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

Abstract

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.

Figure 1. Adult-born SVZ NG2⁺ cells display synaptic currents after migrating into a demyelinated lesion of the corpus callosum

a) Confocal images showing an example of a GFP⁺ cell recorded inside the lesion filled with biocytin (red) during patch-clamp recording and subsequently stained for NG2 (blue). Scale bar = 200 μm. The insert show the location of GFP retrovirus injection in the SVZ (green arrow, 0.5/1.25/2.5 mm) and lysolecithin injection in the corpus callosum (red arrow, 1.3/1.0/2.0 mm). b) Example of current evoked by callosal axon stimulation (arrowhead) in the GFP⁺NG2⁺ cell shown in (b) (Vh = -80 mV) under control conditions, in the presence of 100 μM CTZ and after application of 10 μM CNQX. c) Spontaneous synaptic glutamatergic activity recorded from the same cell under control conditions, in the presence of 100 μM CTZ and after blockade by 10μM CNQX d) Spontaneous excitatory postsynaptic currents recorded from a GFP⁺NG2⁺ cell in the corpus callosum under control conditions and in the presence of the secretagogue ruthenium red (100μM). e) Graph showing the amplitude distribution of the spontaneous events of the cell shown in (e). The insert in (f) shows the events in the histogram. All procedures were approved by the Institutional Animal Care and Use Committee of Children's National Medical Center.

Figure 2. Synaptically connected SVZ–derived OPCs give rise to oligodendrocytes

a) Histograms showing that the percentage of connected GFP⁺NG2⁺ cells in corpus callosum significantly increases from 48 % (12/25) at 2-3 DPL to 91 % (11/12) (*p<0.05; Fisher exact test) at 6-7 DPL. b) Histograms showing the percentage of GFP⁺NG2⁺ oligodendrocyte progenitors and mature GFP⁺CC1⁺ oligodendrocytes within the total GFP⁺ population at 3, 6 and 10 DPL. The remaining GFP⁺NG2^neg and GFP⁺CC1^neg cells are either GFAP⁺ or Dcx⁺ (see also ). Data are shown as mean ± sem (n ≥ 3). The percentage of GFP⁺NG2⁺ cells significantly decreased in favor of mature GFP⁺CC1⁺ cells between 3 and 10 DPL (*p<0.05). c,d) Immunostaining of GFP⁺ cells in corpus callosum with antibodies to NG2 and CC1 at 3 (c) and 10 DPL (d). Empty arrows point to GFP⁺NG2⁺ cells and white arrows point to GFP⁺CC1⁺ cells. Scale bars = 40μm. e) Immunostaining of a biocytin-filled CC1⁺NG2^neg oligodendrocyte displaying glutamatergic synaptic currents shown in (f). Scale bars = 20μm. f) Voltage clamp recording showing spontaneous glutamatergic currents in the presence of 100 μM CTZ and after blockade by 10μM CNQX.

Figure 3. Glutamatergic synaptic transmission between axons and NG2⁺ cells is reduced one week after demyelination

a) Representative western blots of VGLUT-1 and GluR2/3 in control and lysolecithin-injected white matter tissue at 4, 7 and 21 DPL. b) Histograms showing the ratio of VGLUT-1 (top) and GluR2/3 (bottom) protein expression normalized to actin levels at 4, 7 and 21 DPL (mean ± sem n≥3). Expression of both VGLUT-1 and GluR2/3 was significantly decreased at 7DPL (*p<0.05). c) Histograms comparing the average eEPSC amplitude in GFP⁺NG2-DsRed⁺ cells at 7 DPL (mean ± sd, n=16) and in NG2-DsRed⁺ cells in age-matched, uninjected control littermates (n=12). Insert shows a representative average eEPSC in lysolecithin-injected corpus callosum and uninjected controls (*p<0.05).