Oligodendrocyte Progenitor Cells Become Regionally Diverse and Heterogeneous with Age

Abstract

Oligodendrocyte progenitor cells (OPCs), which differentiate into myelinating oligodendrocytes during CNS development, are the main proliferative cells in the adult brain. OPCs are conventionally considered a homogeneous population, particularly with respect to their electrophysiological properties, but this has been debated. We show, by using single-cell electrophysiological recordings, that OPCs start out as a homogeneous population but become functionally heterogeneous, varying both within and between brain regions and with age. These electrophysiological changes in OPCs correlate with the differentiation potential of OPCs; thus, they may underlie the differentiational differences in OPCs between regions and, likewise, differentiation failure with age.

Keywords: bioelectricity; differentiation; electrophysiology; glia; glutamate; ion channels; myelin; neurotransmitter receptors; oligodendrocyte; oligodendrocyte precursor cell.

Graphical abstract

Figure 1

OPCs Acquire Functional Ion Channels at Different Developmental Time Points (A) Schematic diagram of the mouse developmental timeline, indicating developmental and myelination-related milestones over the period studied. (B) Cells were selected by their EYFP expression (NG2-EYFP knockin mice). During whole-cell patch-clamp recording, OPCs were dye-filled with Lucifer Yellow (LY, green) and post hoc-labeled for NG2 (red). Scale bar, 20 μm. (C) Leak-subtracted traces of voltage-gated sodium currents (Na_V) in response to 20-mV steps from a holding potential of −74 mV (inset, voltage pulses from −114 to +26 mV) in OPCs from E13 to 9-month-old-mice. (D) Na_V densities were significantly different between age groups (p = 1.6 × 10⁻¹⁰). Na_V densities peak at P6–P16 (at the time when myelination is at its highest rate; A). The proportion (pie charts) of OPCs with (black) or without (gray) detectable Na_V currents differed (p < 1 × 10⁻¹⁵, χ²) with age. (E) Kainate (30 μM)-evoked currents in OPCs from E13 to 9-month-old-mice. (F) The density of AMPA/kainate receptors (KARs) increased steadily with age (p = 2.1 × 10⁻⁵, ANOVA), and the proportion (pie charts) of OPCs with (black) detectable KA-evoked currents increased (p < 1 × 10⁻¹⁵, χ²) until after birth, when all OPCs had detectable KA-evoked currents. (G) NMDA (60 μM)-evoked currents in OPCs from E13 to 9-month-old-mice. (H) The density of NMDA receptors (NMDARs) in OPCs changed significantly with age (p = 7 × 10⁻⁴, ANOVA). Similarly, the proportion (pie charts) of OPCs with (black) detectable NMDA (60 μM)-evoked currents changed with age (p < 1 × 10⁻¹⁵, χ²). Both current density and OPCs with detectable currents peaked during P6–P35, at the time when myelination is at its highest rate, and declined until becoming undetectable (gray). (I) The fraction of OPCs with detectable voltage-gated potassium (K_V) or Na_V channels and with detectable KAR-evoked and NMDA receptor (NMDAR)-evoked currents across the lifespan. K_V and KA-evoked currents are first detected in OPCs, followed by Na_V; all three remain present in the majority of postnatal OPCs throughout life. In contrast, NMDA-evoked currents are detected last, and only during the period of highest myelination are NMDARs detected in the majority of OPCs. At later ages, NMDARs become undetectable. K_V, p < 1 × 10⁻¹⁵; Na_V, p < 1 × 10⁻¹⁵, χ²; KAR, p < 1 × 10⁻¹⁵, χ²; NMDA, p < 1 × 10⁻¹⁵, χ². The onset of detection between receptors differs (p = 4.3 × 10⁻⁶), and the fraction of OPCs with detectable currents differs across age (p = 2.7 × 10⁻⁵). (J) OPC capacitance peaks with myelination rate and then declines back to perinatal levels. The numbers shown on graphs represent the number of whole-cell patched OPCs from 2–21 animals. Top p values are from ANOVA analyses, whereas bottom p values are from post hoc Holm-Bonferroni analyses. Data are shown ± SEM.

Figure 2

OPC Molecular Signatures Change with Age (A) Volcano plot showing differential expression of significantly altered genes for P12 compared with E16. Light gray circles indicate genes that are more than 4-fold increased between P12 and E16. Significantly altered gene ontology (GO) terms are highlighted (red, migration; dark green, proliferation; light green, differentiation). ^∗selected genes of interest. (B) As for (A) but for P310 compared with P12; all circles denote significantly altered genes. Light gray circles represent genes that are more than 1.5-fold increased. A selection of significantly altered GO terms are highlighted (blue, cell cycle; orange, immune response). ^∗selected genes of interest. (C) Heatmap of genes related to migration, proliferation, differentiation, cell cycle, and senescence across ages. Reads per kilobase million (log2(RPKM)) are plotted. (D) REVIGO semantic clustering visualization of GO analysis, showing all significantly altered terms at P12 compared with E16. The circle size approximately represents the number of genes within a GO term; the color intensity reflects the log10 p value. GO terms of particular interest are highlighted. (E) As for (D) but showing the GO terms at P310 compared with P12.

Figure 3

OPC Cell-Cycle Changes with Age and Na_V Channel density (A) Schematic diagram showing the fluorescent labeling seen at different points in the cell cycle for fluorescent ubiquitination-based cell-cycle indicator (FUCCI) and Ki67-RFP mice. (B) Representative image of flow cytometry analysis of relative DAPI intensity in OPCs, with the color coding of peaks representing different cell cycle stages. (C–E) The percentage of OPCs in G₀/G1 (C), S phase (D), and G2/M phase (E) as measured by flow cytometry. Numbers on graphs represent the number of animals used per time point. (F) Ki67-RFP:NG2-EYFP cortical section showing yellow fluorescent protein (YFP)-positive (green) OPCs with (white arrowheads, in S/G2/M) and without (black filled arrowheads, in G₀/G1) Ki67 (red) expression. Scale bar, 25 μm. (G) FUCCI:NG2-EYFP cortical section showing YFP⁺ OPCs with (white arrowheads, in G₀/G1) and without (black filled arrowheads, in S/G2/M) mCherry labeling. Scale bar, 25 μm. (H) Na_V densities were significantly higher in cycling OPCs compared with non-cycling (cycl, cycling [in S/G2/M]; non cycl, non-cycling [in G₀/G1]), whereas the proportion of OPCs with detectable currents did not change (pie chart, p = 0.50, χ²). (I and J) Neither (I) KAR nor (J) NMDA densities differed between cycling and non-cycling OPCs, nor did the proportions of cells responding (pie charts; KAR, p = 0.86; NMDAR, p = 0.051; χ²). Numbers on graphs represent the number of cell recorded from 18 animals. The p values are from Student’s t tests (H–J) or one-way ANOVA (C–E, top), followed by Holm-Bonferroni post hoc test (bottom). Data are shown ± SEM.

Figure 4

OPCs Become Regionally Diverse after the First Postnatal Week (A) OPCs were whole-cell patch-clamped in the corpus callosum or the cortex, as highlighted in the schematic diagram in purple. (B) Na_V densities (bar graph) and the proportion of OPCs (pie charts) with detectable Na_V currents (black) do not differ between the corpus callosum (CC) or the cortex (CTX), neither during the first postnatal week (p = 0.21, p = 0.94, χ²) nor thereafter (p = 0.61, p = 0.10, χ²). (C) AMPA/KAR densities did not differ between OPCs in the white matter (CC) or gray matter (CTX) at birth (p = 0.79, left) but became significantly different after P8 (p = 4 × 10⁻⁴). Throughout postnatal life, KA (30 μM) evoked currents in OPCs (pie charts underneath the bar graph, with [black] or without [gray] detectable evoked currents). (D) NMDAR densities and the proportion of OPCs with detectable NMDA (60 μM)-evoked currents (pie charts) were not different between OPCs in the CC or the CTX at birth (p = 0.59, p = 0.83, χ²) but became significantly different after P8 (p = 0.02, p = 1.7x10⁻³, χ²), with reduced NMDA-evoked currents in OPCs in the CTX. (E and F) Neither Na_V (E) nor KAR (F) densities nor the proportion of cells with detectable Na_V (p = 0.1, χ²) (E) or KA-evoked currents (p > 0.99, χ²) (F) differed between layer 1 and layer 5 of the CTX. (G) NMDAR densities were higher in layer 5 of the CTX compared with layer 1, but the proportion of OPCs with detectable NMDA-evoked currents did not differ between the two layers (p = 0.1, χ²). (H–J) There was no difference detected between the anterior or posterior CC in receptor densities or the proportion of cells responding to (H) Na_V (p = 0.88, χ²), (I) KAR (p = 0.88, χ²), and (J) NMDAR (p = 0.37, χ²). Numbers shown on bar graphs represent cell numbers from 5–20 animals. Data are shown ± SEM. The p values are from Student’s t tests.

Figure 5

The Age-Related Reduction in Myelination Potential and Ion Channel Expression in OPCs Is Not Reversed by an Altered Environment (A) Schematic of the generation of myelinating OPC-DRG co-cultures. OPCs were isolated by magnetic-activated cell sorting (MACS) from either neonate or adult mice and plated onto neonatal DRG neurons. (B) High-magnification views of a myelinating neonatal oligodendrocyte (top, left) with MBP (green) expressed in processes wrapping axons expressing neurofilament (NF) 160/200 (NF, red, bottom, left), and of an MBP expressing non-myelinating oligodendrocytes from old animals (top, right) where the MBP+ processes are not aligned with axons (bottom, right). Scale bar, 50 μm. (C and D) Quantification of differentiated oligodendrocytes (MBP⁺) in co-cultures comprising neonatal dorsal root ganglion neurons and neonatal or aged OPCs; neonatal OPCs produced more (C) MBP⁺ cells per coverslip and a higher fraction of (D) myelinating cells (right). Numbers represent the number of experiments. (E) Schematic diagram of delivery of GDF11 via minipumps implanted at P150, allowing continuous i.p. infusion of GDF11 until P180, when OPCs were whole-cell patch-clamped. (F–H) Ion channel densities were not significantly different between GDF11 and control-treated animals: (F) Na_V densities (p = 0.44), (G) KAR densities (p = 0.22), and (H) NMDAR densities (p = 0.77). Numbers shown on bar graphs represent cell numbers recorded from 2–3 animals. Data are shown ± SEM. The p values are from Student’s t tests.

Figure 6

Ion Channel Densities in OPCs Change Differently across the Lifespan in the CC, CTX, Cerebellum, and Subventricular Zone (A) Illustration of the brain areas (purple) where OPCs were whole-cell patch-clamped: CC, a highly myelinated region; CTX, a lightly myelinated region; cerebellar molecular layer (ML), a region that is never myelinated in mice; and subventricular zone (SVZ), an area that provides a continuous supply of myelinogenic OPCs throughout life. (B) Na_V densities (bar graph) did not change across postnatal age in the CC (left), CTX (center left), or ML (center right) but did change in the SVZ (right). The proportion of OPCs with detectable Na_V (black) changed throughout postnatal life in the CC (p = 3.1 × 10⁻⁵, χ²), the CTX (p = 2.6 × 10⁻³, χ²), and the ML (p = 6.5 × 10⁻³, χ²), but not the SVZ (p = 0.7, χ²). (C) AMPA/KAR densities increased with age in the CTX and the SVZ but remained stable in other areas throughout life. Throughout postnatal life, KA (30 μM) evoked currents in all OPCs except in the ML, where, in the first 3 months of life, a small proportion of OPCs had no detectable KA-evoked currents, and in the SVZ, where OPCs with no KA-evoked response could be found throughout life. The proportion of OPCs with kainite-evoked currents only changed with age in the hindbrain (CC, p = 1, χ²; CTX, p = 1, χ²; ML, p = 2.5 × 10⁻⁶, χ²; SVZ, p = 0.61, χ²). (D) NMDAR densities significantly declined in all areas except the SVZ across postnatal life, although with a different rate in each area. The proportion of OPCs with detectable NMDA (60 μM)-evoked currents (pie charts) were significantly different across age in all areas except the SVZ, recorded from the following: CC, p < 1 × 10⁻¹⁵, χ²; CTX, p < 1 × 10⁻¹⁵, χ²; ML, p < 1 × 10⁻¹⁵, χ²; SVZ, p = 0.62, χ². (E) The fraction of postnatal OPCs with detectable NMDA-evoked current in the CC), CTX, ML, and SVZ. NMDARs became undetectable at different times in each region but not in the SVZ (p = 0.003, two-way ANOVA, comparison between areas). (F and G) Neither (F) Na_V nor (G) KAR densities differed between parenchymal forebrain OPCs (pFB) and SVZ OPCs. (H) NMDAR density was higher in SVZ OPCs compared with pFB OPCs. Numbers shown on bar graphs represent cell numbers from 1–13 animals. Data are shown ± SEM. Top p values are from ANOVA analyses, and bottom p values are from Holm-Bonferroni post hoc tests.