FUNCTIONAL INTERACTIONS OF Dl AND D3 RECEPTORS: GENERATION AND BEHAVIOURAL ASSESSMENT OF MICE LACKING BOTH RECEPTORS.

A thesis submitted in conformity with the nquirements for the degree of Master of Science Department of Pharmacology University of Toronto

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Master of Science, 2000

Joanna Monika Karasinska Department of Pharxnacology, University of Toronto

ABSTRACT

Dopamine is involved in the control of locomotor activity, reward, cognitive and endocrine hctions. Experimental evidence suggests that D 1 aad D3 dopamine recepton interact in an opposing and synergistic fashion. The objective of this project was to generate mice lacking both receptors in order to investigate D lm3 receptor interactions in behaviour. Dl4*D3'" mice were viable, fertile and showed no gross morphological abnomalities. In a mvel open field, they exhibited lower activity than wild-type, Dl"; and D3" mice. D l"D3" mice perfomed equally poorly in the rotarod and spatial leaming tasks as their Dl4- littermates. Basai locomotor activity and anxiety-like behaviour were normal in D l"D3" mice. Combined deletion of both receptors abolished the exploratory hyperactivity and amiolytic-like behaviour of the D3 receptor mutant phenotype and fcurther attenuated the low exploratory phenotype of DI" mice. These results suggest an interaction of both recepton in the expression of novelty-induced exploratory behaviour and the need for the presence of intact Dl receptor for the expression of certain behaviours manifested by the D3 receptor mutaat pheuotype. In addition, the D1 receptor, but not the D3 receptor, is involved in the ability to perfom on the rotarod as weii as iii

ACKNOWLEDGMENTS

1 would iike to thank my supervisors, Dr. B. F. O'Dowd and Dr. S. R. George, for their guidance, support and constructive cnticim tbroughout my project. 1 would also like to thank my advisor, Dr. P. J. Fletcher, for his helpful advice and discussions on behavioural aspects of my project. 1 am grateful to other members of Dr. O'Dowd's and

Dr. George's laboratory: Mufida El-Ghundi, for her help and guidance during the initial stages of my project, as weil as Tua.Nguyen and Yang Liu for their technical assistance.

This project was supported by the Medical Research Council and the National Idtute on Drug Abuse. TABLE OF CONTENTS

Page List of figures and tables vii List of abbreviations viii CHAPTER 1 INTRODUCTION 1.1. system in the CNS 1.1.1. Historical perspective 1.1.2. The DA receptors (A) Molecular biology of DA recepton 1. Cloning II. Splice variants (B) Structure of DA receptors (C) Distribution of DA recepton 1. D 1 -1ike receptors II. D2-like receptors (D)Function of DA receptors in the CNS 1. Locomotor activity II. Motivation and reward III. Learning and memory 1.2. targeting as a tool in studying receptor fùnction 1.2.1. The mouse knockout mode1 1.2.2. GPCR knockouts 1.2.3. Development of knockout mice to study DA receptor f'unction (A)D 1 receptor knockout (B)D2 receptor knockout (C) D3 receptor knockout @) D4 receptor knockout (E) DS receptor knockout 1-3. Rationale for generation of mice lacking D 1 and D3 receptors 1.3.1. Lessons learned hmDA receptar hockouts 1.3.2. D 1/D3 meptor CO-localisation 25 1.3.3. Evidence for D 1/D3 recepior interactions 25 1.3.4. D 1-1ikelDZ-like receptor interactions in behaviour 26 1.3 .S. Aim of the project 27 CHAPTJCR 2: MATERIALS AND METHODS 28 2.1. Aaimals 29 2.1.1. D 1 and D3 receptor single knockout mice 29 2.1.2. Generation of mice lacking D 1 and D3 teceptors 29 2.1.3. Genotyping by PCR 30 2.2. Behavioural assessrnent 32 2.2.1. Animal housing 32 2.2.2. Experiment 1 : locomotor activity 32 2.2.3. Experiment 2: open field 33 2.2.4. Experiment 3: elevated plus maze 33 2.2.5. Experiment 4: rotarod 33 2.2.6. Experiment 5: water maze 34 2.2.7. Statistical analysis 35 CHAPTER 3s RESULTS 36 3.1. Generation of D 1"D3" mice 37 3.2. Locomotor activity 37 3.3. Open field 37 3.4. Elevated plus maze 42 3.5. Rotarod 45 3.6. Morris water rnaze 45 CEAPTER 4: DISCUSSION AND CONCLUSIONS 51 4.1. Role of Dl and D3 receptors in basal locomotor activity 52 4.2. D l"'D3' mice versus Dl4- and D3" mice in open field and plus maze: indication of D 1/D3 receptor interaction? 53 4.3. The rde of Dl versus D3 receptor in motor control and spatial learning 55 4,4. Adaptive mechanisms in D 1&D3+ mice 58 4.5. Involvement of the 129/Sv background genotype in the behaviod phenotype of DI%* mice 59 4.6. Beyond Dl and D3 receptors 60 4.7. Conclusions 61 CHAPTER 5: REFERENCES 62 vii

LIST OF FIGURES Page Figure 1. The catecholamine biosynthetic pathway. 3 Figure 2. Punnett's square. 30 Figure 3. Genotyping by PCR of D 1 and D3 receptot mutation. 31 Figure 4. Basal locomotor activity. 38 Figure 5. Exploratory behaviour in the open field. 40-4 1 Figure 6. Arm entries in the elevated plus maze. 43-44 Figure 7. Performaace on the rotarod. 46 Figure 8. Performance in the MWM. 49-50

LIST OF TABLES

Table 1. List of GPCR knockouts. Table 2, Phenotypic changes in D 1 and D3 receptor deficient mice. Tabie 3. PCR primer sequences and product sizes. viii

LIST OF ABBREVIATIONS

AC adenylyl cyclase

ANOVA analysis of variance

CAMP cyclic adenosine monophosphate cDNA complementary deoxyribonucleic acid

CNS central nervous system

DA dopamine

DNA deoxyri bonucleic acid

Gi uihibitory G protein G, stimulatory G protein

GPCR G proteinîoupled receptor

ICjM islands of Calleja major

L-DOPA L-dihydroxyp heny lalanine

LTP long-term potentiation mRNA messenger ribonucleic acid

MWM Moms water maze neor neomycin nsistance

NMDA N-methy l-D-aspartate

PCR polymerase chah reaction

TM transmembrane domain wlrr wild-type CWTER 1 INTRODUCTION 1.1. Dopaminergic system in the CNS

The catecholamine dopamine @A) is one of the classical of the central nervous system (CNS). The precursors for the catecholamine biosynthetic pathway are the amino acids and . The fÜst step in the physiological formation of DA is the oxidation of phenyldanine or tyrosine, absorbed fiom diet, into DOPA, in the cell bodies of adrenergic neurons, in melanophore cells, and in the adrenal medullary chromafh cells. Next, the enzyme DOPA decarboxylase catalyses the conversion of WPA into DA. DA can be Mer converted to noradrenaline and adrenaline (Fig. 1) (50). DA in the CNS is involved in the control of locomotor activity, endocrine regdation, reward, cognition and cardiovascular homeostasis.

Imbalances in dopaminergic transmission have been implicated in the pathogenesis of diseases including Parkinson's disease, schizophrenia, Tourette's syndrome, dmg addiction and endoaine abnonnalities.

1.1.1. Historical perspective

For many years the role of DA as a was neglected as DA was considered mainly a step in the major synthetic pathway of two other neurotransmitters, noradrenaline and adrenaline. DA was largely ignored due to its small sympathornimetic de. The interest in DA increased dramatically after the discovery of the eKects of L-DOPA treatment in patients with

Parkinson's disease as well as the properties of DA receptor blockade.

DA was fïrst syuthesised in 1910, and its immediate precursor L-DOPA was synthesised in

1911. Almost twenty years later the evidence for the presence of L-DOPA decarboxylase in

animal tissue was presented, which Ied to the fhdings that decarboxylation of L-DOPA pmduces

DA, and that L-DOPA d DA are intermediates in the biosynthetic pathway of noradredine

and adrenaline. During the 195OVs, the nrst antipsychotic dmgs were introduced and it was F (oxidation)

HO Tyrosine

Tyrosine (oxidation) hydroxylase

DOPA (dihydroxypheny lalanine)

(decarboxy lation) DOPA decarboxylase

Dopamine (dihydroxyphenylethy lamine) observed that patients experienced unexpected side effects upon exposure to these dmgs. The

prolonged exposure to neuroleptics resulted in motor disturbances similar to those observed in

patients in Parkinson's disease. One of the administered neuroleptics was reserpine, which was

later found to deplete the body stores of two monoamines: 5-hydroxytryptamine (serotonin) and

aoradrenaiiie. This led the scientists to the conclusion that reserpine-induced depletion of either

serotonin or noradrenaline may have caused its pharmacological effects. It was however found that the reserpbinduced sedation was reversed only by L-DOPA,but that L-DOPA treatment

did not induce an increase in noradrenaline levels. It was later discovered that L-DOPA treatment

resulted in large accumulatiom of brain DA, and that DA is norrnally present in the brain at

levels simiiar to noradrenaline which suggested that it may have actions of its own. Further

studies demonstrated the wide distribution of DA in the brain. The presence of DA in caudate-

putamen and substantia nigra indicated that DA may be involved in extrapyramidal motor

hctions and in disorders including Parkinson's disease and Huntington's chorea. The first

effects of L-DOPA treatment of patients with Parkinson's disease were observed soon after (5).

Scientists thus began to map out the brain dopamine systems. This led to the identification of

three major dopaminergic pathways: 1) the nigrostriata1 pathway, onginating fiom ce11 bodies of

the mua compacta of the substania nigra with DA containhg nerve tenninals in the stria-, 2)

the mesocorticolhbic pathway, orighting in the ventral tegmental area and terminating in

various parts of the iimbic system, and 3) the tubero~dibularpathway, intrinsic to the

hypothalamus. The aigrostriata1 pathway is involved in extrapyramidai motor fuuction and its

degeneration contributes to the pathogenesis of Parkinson's disease. The mesocorticolimbic

pathway regulates cognition and reward and its disturbances rnay be involved in scbphrenia

and hgabuse. FWy, the tuberoinf'undibdar pathway regdates hormone secretion hm anterior pituitary gland and may be involved endocrine abnonnalities including hy perprolactinemia.

The nrst evidence for the existence of DA receptors in the CNS came in 1972 fiom biochemical studies in which DA was shown to stimulate adenylyl cyclase (AC) in brah tissue homogenates (120), a nsponse that was antagonised by neuroleptic hgs(202). This suggested the presence of DA receptors in the brain coupled to AC, which was stimulated in response to

DA. in 1979 two DA receptor subtypes were proposed, one positively coupled to AC, defined as

D-1 recepton and one independent of, or inhibitory on the CAMP generating pathway, defied as

D-2 recepton (1 19). Later studies confimed that the two receptor subtypes were different pharmacologically, biochemically and had anatomically distinct distributions. In the IWO'S, the

DA recepton were studied with use of receptor autoradiography assays (2 112 12). A decade later, advances in molecular biology have led to the cloning of the five distinct DA receptor subtypes known today.

1.1.2. The DA receptors

The five known DA receptors are divided into two subclasses, the Dl-like and DZlike receptors, based on their , signalling properties and pharmacology. The Dl-

üke famüy includes the Dl and D5 receptor subtypes, which were found to stimulate CAMP formation, increase intracellular calcium stores through stimulation of phosphotidylinositol hydrolysis and protein kinase A activity, as wel1 as inhibit the activity of the Na+Mexchanger.

The D2, D3 and D4 recepton form the D2-like famiiy and they inhibit AC, generally inhibit calcium cumnts, increase outward potassium cturents and arachidonic acid nlease (161). The

DA recepton belong to the superfamily of G protein-coupled receptors (GPCRs) characterised by seven membrane spanning domains (TMs). The GPCR family indudes aImost 2000 known receptors, related by primary stnichite and predicted secondary structural features. The known

GPCRs are grouped into over 100 subfamilies based on sequence homology, stnicme and receptor fiinction, and novel GPCRs are king cloned continuousiy (reviewed in

86,15,113,13 1,143,144). A comprehensive list of GPCRs cm be found on the internet under www.gpcr.org.

(A) Molecular biology of DA receptors

1. Cloning

The first DA receptor to be cloned was the D2 receptor. The cDNA of the rat D2 receptor was isolated in 1988 using the hamster coding sequence as a hybridisation probe to screen a rat genomic library (26). Subsequently, the D2 receptor sequence was later used to screen a rat cDNA library and the D3 receptor was identified (222). The cloning of the Dl receptor was reported by four groups in 1990 using either an oligonucleotide derived nom TM2 of the nit D2 receptor (SS), or by polymerase chah reaction (PCR) with primers correspondhg to

TM3 andTM6 of other catecholamine receptoa (163,227,260). The D4 receptor was identified der a screening of a library fiorn the human neuroblastoma ce11 line SK-N-MC revealed the existence of a novel DA receptor (240). Finally, the second member of the Dl-lüce family was cloned using the Dl receptor sequence by three independent groups, and was referred to as D5

(226), Dl b (235) or Dlbeta (246) receptor. It is now known that Dl and Dlb are the human and rat homologs of the same receptor.

II. Splice varianb

The Dl and D5 receptors are encoded by intronless whereas the D2, D3 and D4 receptor genes contain introns (reviewed in 176), a characteristic shared with the gene for (172). The presence of introns often leads to the biosynthesis of severai splice variants of a receptor. Analysis of gene structure of the D2-like receptors revealed that the D2 receptor gene contains seven introns (177), and soon after the cloning of D2 receptor, the existence of a more abundant longer form of the rat, bovine and human D2 receptor generated by alternative splicing was reported by several laboratones (35,s 1,88,158,164,177,196,2 14). The receptor first cloned by Bunzow et al. (26) was referred to as D, and the longer splice variant containhg an additional stretch of 29 amino acids in the third intracellular loop was named 4,. Only subtle differences between the two receptors have been reported.

The rat and human D3 receptor gene coding regions are intempted by five introns (93,222) and the murine D3 receptor gene contains six introns (75). Alternative splice variants of the rat, murine and human D3 receptor gene have been identified. Giros et al. (87) identified two shorter forms of the rat D3 receptor, including a fnimeshift deletion resulting in a 100-amino acid protein and an Ui-frame 54-bp deletion in sequence encoding TM5. Another tnincation resulting in a

109amino acid protein hss been found in the rat in addition to a sllnilar human variant (221).

Another rat splice variant was found to conta 28 extra amho acids in the first extracellular loop

(186). The mouse D3 receptor exists as two hctional variants that dBer by a stretch of 21 amino acids in the third intracelldar loop (75) Fhally, human D3 receptor variants include a

138-amino acid protein containing ody T'Ml-TM3 found in lymphocytes (169) and a tnincated non-fhctional receptor called D3,, resulting fiom a 98 nucleotide-long deletion in the region encoding the carboxyl tennuial regioa of the third cytoplasmic loop (137). In addition, three shorter variants with either the fht dotthird exon deleted were found in the human brain

(93). The D4 receptor coding region contains thintrons (240) and polymorphic variations within the coding region exist. A 16 amino acid sequence in the third cytoplasmic loop cm exist as repeats leadhg to many D4 receptor variants (205,241).

(B) Structure of DA recepton

The DA receptors display the stniccural characteristics of the GPCRs. They contain seven TM domains with higidy conserved amino acid sequence, an extracellular amino terminal and an intraceiiular carboxyl terminal tail. The D2-like recepton contain a long third intracellular loop, a feature shared by GPCRs coupled negatively to AC through Gi. The Dl-like recepton, like other recepton coupled to G, protein, have a short third intracellular loop (reviewed in 178).

Members of the two DA receptor families are stnicturally homologous, in that Dl and D5 recepton share 80% amino acid identity in their TM domains. Similarly, the D2 and D3, and D2 and D4 have a 75% and 53% TM amho acid identity, respectively. The amho terminus contains similar number of amino acids in al1 receptor subtypes and the carboxyl terminus is about seven times longer in the D 1-1ike receptors than in the D2-like (reviewed in 16 1).

(C) Distribution of DA receptors

1. Dl-like recepton

nie Dl receptor is the most widespread and abundant DA receptor in the brain. Combinations of in situ hybridisation and in vin0 receptor autoradiography revealed that in the rat brain, the Dl receptor is localised at the highest levels in caudate-putamen, nucleus accumbens and olfactory tubede. Dl receptor mRNA was found at lower levels in the cerebrai cortex, other areas of the hbic system, including the islands of Calleja as well as the hypothalamus, thalamus, hippocampus and the arnygdala In addition high levels of D 1 receptor binding sites, but no mRNA, were found in the substaatia nigra pars reticulata, globus pallidus, entopeduncular nucleus, and subthalamic nucleus where DI receptors may be localised on nerve terminais originating in other brain regions such as the basai ganglia (78,142,245). The distribution of the

Dl receptor in the human brain is similar to that in the rat (55).

The D5 receptor is expressed at lower levels in the rat brain tban the Dl receptor. D5 mRNA has been detected in the hippocampus, the lateral marnmiliary nucleus, and the thalamic pazaficicular nucleus, brah regions also expressing low levels of the Dl receptor (1 54). D5 mRNA was also found in the substantia nigra pars compacta, cerebral cortex and the saiatum

(1 3,36). In the periphery, the D 1-like recepton have been found in the cardiovascular system, the

&enal gland and the kidney (1 61).

II. D2-like recepton

The D2 receptor mRNA is expressed in dl major brain regions receiving dopaminergic projections. The highest levels of D2 rnRNA were found in regions of the basal ganglia such as the caudate-putamen, nucleus accurnbens and (1 28). in addition high levels of

D2 mRNA were also found in the dopaminergic neurons of substantia nigra pars cornpacta, ventral tegmental area and the anterior lobe of the pituitary gland (156). Lower levels of D2 mRNA were found in the cortex, central amygdda and hypothalamus (23,245).

The distribution of the D3 receptor mRNA is restricted mainly to the ventral strianim. The highest levels of D3 mRNA cm be found in nucleus accumbens, the granule cells of the islands of Caüeja, and the olfactory tubercie (23,222). Other brain regions reported to exhibit dense expression of D3 mRNA include the bed nucleus of the stria terminalis. hippocampus, mammillary nuclei and lobules 9 and 10 of the cerebellum. Lower levels were detected in the cortex, substantia nigra pars compacts, hypothalamus and the amygdala (2339).

nie D4 nceptor is highly expressed in the frontal cortex, olfactory bulb, hypothalamus and thalamus (182). D4 receptor has aiso been found in the amygdda and hippocampus (240). High Levels of D4 mRNA were detected in the retina (42). The D4 receptor is also found in

GABAergic neurons of globus pailidus, substantia nigni pars reticulata, and thalamic reticular nucleus (167). The D4 receptor is also expressed in the anterior pituitary gland (240).

In the periphery, the D2-Like receptors are localised in the cardiovascular system, the adrenal gland, the kidney and sympathetic ganglia (161).

(D) Funetion of DA receptors in the CNS

Based on the anatomical distribution of the DA receptors as well as pharmacological studies ushg ligands with some selectivity for each receptor, specific roles in the regulation of motor control, reward and motivational behaviour, leaming and memory have been suggested for each receptor subtype.

1. Loeomotor activity

There is abundant evidence for the role of DA receptors in the regulation of locomotor activity. Generally, Dl and D2 receptoa are thought to be stimulatory on locomotor activity. The activation of the Dl or D2 receptors alone increases activity to some degree, however, concomitant activation of Dl and D2 receptors synergistically stimulates locomotor activity

(64,72,73,85,91,243). Recent evidence suggests that the D5 receptor may have an inhibitory effect on locomotor activity, since inhibition of the receptor by an antisense oligonucleotide potentiated the stimdating effect of a D 1-1ike agonist in 6-hydroxydopamine-lesionedrats (67).

However, there is no evidence for such inhibitory effect in mice lacking the D5 receptor.

The activation of presynaptic dopamine D2 receptors (autoreceptors) demases, whereas activation of postsynaptic D2 receptors stimulates locomotor activity (6,115.1 34,171,239).

AAministmtion of D2 receptor antisense oligonucleotides caused a decrease in spontaneous locomotor activity and rotationai behaviour in dents (257359). The D3 receptor has been found to be inhibitory on locomotion. Preferentially selective D3 receptor agonists were reported to decrease locomotor activity (52,54,74,194,230,23 1) and putative D3 antagonists were found to cause an increase in locomotor activity (8 1,244). The inhibitory role of the D3 receptors was

Mercodhed by antisense oligonucleotide administration in rats (155). The D4 receptor does not seem to be involved in locomotor activity. Recent reports however indicate that it may be involved in exploratory activity in mice (65) and novelty-seeking behaviour in humam

(68,î25,237).

II. Motivation and reward

The mesocorticolirnbic DA system has been hplicated in the regdation of motivational and reinforcement processes. Striatal DA neurons show activation in response to the administration of primary reward substances and conditioned reward predicting stimuli (reviewed in

(57,204,209). DA DI -1ike and D2-like receptors in the ventral striatum are known to be involved in the reinforcing properties of dnigs of abuse (reviewed in 216,229). Studies have shown that injections of Dl receptor antagonists into the nucleus accumbens, the amygdala, and the striatum resulted in an increase in cocaine self-administration in rats (28,71,140,153) suggesting that the

Dl receptor mediates the reiaforcing efEects of cocaine. Re-treatment with a Dl-like agonist completely suppressed the initiation of cocaine self-administration in rats and reduced cocaine- seeking behaviour (2 15). Moreover, administration of a D 1-1ike antagonist to humans attenuated the euphoric effects of cocaine (201). The involvement of the Dl receptor in ethanol self- administration has also been docurnented. High preference for ethanol was reduced in rodents after activation of D 1 receptors (66,82,173 218). In addition, ethano1 consumption was attenuated in mice lacking the dopamine D1 receptor (70). Since the Dl teceptor but not the D5 receptor is localised in brain regions associated with reward such as nucleus accumbens, the effects of the

D 1-me ligands are probably mediated through the D 1 receptor.

Stimulation of D2-like receptors was found to enhaace cocaine-seeking behaviow in rats previously exposed to the drug suggesting that D2-like agoni- trigger a relapse in an animal mode1 of cocaine-seeking behaviour (215). Another report also indicated that activation of D2- like recepton enhances the effects of self-administered cocaine and that these recepton may be involved in the abuse-related effects of cocaine (30). A lot of attention has ken given to the role of D3 receptor in the relliforcing properties of cocaine. Studies have shown that agonists with some selectivity for the D3 receptor, when CO-administeredwith cocaine, decreased cocaine self- administration in rats but increased drug self-administration when given alone (27,29,188) indicating that stimulation of the D3 receptor in the nucleus accumbens enhances the rewarding effects of cocaine. In addition, D3 mRNA was found to be upregulated in nucleus accumbens of cocaine overdose victims (147) and the D3 receptor gene rnay be involved in the susceptibility to cocaine dependence (43).

îIï. Learning and memory

Both Dl-like and D2-like receptors in brain areas such as the hippocampus and prefiontal cortex have been implicated in the regulation of cognitive behaviour including leaming and memory. Post-training intrahippocampal, intracaudate and peripheral injections of dopamine Dl and D2 agonists were shown to impmve memory in rats (185,247,248). In addition, in the monkey, local injections of D 1-like antagonists Uito prehntal cortex impaired working memory

(207,208), whenas activation of D2-like receptors in the prehntal cortex redted in cognitive improvement (7). Ventrai hippocampal D2-lïke receptors were show to stimulate spatial workhg memory in rats (249). There is some evidence that the D3 receptors may exert an inhibitory effect on memory consolidation, unlike the D2 receptor, which seems to facilitate memory consolidation (2 17,238). In addition, Dl-like receptor play a critical role in reward- related incentive learning (14) and in fact abnormal associative leaming following repeated stimulation of nucleus accumbens DA system rnay be an important mechanism umierlying addiction (reviewed in 58).

12. Gene targeting as a tool in studying meptor function

1.2.1. The mouse knockout mode1

Although numerous studies in the past 20 years have suggested distinct roles for individual

DA receptors in various aspects of behaviour, the lack of Ligands acting exclusively on each receptor subtype has made it diffcult to assign specific in vivo hctions to one DA receptor and to exclude other members of the same receptor family. Dl and D5 receptors have similar afkities for Dl -like antagonists with the exception of (+)-butaclamoi, for which the Dl receptor displays a slightly higher affinity. They have also similar affbities for most Dl-like agonists.

However, DA itself has a IO-fold higher &ty for the D5 than the Dl receptor (reviewed in

161). D2-like antagonists such as and have a 10- to 20-fold higher affinities for the D2 than the D3 or D4 receptors. , a neuroleptic, has a 10-fold higher affinity for the D4 receptor versus the D2 and D3 ceceptors. Other antagonists with some selectivity for the D3 receptor have ken recently developed including nafadotride, S-14297 and

U-99194A (reviewed in 161). With respect to agonists, DA has 20 times higher affinity for the

D3 receptor than the D2 receptor. 7-OH-DPATand quinphle also bind to the D3 receptor with a

10-fold and 100-fold higher affinity than the D2 receptor, respectively (reviewed in 161). in addition, the recently designed agonist BP897 has a 70-fold higher affinity for the D3 than the

D2 receptor* Different strategies have been used to overcome the problem of non-selective agents. They include local administration of dopaminergic ligands into brah areas expressing only some DA recepton (12) and the use of antisense oligonucleotides targeting a specific DA receptor

(67,155,257,259). However, the advantages of these strategies are limited by the fact that brain regions rarely express a single receptor mbtype, as well as the possibility of diffusion of the dopaminergic ligands Uito neighbouring brain regions, which may express other DA receptoa, and by the fact that the antisense oligonucleotides often affect only a proportion of the targeted receptor population. In the past decade, advances in molecular biology and gene targeting techniques have provided another tool in the investigation of DA receptor functions through the generation of mice lacking a gene encoding the receptor of interest (31,32). In swary, a targeting vector is constnicted in which the coding region of the receptor is disrupted usually by the insertion of the neomycin resistance gene. This new gene constnict is then transfected into pluripotent embryonic stem (ES) cells derived fiom a brown mouse, usuaily a mouse fiom the

129 strain family, and the targeting vector replaces the gene of interest through homologous recombination. ES cells carrying the targeted mutation, selected by neomycin resistance, are next injected into blastocyst-stage embryos usually derived fiom a black-coated fernaie of the

C57BL16 strain. These embryos are then implanted into surrogate mothers and the resuiting male chimetic offspring with mked black-brown coat color are mated to black-coated fernales. Some of the redting offspring will be hetemzygous for the mutation, Le. some mice will contain one copy of the mutant gene. The heterozygous rnice are htercrossed generating liners with one- fourth of offspring being homoygous, i.e. expressing both copies of the mutated gene, according to Mendel's law. The effects of the gene deletion are then studied in these bockout mice using thek wiid-type iittennates as controls. 1.2.2. GPCR knockouts

The mouse knockout model has proven to be a powemii tool in the snidy of function and importance of many G protein-coupled receptors. h fact, there are reports of over 70 single and three double GPCR knockouts (Table 1). The gene targeting technique has been used to investigate the fiinctions of various GPCRs in physiology, development, endocrinology and behaviour. Specitically, the investigation of the effects of receptor gene deletion has led to the discovery of fùnctional differences among GPCR subtypes, especially those belonging to the same receptor families. As sumrnarised in Table 1., some of these knockouts have resulted in pre- or postnatal lethality, including the P, adrenergic, prostaglandin EP4 and knockouts, emphasising the crucial role of these particular recepton in development and survival. Most GPCR knockouts, however, are viable with phenotypes that have often mpported the proposed function of the receptor or have uncovered its yet unknown novel role.

Table 1. List of known GPCR knockout models. PHENOTYPE column represents the observations made during the onginal studies made on the knockout mice. Since the original reports, more studies have ken perforrned which have continued to characterise the mutants.

RECEPTOR PHENOTYPE REFERENCE

- resistance to sehures induced by agonist pilocarpine - ablation of muscarinic receptor controlled MI current potassium charnel regdation - reduction of muscarinic receptor-mediated tremor and salivation, hypothennia and analgesia

- ? basal locomotor actïvity - t locomotor respoasa to D 1 dopamine ceceptor activation PHENOTYPE

- 4 exploratory activity - ? aggressiveness - hypoalgesia - ?' blood pressure - ?' platelet aggregation Adrenaline alb & blood pressure ? sympathetic activity depletion of cardiac tissue noradrenaline down-regulation of cardiac P receptors

no phenotype

prenatai lethality in majority of knockout mice lack of chronotropic and inotropic responses to agonists

vasodilation defect ?' total exercise capacity 4 respiratory change ratio

? fat stores p, mRNA up-regulation

B1/P2 Angiotensin AT1A - juxtoglornerular apparatus hypertrophy ATlA - & systolic blood pressure - impaircd drinking response to water deprivation - & spontaneous movements - t vasopressor response - no phenotype

- abnormal kidney structure - & bfood pressure - mild obesity - hypertension - impairment of glucose metaboiism - & metabolic rate - hyperphagia

GRP-R - loss of bombesin iaduced suppression of giucose inîake RECEPTOR PHENOTYPE REFERENCE

NMB-R - & hvbothermic effect of Bradykinin 82 - loss of bradykinin action in smooth muscle and neurons - Cannabis CB I - ? mortality rate - & iocomotor activity - t ring catalepsy hypoalgesia Chemoattractant C5a - abolition of mucosal host defence in the lung

Chemokine BLRI lack of inguinal lyrnph nodes impaired Iymphocyte migration

CCRl impaired host defence hematopoiesis granulomatous inflammation

CCRl protection fiom panmatitis associated pulmonary inflammation

CCR2 lack of inflammation mediated macrophage recru itrnent impainnent in host defence

impaired monocyte migration & type 1 cytokine responses

impaired macrophage fbnction ? T-cell dependent immune response

delayed antibody response impaired lymphocyte migration Cbolecystokinin CCK-A - abolition of cholecystokiin mediated food intake inhibition

CCK-B/gastrUi - atrophy of gasttk mucosa - severc hv~craastrinemia Corticotropin Releasing Hormone CRHRl - impaired stress response - hnxiety - & exploratory activity Dopamine Dl - dynorphin expression in striatum - ? IofomotorrtMcy - growth retarded RECEPTOR PHENOTYPE 4 rehgbehaviour 4 substance P expression

? enkephalin mRNA expression darker coat colour ? plasma alpha-melanocyta stimulating hormone levels ? expression of pmopiomelanocortin

locomotor impairment L spontaneous movements

chronic hyperprolactinemia anterior lobe pituitary lactotroph hyperplasia

?' behavioural semitivity to concurrent D 1 and D2 receptor stimulation

4 exploratory activity locomotor supersensitivity to ethanol, cocahe and metham phetamine improved rotarod performance

potentiation of D2 knockout locomotor

Endotbelin ET* - severe craniofacial deformities - cardiovmcular defects - intestine distension - spotîed coat color - premature death Castric hhibitory peptide GCPR - glucose intolerance and impaired insulin secretion after oral administration of glucose

Glutamate mGluR1 - severe motor co-otdinatioa and spatial learning deficits - impaireâ synaptic plasticity - & hippocampal Iong-tem potentiation - irnphd context-speciflc associative Ieaming - impairment of hippocampd mossy fiber long-term potentiation

- deficiency on the rotarod test RECEPTOR PHENOTYPE - loss of NMDA mediated LTP in CA 1 newons - deficits in visual transmission - 4 levels of postshock Geezing response

- deficit in conditioned test aversion

Histamine Hl - impaired locomotor activity and exploratory behaviour

- maniritty onset obesity syndrome - hyperphagia - hyperinsulinemia - hyperglycemia Melatonin Me1 la - altered melatonin induced phase-shifts

Neuropeptide Y NPY-Y 1R - & daily food intake - & fast-induced mfeeding - 7 body fat NPY-YSR - mild late-onset obesity Opioid NocicepWorphanin FQ disregulation of hearing ability

& anaigesic eflixts of morphine and its metabolites 4 morphine-induced lethality

'? proliferation of granulocyte-macrophage, erythroid and mdtipotential progenitor celfs & mating acîivity in males $ spam count an motility &littersize

$ tail flick and hot plate test latencies

loss of morphineinduced analgesia, reward and withdrawal

? sensitivity to pain & momhine withdrawal Platekt acüvating factor Pm Pmtagiandïns EE2 - 4 litter size due to ovulation defect - ? systolic blood pressure - salt diet induced systolic hypertension RECEPTOR

- patent ductus artenosus leading to neonatal death - ? swceptibility to thrombosis - 4 hflammatory and pain tesponse Somatostatin Subtype 2 - los of growth hormone induced inhibition of muate neurons Serotonin 5-HTt A - ? anxiety and stress response

- $ exploratory activity - ? anxiety - ? aggressive behaviour

- ? weight due to eating disorder - epilepsy - ? exploratory activity Substance P NK-1 R - lungs protected t)om immune complex injury Thrombin TR - ~renataldeath in 50% of embryos Thromboxane A, TP - prolonged bleeding time - & platelet aggregation

1.2.3. Development of knockout mice to study DA receptor function

Mouse knockout models for al1 five DA receptors have kengenerated. In the case of Dl, D2 and D3 receptors, several models developed by different Iaboratories exist.

(A) Dl receptor knoekout

The Dl receptor was the first DA receptor to be deleted in the mouse. in 1994, two independent groups reported the generation of Dl receptor kwckout mice (62,254). Initial

-dies and observations indicated that these mice are growth retarded and they required a diet supplemented with hydrated food to thive. Behaviod characterisation of the mutants by various Iaboratories produced interesting, aithough sometimes inconsistent redts. One group reported normal locomotion and motor CO-ordination,but decreased rearing, and reduced striatal

substance P expression (62), whereas another group found that the mutants exhibited locomotor

hyperactivity in addition to lower dynorphin expression (254). The attenuation of rearing

behaviour in Dl receptor mutants was conhed by other studies (40,69220). Additiondly,

rnice lacking the D 1 receptor exhibited decreased locomotor activity in an exploratory test (220),

impairments in the Moms water maze (MWM), a test of spatial learning, and deficiency in

movement initiation (69,220). Siftîng and chewing behaviour was reduced in Dl receptor

mutants but grooming behaviour was increased (38). However, the action sequencing during

grooming behaviour was impaired (48). Sensorimotor reflexes, basai locomotion, spontaneous

alternation and contexnial leaming were unchanged in Dl receptor knockout mice (69). Studies

investigating reward in the mutants found that they retain cocainetonditioned place preference

(160) but showed attenuated alcuhol-seeking behaviour (70). Dl receptor mutants exhibited a decrease in amphetamine and cocaine induced locomotor activity (47,251) as well as a reduction

in cocaine-induced Unmediate early gene expression (63). In summary, the findings obtained fiom studies on Dl receptor knockout mice support the receptor's role in cognitive and reward behaviour and in the mediation of psychostimulant dependent effects on locomotor activity and intracelIular processes. In addition to behavioutal chanicterisation of D 1 receptor knockout mice, peripherai eEects of the receptor deletion were investigated and biochemical snidies on signal transduction were performed. The results of these experiments are summarised in Table 2.

(TB) D2 receptor knockout

The nrst D2 receptor knockout mouse mode1 was developed in 1995 by Baik et al (9). Mice

Iackhg the receptor were akinetic and bradykinetic in behavioutal tests and they exhibited decreased Iocomotor activity and renuing as weli as an impairment on the rotarod, a test commonly used to investigate motor coordination (9,39). Another D2 receptor knockout modei was generated two years later (121) and in addition to pituitary lactotroph hyperplasia and hyperprolactinernia observed in the mutants, these mice also exhrbited behavioural changes.

Later tests found that although D2 receptor mutants exhibited reduced locomotor activity, rearùig and hpaired motor function, these impairments were influenced at least to some degree by the genetic background of the 129iSv mouse strain used to develop these knockout mice (122). A study of the effects of D2 receptor deletion on reward found that the rewarding properties of morphine were absent in D2 receptor mutants (141). D2 receptor knockout mice exhibited a loss of autoreceptor function (1 57) and hence a decrease in autoreceptor-controlled DA transporter activity (60) and a reduction in inhibition of DA niease (127). There was an absence of hypolocomotor and hypothermie effects of DZD3 receptor agonists in D2 receptor mutants (20).

Thus, D2 receptor deficient mice have provided Merevidence for the receptor's involvement in motor control and its role as an autoreceptor.

(C) D3 receptor knockout

Soon der the development of Dl and D2 receptor deficient mice, the D3 receptor knockout model was generated. With respect to behavioural changes, one group reported that the D3 receptor mutants were hyperactive in an exploratory test, exhibithg increases in both locomotor activity and rcaring (1). Later, an independent group has generated another D3 receptor biockout model and found that these mice were hyperactive only at the beginning of the exploratory test, and that they exhibited enhanced behaviod sensitivity to cocaine, amphetamine and cornbined administration of Dl-üke and D2-ülre agonists (253). Subsequent behavioural studies reported that mice lacking the D3 receptor exhibited reduced amiety levels in a plus maze test (224).

Interestingly, the mutants showed nomai responses to putative D3 ceceptor preferentid Ligands 23 indicating the lack of selectivity of such ligands in vnto (21,252). Therefore, the results of studies on D3 knockout mice support an inbibitory role of the receptor on some aspects of behaviour.

Although the D3 receptor has been suggested to exist as a presynaptic autoreceptor, this role was not supportad by knockout studies as there were no changes in autoreceptor function in D3 receptor deficient rnice (124). Table 2 summarises the results of additional studies on D3 receptor deficient mice that investigated signal transduction and peripheral effects.

Dl receptor knockout D3 receptor knockout

t systolic and diastolic blood pressure (3) - ? systolic and diastolic blood pressure (8)

4 DA effects on NMDA mediated - no change in clozapine induced c+s responses (133) expression (3 3) lack of cocaine mediated immediate early genes c-fos and zif268 (63)

loss of psychostimulant mediated striatal c-fos and Junl3 expression (1 65)

absence of D 1 receptor mediated CAMP production (79)

no change in Dl receptor mediated inositol phosphate accumulation (79)

& coupling of Dl antagonist binding sites to G, but not G, (79)

absence of late phase hippocampai LTP

Table 2. Redts of studies on Dl and D3 receptor deficient mice investigating signal transduction and peripheral effects of the deletion of each receptor.

@) D4 receptor knockout

Rubinstein et ai. (203) genenited mice lacking the D4 receptor in 1997 and found that the mutants performed better on the mtarod than wild-type mice. In addition, the mutants were supersensitive to the locomotor effects of substances including ethanol, cocaine and methamphetamine. DA synthesis was found to be increased in D4 receptor knockout mice (203).

Subsequent studies found that D4 receptor mutants exhibit decreases in behavioural responses to novelty including a decrease in exploratory activity (65). These findings provide more evidence for the receptor's role in novelty-seeking behaviour and indicate that D4 receptor may be involved in motor CO-ordinationand dmg-stimuiated motor activity.

@) D5 receptor knockout

Although mice lacking the DS receptor have been successfully generated (98)' no reports on their behavioural phenotype have been published as of the present day.

1.3. Rationale for goneration of mice lacking Dl and D3 recepton

1.3.1. Lessons leamed from DA receptor knockouts

The use of single DA receptor kaockout mice has provided important information about the individual roles of each receptor subtype in behavioural processes. Many studies on these knockout mice confinned the existing knowledge of a receptor's function previously based on pharrnacological studies. Some reports, however, unveiled possible novel roles of DA receptoa in behaviour, including the involvernent of the D3 receptor in anxiety-like behaviour (224) and the role of D4 receptor in motor control(203). The advantage of the knockout models was mer emphasised in reports by Xu et al. (252) and Boulay et al. (20)' who found that putative D3 receptor ligands had the same effects on wild-type and D3 receptor deficient mice, stressing the need for caution when ~ssigning specific fiuiction to individual receptors based on pharmacologicai data. Multiple DA receptor hockout models provide a tool to investigate the effects of concurrent inactivation of more than one DA receptor on brain hction. A knockout mouse modei lacking D2 and D3 receptors has been developed (118). Investigation of mice lacking D2 and D3 receptors found a synergistic effect of the deletion of both receptors on locomotor activity decrease.

1.3.2. Dlm3 receptor CO-locaiisatioa

Severai receptor autoradiography and in situ hybridisation histochemistry studies have reported the coexistence of Dl and D3 receptors in single neurons in various brah regions

(129,197,210). ln the islauds of Caileja major (ICjM), a large number of the granule cells co- express Dl and D3 receptor mRNAs. At least 80% of ICjM neurons expressing either the Di or

D3 receptor aiso express the other receptor subtype. In the ventromedial sheil of nucleus accumbens, 45% of Dl receptor neurons CO-expressthe D3 receptor, and about 65% of D3 receptor containhg neurons aiso express the D 1 receptor (1 W,210). Another report indicated that the D3 receptor is CO-expressedwith the D 1 receptor in both the core and shell regions of nucleus accumbens, within a subpopulation of substance P neurons (129). This cellular CO-localisation suggests that DA in the islands of Calleja and nucleus accumbens may mediate its effects on efferent murons through the CO-expressionof Dl and D3 receptors, and that DllD3 receptor hinctional interactions rnay occur at a single neuronal level.

13.3. Evidence for D1m3 receptor interactions

There is growing evidence for the fùnctional interactions of Dl and D3 recepton at both cellular and organismal levels. Activation of either the Dl or D3 receptors iocreased and decreased immediate early gene c-fs mRNA in the rat islands of Calleja, respectively.

Administration of antagonists had effects that were opposite to those of the corresponduig agonists (197). In the ventromedial shell of nucleus accumbens of reserpine-treated rats

(dopaminedepleted rats), a Dl receptor agonist increased substance P mlWA, and a D3 receptor agonist potentiated this effeet (210). In hemipkinsonian rats, the behavioural sensitisation to ievodopa was accompanied by upregulatioa of D3 receptor mRNA in substance Pldynorphin neurons that was fond to be mediated by the Dl receptor (17). In a human medulloblastoma cell line expressing only the Dl and D3 receptor subtypes, activation of the Dl receptor increased both D1 and D3 receptor mRNA levels (132). D3 receptor agonists injected into rat islands of

Calieja decreased body temperature and this effect was potentiated by CO-administrationof a Dl receptor agoaist (12). FinaUy, a snidy using knockout mice reported that the DI receptor mediated c-fos induction was decreased in mice deficient for the D3 receptor (1 17). Hence, it appears that Dl and D3 receptors interact in the regdation of intracellular processes.

Additionally, the reports indicate that the receptors may interact in an opposing or synergistic fashion under different paradigrns. This supports the hypothesis that the two receptors may also interact in their control of DA-rnediated behavioural processes.

13.4. Dl-likJD24ike mptor interactions in behaviour

Due to the co-existence of multiple DA receptors in various brain regions, DA recepton may be postulated to interact in the regdation of DA mediated processes. Interactions between DL like and D2-like receptors in behaviour were reported on locomotor activity, Iearning and reidorcement. Co-administration of Dl-like and D2-like agonists into rat nucleus accumbens at concentrations that had no effect when administered alone synergistically increased locomotor activity (72,192). Moreover, concurrent activation of D 1-1ike and D2-like recepton synergistically impaired passive avoidance learning in mice (105). With respect to reward, concurrent activation of DI-iike and D2-like receptors in the sheii of nucleus accumbens was found to have a co-operative effect on DA-mediated reward processes in rats (106). However,

Dl-Wce and D2-like receptors were fouiid to have opposite effects on cocaine-seeking behaviour in rats, such that Dl-like agonists prevented cocaine-seeking behaviour induced by cocaine itself, whereas D2-üke agonists enhanced it (21 5).

13.5. Aim of the project

Due to the lack of ligands selective for only the D 1 or D3 receptor, the individuai roles of D 1 and D3 recepton in the behavioural interactions of Dl-like and D2-like receptors remah unknown. The objective of this project was to Merinvestigate Dl and D3 receptor functions as well as their interactions in behavioural processes known to be mediated by DA, through the generation of mice deficient for both receptors and through study of mice lacking each and both receptors. The hypothesis was that CO-localisedDI and D3 receptors interact in the regdation of

DA-mediated behaviour in mice, uicluding locomotor activity, leaming and memory and motivation. This hypothesis was tested by generating D 1/D3 receptor double knockout mice and by cornparisons of the changes in DA-mediated behaviour in these mutants to mice lacking either the Dl or D3 receptor done, and mice with both fiinctional receptors. Specifically, this study investigated the effects of deletion of both recepton on locomotor activity, exploratory behaviour, anxiety, motor control and spatial learning. Spontaneous locomotor activity was measined after over a one hour period following habituation. The behaviour of Dl"D3" mice was also assessed in the novel open field and elevated plus maze tests, which are well established animai models of explorritory activity (56.6 1,99,13 8242) and anxiety-We behaviour (49,53), respectively. The rotating rod was used to assess motor control(16,112,116,190), and hally the

Moms water maze was used to hvestigate spatial learning and memory (166.183). CHGPTISR 2

MATERIALS AND METHODS 2.1. Animais

2.1.1. Dl rad D3 receptor single knockout mice

The generation of Dl receptor knockout (Dl4) and D3 receptor knockout (~3"~)mice through homologous recombination hm ken described previously by our collaborator (1,62). Briefly, a targeting vector containing the genomic sequence of the Dl receptor with neor gene inserted into the coding region of the fiAh TM, and excised sequence encoding the third intracellular loop, was transfected into ES cells derived fiom 129ISv Jae mouse strain. Positive clones were used to generate chimeric male mice, which were then crossed with female C57 BL16 mice to obtain heterozygotes. The Dl" heteroygotes were intercrossed to produce mice homoygous for the

Dl receptor mutation (62). The Dl receptor knockout mice were generated at the National

Institutes of Health 0,Bethesda, MD, USA. To generate D3 receptor knockout mice, a targeting vector containing a sequence fiagrnent encoding exon 2 of the receptor gene intempted by the neor gene cassette was transfected into ES cells derived from the 1291Sv Joe mouse strain.

The resulting male chimeric mice were mated with CS7 BLl6 females to generate D3'" heteroygotes. Mice heterozygous for the mutation were intercrossed to obtain homoygous D3" mice (1).

2.1.2. Generation ofmiee lacking Dlaad D3 recepton

The Dl" mice were a gift fkom Dr. J. Dmgo (NIH, Bethesda, USA) and D3" mice were obtained fkom Jacksoa Laboratones (Bar Harbor, Maine, USA). Dl" mice were mated to D3" mice to obtain the F 1 generation of D l+&~3+/'mice. The D l+'*D3+'-mice were subsequentiy bred to produce the F2 generation with predicted fkquencies of 1116 of each wild-type, DI'*, ~3"and

DlJP3" offiring, according to Mendel's Iaw (Fig. 2). To inmase the fkquencies of each genotype, F2 Dt4D3''* mice were interctossed to obtain D14~3" and DI" offspring with predicted frequencies of 25% in F3. The ~3"and wild-type controls were generated by breeding

mice, derived fiom the same onginal D3 receptor knockout mice as the F2 D14D3+'- mice.

Mother + D 1' D3' D 1' D3' DI' D3' Dl' D3' Father D l'D3' DlJ'D3"" Dl"D3* D1"D3J' DlJ'D3" DI' D3' D 1"D3"' D l"~3'~ D IJ*D3+'- 1 D IhD3"'+

Figure 2. Punnett's square. Possible offspring genotypes resulting from hetero ygous Dl"D3" breedhgs. Top row represents the possible combinations of Dl -id D3 alleles inherited fkom the mother and left column represents al1 possible Dl and D3 alleles that can be inherited fiom the father.

2.13. Genotyping by PCR

Mice were genotyped for Dl and D3 receptor mutations by PCR, using the Qiagen Taq polymerase kit, on genornic DNA derived fiom tail biopsies (Puregene DNA Isolation Kit,

Gentra Systems, Minneapolis, MN) (Fig. 3). The primer sequences and conditions required for genotyping the Dl receptor mutation are available on the Jackson Laboratory web page

(http://www.jax.org). The PCR protocol for genotyping D3 mutants was developed by Dr. I.

Drago (Monash University, Clayton, Audia). Primer sequences and siw of the amplüied bands correspondhg to wild-type and mutant alleles of Dl and D3 recepton are summariummarised in

Table 3. Dl wild-type Dl mutant 100 bp +/+ +/- 4- DBKO +/+ +/- 4- DBKO

D3 lOObp +/+ +!- -1- DBKO

Figure 3. Genotyphg by PCR of Dl and D3 receptor mutations. +/+, +/-, and -1- correspond to wild-type, heterozygous and hornozygous mutant DNA controls, respectively. lOObp is a DNA ladder used as marker. DBKO corresponds to a ~1%~3" DNA sample. (a) Analysis of Dl receptor gene. Dl wild-type dele: 15 1 bp band, D1 mutant dele: 350 bp band. (b) Analysis of D3 receptor gene. Both mutant and wild-type alleles were analysed in one PCR reaction. D3 wild-type aüele: 137 bp band. D3 mutant allele: 200 bp band. Receptor gene Primer sequence - End size D 1 wiid-type Forward: S'CCCGTAGCCATïATGATCGT3' 151 bp alleie Backward: S'ATTGAGAGCATTCGACAGGG3' Dlmutant Forward:S'TGGATGTGGAATGTGTGCGAG3' 350 bp allele Backward: S'CTGATTAGCGTAGCATGGACTTTGTC3' D3 wild-type Forward: S7GCAGTGGTCATGCCAGTTCACTATCAG3' 137 bp ailele Backward: S'CCTGTTGTGCTGAAACCAAAGAGGAGGAGAGG3' D3 mutant Forward: S'TGGATGTGGAATGTGTGCGAG3 ' 200 bp allele Backward: S'GAAACCAAAGAGGAGAGGGCAGGAC3'

Table 3. Fornard and backard primer sequences required to analyse mouse genomic DNA for Dl and D3 receptor mutations. Also shown are the resulting products of the PCR reactions. Separate PCR reactions for Dl mutant and wild-type alleles are required. However, the D3 receptor gene cm be analyscd in one reaction.

2.2. Behavioural assessment

23.1. Animal bousing

The animals were group housed and kept on a 12h light-12h dark cycle (lights on at 0700 hours) under standard conditions in accordance with the guidelines of the Canadian Council on

Animal Care. Male wild-type, Dl", D3" and D14D3" mice were used br al1 tests. The sarne subjects were used in al1 experiments. Behavioural testing began when mice were between 12 and 19 weeks old and lasted for 6 months. Al1 testing took place during the light phase of the

Light-dark cycle.

23.2. Experiment 1: locomotor acmtty

The assessment of basal horizontal locomotion was conducted in clear, rectangular plastic cages of 27x15 cm and 17 cm high. The cages were identical to the home cages, except that they did not contain floor bedding, water or food. The test cages were placed in -es equipped with six photoceii beams on egch of die opposite Longer sides. Before the test session, mice were habituated to the cages in 15 min sessions on three consecutive &YS. One week after the habituation sessions the test session was conducted and photobeam breaks were recorded for 60 min, as a measure of basal locomotor activity.

2.23. Experiment 2: open field

To measure exploratory activity, each mouse was tested in a novel open field. The apparatus consisted of a square enclosed Plexiglas box of 60x60 cm and 30 cm high. The floor was white and divided into nine squares of 20x20 cm each. To begin testing, each mouse was placed in the central square of the field and allowed 15 min exploration. A video camera was used to record the sessions, and the peripheral and central square crossings as well as the number of rearing events were counted fiom the videotape.

234. Experiment 3: elevated plus maze

Mice were tested on the elevated plus maze, a test for anxiety. The maze consisted of two opposite open anns, 30 cm long and 5 cm wide, and two opposite closed amis of same dimensions, surrounded by 15 cm high walls. The four amis were attached to a central square platfonn of 5x5 cm. The plus maze was elevated 70 cm above the floor. At the beginning of the test session, each mouse was placed on the central platforrn facing one of the closed arms and allowed 15 min exploration. The sessions were videotaped and the number of closed arm entries with four paws, as well as the number of open ami entries with four paws and two forepaws, and the time spent in the open arms were recorded.

2.2.5. Expriment 4: rotarod

To investigate the effects of deletion of Dl and D3 receptors on motor control and co- ordination, the mice were tested on the rotamd. The rotarod was a 3 cm diameter cylinder rotating at 10 rpm. Testing on the rotamd took place on two consecutive days. On day one, each mouse was given a 10 min training session. The mouse was placed on the immobile rod and the speed was turned on to 10 rpm. If the mouse fell from the rotarod, it was placed back on. Two hours after the training session, performance was tested in a 3 min session. The latency to Ml off the rotarod was measured. On day two, mice that performed poorly on the first probe trial

(latency < 30 sec), were given another 10 min training session. Two hours later, a 3 min test session was conducted for al1 mice.

2.2.6. Experiment 5: water mazc

The Morris water maze (MWM) is a test for spatial learning and memory. The mice were tested in the hidden and cued venions of MWM. The apparatus consisted of a white circular tank of 120 cm diameter and 30 cm high, filled with water to a depth of 23 cm. The water was rendered opaque by the addition of a non-toxic white paint, and maintained at room temperature. in the hidden version of the task, a platform 12 cm in diameter was submerged 1 cm below the water surface. in the cued version, a 15 cm high and 2.5 cm diarneter red cylinder was attached to the centre of the platform.

Hidden plalform. During place training, the platform was located in middle of one of the four quadrants, labelled NE, NW, SE and SW. The location of the platfonn was chosen randomly and kept constant for each mouse. Training took place on three consecutive days and consisted of four trials in three blocks pet day for a total of 36 trials. On day one, before the fitrial, each mouse was allowed a 30 sec fiee swim without the platform to allow acclimation to the tank. At the beginning of each trial, the moue was placed in the tank facing the wd, at one of fout start locations labelled N, S, E and W. The oder of the start location was random withlli a block of triais. The mouse was allowed to swim for 60 sec and the latency to find the plattom was recorded. If the platform was not located during 60 sec, the animai was manually guided towards it and given a score of 61 sec, Each mouse was aiiowed to stay on the platfom for 60 sec in- between trials. The the interval between blocks was two and a half hours. On day three, one hour after the last trial, the platform was removed and a 60 sec probe trial was conducted. Tirne spent in each quadrant and annulus crossings, i.e. crossings thmugh platfonn location, were recorded from a videotape of the probe trial.

Cued version. The procedure was identical to the hidden version, except that platform location was changed between the NE, NW, SE and SW quadrants within a block, and no probe trial was conducted.

2.2.7. Statistical analysb

h al1 experiments, data was recorded for each mouse and expressed as mean of each group f

SEM. For most comparisons, one-way ANOVA foilowed by Newman-Keuls post hoc test (a=

0.05) was used to assess genotype differences. Thecourse horizontal activity in the open Field, rotarod fa11 latencies, water maze escape latencies and quadrant preference were analysed by two-way ANOVA using genotype and thne blockfdayltrial block/quadrant as factors. The ciifferences in rearing between Dl" and D14D3" rnice and quadrant bias were analysed by

unpaired t-test. CHAPTER 3 RESULTS 3.1. Generation of ~l&D3&mice

Upon breeding of F2 ~1~~3"mice, 13% of the offspring expressed the Dl-'-~3"genotype and 35% expressed the D 1" genotype. The fkquencies were different thau expected (25% each

~1~D3"and Dl'") probably due to the low number of breeding pairs used (5 pairs). The

Dl-3" mutants were healthy, and had no gross physical abnomalities. Their home cage behaviour appeared to be normal. They were fertile, producing Iitters of expected size and sex distribution. Dl4-D3" mice were small in size, which is consistent with previous reports of the smaller size of Dl" mice compared to wild-type mice (62,254). Since Dl" mice require a diet of hydrated food to thnve aftet weaning (62,70), the Dl"D3" mice received mashed pellets upon weaning. Weight measurements during the experiments indicated that Dl'" and DlAD3" mice had body weights 10-20% lower than the wild-type and D3" mice.

3.2. Locomotor activity

To investigate if the presence of Dl and D3 recepton is necessary for normal spontaneous locomotor activity, forward locomotion was assessed in the double mutants and compared to

DI"; D3& mice and wild-type controls, using automated photocell beam boxes. No significant différences were observed among the four genotypes in horizontal activity in the 60 min session

(F ,, 39 = 2.65) (Fige 4)-

3.3. Open field

h order to determine whether the deficiency of both Dl and D3 receptors afEected exploratory behaviour in a novel environment, horizontal (square crossings) anci vertical (rearing) activity was measured in a large open field. Th= was a sipnincant Merence among the groups in total horizontal crossings (F a, ,, = 12.66; p < 0.0001) (Fig. 5A). Further analysis Figure 4. Balal locomotor activity of wüd-type (n = IO), Dl "(n = 8), D3 4 (n = 10) and DI'lM4 (n = Il) mice. Each mouse was placed in the activity box and photobeam breaks were measured for 60 rnin.Values represent mean sfSEM. One-way ANOVA did not reveal sigaificant differences among the genotypes. indicated that D 14D3* mice exhibited a decreased number of crossings compared to wild-type @

<0.001), DI' (p < 0.05) and D3" mice @ < 0.001). In addition, D 1" mice were significantly less

active than either wild-type or D3" mice @ < 0.05). There was no significant difference in the

total numbet of square crossings between wild-type and D3" mice over the 15 min session.

A detailed thne course analysis indicated a significant main effect of genotype (F ,, = 27.13; p < 0.000 1) and the block (F ,,,, = 25.45; p < 0.000 1) but no interaction (genotype x time

block). ANOVA of each five min time block revealed significant differences in square crossings

(1-5 min: F ,, ,, = 13.59; p < 0.0001,6-10 min: F ,,, = 10.85; p < 0.0001, 11-15 min: F ,, ,, =

6.04; p < 0.01). Subsequent analysis indicated that Dl4-D3'- mice exhibited lower activity than

wild-type @ < 0.001), Dl" @ < 0.05) and D3" mice (p < 0.001), in the first five min block of the

test session (Fig. SB). in the 6-1 0 min and 11-1 5 min blocks, the activity levels of D 1"D3'- mice

were not significantly different fiom theù D 1" littermates, but were significantly lower than the

levels of wild-type and D3" mice @ < 0.01). The decrease in horizontal activity of Dl" mice, as

compared to wild-type and D3* mice, was observed only in the 1-5 min time block.

Interestingly, in the 6-10 minute block, ~3"mice showed an increase in square crossings when

compared to wild-type mice @ < 0.05). This hyperactivity was not observed in the 1-5 min and

11- 15 min blocks.

The distribution of horizontal activity between the peripheral and central squares of the open

field did not Mer significantly among the genotypes. Normalised centre square entries (centre

entriedtotal crossings) indicated that aithough Dl> D3& and D14D3" mice tended to enter the

centre square more often than wild-type mice, these ciiflierences were not sigaificant (Fig. SC).

Moreover, a tirne course analysis of centre entries did not reveal any dBerences among the

groups in any of the theblocks (data not sbown). W/C ~1~'~3'- ~1~~3~' Genotype

lime block (min)

Figure 5. ExpIontoy behaviour in the open field ofWlT (n = 10, white ban), olJ*(n = 7, diagonal ban), D3 &(n = 10, checkereâ ban) and Dl 'lD3& (n = 11, black bars) mire. Each mouse was placed in the centre and the activity was recorded for 15 min. Values represent gmup means f SEM. (a) Total horizontal square crossings. *, p < 0.05 compaml to +type and ~3 ". il, p < 0.00 1-0.05 comptued to W/T, DI& and ~3'~.(b) Tirne course andysis of total horizontal activity. 1-5 min. *, p

< 0.01- 0.05 compared to W/T and D3 'O.#, p < 0.001-0.05 compared to W/T, Dl ''O and ~3". 6-10 min: *, p < 0.01-0.0s comparai to W/T and D 1 O, p < 0.001-0.0 1 compared to WR and ~3": 11-12min: #, p < 0.01 compareci to W/r. and ~3. D 1'- ~3'- D 1'*~3~- Genotype

Figure S cont'd. (c) Normalized centre crossings (centre/total crossings). One-way ANOVA did not reveal significant Merences among the genotypes. (d) Numkt of rem in IS min. *, p < 0.001 compared to W/T and D3 ". A, p < 0.00 1 compared to WIr. #, p < 0.001 wmpdto Wîï and ~3"and p = 0.22 compared to D 1" (unPairai t-test). Analysis of vertical activity imücated significant differences arnong the genotypes (F ,, =

55.58; p < 0.0001). Dl" mice reared less, and ~3"mice reared more than wild-type rnice @ <

0.001) (Fig. SD). Dl'D3" mice reared significantly less than either wild-type or D3'- mice @ <

0.001). Although Dl4-D3"mice appeared to rear less Uian Dl" mice, ANOVA did not reveal a statistically significant dflerence. Nevertheless, a simple cornparison of the two groups by an unpaired t-test yielded a significant difference (t = 2.48, p = 0.022), indicating a tendency towards decmed rearing in DlAD3" mice.

3.4. Elevated plus maze

in order to study the effects of combined lack of Dl and D3 receptor on anxiety-related behaviour, we examined the performance of the mice in the elevated plus maze task. The entries into open amis with two forepaws versus al1 four paws was recorded to observe if mice that entered open arms with al1 paws, a measure of decreased anxiety, also tended to cross more often

into the open arms with the two forepaws. No differences in closed arm entries (F ,n -

2.29)(Fig. 6A) and open arm entries with two forepaws (F (,, ,q = 1.25)(Fig. 6B) were observed arnong the genotypes. D14D3" mice did not differ in their entries into the open arms with four paws fiom either wild-type or Dl" mice. As expected, D3" mice entered the open arxns with four paws more often than any other genotype, when expressed as either the number of open arm entries (Fig. 6C), or as a percentage of al1 entries (data not show) (p < 0.05). The increased exploration of the open arms with four paws observed in D3" mice was abolished by the

concunent deletion of the Dl receptor, as obsewed in D14D3" mice. No ciifference in time spent

in the open arms with either two or four paws was observed among the genotypes. Although D3%

miee appeared to spend more time in the open amis with four paws than any Dl" Genotype

Geaotype

Figure 6. AR entria of W/T (II = IO), Dl 4 (n = 8), ~3~ (IL= 10) and Ill4* (n = 11) mice in the ekvateà plu8 mw. Each mouse was placed on the centre platEorm and the numkof ann entries was counted for 15 min. Values represent group means i SEM. No sigiiincant di&rences were found in the number of (a) cIosed ami entries and (b) open arm entries wih two fonpaws. W/T D 14' ~3" ~1~1)3'- Genotype

Figure 6 coiit'd. (c) ~3~-mice entered the open arms with four paws more often than other genotypes. *, p < 0.05 compared to W/T, DI" and ~1-'~3'~. other genotype, this difference failed to show statistical significance. Moreover, when the variable was expressed as tirne spent per each entry, no clifference was observed among the four groups (data not show).

3.5. Rotarod

Motor control of DI"D~" mice was investigated using the rotarod. Al1 mice were able to stay on the immobile cylinder for three minutes. A significant main effect of genotype (F ,,, ,,, -

67.56; p c 0.000 1) but no significant main effect of day (F ,,,,, = 0.07) and no interaction

(genotype x day) were observed. D14D3" mice performed very poorly on the task on both days of testhg (Fig. 7). The latency to fdl off the rotating cylinder in the double mutants was significantly lower than in either the wild-type or the D3" rnice on each day @ < 0.001). The performance of D14D3" mice, however, did not differ from Dl+ mice. Dl" and D 14D3" rnice feil off the cylinder imrnediately der it started rotating, during training and trial sessions. Their performance did not improve on the second &y despite another 10 min training session. The latency to faIl was the same in wild-type and D3' mice on day one. On day two, however, D3" mice perfomed better than wild-type mice, staying longer on the rotarod O, < 0.05).

3.6, Morris water maze

We tested the ability of Dl4-D3"mice to locate an escape platfonn submerged in water using distai (outside of tank) and proximal (marked platfom) eues, to investigate if deficiency of both recepton affects the performance in the task. No differences in swim speeds were found among wild-type (4.9 1 cm/s f: 0.26). D 1" (4.53 cmls i 0.50) and D3" mice (4.54 cm/s * 0.23). D 14'D3" mice, however, dernonstrated significantly lower swim speeds (3.06 cm/s f 0.27) than the other three pups (F , ,, = 7.83; p < 0.01). In addition, observation of the swim paths Genoîype

Figure 7. Pedormance of WA' (n = 10) ~1'"(n = 8), ~3~ (n = 9) and ~î''p3~(n = 10) mice on the rotarod. Each mouse was placed on the rotarod and the speed was hÿned on to 10 rpm. The latency to fdl was memdand maximum time ailowed was 3 min. Values represent group means f SEM during probe nids on day 1 and day 2. *, p < 0.001 and #, p < 0.001 compared to W/T and ~3'~.A, p < 0.05 compared to W/T. reveded that ~1~~~3"and DI" mice often floated in the water and teaded to swim amund the walls of the tank (thigmotaxis).

In the hidden platfom version of the water mue, a significant main effect of genotype (F ,, ,,

= 132.2; p < 0.0001) and trial block (F , ,,,, = 522; p < 0.000 1) were obsewed as well as an interaction (genotype x trial block, F 313 = 3.24; p < 0.000 1). Both D 1" and D 1%3" mice exhibited longer latencies to find the pladorm than wild-type mice by trial block 2, and D3" mice by trial block 3, and al1 blocks thereafter (Fig. 8A). The escape performance of wild-type and ~3"mice improved over training as indicated by the decreasing latencies to reach the platform. The analysis of the data fiom the probe trial reveded no main effect of genotype but a sipnificant main effect of quadrant (F ,,, ,, = 16.78; p < 0.001) and an interaction (genotype x quadrant, F ,,, = 7.16; p < 0.00 1). A simple anaiysis of the swim paths indicated that only wild- type and D3" mice spent more time in the target quadrant versus the opposite quadrant (t = 2.73; p = 0.0231 and t = 4.37; p = 0.0018, respectively, paired t-test) (Fig. 8B). Dl" and D l'"D3'- mice spent similar amounts of time in the target and opposite quadrants, indicating lack of leamhg of the escape platform location. A significant difference among the genotypes was observed in annuius crossings (F ,,. u, = 15.26; p < 0.000 1). Wild-type and D3"-mice crossed the location of the escape platform more of'ten than either D 1" or D1 "D3" mice (p < 0.00 1), indicating retention of memory of the platfom location (Fig. 8C). No dserences in annulus crossings were observed between wild-type and ~3~ mice, and between D lCand D 14D3" mice.

In the cued version of the task, ignincant main effects of genotype (F ,) = 742.7; p <

0.000 1) and trial block (F,, ,, = 7.86; p < 0.0001) and an interaction (genotype x trial block, F cK ts= 1-55, p < 0.05) were observed. The escape latencies of wild-type and D3" mice decreased rapidly over the trials (Fig. 8D). Both Dl" and ~1'-D3" mice, however, showed no improvement in locating the visible platform over the trials as their latencies remained higher in dl trial blocks.

No difference in latencies were observed between wild-type and D3" mice as well as between

Dl" and Dl4-D3'- mice in ail trial blocks. O! oi2j4Liià9ioI Block of trials

Genotype

Figure 8. Performance of WlT (n = IO), DI" (n = 3, ~3'- (a = 10) and ~1'1)3~- (n = 11) mire in the Morris water mue. Each mouse was placed iato the tank and behaviour was obsmed for up to 60 sec. Data are represented as means * SEM. (a) Latency to find Wenplatform. Dl " and ~l''Tl3" had higher lamies than W/T or ~3. (b) Target versus opposite quadrant preference during the probe trial when platform was removd Wfî: +,p < 0.05 compareâ to target quadtant, D3 ": #, p < 0.0001 cornpared to target qtiadtant. No sionificant Merences in quadrsnt preference weE observed for D 1" and DI%^". DI'- ~3'- ~1~~3" Geno type

Block of trials

Figure 8 cont'd. (c) Annuius crossings duriag the probe eial. *, p < 0.00 1 and W, p <

0.001 compared to WiT and D3 'O. (d) Escape latcncies to visible pladonn in cued version of the task W/T and ~1"~erfonnedbetter than D 1" and ~1~~3~~ CKAPTER 4

DISCUSSION AND CONCLUSIONS To investigate brain functions of DI and D3 receptors and theu interactions in DA-mediated behaviours, mice lacking both receptors were generated and their performance in various behavioural tests was studied in cornparison to mice lacking each receptor alone, and mice with both bctional recepton. The major fhdings of these tests suggest that: 1) the combined lack of

Dl and D3 receptors fiuther attenuated the low exploratory phenotype resulting from Dl receptor mutation, 2) the presence of intact Dl receptors is necessary for the expression of some aspects of the D3 receptor knockout phenotype including increases in reariag in the open field and open amis entries in the plus man, and 3) impairments in behavioural tests observed in

DI"- mice, such as the rotarod and water maze, were not affected by concurrent deletion of the

D3 receptor.

4.1. Role of Dl and D3 mepton in basal locomotor activity

Dl"D3" mice exhibited normal spontaneous Iocomotor activity in a familiar envuonment,

when compared to mice with f'unctiond Dl and D3 receptors. These 'ndings suggest that the

presence of either, or both receptors, is not required for the expression of normal basal foiward

locomotion. Lack of significant change in basal locomotor activity in either Dl4 mice, as

demoastrated in the present and past studies (69). or D3C mutants Mersupports these fmdings.

Phannacological studies ushg Dl-like and D2-like ligands are in keeping with these fïndings,

having shown that activation of both receptor subtypes synergistically increased locomotor

activity (64,72,73,85,9 1,192,243), whereas inhibition of both receptocs synergistically induced

catalepsy (25). Our fïndings indicate that the D3 receptor is not involved in this synergism, since

the absence of the receptor alone and together with the Dl receptor had no effect on spontaneous

Locomotor activity. Thus, it seems that a synergistic interaction between the Dl and D2 receptors

but not between the Dl and D3 receptors, controls forward locomotion. The abundance of both Dl and D2 receptors in the caudate-putamen (245), a brah region involved in locomotor control and the locomotor impairment of mice lacking the D2 receptor (9,118) mersupport this conclusion. Certainly, it would be usefbi to investigate spontaneous locomotor activity in mice lacking DI and D2 receptors to demonstrate if indeed the two receptors are necessary for basal fomdlocomotion.

4.2. D14D3+ mhe versus DlC and DS4* mice in open fkld and plus maze: indication of

D1/D3 receptor interaction?

D 1'"03'- mice exhibited significantiy lower locomotor activity than wild-type, D 1" and D3" mice in the open field, a test for exploratory behaviour and anxiety. Dl receptor knockout mice have been reported to show lower locomotor activity and rearing in an open field (62,220) and

D3 receptor knockout mice were found to be hyperactive in the same test (1). The present findings that deletion of Dl receptor attenuates, while deletion of D3 receptor stimulates exploratory behaviour are therefore in agreement with these reports. However, an increase in locomotor activity in Dl receptor mutants was reported by other groups (38,254), including one that used mice generated by the same laboratory as ours (38). The differences in resuits could be attnbuted to factors including environmentai differences and variations in test protocols. As indicated by the present results, a distinction should be made between basal locomotion tests, which involve long sessions and aiiow habituation, and tests of exploratory activity, which is

Sected by the novelty of the environment. Thus, the difference in the activity in the locomotion and open field tests of ~l~D3"mice and their DI" littemates may be atüibuted to the different types of behaviour measured by these tests. In the locomotion test, mice were habituated to the acfivity cages prior to the test session in order to reduce the element of mvelty, and thus were fdiliar with the test environment. In addition, the test cages were dand they resembled the home cages. The open field, however, was a large bright novel space to which the mice were exposed for the first time during the test session and was hence used to study exploratory behaviour, Le. the animal's tendency to explore a novel environment.

Concurrent deletion of Dl and D3 receptors Merattenuated the aiready low exploratory activity observed in D 1" mice. According to the present results, a reduction in locomotor activity of D ID3 double mutants in response to novelty does not reflect deficits in general locomotor activity but indicates an impairment in either movement initiation or approach response, i+. exploration. In fact, Dl'"D3" mice showed impairment in response initiation, as indicated by the lowest activity in the initial five minutes of the open field test as well as the fact that some Dl"

D3" mutants stayed in the central square for a few minutes before initiating movement (personal observation). The present fmding that mice lacking Dl and D3 receptors showed an impairment in novelty-induced locomotor activity is supported by previous reports. Dopamkergic and

GABA tnuismission in the ventral tegmental are4 nucleus accumbens and ventral pallidum have been implicated in novelty-seeking behaviour (1 1,100). Administration of DA antagonists or

GABA agonists into these areas prevented novelty-induced motor activation without suppressing the activity of habituated rats (100). According to previous and present fmdings, inactivation of

D 1 receptor decreased and inactivation of D3 receptor increased exploratory behaviour expressed by locomotor activity and rearing in an open field (1,62220). The concomitant inactivation of both ceceptors potentiated the Dl receptor mutant hypoactivity. The results obtained nom Dl" and D3'- mice suggest that the two receptors exert oppsite effects on the expression of normal novelty-induced locomotor activity. The hdings obtained fiom Dl4-D3" mice indicate that both receptors acting concurrendy are required for nodexploration. Hence, the Dl and D3 receptors may interact with each other in the regdation of expIoratory activity in an open field. In addition to evoking approach or exploratory behaviour, novel stimuli may also initiate avoidance or aauiety-related behaviour. The low locomotor activity of D 1"D3" and D 1" mice in the open field test could not only reflect the attenuation of exploratory activity, but also changes in anxiety state. Anxiety-related behaviour has been shown to be afKected by D2-like ligands, with D2-like agonists and antagonists havhg anxiogenic-like and anxiolytic-like effects, respectively (46,l9 1,198). However, results obtained fian the plus maze test indicated that D 1 and D1/D3 receptor knockout rnice did not show changes in the open ami entries, which suggests that they exbibit normal anxiety levels when compared to wild-type mice. However, the number of open ann entries of Dl" and Dl"D3" mice was very low possibly creating s floor effect, such that Merdecrease in ami entries due to D3 receptor deletion may have been impossible to observe. There were also no changes in the centre square entries in the open field, a rneasure of anxiety level, between D14D3& and wild-type mice. According to the present fuidngs, the exploratory hyperactivity (Le. the increase in both rearing and horizontal activity in the 6-10 min block) and anxiolytic-like effect (increase in open arm entries) of the D3 receptor mutation were eliminated after concurrent deletion of Dl receptor. This suggests that the Dl receptor, in the absence of the D3 receptor, may facilitate the stimulation of these behaviours and hence the D3 receptor may exert an inhibitory effect on these aspects of DL receptor function.

43. The role of Dl versus D3 receptor in motor control and spatial learning

The inability of DI~D~"mice to perform on the rotami indicates a possible motor control deficit or ataxia In order for mice to remain on the rotating roâ, normal CO-ordinationbetween the fiont and hdpaws is required in addition to the ability to lem the task. However, Dl4-D3"' mice and thek DI" littermates fell off the rod immediately ahr it started rotating, wbich indicates that they faüed to tespond to the stimulus, Le. die rotating cyünder. in faet, since Dl and DIID3 receptor mutants did aot appear to try to walk on the rotarod, theù low fail latencies do not indicate a motor control deficit but an impairment in movement initiation. Previous studies reporthg that Dl" mice did not exhibit changes in motor CO-ordination(62.69) support these hdings.

One of the major findings obtaùied in the water maze was that in the present study Dl4-mice showed impairment, a thding that is consistent with that reported by Smith et al. (220).

However, this spatial leamhg deficit was more severe than that reported previously by our laboratory (69). According to El-Ghundi et al. (1999), although Dl4 rnice were impaired in the water maze, they showed an improvement over the trials as theu escape latencies declined in both the hidden and cued versions of the task. Variations in the procedure applied in this project could explain these different patterns of resuits. In cornparison to the 60 sec trial maximum used in this study, El-Ghundi et al. (1999) used a 90 sec trial maximum. Thus, the shorter trial length could affect the chances of the impaired Dl" mice to locate the pladocm. Additionally, in the present study mice were tested in consecutive trials with a 60 sec intertrial interval on the platfonn, while in the previous study by El-Ghundi et al. (1999), an intemiai interval of approximately 6 min was allowed, during which mice were retumed to their home cages. It is possible that the massed trial procedure used in this project may have been more stressful to the mice than one in which long intervals intervened between successive trials. Since DA in the prefbntal cortex is thought to be involved in mediating responses to stress (102), it is possible that any differences in stress levels experîenced by DlC mice when subjected to different procedures may have affected performance and/or Iearning in these animals.

DI4*D3" mice exhibitcd impallments in the water maze that were indistinguishable hmtheü

Dl" littermates. The inabiiity of Dl" and Dl4-D3" mice to locate the escape plahin both the hidden and cued versions of the task supports the role of Dl receptor in cognitive processes as suggested by its distribution in the hippocampus, an area associated with ieaming and memory.

The hippocampus is a brain region important in the processing of spatial memory (95). The dopamhergic system in the nucleus accumbens has also been shown to affect spatial leaming

(193). However, in addition to a spatial leaming deficit, i.e. the inability to learn to associate extemal cues with the location of the escape platfonn, the impairment in the water maze may be a result of other factors. The impairnient could have resulted from increased thigmotaxis as well as penods of floating instead of swimming observed in Dl and D1D3 receptor mutants.

Although D14D3" mice exhibited lower shspeeds, this was unlikely to have caused their poor performance, since the irnpaired Dl mutants showed normal swim speeds. The rotarod results may again suggest motor deficits, which may have afKected the swimming performance and thus the ability of ~1~~3'"rnice to locate the escape platform. Swimming disability as a result of motor incoordination was not the reason for impainnents in the water maze, since Dl" mice developed normal swim speeds compared to wild-type mice, and both D 1" and DI4D3" mice were able to climb onto the platform. Visual impaimients were unlikely to have caused the poor performance of Dl "D3" mice, since naïve Dl" mice were able to locate the cued platform

(69). Moreover, according to studies by another group, initial processing of visual Uifonnation is unchanged in Dl receptor knockout mice (220). It is possible that Dl'"D3+ mutants failed to leam the association between the platform and escape fiom water. One of the limitations of the water maze tasic, is its restriction to aversively motivated behaviour (166) which is mediated in part by nucleus accumbens dopaminergic system. DA release in nucleus accumbens is stimulated by aversive and stressfùi stimuli including footshock or tail pinch as weii as environmental cues associateci with aversive events (reviewed in 107). Thetefore, imunpaients in aversive motivational processes, in addition to impaired spatial leamhg and response initiation (floating) as well as thigmotaxis, may dl be variables contributhg to the poor performance of Dl'"~3" mice in the water maze. Future studies are needed to merinvestigate the role of both Dl and

D3 receptors in specific aspects of motivation and learning.

Although D14D3" mice perfonned poorly on both the rotarod and water maze tasks, their impairnient was identical to that of D 1" mice. These fïndings suggest the involvement of Dl but not D3 receptors in the ability to perform on the rotarod and to locate the escape platform in the water maze, since D3" rnice perfonned uonnally in both tasks. It should be noted, however, that the impairments of Dl" and D14D3" mice resulted in very low fall latencies in the rotarod test and very high latencies in the water maze, possibly creating a floor and ceiling effect, respectively. Therefore a potential merdecrease in perfomance due to D3 receptor deletion may have been impossible to measure.

4.4. Adaptive mechanisms in D14D3& mice

One of the limitations of the gene knockout mode1 is the possibility of adaptive mechanisms developed by the mutant rnice to compensate for the absent proteins. The possibility of cornpensatory changes by other dopamine receptors as well as other brain systems in Dl"D3" mutants has to be considered. It has been reported that D2 receptor mutants exhibit increased accumulation of DA metabolites in the dorsal striatum as well as increased expression of the D3 receptor which rnay in fact compensate for the absent D2 receptor hctions (1 18). The same study, however, reported no changes in D2 receptor expression in D3 receptor mutants, which is consistent with previous reports (1,253). Dopaminergic nemm and Dl-like binding also appeared normal in D3 receptor biockout mice (253). Dl receptor mutants were reported to exbibit a reduction in dynorphin and substance P expression (62,254). However, DA containhg systems and D2-like binding sites were preserved in Dl" mutants (70,254). Hence, it is unlikely that the DIAD3-'-mice exhibit changes in the expression of other DA receptor subtypes. They may, however, display chauges in the expression of other proteins and peptides, Uicluding dynorphin and substance P. Fuhue studies should therefore include the characterisation of the expression of other proteins known to be associated with the dopaminergic system in the brains of Dl4-D3" mice and the effects of any observed changes on the behavioural phenotype of the double mutants.

4.5. lnvolvement of the 1291Sv background genotype in the behaviounl pheaotype of

D14D3& mice

Another limitation of the knockout mode1 is the presence of potential differences in behavioural characteristics between the dBerent mow strains used to produce knockout mice

(84). The Dl and D3 receptor knockout mice used to generate D14D3" mice had a mixed genetic background containing genes from the 129ISv Jae and C57 BL/6 mouse mbstrains (see Section

2.1.). Since rnice of the 1291Sv stmh have shown impairments in some behavioural tasks

(138,183), there is the possibility that the phenotypic impairments observed in D 1" and Dl"D3" mice may be caused at least in part by the presence of 129ISv genes and not by the deletion of the Dl receptor gene. Although mice of the 129/Sv Jae substrain have not been behaviourally characterised, reports indicate that some 1291Sv substrains exhibit low locomotor activity and perfonn porly on the water maze and rotarod tasks (122,138,159,183). However, several findings obtained during this project as weU as reports by other labonitories indicate that the phenotypic diffierences of the DVD3 receptor double mutants were a result of gene deletion and not background genotype. Dl4*, D3" and ~l~D3"mice aii contained a hybrid genotype but they exhited normal locomotor activity compared to wiId type mice and althoagh the activity in the open field test was low in DlC and D14D3" mice, D3" rnice exhibited increased horizontal activity in one of the tirne blocks and increased rearhg. Similady, D3'" mice perfomed normaily in the water maze and rotamd tasks uniike Dl" and Dl"D3" mutants. Moreover, studies on mice obtained fiom crossings of mice fiom the CS7 BL/6 and various 129/Sv stmins indicated that the performance of the Fl hybrids in locomotor activity and water maze tasks was similar to that of the C57 BL/6 progenitor and not the 129/Sv seain (138,159,183,230). Nevertheless, more steps could be taken in the fiitwe studies to fWther elimhte the possibility of the involvement of

129/Sv Jae strain in the phenotype of the mutants. Fht, mice can be backcrossed to the C57

BL/6 strain to Merdilute the number of 1291Sv Jae genes. Second, a larger nurnber (n=20) of animais could be used to compensate for genetic variability.

4.6. Beyond Dl and D3 recepton

By deleting the Dl and D3 receptor genes, in addition to elirninating DlD3 receptor interactions, other interactions that the two receptors may undergo with other DA recepton and their splice variants as well as other brain systems are abolished. In addition to Dl-like and D2- like receptor interactions, there is cross-talk between DA and other neurotransmitters, including noradrenaliw, serotonin, GABA, opiates, excitatory amino acids, acetylcholine and others

(reviewed in 11 1). For instance, the muscarinic m4 receptor was reported to exert an inhibitory effect on Dl receptor induced locomotor activity (90). Activation of the Dl receptor stimulates the release of glutamate in hippocampus (22), which in turn is involved in leamhg and locomotor activity (184, reviewed in 145). One report demonstrated that modulatory actions of

DA on NMDA receptor-mediated responses were reduced in Dl receptor mutants (1 33). NMDA receptor activation is necessary for induction of LTP, a mediator of leaming and memory (174) and NMDA is also involved in DA stimuiated locomotor activity (10). Hence, the phenotypic changes observed in Dl4-D3" mice, may be due in part to changes in other brain systems interacting with the DA systern, in addition to the lack of direct fictions of Dl and D3 receptors.

4.7. Conclusions

In conclusion, studies of the DI'"D~' mice have demonstrated a significant additive role of

D1 and D3 receptoa in at least one aspect of behaviour, the expression of normal novelty- induced exploratory activity. The results of this project have shown that the exploratory Dl receptor knockout phenotype was potentiated in DI"D3"- mice and that the concurrent deletion of Dl receptor prevented the expression of the phenotype observed in D3" mice. The present hdings therefore suggest a D 1/D3 receptor interaction in the regdation of exploratory activity in mice. The observations made in open field and plus maze tasks imply an inhibitory role of D3 receptor on Dl receptor hction. Moreover, the importance of Dl receptor but not the D3 receptor was revealed in spatial learning and performance on the rotarod. Thus, the phenotype of

Dl4-D3" mice consists of a response initiation deficit, demeased exploratory behaviour and impaired spatial learning, a phenotype that is qualitatively sllnilar to, but more severe than that of

Dl" mutants. Future studies are needed to Merinvestigate the roles of Dl and D3 recepton and their interactions in leaniùig and mernory, motivation and rewd. CHAPTER 5

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