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Alteration of synaptic connectivity of oligodendrocyte precursor cells following demyelination.

Sahel A, Ortiz FC, Kerninon C, Maldonado PP, Angulo MC, Nait-Oumesmar B - Front Cell Neurosci (2015)

Bottom Line: A reduction in synaptic connectivity was confirmed by the lack of VGluT1+ axon-OPC contacts in virtually all rapidly proliferating OPCs stained with EdU (50-ethynyl-20-deoxyuridine).At the end of the massive proliferation phase in lesions, the proportion of innervated OPCs rapidly recovers, although the frequency of spontaneous synaptic currents did not reach control levels.In conclusion, our results demonstrate that newly-generated OPCs do not receive synaptic inputs during their active proliferation after demyelination, but gain synapses during the remyelination process.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1127, Institut du Cerveau et de la Moelle Epinière Paris, France ; Université Paris 6, Sorbonne Paris Cité, UMR-S1127 Paris, France ; Centre National de la Recherche Scientifique UMR 7225 Paris, France.

ABSTRACT
Oligodendrocyte precursor cells (OPCs) are a major source of remyelinating oligodendrocytes in demyelinating diseases such as Multiple Sclerosis (MS). While OPCs are innervated by unmyelinated axons in the normal brain, the fate of such synaptic contacts after demyelination is still unclear. By combining electrophysiology and immunostainings in different transgenic mice expressing fluorescent reporters, we studied the synaptic innervation of OPCs in the model of lysolecithin (LPC)-induced demyelination of corpus callosum. Synaptic innervation of reactivated OPCs in the lesion was revealed by the presence of AMPA receptor-mediated synaptic currents, VGluT1+ axon-OPC contacts in 3D confocal reconstructions and synaptic junctions observed by electron microscopy. Moreover, 3D confocal reconstructions of VGluT1 and NG2 immunolabeling showed the existence of glutamatergic axon-OPC contacts in post-mortem MS lesions. Interestingly, patch-clamp recordings in LPC-induced lesions demonstrated a drastic decrease in spontaneous synaptic activity of OPCs early after demyelination that was not caused by an impaired conduction of compound action potentials. A reduction in synaptic connectivity was confirmed by the lack of VGluT1+ axon-OPC contacts in virtually all rapidly proliferating OPCs stained with EdU (50-ethynyl-20-deoxyuridine). At the end of the massive proliferation phase in lesions, the proportion of innervated OPCs rapidly recovers, although the frequency of spontaneous synaptic currents did not reach control levels. In conclusion, our results demonstrate that newly-generated OPCs do not receive synaptic inputs during their active proliferation after demyelination, but gain synapses during the remyelination process. Hence, glutamatergic synaptic inputs may contribute to inhibit OPC proliferation and might have a physiopathological relevance in demyelinating disorders.

No MeSH data available.


Related in: MedlinePlus

Immuno-characterization and electrophysiological properties of OPCs, oligodendrocytes and microglia in transgenic lines following demyelination. (A) OPCs were identified as DsRed+/GFP+ cells (arrows) in the NG2-DsRed;CNPase-GFP double transgenic mouse. (B) Mature oligodendrocytes were identified as CC1+ cells (blue) and also expressed GFP (arrows) but not DsRed. (C) Iba1+ resident microglia/macrophage expressing DsRed in the NG2-DsRed mouse strain. (D) Bar plot showing the percentage of NG2+ and CC1+ cells within control and lesioned corpus callosum in CNPase-GFP animals (N = 3 mice). (E) Currents elicited by voltage steps from +40 mV to −120 mV in a DsRed+/GFP+ OPC. held at −90 mV and recorded inside a lesion. Note the presence of INa+ (inset). (F) Currents induced by voltage steps from +40 mV to −120 mV in a DsRed−/GFP+ oligodendrocyte at 14 dpi held at −90 mV. (G) Currents induced by voltage steps from +40 mV to −120 mV in a DsRed+/GFP− microglia held at −90 mV. Note the absence of INa+ (inset). We confirmed that DsRed was expressed in activated microglia inside lesions by crossing NG2-DsRed line with the CX3CR1-EGFP strain in which microglia/macrophages express GFP (see Figure 1H) (Avignone et al., 2008). (H) Bar plot for the proportion of OPCs (O), microglia (M) and oligodendrocytes (OL) identified by their electrophysiological profiles and recorded in different mouse strains following demyelination. Scale bars for insets: 10 μm. **p < 0.01, ***p < 0.001 respect to Olig2 expression.
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Figure 2: Immuno-characterization and electrophysiological properties of OPCs, oligodendrocytes and microglia in transgenic lines following demyelination. (A) OPCs were identified as DsRed+/GFP+ cells (arrows) in the NG2-DsRed;CNPase-GFP double transgenic mouse. (B) Mature oligodendrocytes were identified as CC1+ cells (blue) and also expressed GFP (arrows) but not DsRed. (C) Iba1+ resident microglia/macrophage expressing DsRed in the NG2-DsRed mouse strain. (D) Bar plot showing the percentage of NG2+ and CC1+ cells within control and lesioned corpus callosum in CNPase-GFP animals (N = 3 mice). (E) Currents elicited by voltage steps from +40 mV to −120 mV in a DsRed+/GFP+ OPC. held at −90 mV and recorded inside a lesion. Note the presence of INa+ (inset). (F) Currents induced by voltage steps from +40 mV to −120 mV in a DsRed−/GFP+ oligodendrocyte at 14 dpi held at −90 mV. (G) Currents induced by voltage steps from +40 mV to −120 mV in a DsRed+/GFP− microglia held at −90 mV. Note the absence of INa+ (inset). We confirmed that DsRed was expressed in activated microglia inside lesions by crossing NG2-DsRed line with the CX3CR1-EGFP strain in which microglia/macrophages express GFP (see Figure 1H) (Avignone et al., 2008). (H) Bar plot for the proportion of OPCs (O), microglia (M) and oligodendrocytes (OL) identified by their electrophysiological profiles and recorded in different mouse strains following demyelination. Scale bars for insets: 10 μm. **p < 0.01, ***p < 0.001 respect to Olig2 expression.

Mentions: All patch-clamp recordings were performed inside the lesion core in adult NG2-DsRed;CNPase mice. This double transgenic line allowed us to unambiguously discriminate OPCs and oligodendrocytes in brain slices and compared their electrophysiological properties in lesions, as all cells of the oligodendroglial lineage express GFP in the CNPase-GFP mouse line (Yuan et al., 2002). The large majority of DsRed+/GFP+ cells in the lesion had the immunohistochemical phenotype of NG2+ OPCs (Figure 2A) while DsRed−/GFP+ cells were labeled for CC1, a specific marker of differentiated oligodendrocytes (Figure 2B). As expected in the LPC model of demyelination (Watanabe et al., 2002), the population of DsRed+/GFP+ OPCs was largely increased at 7 dpi whereas a clear increase in differentiated DsRed−/GFP+/CC1+ oligodendrocytes was detected at 14 dpi (Figure 2D). In addition to DsRed+/GFP+ OPCs and DsRed−/GFP+ oligodendrocytes, we observed the presence of DsRed+/GFP- cells with large somata and thick primary processes inside the lesion, characteristic of activated microglia. To confirm the expression of DsRed by microglia in LPC-induced lesions, we used the microglial marker Iba1 and confirmed the expression of DsRed in Iba1+ cells (Figure 2C, see also Bu et al., 2001). Hence, in LPC-induced lesions, the NG2-DsRed;CNPase-GFP mouse line allows for the unequivocal identification of OPCs from mature oligodendrocytes and microglia by the simultaneous expression of both DsRed and GFP.


Alteration of synaptic connectivity of oligodendrocyte precursor cells following demyelination.

Sahel A, Ortiz FC, Kerninon C, Maldonado PP, Angulo MC, Nait-Oumesmar B - Front Cell Neurosci (2015)

Immuno-characterization and electrophysiological properties of OPCs, oligodendrocytes and microglia in transgenic lines following demyelination. (A) OPCs were identified as DsRed+/GFP+ cells (arrows) in the NG2-DsRed;CNPase-GFP double transgenic mouse. (B) Mature oligodendrocytes were identified as CC1+ cells (blue) and also expressed GFP (arrows) but not DsRed. (C) Iba1+ resident microglia/macrophage expressing DsRed in the NG2-DsRed mouse strain. (D) Bar plot showing the percentage of NG2+ and CC1+ cells within control and lesioned corpus callosum in CNPase-GFP animals (N = 3 mice). (E) Currents elicited by voltage steps from +40 mV to −120 mV in a DsRed+/GFP+ OPC. held at −90 mV and recorded inside a lesion. Note the presence of INa+ (inset). (F) Currents induced by voltage steps from +40 mV to −120 mV in a DsRed−/GFP+ oligodendrocyte at 14 dpi held at −90 mV. (G) Currents induced by voltage steps from +40 mV to −120 mV in a DsRed+/GFP− microglia held at −90 mV. Note the absence of INa+ (inset). We confirmed that DsRed was expressed in activated microglia inside lesions by crossing NG2-DsRed line with the CX3CR1-EGFP strain in which microglia/macrophages express GFP (see Figure 1H) (Avignone et al., 2008). (H) Bar plot for the proportion of OPCs (O), microglia (M) and oligodendrocytes (OL) identified by their electrophysiological profiles and recorded in different mouse strains following demyelination. Scale bars for insets: 10 μm. **p < 0.01, ***p < 0.001 respect to Olig2 expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Immuno-characterization and electrophysiological properties of OPCs, oligodendrocytes and microglia in transgenic lines following demyelination. (A) OPCs were identified as DsRed+/GFP+ cells (arrows) in the NG2-DsRed;CNPase-GFP double transgenic mouse. (B) Mature oligodendrocytes were identified as CC1+ cells (blue) and also expressed GFP (arrows) but not DsRed. (C) Iba1+ resident microglia/macrophage expressing DsRed in the NG2-DsRed mouse strain. (D) Bar plot showing the percentage of NG2+ and CC1+ cells within control and lesioned corpus callosum in CNPase-GFP animals (N = 3 mice). (E) Currents elicited by voltage steps from +40 mV to −120 mV in a DsRed+/GFP+ OPC. held at −90 mV and recorded inside a lesion. Note the presence of INa+ (inset). (F) Currents induced by voltage steps from +40 mV to −120 mV in a DsRed−/GFP+ oligodendrocyte at 14 dpi held at −90 mV. (G) Currents induced by voltage steps from +40 mV to −120 mV in a DsRed+/GFP− microglia held at −90 mV. Note the absence of INa+ (inset). We confirmed that DsRed was expressed in activated microglia inside lesions by crossing NG2-DsRed line with the CX3CR1-EGFP strain in which microglia/macrophages express GFP (see Figure 1H) (Avignone et al., 2008). (H) Bar plot for the proportion of OPCs (O), microglia (M) and oligodendrocytes (OL) identified by their electrophysiological profiles and recorded in different mouse strains following demyelination. Scale bars for insets: 10 μm. **p < 0.01, ***p < 0.001 respect to Olig2 expression.
Mentions: All patch-clamp recordings were performed inside the lesion core in adult NG2-DsRed;CNPase mice. This double transgenic line allowed us to unambiguously discriminate OPCs and oligodendrocytes in brain slices and compared their electrophysiological properties in lesions, as all cells of the oligodendroglial lineage express GFP in the CNPase-GFP mouse line (Yuan et al., 2002). The large majority of DsRed+/GFP+ cells in the lesion had the immunohistochemical phenotype of NG2+ OPCs (Figure 2A) while DsRed−/GFP+ cells were labeled for CC1, a specific marker of differentiated oligodendrocytes (Figure 2B). As expected in the LPC model of demyelination (Watanabe et al., 2002), the population of DsRed+/GFP+ OPCs was largely increased at 7 dpi whereas a clear increase in differentiated DsRed−/GFP+/CC1+ oligodendrocytes was detected at 14 dpi (Figure 2D). In addition to DsRed+/GFP+ OPCs and DsRed−/GFP+ oligodendrocytes, we observed the presence of DsRed+/GFP- cells with large somata and thick primary processes inside the lesion, characteristic of activated microglia. To confirm the expression of DsRed by microglia in LPC-induced lesions, we used the microglial marker Iba1 and confirmed the expression of DsRed in Iba1+ cells (Figure 2C, see also Bu et al., 2001). Hence, in LPC-induced lesions, the NG2-DsRed;CNPase-GFP mouse line allows for the unequivocal identification of OPCs from mature oligodendrocytes and microglia by the simultaneous expression of both DsRed and GFP.

Bottom Line: A reduction in synaptic connectivity was confirmed by the lack of VGluT1+ axon-OPC contacts in virtually all rapidly proliferating OPCs stained with EdU (50-ethynyl-20-deoxyuridine).At the end of the massive proliferation phase in lesions, the proportion of innervated OPCs rapidly recovers, although the frequency of spontaneous synaptic currents did not reach control levels.In conclusion, our results demonstrate that newly-generated OPCs do not receive synaptic inputs during their active proliferation after demyelination, but gain synapses during the remyelination process.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1127, Institut du Cerveau et de la Moelle Epinière Paris, France ; Université Paris 6, Sorbonne Paris Cité, UMR-S1127 Paris, France ; Centre National de la Recherche Scientifique UMR 7225 Paris, France.

ABSTRACT
Oligodendrocyte precursor cells (OPCs) are a major source of remyelinating oligodendrocytes in demyelinating diseases such as Multiple Sclerosis (MS). While OPCs are innervated by unmyelinated axons in the normal brain, the fate of such synaptic contacts after demyelination is still unclear. By combining electrophysiology and immunostainings in different transgenic mice expressing fluorescent reporters, we studied the synaptic innervation of OPCs in the model of lysolecithin (LPC)-induced demyelination of corpus callosum. Synaptic innervation of reactivated OPCs in the lesion was revealed by the presence of AMPA receptor-mediated synaptic currents, VGluT1+ axon-OPC contacts in 3D confocal reconstructions and synaptic junctions observed by electron microscopy. Moreover, 3D confocal reconstructions of VGluT1 and NG2 immunolabeling showed the existence of glutamatergic axon-OPC contacts in post-mortem MS lesions. Interestingly, patch-clamp recordings in LPC-induced lesions demonstrated a drastic decrease in spontaneous synaptic activity of OPCs early after demyelination that was not caused by an impaired conduction of compound action potentials. A reduction in synaptic connectivity was confirmed by the lack of VGluT1+ axon-OPC contacts in virtually all rapidly proliferating OPCs stained with EdU (50-ethynyl-20-deoxyuridine). At the end of the massive proliferation phase in lesions, the proportion of innervated OPCs rapidly recovers, although the frequency of spontaneous synaptic currents did not reach control levels. In conclusion, our results demonstrate that newly-generated OPCs do not receive synaptic inputs during their active proliferation after demyelination, but gain synapses during the remyelination process. Hence, glutamatergic synaptic inputs may contribute to inhibit OPC proliferation and might have a physiopathological relevance in demyelinating disorders.

No MeSH data available.


Related in: MedlinePlus