Etude de la connectivité GABAergique des précurseurs d’oligodendrocytes durant le développement cortical

par Maddalena Balia

Thèse de doctorat en Neurobiologie

Sous la direction de María Cecilia Angulo Jaramillo.

Soutenue le 25-11-2016

à Sorbonne Paris Cité , dans le cadre de École doctorale Bio Sorbonne Paris Cité (Paris) , en partenariat avec Université Paris Descartes (1970-2019) (établissement de préparation) .

  • Titre traduit

    GABAergic connectivity of oligodendrocyte precursors cells during cortical development

  • Résumé

    Oligodendrocyte precursors cells (OPCs) are a major pool of progenitors during early development, but also persist in the adult. In 2000, a major dogma in neuroscience was broken when the existence of bona fide synapses between neurons and OPCs, a non-neuronal cell in the brain, was demonstrated. Now, it is known that OPCs can be contacted either by glutamatergic or GABAergic synapses in grey and white matter regions. However, the role of these synapses, especially of GABAergic synapses, in OPC physiology is still unclear. Our team previously demonstrated that synaptic inputs received by OPCs in the deep layer of the somatosensory cortex are primarly GABAergic. It shows that these synapses are transient, reaching a peak of connectivity in the second postnatal week (2PNW) and disappearing in the fourth postnatal week (4PNW). Nevertheless, a different mode of GABAergic transmission still persists in the 4PNW in a form of an extrasynaptic transmission relying solely on GABA spillover from nearby neurons. In the first study of this thesis, I demonstrated that the developmental switch of transmission from synaptic to extrasynaptic between GABAergic interneurons and OPCs is accompanied by molecular changes in the subunit composition of the GABAA receptors (GABAARs) of OPCs. These changes are mainly characterized by the downregulation of the γ2 and the α5 subunits between the 2PNW and the 4PNW. Interestingly, the γ2 subunit, known as a hallmark of synaptic GABAARs in neurons, is downregulated in concomitance with the loss of synaptic inputs of cortical OPCs. Pharmacology specific for γ2 showed that the switch of transmission starts at the end of the 2PNW, with a gradual loss of sensitivity to diazepam, and a decrease in the amplitude of the miniature GABAergic evoked events. The leading hypotheses regarding the role of OPC synapses include proliferation and differentiation of OPCs as well as myelination. However, none has been formally demonstrated. Since γ2 is expressed exclusively at synaptic sites in OPCs, I targeted this subunit to inactivate γ2-mediated synapses and unravel their fonction in OPCs during postnatal development. The inducible deletion of γ2 in OPCs decreased by more than a half their GABAergic synaptic activity during the 2PNW, indicating that this model constitutes a suitable tool to inactivate cortical GABAergic OPC synapses. Contrary to initial hypotheses, including those of my team, we did not observe any change in proliferation, differentiation or developmental myelination pattern of the somatosensory cortex in the knockout mouse. In addition, two-photon calcium imaging allowed us to demonstrate that evoked γ2- mediated synaptic signaling does not involved calcium signaling. Nevertheless, we observed a decrease in the number of OPCs at P30 in the knockout mouse, suggesting that these synapses regulate the self-maintenance capacity of OPCs rather than oligodendrogenesis or myelination. To confirm that the reduction of OPC density is caused specifically by the inactivation of γ2-mediated synapses, we examined the proportion of recombinant OPCs and OLs, and used non-recombinant cells as internal controls. We observed a significant 32% decrease of recombined OPCs in the knockout at P30 that was not compensated by recombinant OLs or non-recombinant cells. Hence, postsynaptic γ2-mediated GABAARs play a role in adjusting OPC density during postnatal development. In conclusion, during my thesis I have demonstrated that the postnatal switch of transmission from synaptic in the 2PNW to extrasynaptic at the 4PNW between interneurons and OPCs is accompanied by the down-regulation of the γ2 subunit of GABAARs in cortical OPCs. Impairing the γ2-mediated synaptic GABAergic signaling in OPCs did not result in drastic changes in the proliferation of differentiation of these cells. Instead, our results rather indicate a role in the density homeostasis of OPC population during cortical development.

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