Neurochemical changes in dopamine D1, D3 and D1/D3 receptor knockout mice

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Abstract

Neurochemical changes were examined in dopamine D1 receptor knockout (D1−/−), dopamine D3 receptor knockout (D3−/−) and dopamine D1/D3 receptor double knockout (D1−/−D3−/−) mice. The level of dopamine D1- and D2-like receptors and γ-aminobutyric acid (GABAA) receptor was assessed by ligand autoradiography and dopamine D1- and D2 receptor, enkephalin, dynorphin and substance P transcripts measured by in situ hybridization. D1−/− mice had normal GABAA receptor levels, reduced dynorphin and substance P, and increased enkephalin mRNA and dopamine D2-like binding. D1−/−D3−/− mice evidenced decreased dynorphin and substance P but normal enkephalin expression, whereas dopamine D2-like and GABAA receptor binding were increased. Major changes occur in substance P and dynorphin expression in D1−/− mice and these changes are unaffected by loss of dopamine D3 receptors. Upregulated dopamine D2-like binding and enkephalin in D1−/− mice may be due to decreased dopamine turnover. Upregulated enkephalin in D1−/− mice is dependent on functional dopamine D3 receptors.

Introduction

The neurotransmitter dopamine plays a pivotal role in the regulation of a diverse array of neural processes that include motor control, reward, neuroendocrine and cardiovascular regulation, cognition (Jaber et al., 1996) and the control of respiration (Mueller et al., 1982). The effects of dopamine on behaviour and drug responses are mediated by two subfamilies of G-protein-coupled receptors (Sibley and Monsma, 1992). The dopamine D1 receptor subfamily consists of dopamine D1 and D5 receptors (also called D1A and D1B dopamine receptors, respectively) while the dopamine D2 receptor subfamily consists of dopamine D2, D3 and D4 receptors. High levels of dopamine D1 receptors are detected in the caudate putamen, nucleus accumbens, olfactory tubercle and Islands of Calleja, and lower levels are found in the substantia nigra pars reticulata/entopeduncular nuclear complex and the ventral tegmental area. The localisation of dopamine D1 receptor mRNA generally correlates well with the regional distribution of the dopamine D1 receptor, although dopamine D1 receptor mRNA is not found in the substantia nigra Meador-Woodruff et al., 1991, Sibley and Monsma, 1992, Gingrich and Caron, 1993. Low levels of dopamine D1 receptor mRNA expression (Le Moine et al., 1991) have been found in striatal cholinergic interneurons (Kawaguchi et al., 1995) and striatal interneurons that make the inhibitory neurotransmitter γ-aminobutyric acid (GABA).

The dopamine D3 receptor has a 52% overall amino acid homology with the dopamine D2 receptor (Sibley and Monsma, 1992). Dopamine D3 receptor mRNA shows a distinctive distribution with relatively high levels in the limbic areas of the brain including the ventral striatal complex consisting of the nucleus accumbens, olfactory tubercle, Islands of Calleja and ventral pallidum as well as high level expression in the ventral tegmental area Sokoloff et al., 1990, Landwehrmeyer et al., 1993; brain regions implicated in the regulation of motivation and emotion. The dopamine D3 receptor is also expressed at low levels in dopaminergic neurons within the substantia nigra, suggesting that it may have a presynaptic function (Sokoloff et al., 1990).

Dopamine receptor and neuropeptide expression in the normal brain is complex, with interactions between receptor systems within neurons and between neurons ultimately impacting on behaviour and responses to drug treatment. The lack of ligands with absolute receptor specificity stimulated the use of gene targeting approaches to examine the in vivo function of the dopamine D1 receptor Drago et al., 1994, Drago et al., 1996, Xu et al., 1994a, Xu et al., 1994b, Miner et al., 1995, Levine et al., 1996, Moratalla et al., 1996, Crawford et al., 1997, Friedman et al., 1997, dopamine D2 receptor Baik et al., 1995, Maldonado et al., 1997, dopamine D3 receptor Accili et al., 1996, Steiner et al., 1997, dopamine D4 receptor (Rubinstein et al., 1997), dopamine D5 receptor (Sibley et al., 1998) and dopamine transporter (Giros et al., 1996). The generation of dopamine receptor mutants offers a powerful opportunity to evaluate the roles of these receptors in dopamine-mediated behaviours and transcriptional regulation. Indeed, studies on both lesioned and genetically manipulated animals suggest that dopamine has a pivotal role in the regulation of gene transcription. Recently, Karasinska et al. (2000) generated dopamine D1/D3 receptor double knockout (D1−/−D3−/−) mice and suggested that dopamine D1/D3 receptor interaction was involved in the regulation of exploratory activity in mice. Recent data suggest cellular dopamine D1/D3 receptor interactions Levavi-Sivan et al., 1998, Ridray et al., 1998, Jung et al., 1999; also, a role for the dopamine D3 receptor in regulating dopamine D1/D2 receptor interactions, particularly in terms of a possible inhibitory effect on dopamine D1/D2 receptor interactions at both electrophysiological and behavioural levels (Xu et al., 1997), has been proposed. While mice with deletion of the dopamine D1 receptor or of the dopamine D3 receptor have been studied, the neurochemical properties of dopamine D1 and D3 receptors and their interactions would be illuminated most powerfully by their co-deletion. We have dopamine D1 receptor knockout (D1−/−) and dopamine D3 receptor knockout (D3−/−) mice in house and have independently generated a dopamine D1/D3 receptor double knockout (D1−/−D3−/−) mouse line. The aim of this study was to characterize the neurochemical changes in D1−/−, D3−/− and D1−/−D3−/− mice in an effort to understand the neuroregulatory role of dopamine D1 receptors and any facilitatory role that dopamine D3 receptors may play in dopamine D1 receptor-mediated processes. Quantitative ligand autoradiography was undertaken for striatal dopamine D1- and D2-like receptors, muscarinic acetylcholine receptors, GABAA receptors and the dopamine transporter. In addition, striatal mRNA expression was also quantified for dopamine D1 receptors, dopamine D2 receptors and the neuropeptides enkephalin, dynorphin and substance P.

Section snippets

Animals

The generation of D1−/− and D3−/− mutants was as reported previously Drago et al., 1994, Accili et al., 1996. Heterozygous founders for both the D1−/− and D3−/− genotype were transported from our original colonies at the National Institutes of Health and maintained in a hybrid C57/BL6×129/Sv genetic background Drago et al., 1994, Accili et al., 1996. D1−/−D3−/− mice were obtained by crossing D1−/− and D3−/− mice. The resulting F1 heterozygous D1+/−D3+/− mice were bred to produce F2 generation,

Dopamine D1 receptor mRNA expression

Dopamine D1 receptor mRNA was identified in the striatum, nucleus accumbens, Islands of Calleja and olfactory tubercle of WT mice (Fig. 1A) but was not detected in the striatum of D1−/− mice (Fig. 1G) or D1−/−D3−/− mice (Fig. 1S). The signal obtained using both labelled and unlabelled excess D1.3 oligonucleotide in normal mice was identical to the signal obtained when labelled D1.3 oligonucleotide was hybridized to D1−/− or D1−/−D3−/− mice, suggesting that this signal intensity represented

Discussion

At a neuroanatomical/neurochemical level, the dual pathway model of basal ganglia circuitry assumes the segregation of dopamine D1 and dopamine D2 receptors Albin et al., 1989, Kawaguchi et al., 1990. In situ hybridization studies have shown that dopamine D1 receptors are preferentially expressed on substance P/dynorphin positive neurons which project directly to the substantia nigra pars reticulata/entopeduncular nuclear complex. The same studies also revealed that enkephalin-positive dopamine

Acknowledgements

The authors thank Sara Fuchs and Domenico Accili for providing the breeding colony of D3 mutants. This work was supported in part by the National Health and Medical Research Council of Australia. J.J.C. was supported by the Australian National Health and Medical Research Council Brain Network into Mental Diseases; J.D. is an Australian National Health and Medical Research Council Practitioner Fellow; J.J.C. and J.L.W. are supported by the Higher Education Authority of Ireland, a Galen

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