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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

Spontaneous glutamatergic synaptic currents of OPCs in demyelinated lesions. (A–C) Spontaneous synaptic currents of recorded OPCs held at −90 mV from a control (A), at 4 (B) and 14 dpi (C). Note the fast rise and decay times of individual currents (*) in all conditions (insets, see also Figures 7A,C). (D) Histogram of the percentage of synaptically connected OPCs in control and at 4, 7, and 14 dpi. (E) Bar plot of the frequency of spontaneous synaptic currents observed in connected OPCs in control and at 4, 7, and 14 dpi. Cells without synaptic currents were excluded. *p < 0.05, **p < 0.01, ***p < 0.001 respect to the control (F) Triple immunolabeling for NG2 (red, arrows), EdU (white) and VGluT1 (green) in a LPC-induced lesion at 4 dpi. (G) 3D reconstruction of a typical NG2+ (red), EdU+ (white) cell lacking VGluT1 contacts (green) within the lesion at 4 dpi. (H) 3D reconstruction of an NG2+ EdU- cell in the non-demyelinated area of the corpus callosum (normal appearing white matter, NAWM). Nuclei were stained with Dapi.
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Figure 6: Spontaneous glutamatergic synaptic currents of OPCs in demyelinated lesions. (A–C) Spontaneous synaptic currents of recorded OPCs held at −90 mV from a control (A), at 4 (B) and 14 dpi (C). Note the fast rise and decay times of individual currents (*) in all conditions (insets, see also Figures 7A,C). (D) Histogram of the percentage of synaptically connected OPCs in control and at 4, 7, and 14 dpi. (E) Bar plot of the frequency of spontaneous synaptic currents observed in connected OPCs in control and at 4, 7, and 14 dpi. Cells without synaptic currents were excluded. *p < 0.05, **p < 0.01, ***p < 0.001 respect to the control (F) Triple immunolabeling for NG2 (red, arrows), EdU (white) and VGluT1 (green) in a LPC-induced lesion at 4 dpi. (G) 3D reconstruction of a typical NG2+ (red), EdU+ (white) cell lacking VGluT1 contacts (green) within the lesion at 4 dpi. (H) 3D reconstruction of an NG2+ EdU- cell in the non-demyelinated area of the corpus callosum (normal appearing white matter, NAWM). Nuclei were stained with Dapi.

Mentions: To test whether synaptic properties of reactivated OPCs are modified in LPC lesions, we recorded spontaneous currents of OPCs in slices from control animals and at 4, 7, and 14 dpi which correspond to major phases of OPC proliferation and differentiation inside the lesion (Nait-Oumesmar et al., 1999). At 4 dpi, most cells were weakly or not connected, suggesting that newly generated OPCs may receive few or no synaptic contacts inside the lesion (Figures 6A,B). The proportion of innervated OPCs in control brain slices was 97% (Figure 6D). Interestingly, this proportion fell to 38% at 4 dpi (Figure 6D) and the frequency of spontaneous synaptic events recorded in innervated OPCs at this stage was also greatly reduced (Figures 6B,E). It is also noteworthy that the reduction in synaptic activity at 4 dpi might be caused by a decrease of membrane time and space constants and therefore by a filtering of postsynaptic currents in recorded cells. However, in our recording conditions, we did not observe any change in capacitance, membrane time constant or input resistance with respect to controls and no correlation was obtained between synaptic current frequency and input resistance (Spearman r: −0.116; p > 0.05). In addition, bath application of 75 μM ruthenium red alone did not reveal any synaptic current in OPCs lacking synaptic activity at 4 dpi (n = 5). Hence, to determine whether this loss of synaptic activity of OPCs at 4 dpi is correlated with active proliferation of endogenous OPCs, lesioned mice were injected with EdU (5 injections of EdU at 2 h intervals before sacrifice) in order to label actively proliferating cells. Our results show that all EdU+/NG2+ OPCs within the lesion virtually lacked VGluT1+ contacts (Figures 6F,G). In contrast, NG2+ OPCs with VGluT1 puncta detected in the normal appearing white matter were not labeled with EdU (Figure 6H). Altogether, these results show a strong down-regulation of synaptic inputs in actively proliferating OPCs in demyelinated lesions.


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)

Spontaneous glutamatergic synaptic currents of OPCs in demyelinated lesions. (A–C) Spontaneous synaptic currents of recorded OPCs held at −90 mV from a control (A), at 4 (B) and 14 dpi (C). Note the fast rise and decay times of individual currents (*) in all conditions (insets, see also Figures 7A,C). (D) Histogram of the percentage of synaptically connected OPCs in control and at 4, 7, and 14 dpi. (E) Bar plot of the frequency of spontaneous synaptic currents observed in connected OPCs in control and at 4, 7, and 14 dpi. Cells without synaptic currents were excluded. *p < 0.05, **p < 0.01, ***p < 0.001 respect to the control (F) Triple immunolabeling for NG2 (red, arrows), EdU (white) and VGluT1 (green) in a LPC-induced lesion at 4 dpi. (G) 3D reconstruction of a typical NG2+ (red), EdU+ (white) cell lacking VGluT1 contacts (green) within the lesion at 4 dpi. (H) 3D reconstruction of an NG2+ EdU- cell in the non-demyelinated area of the corpus callosum (normal appearing white matter, NAWM). Nuclei were stained with Dapi.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 6: Spontaneous glutamatergic synaptic currents of OPCs in demyelinated lesions. (A–C) Spontaneous synaptic currents of recorded OPCs held at −90 mV from a control (A), at 4 (B) and 14 dpi (C). Note the fast rise and decay times of individual currents (*) in all conditions (insets, see also Figures 7A,C). (D) Histogram of the percentage of synaptically connected OPCs in control and at 4, 7, and 14 dpi. (E) Bar plot of the frequency of spontaneous synaptic currents observed in connected OPCs in control and at 4, 7, and 14 dpi. Cells without synaptic currents were excluded. *p < 0.05, **p < 0.01, ***p < 0.001 respect to the control (F) Triple immunolabeling for NG2 (red, arrows), EdU (white) and VGluT1 (green) in a LPC-induced lesion at 4 dpi. (G) 3D reconstruction of a typical NG2+ (red), EdU+ (white) cell lacking VGluT1 contacts (green) within the lesion at 4 dpi. (H) 3D reconstruction of an NG2+ EdU- cell in the non-demyelinated area of the corpus callosum (normal appearing white matter, NAWM). Nuclei were stained with Dapi.
Mentions: To test whether synaptic properties of reactivated OPCs are modified in LPC lesions, we recorded spontaneous currents of OPCs in slices from control animals and at 4, 7, and 14 dpi which correspond to major phases of OPC proliferation and differentiation inside the lesion (Nait-Oumesmar et al., 1999). At 4 dpi, most cells were weakly or not connected, suggesting that newly generated OPCs may receive few or no synaptic contacts inside the lesion (Figures 6A,B). The proportion of innervated OPCs in control brain slices was 97% (Figure 6D). Interestingly, this proportion fell to 38% at 4 dpi (Figure 6D) and the frequency of spontaneous synaptic events recorded in innervated OPCs at this stage was also greatly reduced (Figures 6B,E). It is also noteworthy that the reduction in synaptic activity at 4 dpi might be caused by a decrease of membrane time and space constants and therefore by a filtering of postsynaptic currents in recorded cells. However, in our recording conditions, we did not observe any change in capacitance, membrane time constant or input resistance with respect to controls and no correlation was obtained between synaptic current frequency and input resistance (Spearman r: −0.116; p > 0.05). In addition, bath application of 75 μM ruthenium red alone did not reveal any synaptic current in OPCs lacking synaptic activity at 4 dpi (n = 5). Hence, to determine whether this loss of synaptic activity of OPCs at 4 dpi is correlated with active proliferation of endogenous OPCs, lesioned mice were injected with EdU (5 injections of EdU at 2 h intervals before sacrifice) in order to label actively proliferating cells. Our results show that all EdU+/NG2+ OPCs within the lesion virtually lacked VGluT1+ contacts (Figures 6F,G). In contrast, NG2+ OPCs with VGluT1 puncta detected in the normal appearing white matter were not labeled with EdU (Figure 6H). Altogether, these results show a strong down-regulation of synaptic inputs in actively proliferating OPCs in demyelinated lesions.

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