Experience-dependent control of both GABAergic connectivity of oligodendrocyte precursors and myelination in the somatosensory cortex – OLIGOSENSOR

The main objective of the present proposal is to demonstrate whether GABAergic interneurons influence cortical myelination by regulating the proliferation and/or the differentiation of OPCs in an activity-dependent manner in the somatosensory (barrel) cortex. To reach this goal, we use a multidisciplinary approach combining electrophysiology, two-photon calcium imaging, holographic photolysis, immunocytochemistry in control and demyelinated mice showing a deletion of a subunit of GABA-A receptors, specifically in OPCs.

First, paired recordings and holographic photolysis are used to determine the spatial organization of interneuron-OPC connections in the network and the identity of GABAergic interneurons synaptically connected to OPCs that might regulate oligodendrogenesis. Holographic photolysis is an optical method that allows for the generation of flexible and precise light patterning enabling the photolysis of caged neurotransmitters. In our case, we use this technique to photostimulate interneurons at single cell resolution with caged glutamate and defined the spatial organization of the GABAergic connectivity of OPCs.

Second, GABA-A receptor subunit deletion in OPCs should impair synaptic activity in normal conditions and in demyelinated mice. This strategy should allow us to unravel whether GABAergic synaptic connectivity of OPCs directly regulates oligodendrocyte differentiation and myelination. The effect of GABA-A receptor subunit deletion in OPCs is assessed with electrophysiology and two-photon calcium imaging while the impact on OPC development and myelination is analyzed by immunostainings against different markers of OPCs, oligodendrocytes and myelin.

Our data already revealed that distinct types of interneurons innervate OPCs in a highly regulated manner. Different types of interneurons discriminate OPC postsynaptic sites, targeting anatomically segregated subcellular domains, containing distinct GABA-A receptors. OPCs thus compartmentalize input regions according to the identity of the interneuron. Surprisingly, our results also revealed that interneurons and OPCs form a specific network characterized by a very local microarchitecture. Finally, we also showed that interneuron-OPC synaptic connectivity reaches its peak in correlation with a switch to a massive OPC differentiation onto myelinating oligodendrocytes, suggesting a role of these synapses in oligodendrogenesis (Orduz et al., 2015). To directly assess the functional role of OPC synaptic activity in oligodendrogenesis and myelination, we already generated a conditional null mouse with loss of function of a synaptic GABA-A receptor subunit in OPCs. Patch-clamp recordings already showed a half decrease in the amplitude of evoked GABAergic synaptic currents and in the frequency of spontaneous synaptic activity of OPCs in the knockout mouse during the peak of interneuron-OPC connectivity. We are presently analysing by immunostainings the consequence of the subunit deletion in OPC proliferation, differentiation and in myelination during the first postnatal month. Moeover, we are analysing the expression profile of the subunit of the GABA receptor following focal demyelination and the consequences of its deletion on remyelination.

The demonstration that OPC GABAergic synaptic activity regulates oligodendrogenesis may have profound consequences for the design of innovative therapies promoting myelination and oligodendrocyte regeneration in myelin disorders. Indeed, if the involvement of synaptic inputs onto OPCs influences the generation of myelinating oligodendrocytes, pharmacological strategies aiming at modulating the synaptic activity of OPCs could be envisaged.

Publication

Orduz D, Maldonado PP, Balia M, Vélez-Fort M, de Sars V, Yanagawa Y, Emiliani V, Angulo MC (2015) Interneurons and oligodendrocyte progenitors form a structured synaptic network in the developing neocortex. eLife 4:e06953.

Cognitive, motor and sensory functions depend on the proper maturation of neuronal signals and their integration across different brain regions. This is partly achieved by myelination of neuronal fibers, a process required to speed neuronal transmission by insulating axons from current leakage. Myelination is a critical developmental process just as it is vulnerable. Major developmental brain disorders induce irreversible myelination defects. One possibility to overcome myelination impairment is to stimulate the production of mature oligodendrocytes from endogenous oligodendrocyte precursor cells (OPCs). To reach this aim, however, it is first necessary to understand the cellular and molecular signals that control OPC proliferation and differentiation during postnatal development.
Recent advances have revealed functional synaptic connections between neurons and OPCs which role remains elusive. Synaptic neurotransmitter release onto OPCs constitutes a suited mechanism to control the fate of these cells. We recently demonstrated that spontaneous GABAergic synaptic activity in cortical OPCs is higher when the production of pre-myelinating oligodendrocytes reaches a peak and disappears after. This loss of GABAergic synapses is accompanied by dramatic changes in the subunit composition of GABAA receptors (GABAARs) of OPCs which are mainly characterized by the downregulation of the ?2 subunit expression. This correlation between transient synaptic innervation, ?2 subunit downregulation and oligodendrogenesis suggests that GABAergic inputs influence OPC differentiation and thus myelination.
The main goal of this proposal is to unravel whether GABAergic connectivity of OPCs has an impact on OPC differentiation and myelination during postnatal development of the somatosensory (barrel) cortex. To assess the functional role of gamma2 subunit in these processes, we already generated a conditional null mouse with loss-of-function of GABAAR-gamma2 subunit specifically in OPCs. Since this subunit is an important component of GABAARs at interneuron-OPC synapses, this strategy will allow us to address the role of GABAergic synapses of OPCs in their proliferation, differentiation and myelination. As a complementary approach, we will alter intra-cortical inhibition and interneuron activity by using a protocol of sensory deprivation. The effect of sensory deprivation on GABAergic synaptic transmission, oligodendrogenesis and myelination will be assessed by patch-clamp recordings and immunohistochemistry in control and GABAAR-gamma2 conditional knockout mice. We expect that this model will allow us to demonstrate an experience-dependent control of the GABAergic connectivity on OPCs and to provide a demonstration that oligodendrogenesis depends on the activity of interneurons. Finally, one key unresolved question is the identity of interneurons synaptically connected to OPCs that might control their development. Another innovated aspect of our proposal is to combine paired-recordings with holographic photolysis to map interneuron-OPC connectivity. While we will use holographic photolysis to define the spatial organization of the GABAergic connectivity of OPCs, paired recordings will allow us to analyze, with high degree of detail, the presynaptic and postsynaptic compartments of unitary interneuron-OPC connections. The comparison of the properties of unitary connections in control and whisker-deprived mice should reveal the impact of specific interneurons on GABAergic connectivity and on oligodendrogenesis.
The demonstration that OPC GABAergic synaptic activity regulates oligodendrogenesis may have profound consequences for the design of innovative therapies promoting myelination and oligodendrocyte regeneration in myelin disorders. Indeed, if the involvement of synaptic inputs onto OPCs influences the generation of myelinating oligodendrocytes, pharmacological strategies aiming at modulating the synaptic activity of OPCs could be envisaged.