Lack of autoreceptor-mediated inhibitory control of dopamine release in striatal synaptosomes of D2 receptor-deficient mice
Introduction
Dopamine acts through the binding to five distinct dopaminergic receptors (DR). These receptors have been classified in two families, D1R and D2R, upon pharmacological, biochemical and structural criteria. The D1R family comprises the D1R and D5R, while the D2R contains the D2R, D3R and D4R. The D2R is particular in that it exists in two molecular distinct species: D2S (short) and D2L (long). Both are generated from the same gene by differential splicing. D2L contains an additional insert of 29 amino-acids in the third cytoplasmic loop as related to D2S 41, 55. In spite of the numerous available dopaminergic agonists and antagonists within each dopaminergic receptor sub-family, highly specific drugs for each type of receptors are still lacking [55]. This has limited the precise determination by pharmacological studies, of the functional characteristics of the different types of receptors. Genetically modified mice lacking components of the dopaminergic transmission have been generated by homologous recombination. The study of these animals has greatly enhanced our understanding of the physiological function of the different receptors in vivo. Indeed, D1R, D2R, D3R and D4R-deficient mice have been generated. Interestingly, these animals present specific phenotypes which have given insights into the specific function of each receptor 1, 6, 10, 22, 29, 34, 37, 38, 39, 43, 46, 60.
We analysed D2R-deficient mice 6, 10, 34, 37, 48in order to confirm the role of D2Rs as autoreceptors and to look for an eventual role of the D3R in this process in the absence of D2R. Indeed, in transfected cells both D2Rs and D3Rs have been reported to act as autoreceptors [57]. As for dopamine, most D2 agonists used in release studies to investigate the properties of dopaminergic autoreceptors, were found to have higher affinity for D3R than for D2R [55]. In addition, controversies exist concerning the presence of D3R in nigral dopaminergic neurones and the existence of species differences 4, 8, 17, 19, 20, 40, 54. No significant difference in the pattern of expression of the D3R and D4R genes was found in D2R-deficient mice. Furthermore, the number and affinity of the D1Rs are not affected [6]. Recent studies using these mice have shown a very weak but specific residual [125I]-iodosulpride labelling in the substantia nigra–ventrotegmental area of D2R-deficient mice. From this study, it was concluded that D2Rs and D3Rs share a common presynaptic localisation in dopaminergic neurones, although D2Rs are the most abundant [37]. In this article, we confirm the previously obtained results and we show that both NPA (100 nM) and PD-128,907 (100 nM) (a D3 specific agonist)-evoked responses were completely suppressed in D2R-deficient mice.
Section snippets
Materials
Initial experiments were performed on male C57BL/6 mice from Charles River (France). Animals were housed in a temperature-controlled room (22–23°C) in which the light was on from 0600–2000 h. Mice lacking D2R (−/−) [6]and respective control littermates (+/+) were obtained by matings of heterozygous mice. All experiments were conducted in conformity with the French publication on animal experimentation (no. 87-848), the U.K. Animals (Scientific Procedures) Act (1986) and the European Communities
Quinpirole-induced inhibition of dopamine release under different conditions of depolarisation
Initial experiments were performed with striatal synaptosomes from C57BL/6 mice by measuring the release of [3H]-DA continuously synthesised from [3H]-tyrosine. The three depolarising agents, potassium chloride (25 mM), veratridine (1 μM) and 4-AP (100 μM) which act through different processes were selected to evoke the release of newly synthesised [3H]-DA. As illustrated in Fig. 1, at the respective concentrations used, these agents stimulated the release of [3H]-DA with a similar amplitude.
Discussion
Compelling evidence indicates that in the striatum, D2Rs are located on populations of striatal cells and on terminals of the corticostriatal projections 15, 16, 27, 28, 32, 33, 50, 53. D2Rs are also present on synaptic terminals, soma and dendrites of the nigrostriatal dopaminergic neurones 2, 5, 27, 28, 35, 44, 53. It was originally thought that an inhibitory control of DA synthesis was responsible for the decreased release of the transmitter. Indeed, on the basis of experiments performed on
Acknowledgements
A.C. is a research fellow of Rhône-Poulenc Rorer. This study was supported by INSERM, Collège de France and Rhône-Poulenc Rorer.
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