Research reportDopamine receptor-modulated []GTPγS binding in striatum of 6-hydroxydopamine-lesioned rats
Introduction
Neurotransmitter receptors are subject to various forms of regulation that allow their physiological responses to be modified appropriately in response to changes in the environmental conditions. Such mechanisms affecting dopamine receptors have been implicated in the appearance of motor disorders arising after chronic neuroleptic treatment or in Parkinson's disease. Indeed, these two situations led to a dramatic decrease in the stimulation of dopamine receptors within the nigrostriatal pathway. Although there is a large body of work describing the regulation of dopamine receptors (for review, see Ref. [29]), the functional consequences at the biochemical level are not fully understood.
Experimentally, alteration in the dopamine transmission can be obtained after intranigral microinjection of 6-hydroxydopamine (6-OHDA), that results in dopamine depletion in the corresponding striatum. In this well-known model of hemi-Parkinsonism [33], the density of striatal D2 dopamine receptors is generally found to be increased 1, 8. This is accompanied by enhanced functional responses to dopamine agonists, as measured by behavioural [34], electrophysiological [14]and biochemical 9, 22techniques. It is, however, noteworthy that the time course and the amplitude of changes in dopamine receptors density were not clearly found to be consistent with those of hypersensitivity to dopamine agonists 23, 30. Moreover, in several models of dopaminergic hypersensitivity, an increase in functional response is not always correlated with D2 dopamine receptor upregulation [17]. In particular, rats with a unilateral 6-OHDA lesion chronically treated with eticlopride rotate contralateral to the lesion in response to quinpirole despite showing striatal symmetry of D2 receptor binding [16]. Concerning the D1 dopamine receptor density after dopamine depletion, the results described in the literature are conflicting (for review, see Ref. [29]), with most of the data indicating a decrease in D1 receptor expression. Some authors have proposed that modulation at the level of the signal transduction could explain the dopaminergic hypersensitivity following dopamine depletion or chronic dopamine receptor blockade. Thus, changes in striatal G-protein densities or functions have been proposed to be involved in the enhancement of the functional responses to dopamine agonists 4, 5, 20, 28, 31, 32.
The aim of our study was to evaluate the role played by the modulation of dopamine receptor–G protein coupling in the development of dopaminergic hypersensitivity accompanying the unilateral nigral 6-OHDA lesion. For this purpose, we compared dopamine agonist-induced binding of guanosine 5′-O-(γ[]thio)triphosphate ([]GTPγS) in membranes of denervated and control striata from the same animals. Agonist-induced binding of non-hydrolysable analogues of GTP was proven to be useful to study the functional coupling of a large variety of G protein-coupled receptors, in different models of transfected cells as well as in tissues 18, 19, 35. This technique was previously optimised for the study of dopamine receptor–G protein coupling in membranes of rat striatum 11, 27.
In order to take more than one stage of the pathological state into account, the analysis was performed after different periods of time (1–5 weeks) following the lesion. Indeed, most data from the literature focus on a single time interval after the lesion resulting in some conflicting observations. Together, our results show that dopamine-stimulated []GTPγS-specific binding was increased following the lesion as compared to the contralateral striata. Pharmacological characterization of the response to dopamine revealed the involvement of both D1 and D2 dopamine receptors. A preliminary report of some of these data has been presented at the XIIIth International Congress of Pharmacology [10].
Section snippets
Surgery
Under chloral hydrate anaesthesia (400 mg/kg), male Wistar rats (250–300 g) were placed in a David Kopf stereotaxic frame. A unilateral injection of 6-OHDA (4 μl of a 3 mg/ml solution of 6-OHDA hydrobromide in 0.9% NaCl and 0.1% sodium metabisulfite) was delivered into the substantia nigra pars compacta at stereotaxic coordinates measured from bregma: lat=−1.8 mm; post=−5.2 mm; prof=−7.8 mm [25]. Theoretical coordinates were previously adjusted by stereotaxic microinjection of India ink in the
Characterization of dopamine receptor-mediated []GTPγS binding
As previously reported [11], the specific binding of []GTPγS to rat striatal membranes was found to be increased in the presence of dopamine. Thus, dopamine increased the []GTPγS binding by up to ∼50% after 60 min incubation. Indeed, both basal and dopamine-induced specific binding of []GTPγS progressively increased during the incubation, but their ratio was found to be relatively constant between 15 and 60 min (not shown). To a lesser extend (∼30% above basal), the effect of dopamine
Discussion
The aim of the present work was to evaluate alteration in the functional coupling of dopamine receptors in rat striatum after prolonged dopamine depletion within the nigrostriatal pathway. Dopamine receptor agonist-modulation of []GTPγS binding was measured in order to study the first step in the signalling pathway activated by dopamine receptors. We have previously reported the optimization of experimental conditions of this assay for the characterization of the pharmacological properties
Acknowledgements
The authors wish to thank A. Lebbe and H. Lenaert for their excellent technical assistance and Dr. C. Godfraind (from the Laboratory of Special Pathological Anatomy of the Catholic University of Louvain) for her help in adjusting histologically accurate stereotaxic coordinates. This work was supported by the National Fund for Scientific Research (F.N.R.S., Belgium, Convention FRSM 3.453.997F), by a Grant of Ministry of Scientific Policy (Belgium) (ARC 95/00-188), and by the Belgian Queen
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