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.
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At a glance
: Adult-born SVZ NG2-positive cells display synaptic currents after migrating into a demyelinated lesion of the corpus callosum.
a) Confocal images showing an example of a GFP-positive cell recorded inside the lesion filled with biocytin (red) during patch-clamp recording and subsequently stained for NG2 (blue). Scale bar represents 200 μm. The insert shows the location of GFP retrovirus injection in the SVZ (green arrow; 0.5, 1.25 and 2.5 mm, anteroposterior relative to bregma, mediolateral and dorsoventral from surface of the brain) and lysolecithin injection in the corpus callosum (red arrow; 1.3, 1.0 and 2.0 mm, anteroposterior relative to bregma, mediolateral and dorsoventral from surface of the brain). ( b) Example of a current evoked by callosal axon stimulation (arrowhead) in the GFP- and NG2-positive cell shown in a ( V h = −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 10 μM CNQX. ( d) Spontaneous excitatory postsynaptic currents recorded from a GFP- and NG2-positive cell in the corpus callosum under control conditions and in the presence of the secretagogue ruthenium red (100 μM). ( e) Graph showing the amplitude distribution of the spontaneous events of the cell shown in d. The insert shows the events in the histogram. All procedures were approved by the Institutional Animal Care and Use Committee of Children's National Medical Center.
: Synaptically connected SVZ-derived OPCs give rise to oligodendrocytes.
a) Histogram showing that the percentage of connected GFP +NG2 + cells in corpus callosum significantly increased from 48 % (12 of 25) at 2–3 DPL to 91 % (11 of 12) ( * P < 0.05, Fisher exact test) at 6–7 DPL. ( b) Histograms showing the percentage of GFP- and NG2-positive oligodendrocyte progenitors and mature GFP- and CC1-positive oligodendrocytes in the total GFP-positive population at 3, 6 and 10 DPL. The remaining GFP-positive, NG2-negative cells and GFP-positive, CC1-negative cells were either GFAP or Dcx positive (see also ref. 4). Data are shown as mean ± s.e.m. ( n ≥ 3). The percentage of GFP- and NG2-positive cells significantly decreased in favor of mature GFP- and CC1-positive cells between 3 and 10 DPL. ( c, d) Immunostaining of GFP-positive cells in corpus callosum with antibodies to NG2 and CC1 at 3 ( c) and 10 DPL ( d). Empty arrows point to GFP- and NG2-positive cells, and white arrows point to GFP- and CC1-positive cells. Scale bars represent 40 μm. ( e) Immunostaining of a biocytin-filled CC1-positive, NG2-negative oligodendrocyte with the glutamatergic synaptic currents shown in f. Insert represents a current-voltage profile of the cell (voltage steps from −100 to 30 mV in 10-mV increments, vertical bar represents 500 pA and horizontal bar represents 200 ms). Scale bar represents 20 μm. ( f) Voltage-clamp recording showing spontaneous glutamatergic currents in the presence of 100 μM CTZ and after blockade by 10 μM CNQX.
: Glutamatergic synaptic transmission between axons and NG2-positive cells is reduced 1 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 ± s.e.m., n ≥ 3). Expression of both VGlut-1 and GluR2/3 was significantly decreased at 7 DPL ( * P < 0.05). ( c) Histograms comparing the average eEPSC amplitude in GFP- and NG2-DsRed–positive cells at 7 DPL (mean ± s.d., n = 16) and in NG2-DsRed–positive cells in age-matched, uninjected control littermates ( n = 12). Insert shows a representative average eEPSC in lysolecithin-injected corpus callosum and uninjected controls.