Muriel Geurts

Altered expression of regulators of G-protein signaling (RGS) mRNAs in the striatum of rats undergoing dopamine depletion

Quantitative in situ hybridization was used to investigate the effect of prolonged striatal dopamine or monoamine depletion on the mRNA density of regulators of G-protein signaling (RGS) 2-12 proteins. Two types of treatments were studied: a 6-hydroxydopamine-induced unilateral lesion of the nigrostriatal pathway and a 5-day reserpine treatment. The results clearly show a selective increase in the mRNA levels of RGS2, 5 and 8 and a decrease in RGS4 and 9 mRNA levels following nigrostriatal denervation. In this model, we observed no change in the mRNA levels of RGS10 and other RGS proteins that are weakly expressed in the striatum (RGS3, 6, 7, 11 and 12). On the other hand, the mRNA levels RGS2, 4, 5, 8, 9 and 10 were found to be significantly decreased after prolonged reserpine treatment. In contrast, the densities of these transcripts (in particular, RGS2, 4 and 10) tend to increase after an acute administration of reserpine, used as control. These results provide further evidence for the influence of dopamine and/or other monoamines in the regulation of RGS protein expression in the striatum. In connection with the previously documented acute regulation of RGS proteins after modulation of the dopaminergic transmission [Geurts et al., Neurosci Lett 2002;333:146-50], the present study demonstrates that alteration in their genetic expression can be long-lasting and this could reflect the adaptation processes that occur in certain pathological states, such as Parkinsons disease.

Opposite modulation of regulators of G protein signalling-2 (RGS2) and RGS4 expression by dopamine receptors in the rat striatum

The role of dopaminergic transmission on striatal mRNA levels of regulators of G protein signalling proteins RGS 2-12 was evaluated by quantitative in situ hybridisation. A single injection of L-dopa (50 mg/kg i.p.) significantly increased the RGS2 mRNA level (by 25%), an effect that was specifically abolished by the D1 dopamine receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (R(+)-SCH23390), but not changed by S(-)-eticlopride. Interestingly, the administration of this D2 dopamine receptor antagonist alone markedly enhanced the expression of RGS2 (by 71%), which suggests a constitutive inhibition of RGS2 expression by D2 dopamine receptors. Opposite results were obtained concerning the regulation of RGS4 since L-dopa alone was without effect whereas co-administration of L-dopa and R(+)-SCH23390 significantly enhanced the RGS4 mRNA levels (by 38%). In conclusion, D1 and D2 dopamine receptors appear to mediate opposite regulatory effects on RGS2 and RGS4 expression in the striatum.

Regional distribution of manganese found in the brain after injection of a single dose of manganese-based contrast agents

Manganese (Mn) complexes are unstable and dissociate in vivo. Because of the release of this metal, there exists some concern about the potential long-term neurotoxicity associated with the use of Mn-based contrast agents. This latter problem arises because manganese is known to accumulate in specific regions of the brain of people intoxicated by this metal. It was previously demonstrated that Mn can accumulate in the mice brain after administration of 5 micromol/kg of MnCl2, Mn-diethylenetriaminepentaacetate (Mn-DTPA), or Mn-dipyridoxal diphosphate (Mn-DPDP). In order to better characterize the behavior of Mn complexes after administration, this study assesses the regional distribution of Mn in the brain after i.v. injection of a single dose of MnCl2 or Mn-DTPA. Male Wistar rats received an i.v. injection of 5 micromol/kg of 54Mn as MnCl2 or Mn-DTPA. The rats were killed at one and two weeks post exposure. The distribution of the radioactivity in the slices was monitored by autoradiography. For both MnCl2 or Mn-DTPA, we observed that the radioactivity was dispersed in the entire brain, but the radioactivity was higher in several regions. No difference was observed between MnCl2 or Mn-DTPA in the regional distribution of Mn, and no difference was observed between the two times of exposure (1 week or 2 weeks). The uptake of Mn was minimal in corpus callosum. Maximal Mn concentration was observed in the hippocampal region, thalamus, colliculi, amygdala, olfactory nuclei, and cerebellum.

Non invasive quantification of manganese deposits in the rat brain by local measurement of NMR proton T1 relaxation times

Up to now, there is no reliable non invasive biomarker for the concentration of manganese (Mn) in the brain after intoxication to this metal. The aim of the present experimental study was to determine the predictive value of the localized measurement of the proton NMR relaxation time T1 as a quantitative estimation of the concentration of Mn in brain. The relationship of the proton relaxation rates (1/T1) was established in rat brain homogenates as a function of the Mn, iron, and copper concentration. Subsequently, an experimental model of Mn neurotoxicity was used: rats were stereotactically injected with increasing amounts of Mn2+ (as MnCl2) in the ventricles. After 3 weeks, local measurements of T1 were carried out in live rats. They were then sacrificed in order to sample the striatum, the cortex, and the cerebellum from the brain and to perform a quantitative determination of the concentration of Mn in these tissues by atomic absorption spectrometry (AAS). The results indicate excellent correlation coefficients between relaxation rates and tissue Mn concentrations (r= 0.84, 0.77 and 0.92 for the striatum, the cortex and the cerebellum, respectively). This methodology offers a unique toolfor monitoring the degree of Mn concentration in different areas of the brain in animal models of Mn intoxication. It will be useful for evaluating the efficacy of treatments aimed at decreasing the metal in the brain. The method could be potentially useful for being transposed in the clinical situation for monitoring Mn-exposed workers.

Down-regulation of neurotensin receptors after ligand-induced internalization in rat primary cultured neurons.

When rat cultured neurons were incubated with unlabelled neurotensin (3 nM) for 1 or 24 h at 37 degrees C, the [3H]-neurotensin specific binding measured in cell homogenates was decreased to about 35 and 65% of control values, respectively. In these experiments, the decreases in binding corresponded to reductions of Bmax values without changes in the affinity. The slow neurotensin-induced receptor down-regulation is thought to result from receptor degradation since it was reduced by the lysosomotropic drugs chloroquine and methylamine and because no change in neurotensin mRNA level could be measured after the neurotensin stimulation. After their internalization, receptors slowly reappeared at the cell surface after further incubation in the absence of the peptide. Such receptor reappearance was prevented in the presence of the protein synthesis inhibitor cycloheximide and is therefore thought to result from new synthesis and not from recycling of internalized receptors. These results indicate that the neurotensin-induced receptor internalization in cultured neurons is irreversible and that it is followed by a down-regulation of the receptor through a degradative process.

Chronic treatment with certain antipsychotic drugs preserves upregulation of regulator of G-protein signalling 2 mRNA in rat striatum as opposed to c-fos mRNA

Quantitative in situ hybridization on rat coronal brain sections with radiolabelled oligonucleotide probes was performed to investigate the effects of antipsychotic drugs on mRNA levels of regulator of G-protein signalling (RGS) 2 and c-fos. This study demonstrated a similar increase of RGS2 mRNA level in the striatum upon both a single and a 21-day treatment with either haloperidol (2 mg/kg) or risperidone (7.5 mg/kg) in contrast to clozapine (20 mg/kg). Otherwise, the acute c-fos mRNA induction in the striatum was abolished by 74 to 89% upon chronic treatment with either haloperidol or risperidone. In conclusion, the induction of RGS2 mRNA in the striatum, in contrast to the immediate early gene c-fos mRNA, is preserved upon chronic treatment with haloperidol and risperidone.

Agonist and antagonist modulation of [35S]‐GTPγS binding in transfected CHO cells expressing the neurotensin receptor

1 The functional interaction of the cloned rat neurotensin receptor with intracellular G‐proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [35S]‐GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells. 2 The agonist‐induced binding of [35S]‐GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [3H]‐neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC50 value of 2.3±0.9 nM). 3 The stimulation of [35S]‐GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV‐449 (EC50 value of 1.7±0.4 nM) and the neurotensin related peptide neuromedin N (EC50 value of 21±6 nM). 4 The NT‐induced [35S]‐GTPγS binding was competitively inhibited by SR48692 (pA2 value of 9.55±0.28), a non‐peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [35S]‐GTPγS. 5 The response to neurotensin was found to be inhibited by the aminosteroid U‐73122, a putative inhibitor of phospholipase C‐dependent processes, indicating that this drug may act at the G‐protein level. 6 Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G‐protein level that is induced by the neuropeptide neurotensin after binding to its receptor.

Selective destruction of nigrostriatal dopaminergic neurons does not alter [3H]-ryanodine binding in rat striatum

Dopamine nigrostriatal neurons are important for motor control and may contain a particularly dense population of ryanodine receptors involved in the control of dopamine release. To test this hypothesis, we used a classical model of unilateral selective lesion of these neurons in rats based on 6-hydroxydopamine (6-OHDA) injection into the substantia nigra. Binding of [3H]-GBR 12935, used as a presynaptic marker since it labels specifically the dopamine uptake complex, was dramatically decreased by 83-100% in striatum homogenates after 6-OHDA lesion. On the contrary, no reduction of [3H]-ryanodine binding was observed. The present data indicate that [3H]-ryanodine binding sites present in rat striatum are not preferentially localized in dopaminergic terminals.