mutants to WTC57BL/6 for 14 generations. Homozygous KO mice (D1

-/- and D2-/-) and WT (D1

+/+ andD2

+/+) littermates were bred by heterozygous intermatings of the incipient congenicheterozygote mutants imported from Royal College of Surgeons, Dublin, Ireland. Congenicstrains have significant advantages over mixed background strains in reducing inter-animalvariability and maximizing the specificity of the phenotype to the targeted gene (Gerlai,1996; Silva et al., 1997). D1 KO mice had low body weight and poor growth and wereweaned later than D2 KO mice to facilitate growth and survival. They were given a wetmash diet in the home cages after weaning, this has been reported previously in these mice(Doherty et al., 2008). D1 KO mice were bred with the kind permission of Professor JohnDrago, University of Melbourne. D2 KO mice were bred with the kind permission ofProfessor Malcolm Low, Oregon Health and Science University, USA.

Male and female KO mice and WT littermates aged at least 10 wk were used in theexperiments. Animals were housed 1-4 per cage under a 12-h light-dark cycle (lights on07:00 hours), at constant temperature and humidity with food available ad libitum.Experiments were performed during the light period. Mice used for the suppression oflicking model were subjected to water restriction periods of 23 h. All experiments wereperformed in accordance with local and national rules on animal experimentation, and withappropriate personal and project licence authority under the Animals (Scientific Procedures)Act, UK 1986 (PPL no: 40/2883).

GenotypingGenotyping was performed by PCR using genomic DNA extracted from ear biopsies by the‘hot sodium hydroxide and tris’ (hotSHOT) method (Truett et al., 2000). The reactionsdetermined the presence of both the neomycin phosphotransferase (NEO) gene and presenceof either the D1 gene or the D2 gene. The following primers were used for the D1 reaction:

NEOf: 5′-CTG AAT GAA CTG CAG GAC GA-3′,

NEOr: 5′-ATA CTT TCT CGG CAG GAG CA-3′,

D1f: 5′-CAA TCA GAA AGT TCC TTT AAG ATG TCC-3′,

D1r: ATG GTG GCT GGA AAA CAT CAC AGC CCC-3′,

and for the D2 reaction:

NEOf: 5′-CTT GGG TGG AGA GGC TAT TC-3′,

NEOr: 5′-AGG TGA GAT GAC AGG AGA TC-3′,

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D2f: 5′-TGT GAC TGC AAC ATC CCA CC-3′,

D2r: 5′-GCG GAA CTC AAT GTT GAA GG -3′.

PCR products were separated on a 1.5 % agarose gel. For D1 gene, amplification of a 172-bpPCR product indicated the presence of at least one transgenic allele and amplification of a353-bp PCR product indicated the presence of at least one WT allele. For the D2 gene,amplification of a 280-bp band indicated the presence of at least one transgenic allele and a105-bp band indicated the presence of at least one WT allele.

Instruments and proceduresLatent inhibition—Training and testing occurred in six identical conditioning chambers(21.6 cm×17.8 cm×12.7 cm, ENV-307W; Med Associates Inc., St Albans, VT, USA)housed in light and sound-attenuating boxes (ENV-022V, Med Associates Inc.). Theconditioning chambers were composed of Plexiglas walls in the front and back, stainlesssteel sides, and a metal grid floor connected to a shock scrambler and generator. Each boxcontained a ventilation fan, mounted at the back to provide air exchange and backgroundnoise (69 dB white noise), and a sonalert mounted on the right wall for delivering theconditioned stimulus (CS) (85 dB) (ENV-323AW, Med Associates Inc.). Each chamber wasequipped with a removable drink spout located in the left wall of the chamber. The lickspout was connected to a lickometer (ENV-250, Med Associates Inc.) which recorded thenumber of licks. The chambers were interfaced with a PC computer running MED-PCsoftware (SOF-735, Med Associates Inc.) to control stimulus presentation and record data.

The LI protocol consisted of six stages: water restriction, pre-training, pre-exposure/no pre-exposure, conditioning, re-establishment of drinking, and testing.

Water restriction (days 1-7): Mice were placed on a 23-h water restriction schedule 7 dbefore pre-training began. Water in the test apparatus was given in addition to the dailyration of 1 h delivered in the home cages. This regimen was maintained throughout theexperiment.

Pre-training (days 8-13): Mice were placed in the conditioning chambers and allowed todrink freely from a water sipper for 15 min. The number of licks made and latency to lickwas recorded.

Pre-exposure (day 14): Mice were placed in the conditioning chambers without access tothe water sipper. They were given 40 (expts 1 and 3), 60 (expt 2), 5 (expt 4) or 10 (expt 4)presentations of an 85-dB 5-s tone with a 15-s interstimulus interval. NPE control mice wereplaced in the chambers for the same amount of time but received no pre-exposures to thetone.

Conditioning (day 15): Mice were placed in the experimental chambers without access tothe water sipper. After 5 min, two tone-footshock pairings were presented. Each tone (CS)was of 5-s duration and was followed by a 1-s 0.38-mA US footshock. There was a 2.5-mininterval between pairings and mice remained in the chamber for 5 min following the secondtone-shock presentation.

Lick training (days 16-17): Mice were placed in the conditioning chambers for 15 min andgiven free access to the water sipper to re-establish licking in the chamber prior to testing.Mice that did not lick consistently were omitted from the experiment at this stage (n=2 forexpts 1-4).

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Testing (day 18): Mice were placed in the conditioning chambers with access to the watersipper. The number of licks and the time to complete licks 80-90 (A) and 90-100 (B) wererecorded electronically. After the first 90 licks, the tone CS was presented until the mousereached lick 100. The standard measure of conditioned suppression was the time taken tocomplete licks 90-100 in the presence of the CS. A suppression ratio (SR) was calculatedaccording to the formula A/(A+B) yielding a scale of 0-0.5. Low SR indicates good learningwhile high SR indicates poor learning of the association between the tone and footshock. LIis demonstrated as higher SR in the PE group compared to the NPE group.

Spontaneous locomotor activity—Spontaneous locomotor activity was recorded usingthe open-field test. The open-field apparatus consisted of six Plexiglas boxes measuring 30cm×17 cm with 25 cm walls. All walls were dark except for one transparent wall in eachbox allowing light to enter. Animals were placed in the boxes to habituate for 30 min.Twenty-four hours after the habituation session the animals were again placed into the open-field boxes for 30 min and total distance moved was recorded and analysed using acomputerized video-tracking program EthoVision (version 3.1, Noldus, The Netherlands).Six animals were tested simultaneously in six separate open-field arenas. Between mice, allarenas were thoroughly cleaned with water and odour-free soap.

Motor coordination—The rotarod test was used as a measure of motor coordination.Animals were placed on a plastic rod rotating at 3 rpm (Rota-Rod/RS, LSI, Letica,Barcelona, Spain). Latency (s) to fall from the rod up to a maximum of 300 s was manuallyrecorded. If a mouse fell within the first 5 s, the trial was restarted.

Experimental procedureExperiments were run in the following order:

Expt 1—LI at 40 PE in D2 KO mice (n=19 WT, n=24 D2 KO). Mice were tested first onrotarod activity, 4 d later on locomotor activity and 14 d later began water deprivationschedule for LI. The LI procedure was run using 40 PE to produce low levels of LI in WTmice, this number was determined by pilot experiments in our laboratory.

Expt 2—LI at 60 PE in D2 KO mice (n=33 WT, n=25 D2 KO). The LI procedure was runusing 60 PE to produce LI in WT mice.

Expt 3—LI at 40 PE in D1 KO mice (n=24 WT, n=21 D1 KO). Mice were tested first onrotarod activity, 2 d later on locomotor activity and 18 d later began water deprivationschedule for LI. The LI procedure was run using 40 PE.

Expt 4—This experiment was performed to further investigate LI in D1 KO males as expt 3showed that LI was still high at 40 PE in male D1 KO WT mice. LI was tested at 5 PE and10 PE in male D1 KO mice (n=36 male WT, n=15 male D1 KO) to produce low LI in WTmice.

StatisticsStatistics were performed using SPSS software, version 15 (SPSS Inc., Chicago, IL, USA).For LI experiments independent analysis of variance (ANOVA) was used with SR asdependent variable and exposure (NPE/PE) genotype (WT/KO) and sex (male/female) asfactors. For post-hoc comparisons and other behavioural experiments Student’s independentt tests were used. Five extreme values (>3 standard deviations of the mean) occurred in expt3 and were removed from analysis: one WT PE female, one WT NPE female, one KO NPEmale, two WT NPE males. No extreme values were found in any other experiment. Unless

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otherwise indicated, data were collapsed across sex when there was no effect of sex and nointeraction between sex and exposure or genotype.

ResultsLI in D2 receptor KO mice

There was a clear enhancement of low LI (40 PE) in D2 KO mice compared to WTlittermates (Figure 1a). A two-way ANOVA with exposure, genotype and sex as factorsdemonstrated a significant effect of exposure [F(1, 34)=6.113, p=0.01], genotype [F(1,34)=6.812, p=0.01] and a significant exposure×genotype interaction [F(1, 34)=5.667,p<0.05]. No sex differences were found [F(1, 34)=0.001, n.s.]. Post-hoc t tests confirmedthat D2 KO animals showed a significant difference between NPE and PE groups (denotingLI) whereas WT animals did not [D2 KO NPE vs. D2 KO PE: t(21)=−2.853, p<0.01; WTNPE vs. WT PE: t(14)=−1.139, n.s.]. Moreover, PE groups were significantly differentbetween genotypes whereas NPE groups were not [WT PE vs. D2 KO PE: t(17)=2.448,p<0.05; WT NPE vs. D2 KO NPE: t(18)=1.856, n.s.]. Figure 1(b, c) shows the same animalsfrom expt 1 (40 PE) divided by sex.

Figure 2 shows that when WT mice show robust LI (at 60 PE) there is no difference betweengenotypes, with both groups showing clear LI. There was a significant effect of exposure[F(1, 46)=9.481, p<0.01] indicating the presence of LI but no effect of genotype [F(1,46)=0.948, n.s.] and no exposure×genotype interaction [F(1, 47)=0.561, n.s.]. No sexdifferences were found [F(1, 46)=0.080, n.s.] and therefore, in post-hoc analysis the sexeswere pooled. Post-hoc t tests confirmed an exposure effect (i.e. the presence of LI) in bothgenotypes [WT NPE vs. WT PE: t(28)=−2.545, p=0.01; D2 KO NPE vs. D2 KO PE: t(19)=−2.409, p<0.05]. Post-hoc t tests showed no differences between NPE groups or PE groups[WT NPE vs. D2 KO NPE: t(23)=1.230, n.s.; WT PE vs. D2 KO PE: t(27)=0.897, n.s.].

Baseline differences in drinking rates during pre-training between genotypes were not foundin any experiment. Repeated-measures ANOVA on drinking rates in expt 1 with pre-trainingday and genotype as factors showed a significant effect of pre-training day [F(5, 35)=4.840,p<0.01], no effect of genotype [F(5, 39)=0.123, n.s.] and no pre-training day×genotypeinteraction [F(1, 35)=1.911, n.s.]. The same was found for expt 2: a significant effect of pre-training day was found [F(5, 52)=8.446, p<0.001], no significant effect of genotype [F(1,56)=0.136, n.s.] and no pre-training day×genotype interaction [F(5, 52)=1.111, n.s.]. Similarresults were found for expts 3 and 4 (data not shown). No baseline changes in drinkingbehaviour were seen in the test phase of the experiment either as measured by the time Ascores. Expt 1: no effect of group [F(1, 34)=1.580 n.s.], genotype [F(1, 34)=0.710, n.s.], orsex [F(1, 34)=0.470, n.s.]. Expt 2: no effect of group [F(1, 46)=0.394, n.s.], genotype [F(1,46)=0.313, n.s.] or sex [F(1, 46)=0.434, n.s.]. Expt 3: no effect of group [F(1, 33)=0.641,n.s.], genotype [F(1, 33)=1.742, n.s.] or sex [F(1, 33)=1.373, n.s.]. Expt 4: no effect of group[F(1, 50)=1.01, n.s.] or genotype [F(1, 50)=1.05, n.s.].

No effect of sex or genotype was found for NPE control learning groups in any of the fourexperiments (all F values <1).

Spontaneous locomotor activity and motor coordination in D2 receptor KO miceFigure 3a shows that D2 KO animals are hypoactive compared to WT littermates [F(1,40)=90.840, p<0.001] in the open-field test where spontaneous locomotor activity over 30min was measured. A sex difference was also found [F(1, 40)=10.859, p<0.01], where malestended to be hyperactive compared to females. Post-hoc t tests showed significant sexdifferences between D2 KO animals [male D2 KO vs. female D2 KO: t(23)=2.923, p<0.01]but not between WT animals [male WT vs. female WT: t(17)=1.927, n.s.]. The significant

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genotype effect was found in both males and females [male WT vs. male D2 KO: t(19)=−5.895, p<0.001; female WT vs. female D2 KO: t(21)=−7.973, p<0.001].

Figure 3b shows that D2 KO mice demonstrate impaired motor coordination as measured bythe rotarod test when compared to WT littermates [F(1, 42)=12.328, p<0.001]. No sexdifferences were found [F(1, 42)=0.969, n.s.]. Post-hoc t tests confirmed the genotype effect[male WT vs. male D2 KO: t(21)=−2.905, p<0.01; female WT vs. female D2 KO: t(21)=−2.005, p=0.05].

D1 receptor KO mice and LIFigure 4a indicates slightly enhanced LI in D1 KO mice. There was a significant effect ofexposure [F(1, 67)=7.223, p<0.01] and a significant effect of genotype [F(1, 67)=4.716,p<0.05], but no exposure×genotype interaction suggesting the effect is not specific to the PEgroup. There was a significant effect of sex [F(1, 52)=7.159, p<0.01] in WT mice.Subsequent ANOVAs for separate sexes with exposure and genotype as factors showed asignificant effect of exposure [F(1, 34)=9.045, p<0.01] and importantly no effect ofgenotype [F(1, 34)=0.036, n.s.] in males (Figure 4b) suggesting no potentiation in male KOmice. Females showed a significant effect of exposure [F(1, 29)=5.472, p<0.05] but incontrast with data in male mice, significant enhancement of LI was found in female KOmice (Figure 4c). In females there was a significant effect of genotype [F(1, 29)=15.649,p<0.001] and a significant exposure×genotype interaction [F(1, 29)=5.754, p<0.05]. Post-hoc t tests confirmed a genotype difference in females in the PE group [female WT PE vs.female D1 KO PE: t(14)=3.574, p<0.01] but not the NPE group [female WT NPE vs. femaleD1 KO NPE: t(15)=1.768, n.s.]. Post-hoc tests revealed no effect of exposure [female WTNPE vs. female WT PE: t(22)=0.317, n.s.; female D1 KO NPE vs. female D1 KO PE: t(7)=−1.379, n.s.]. In male animals no genotype differences were found [male WT PE vs. maleD1 KO PE: t(18)=0.274, n.s.; male WT NPE vs. male D1 KO NPE: t(15)=−0.789, n.s.] but asignificant effect of exposure was found in WT but not D1 KO animals [male WT NPE vs.WT PE: t(26)=−3.099, p<0.01; male D1 KO NPE vs. male D1 KO PE: t(7)=−1.515, n.s.].This suggests potentiation of LI in female but not male D1 KO mice.

There was no significant effect of D1 genotype on baseline drinking rate. Repeated-measuresANOVA showed a significant effect of pre-training day [F(5, 36)=6.047, p<0.001], no effectof genotype [F(2, 40)=2.236, n.s.] and no genotype×pre-training day interaction [F(10,74)=1.852, n.s.].

To investigate further the suggestion from the above data that male D1 KO mice do notshow enhanced LI a further experiment was performed investigating LI at very low numbersof exposures such that WT mice do not show LI. Figure 6 shows that at 5 PE where WTmale mice do not show LI, there is similarly no LI in D1 KO male mice. Furthermore, at 10PE there is a trend towards disruption in D1 KO mice. There was a significant effect ofexposure [F(1, 47)=11.76, p<0.0001], no effect of genotype (F<1), nor a genotype×exposureinteraction [F(2, 47)=1.573, n.s.] t test-confirmed presence of LI in WT at 10 PE [NPE vs.10 PE (t=7.9, p<0.001)].

Spontaneous locomotor activity and coordination in D1 receptor KO miceFigure 5a shows that in the open-field test of spontaneous locomotor activity, D1 KOanimals are hyperactive compared to WT littermates with a significant effect of genotype[F(1, 41)=40.596, p<0.001] and no effect of sex [F(1, 41)=0.471, n.s.].

Figure 5b shows no effect on motor coordination in D1 KO mice of either sex. No effect ofgenotype [F(1, 41)=0.097, n.s.] or sex [F(1, 41)=0.685, n.s.] was found.

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DiscussionThese data demonstrate that D2 KO mice show markedly enhanced LI in low PE conditionswhere WT mice do not show LI. This profile reproduces the established profile of a range ofantipsychotic drugs such as haloperidol, clozapine, remoxipride in LI in rats and mice(Mongeau et al., 2007; Moran et al., 1996; Moser et al., 2000; Trimble et al., 1997;Warburton et al., 1994; Weiner et al., 2003). These data for the first time confirm using agenetic approach, a specific role for the D2 receptor in the modulation of LI. Furthermore,our results suggest that the enhancement of LI characteristic of antipsychotics can be elicitedby deletion of the D2 receptor subtype alone, and is therefore probably not dependent uponD3 and D4 receptors for which antipsychotic drugs also have affinity. In conditions where LIis present in controls, i.e. high number of preexposures (60 PE), D2 receptor gene deletion iswithout effect confirming that LI expression does not require the D2 receptor in conditionswhere WT mice show robust LI. This reproduces well-established effects of D2 receptorantagonists on LI (Moran et al., 1996; Ruob et al., 1998; Shadach et al., 2000).

It has been suggested that D2 KO mouse models possess limited value in modellingantipsychotic drug effects as, with the notable exception of side-effect-relevant behaviourssuch as motor deficits, they have not straightforwardly reproduced the behavioural effects ofD2 antagonist antipsychotic drugs (Chen et al., 2006; Gainetdinov et al., 2001; Holmes et al.,2004; Waddington et al., 2005). For example D2 KO mice do not show similar effects toantipsychotic drugs in sensory gating in the pre-pulse inhibition (PPI) model, althoughamphetamine abolition of PPI is prevented in these mice (Ralph et al., 1999). The finding ofa similar profile of D2 receptor knockout and antipsychotic drugs in LI suggest that, for theconstituent KO model, such a conclusion is premature. It is noteworthy that in theconditioned avoidance response model which is also sensitive to the effects of antipsychoticdrugs, D2 KO mice similarly reproduce the effects of antipsychotic drugs, although sexdifferences in this model have not been addressed (Smith et al., 2002).

The present study also confirms that D2 gene deletion produces hypoactivity and impairedmotor coordination as has been shown previously (Aoyama et al., 2000; Baik et al., 1995;Chausmer et al., 2002; Clifford et al., 2000; Fowler et al., 2002). This confirms that in thiscongenic line the established locomotor activity and motor coordination-impairing profile ofD2 antagonists is also reproduced (Aoyama et al., 2000).

In D1 KO mice we unexpectedly found significant enhancement of low LI in female but notmale mice. However, at 40 PE in WT males there was a higher baseline LI than in thefemale WT cohort perhaps allowing enhancement to be seen more readily in D1 KOfemales. We have subsequently replicated this higher LI in male WT D1 mice at 20, 40 and60 PE suggesting it is a robust effect (data not shown) and expt 4 has shown that we still seeLI at 10 PE in male WT animals although this is not seen at 5 PE. We cannot fully explainthis difference at present, however, it is possible that longer weaning in D1 WT mice mayhave exerted a beneficial cognitive effect on males as it is known that curtailment ofweaning in male mice has negative behavioural consequences and induces corticosteroneabnormalities (Wurbel and Stauffacher, 1997), this may also explain the baseline differencesin WT activity and rotarod that we observed. It is clear that the amount of pre-exposurerequired to produce LI in WT animals differs between the sexes in these mice, with malesshowing no LI at 5 PE and females no LI at 40 PE. These differences are, however,consistent with studies of LI in humans where females show reduced LI compared to males(Lubow and De la Casa, 2002). Despite this higher baseline in males there is still aconsiderable margin to detect enhancement of LI if it existed and we see no evidence forthis. At 5 PE we see no LI in either WT males or D1 KO males and a trend towards reducedLI in D1 KO males at 10 PE. This supports the suggestion that D1 KO males do not show

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enhanced LI compared to WT males. Further studies using larger cohorts than used in thepresent study would be desirable before concluding definitively that D1 KO males do notshow enhanced LI under any experimental conditions. At 10 PE there is some suggestionthat D1 KO males may show a deficit in LI produced by 10 PE, although this is notsignificant in the present study. This possibility will need to be replicated and extended tolarger cohorts in follow-up studies. These data nonetheless suggest that D1 receptor deletionmay enhance LI exclusively in females. This is contrary to prior studies in rats showing noeffect of D1 antagonists on low LI (Trimble et al., 2002). It is notable that these studies incommon with the majority of behavioural studies in rodents were carried out exclusively inmales (Hughes, 2007).

D1 KO mice showed hyperactivity and no deficits in rotarod motor coordination that wassimilar between the sexes confirming previous studies in these mice (Holmes et al., 2004).The lack of a similar profile of sex difference in activity and motor coordination additionallysuggests that the LI sex difference we have identified is highly specific to LI and notattributable to generalized behavioral/motor compromise in these mice. This is furthersupported by the fact that there is no effect of genotype or sex on the NPE learning groups.

Clinical trials reporting no benefit of D1 antagonists in schizophrenia patients have beenprimarily carried out with male patients (de Beaurepaire et al., 1995; Den Boer et al., 1995).The present data might help to explain this lack of efficacy. These data indicate thatenhancement of LI may involve differential regulation by D1 and D2 receptor subtypes inmales and females.

The existence of sex differences in phenomenology, epidemiology, neuroanatomy andresponse to antipsychotic drugs in schizophrenia has been well described (Goldstein et al.,2002; Lewine et al., 1996; Usall et al., 2007). Recently, Rubin et al. (2008) found sexdifferences in cognitive responses to antipsychotic drugs in patients with schizophrenia. Thepresent results may help to explain sex differences in response to antipsychotic drugs interms of dissociable dopaminergic substrates; one possible mechanism is an interaction withsex hormones. It is well established that the oestrous cycle influences variation in basalextracellular concentration of striatal DA, both in drug-stimulated DA release and in DA-mediated behaviours (Becker, 1999). Oestrogen directly acts on the striatum and nucleusaccumbens to enhance DA release and DA-mediated behaviours (Le Saux et al., 2006).

High levels of oestrogen have been shown to increase mesolimbic DA transmission (knownto disrupt LI) (Gray et al., 1995, 1997, 1999; Joseph et al., 1993; Van Hartesveldt and Joyce,1986) while ovariectomy reduces basal extracellular DA, amphetamine-induced striatal DArelease, and behaviours mediated by the striatal DA system (Becker, 1999).

Sex differences have also been reported in rodents in behavioural response to drugs of abusesuch as cocaine, ethanol and MDMA; these differences have been shown to be mediated inpart by the D1 receptor (Ferris et al., 2007; Festa et al., 2006; Melnick and DowEdwards,2001). Behavioural responses to D1 agonists and antagonists have also been demonstrated todiffer between the sexes (Nazarian et al., 2004).

Sexually dimorphic behavioural phenotypes in mice with mutations for genes mediatingdiverse aspects of DA neurotransmission have also been reported including DARPP-32 andcatechol-O-methyltransferase (COMT) function (Babovic et al., 2007; Harrison andTunbridge, 2007; Waddington et al., 2005).

In all KO mouse studies where the genetic deletion is present from birth there is thepotential for compensatory developmental changes to occur. While this cannot be excludedhere, this is currently the only approach to differentiate the impact of the absence of specific

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DA receptor subtypes in isolation. The recent development of inducible D1 mutants will beof value in future exploration of sexually dimorphic contributions of different DA receptorsubtypes to salience allocation processes and their aberrant states in psychosis (e.g. Gantoiset al., 2007). The present results are, however, consistent with predictions frompharmacological studies in LI for the D2 receptor, while the novel sex differences for D1suggested will generate further investigation using pharmacological approaches and a widerrange of behavioural models known to be sensitive to antipsychotic drug effects.

In summary we have shown that the antipsychoticlike profile of enhanced LI can be elicitedby D2 but not D1 receptor KO in males but by both D1 and D2 receptor KO in females. Thissuggests the possibility that the mechanism of action of antipsychotic drugs may differbetween the sexes and that the D1 receptor may be an important factor in dissociable maleand female responses to antipsychotic drugs.

AcknowledgmentsWe acknowledge the kind permission of Professor John Drago University of Melbourne, Australia and ProfessorMalcolm Low, Oregon Health and Science University, USA to use the KO mice. We are grateful for the support ofthe University of Nottingham New Researchers Fund, Science Foundation Ireland, and the Wellcome Trust.

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Figure 1.Latent inhibition (LI) in D2 KO mice with 40 pre-exposures. Columns represent mean±S.E.M.(a) LI in males and females (n=9-12 per group). Panels (b) and (c) show the data from panel(a) divided into (b) males and (c) females. ** p<0.01 compared to no pre-exposure (NPE)for same genotype; § p<0.05 compared to pre-exposed (PE) for wild-type (WT) mice.Enhanced LI is found in D2 KO mice at 40 PE where LI is not present in WT littermatecontrols.

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Figure 2.Latent inhibition (LI) in D2 KO mice (males and females) with 60 pre-exposures (PE)(n=9-16 per group). Columns represent mean±S.E.M.* p<0.05, ** p<0.01 compared to no pre-exposure (NPE) for same genotype. At 60 PE where LI is present in wild-type (WT)littermate controls the D2 KO is without effect.

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Figure 3.(a) Locomotor activity in D2 KO mice (n=9-13), (b) motor coordination in D2 KO mice(n=10-13). Columns represent mean±S.E.M.* p<0.05, ** p<0.01, *** p<0.001 compared towild-type (WT) littermate controls of same sex. Both male and female D2 KO mice showreduced spontaneous locomotor activity in the open-field test and impaired motorcoordination on the rotarod.

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Figure 4.Latent inhibition (LI) in D1 KO mice with 40 pre-exposures (PE). Columns represent mean±S.E.M. (a) LI in males and females (n=11-12 per group). Panels (b) and (c) show the datafrom panel (a) divided by sex: (b) LI males (n=5-6), (c) LI females (n=6). ** p<0.01compared to no pre-exposure (NPE) for same genotype; §§ p<0.01 compared to wild type(WT). No effect of D1 KO on LI is found at 40 PE in males. Female D1 KO animals show aclear enhancement of LI compared to WT littermate controls.

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Figure 5.(a) Locomotor activity in D1 KO mice (n=10-12), (b) motor coordination in D1 KO mice(n=10-12). Columns represent mean±S.E.M. *** p<0.01 compared to wild-type (WT)littermate controls of same sex. D1 KO mice of both sexes are hyperactive compared to WTlittermate controls as measured in the open-field test. The D1 KO had no effect on motorcoordination in either sex measured on the rotarod.

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Figure 6.The effects of D1 KO on latent inhibition (LI) at 5 and 10 pre-exposure (PE) in wild-type(WT) male mice only (n=5-8). Columns represents mean±S.E.M.** p<0.001 compared to nopre-exposure (NPE) for same genotype. WT mice show no LI at 5 PE but significant LI at10 PE. D1 KO mice do not show LI at either 5 or 10 PE.

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