Adult-born SVZ progenitors receive transient synapses during remyelination in corpus callosum.

We found that demyelinated axons formed functional glutamatergic synapses onto adult-born NG2(+) oligodendrocyte progenitor cells (OPCs) migrating from the subventricular zone after focal demyelination of adult mice corpus callosum. This glutamatergic input was substantially reduced compared with endogenous callosal OPCs 1 week after lesion and was lost on differentiation into oligodendrocytes. Therefore, axon-oligodendrocyte progenitor synapse formation is a transient and regulated step that occurs during remyelination of callosal axons.

After demyelination of the corpus callosum, repopulation of the lesion and remyelination are not only ensured by local oligodendrocyte progenitor cells (OPCs)[1], but also by NG2-expressing OPCs generated de novo in the subventricular zone (SVZ) and migrating into the lesion[2,3,4]. During development, unmyelinated axons can form glutamatergic synapses onto NG2+ cells present in the white matter after the first postnatal week[5,6]. However, in the context of demyelinating disorders, it is unknown whether demyelinated axons in the adult brain retain the potential to form new synapses with OPCs generated in the SVZ and repopulating a lesion and how these synapses are regulated during the remyelination process.

In order to answer these questions, we performed whole-cell recordings on SVZ-derived progenitors selectively label with a GFP retrovirus at different time points after focal demyelination of the adult mouse corpus callosum (). Virtually all infected cells were restricted to the SVZ (), as predicted by the low number of Ki67+ cycling cells in the adult white matter compared to the SVZ[4] (). Two days after viral injection, we induced a demyelinating injury in the anterior corpus callosum by the injection of lysolecythin (). We confirmed that SVZ progenitors are indeed strongly recruited by a demyelinating lesion compared to a control saline injection ().

At three days post lesion (3DPL), we observed GFP+ cells not only in the SVZ, but also migrating at the border between the SVZ and the corpus callosum, and inside the corpus callosum itself ( and ). At this stage, we could distinguish two main populations of GFP+ cells (). One population of GFP+ cells expressed doublecortin (Dcx), a marker of migrating neuroblasts, and was mainly found in the SVZ (). The other population expressed Olig2 and the proteoglycan NG2, a marker of OPCs, and was mainly found within the corpus callosum (). NG2+ cells could be distinguished from Dcx+ cells based on their membrane properties and the presence of a large transient K+ current (Ka) ().

We then investigated whether these two progenitor populations received glutamatergic synapses from callosal axons. We found that, as early as 2-3 DPL, approximately 30% of GFP+NG2+ cells at the border (n = 3/9) and 48% of GFP+NG2+ cells in the corpus callosum (n = 12/25) exhibited evoked EPSCs (eEPSCs; ) and spontaneous EPSCs (sEPSC; ). The average amplitude of eEPSCs was –37 ± 7 pA, with an average decay time constant of 1.4 ± 0.1 ms. Average amplitude of sEPSCs was 15.9 ± 1 pA, with an average decay time constant of 1.2 ± 0.1 ms and an average frequency of 0.076 ± 0.017 Hz. Both evoked and spontaneous EPSCs were blocked by CNQX, a specific antagonist of AMPA/Kainate receptors (). Moreover, sEPSC decay time constant was increased by 162 ± 10 % in presence of 100 μM cyclothiazide, a specific modulator of AMPARs (n = 3) (). Finally, the frequency of sEPSCs was increased by 263 ± 18 % in the presence of ruthenium red, a secretagogue known to increase vesicular neurotransmitter release (n = 7) (). By contrast, we never observed any evoked or spontaneous EPSCs in GFP+Dcx+ cells (n = 16), even in the presence of ruthenium red (n = 3) (). These results demonstrate that synapse formation specifically occurs onto GFP+NG2+ OPCs.

We next investigated whether synaptically-connected, SVZ-derived OPCs could give rise to myelinating oligodendrocytes. The percentage of GFP+NG2+ cells exhibiting sEPSCs significantly increased from 48% (n=12/25) to 91% (n=11/12) between 2-3 DPL and 6-7 DPL (), indicating that virtually all GFP+NG2+ cells became synaptically connected by 6-7 DPL. We observed a significant decrease in the percentage of GFP+NG2+ OPCs in favor of GFP+CC1+ oligodendrocytes between 6-7 and 10 DPL (; see also ref. 4), suggesting that synaptically connected GFP+NG2+ cells do convert into CC1+ cells. Moreover, since the percentage of GFP+CC1+ cells whose processes aligned with callosal axons remained stable between 6-7 and 10 DPL (82% vs. 79%), at least a fraction of CC1+ cells generated from synaptically connected NG2+ cells should be actively remyelinating.

We then investigated whether GFP+CC1+ oligodendrocytes would still exhibit functional glutamatergic synapses. As previously reported[7], we found that differentiated GFP+CC1+ oligodendrocytes displayed a significantly higher membrane capacitance (77 ± 7 pF; n=14) compared to GFP+NG2+ cells (26 ± 3 pF; n=7, p<0.001) at 6-7 DPL. More importantly, we observed that approximately 28% of GFP+CC1+ oligodendrocytes (n = 4/14) exhibited spontaneous EPSCs (). The low proportion of GFP+CC1+ cells exhibiting synaptic activity at 7 DPL, as compared to GFP+NG2+ OPCs at the same time point (91%, n=11/12), suggests that synaptic integration is a transient property of differentiating OPCs. While only a very small fraction of GFP+ cells co-expressed NG2 and CC1 around 7 DPL, we found that one such GFP+NG2+CC1+ cell clearly exhibited EPSCs in response to callosal stimulation (), further confirming the ability of NG2+ cells to differentiate into CC1+ oligodendrocytes while receiving glutamatergic synapses.

Finally, we explored how axon-OPC glutamatergic synapses were affected during the demyelination/remyelination process. First, we confirmed the presence of VGLUT-1 puncta in the vicinity of SVZ-born GFP+ OPCs (), as previously reported for endogenous NG2-DsRed+ OPCs in the corpus callosum[6]. We also observed that both SVZ-born GFP+ OPCs and NG2-DsRed+ cells were immunoreactive for the postsynaptic AMPA receptor subunit GluR2/3 (). Western blot analysis demonstrated that VGLUT-1 and GluR2/3 expression were strongly reduced in corpus callosum at 7 DPL, compared both to 4 DPL or to control NaCl injection (). To confirm that decrease at the functional level, we recorded EPSCs evoked by callosal stimulation and observed a significantly lower eEPSC average amplitude in GFP+DsRed+ cells at 7 DPL (-20± 12 pA; n=16) than in either control callosal NG2-DsRed+ cells (-34 ± 13 pA, n=12, p<0.05) or in NG2+GFP+ cells at 2-3 DPL (–37 ± 7 pA, n=12, p<0.05)(). While proteins involved in vesicular release, including VGLUT-1, are expressed in astrocytes[8], the high glutamate concentration needed for the fast EPSCs recorded in NG2+ cells are unlikely to be due to glutamate released from astrocytes.

We demonstrate here that new axon-NG2+ cell synapses are actively formed during the early stages of the remyelination process. The finding that these synapses are selectively formed on OPCs present in the demyelinating lesion and not on Dcx+ neuronal precursors suggests a specific function of glutamatergic synapses in axon-OPC recognition. We also provide evidence that axonal glutamatergic synapses contact NG2+ cells that give rise to mature oligodendrocytes, although synaptic connectivity appears to be lost during the subsequent stages of differentiation. Finally, we found that while synapse formation occurs during early phases of axon-OPC recognition, the subsequent reduction in glutamatergic activity observed one week after the lesion may be a necessary condition to allow OPCs to differentiate into oligodendrocytes, as predicted by the inhibitory influence of glutamate on OPC differentiation[9,10]. Future experiments would be needed to determine the exact function of transient glutamatergic synapses during the remyelination process.