Modulation de l’expression du transporteur vésiculaire du glutamate : implication dans la plasticité des neurones dopaminergiques

Numerous studies have established that the majority of neurons release more than one chemical substance. It is well known that neurons can co-express and co-release neuropeptides in addition to their neurotransmitter, but evidence of co-release of two small and fast-acting neurotransmitters has been accumulated recently.  Electrophysiological recordings have also shown that isolated serotonine and dopamine neurons can release glutamate as a co-transmitter when they are placed in culture. Furthermore, the presence of glutamate and glutaminase has been detected in serotonine, dopamine and noradrenaline neurons by immunolabelling in brain slices. Unfortunately, considering the metabolic role of glutamate, its immunodetection is not sufficient to assert the glutamatergic phenotype of a neuron. Recently, the discovery of three vesicular glutamate transporters (VGLUT1-3) has greatly facilitated the identification of glutamate neurons. These transporters are necessary for the glutamate release by neurons and constitute the first molecular markers of a glutamatergic phenotype. Interestingly, it was demonstrated that some noradrenaline neurons express VGLUT2 and that some serotonin neurons express VGLUT3. But no evidence for expression of any VGLUT subtypes was initially reported for dopamine neurons. The goal of our work was to identify which VGLUT subtype is expressed by mesencephalic dopamine neurons, and to determine if the glutamatergic phenotype of these neurons can be modulated under specific conditions. First, we used microcultures to isolate dopamine neurons and double immunocytochemistry to visualize VGLUT expression in tyrosine hydroxylase (TH) positive neurons. We showed that the majority (80%) of isolated TH+ neurons express specifically VGLUT2. This expression occurred early during in vitro development and was limited to axonal projections of dopamine neurons. However, this strong expression in vitro contrasted with the lack of detection of this transporter in adult rats in vivo. We next decided to investigate if VGLUT2 expression could be regulated during brain development of young rats and under traumatic conditions, using double in situ hybridization. At embryonic days 14 to 16, VGLUT2 and TH labelling displayed significant overlap which was no longer found at later stages. In postnatal mesencephalon of young rats, we detected VGLUT2 mRNA in approximately 1-2% of neurons expressing TH mRNA in the substantia nigra and in ventral tegmental area (VTA). To explore the regulation of VGLUT2 expression under traumatic condition, we used 6-hydroxydopamine (6-OHDA) to damage dopamine neurons in young rats. Ten days post-surgery, we found that 27% of surviving dopamine neurons in the VTA expressed VGLUT2 mRNA in 6-OHDA animals. Finally, we observed the colocalisation of VGLUT2 protein in TH positive terminals by electron microscopy. In normal rats, VGLUT2 protein was found in 28% of TH positive axon terminals in nucleus accumbens. In 6 OHDA-lesioned rats, we observed a considerable reduction of TH positive terminals, and an increase in the proportion (37%) of dopamine terminals displaying VGLUT2. Our results suggest that the glutamatergic phenotype of dopamine neurons is developmentally regulated, can be reactivated under pathological states, and that these neurons are able to release glutamate under specific conditions.

Keyphrases: dopamine, glutamate, VGLUT2, Parkinson, 6-OHDA, mesencephalon, VTA