Distribution and phenotype of TrkB oligodendrocyte lineage cells in the adult rat spinal cord

Abstract

The distribution and phenotype of a previously undescribed population of nonneuronal cells in the intact spinal cord that expresses TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF) and neurotrophin 4 (NT-4), were characterized by examining the extent of co-localization of TrkB with NG2, which identifies oligodendrocyte progenitors (OPCs) or CC1, a marker for mature oligodendrocytes (OLs). All TrkB nonneuronal cells expressed Olig2, confirming their role in the OL lineage. Similar to OPCs and OLs, TrkB cells resided in gray and white matter of the spinal cord in similar abundance. Less than 2% of TrkB cells expressed NG2, while over 80% of TrkB cells in the adult spinal cord co-expressed CC1. Most OPCs did not express detectable levels of TrkB, however a small OPC pool (~5%) showed TrkB immunoreactivity. The majority of mature OLs (~65%) expressed TrkB, but a population of mature OLs (~36%) did not express TrkB at detectable levels, and 17% of TrkB nonneuronal cells did not express NG2 or CC1. Approximately 20% of the TrkB nonneuronal population in the ventral horn resided in close proximity to motor neurons and were categorized as perineuronal. We conclude that TrkB is expressed by several pools of OL lineage cells in the adult spinal cord. These findings are important in understanding the neurotrophin regulation of OL lineage cells in the adult spinal cord.

Keywords: NG2 cells; OPCs; Oligodendrocyte precursor cells; Perineuronal oligodendrocytes; Satellite oligodendrocytes.

Figure 1

Localization and identification of OL lineage cells in the adult rat spinal cord. A. Diagrammatic representation of the adult rat spinal cord showing the location of the measuring boxes that were used in this study. DH, dorsal horn; LH, lateral horn; VH, ventral horn; CC, central canal; DF, dorsal funiculus; LF, lateral funiculus; VF, ventral funiculus. B. Confocal micrographs depicting the characteristic immunolabeling used to identify TrkB, CC1, and NG2 cells in the spinal cord. C. Double immunolabeling with TrkB (red) and Olig2 (green) revealed that all nonneuronal TrkB-immunoreactive (-ir) cells also expressed Olig2 (white arrows). Olig2 cells that were not TrkB-ir (yellow arrow) may represent mature OLs (CC1⁺/TrkB⁻) and OPCs (NG2⁺/TrkB⁻). TrkB-ir neurons (yellow asterisks), identified by larger cell volume, were observed in the gray matter and did not express Olig2. In merged images (far right, B., C.), DAPI was used to demonstrate the cellular nature of the staining. Scale bar in B. = 5μm; Scale bar in C. = 20 μm.

Figure 2

Density of OL lineage cells within white matter (WM) and gray matter (GM) in cervical (C4) and thoracic (T1) segments of the adult rat spinal cord. Comparison of cell densities between the WM and GM within segments revealed no differences in the number of TrkB (A.), CC1 (B.), or NG2 (C.) cells. In addition, no differences were observed in the number of TrkB and CC1 cells when the cervical and thoracic segments were compared. However, the density of NG2 cells in the GM in the thoracic segment was significantly higher when compared with GM density in the cervical segment. Data shown as mean +/− SEM. *, significance reported at p<0.05. WM, white matter, GM, gray matter.

Figure 3

The majority of mature OLs expressed TrkB at detectable levels. A.-A.” Confocal micrographs of coronal sections through the T1 segment of the spinal cord reveal the co-localization of CC1 and TrkB in nonneuronal cells (yellow arrows). Note one cell expressing CC1 only (green arrows), but only faintly immunoreactive for TrkB. Scale bar = 50 μm. B.-B.” Most cells expressed both CC1 and TrkB (yellow arrows), yet cells expressing CC1 only (green arrows) also were observed. DAPI was used to demonstrate the cellular nature of the staining. Scale bar = 20 μm. C. Basic Venn diagrams (left panel) represent the percentage of cells that expressed CC1 only (green), TrkB only (red), or CC1 and TrkB (yellow) in cervical (C4) and thoracic (T1) segments. Stacked Venn diagrams (right panel) reveal that ~81% of the TrkB cells also expressed CC1 in both of the segments.

Figure 4

Little overlap between OPC and TrkB populations in the intact spinal cord. A.-A.” Confocal micrographs of coronal sections at T1 segment reveal the lack of co-localization between NG2 and TrkB cells. NG2 cells (green arrows) and TrkB cells (red arrows) showed no overlap. DAPI was used to demonstrate the cellular nature of the staining. Scale bar = 50 μm. B. Basic Venn diagrams (left panel) represent the percentage of cells expressing NG2 only (green), TrkB only (red), or NG2 and TrkB (yellow) in the spinal cord in cervical (C4) and thoracic (T1) segments. Stacked Venn diagrams (right panel) reveal that ~2% of the TrkB cells also expressed NG2 in both of segments.

Figure 5

Clusters of TrkB and CC1 cells were observed primarily in the GM of the spinal cord. A.-A.” Both TrkB (red) and CC1 (green) cells in the ventral horn (delineated by yellow lines) frequently were observed in aggregates or clusters of two or more cells (white arrows). Motor neurons (yellow asterisks) abutted by CC1⁺/TrkB⁺ cells were present. DAPI used to demonstrate the cellular nature of each cell type. Scale bar = 50 μm. B. Graphs showing the number of TrkB (upper) and CC1 (lower) cells that were observed in clusters at cervical (C4) and thoracic (T1) segments of the spinal cord. In the thoracic segment, the number of CC1 and TrkB clusters was significantly higher in the GM compared with WM. Data shown as mean +/− SEM. *, significance at p<0.05 when comparing WM and GM at T1.

Figure 6

A population of OL lineage cells resided in close apposition to motor neurons in the ventral horn of adult rat spinal cord and was categorized as perineuronal. A.-A.” The majority of perineuronal cells expressed both CC1 and TrkB (yellow arrows). CC1 cells that were not in close proximity to motor neuron cell bodies (*) also were observed (white arrows). B.-B.” TrkB perineuronal cells (white arrows) are shown in close proximity to ChAT-ir ventral horn motor neurons (*). Yellow arrows indicate the TrkB nonneuronal cells that were not in direct apposition to neuronal cell bodies. C.-C.” NG2 cell cell bodies (yellow arrows) rarely were observed in direct apposition to ventral horn neuronal cell bodies (i.e. perineuronal), yet NG2-ir processes (white arrows) frequently were observed in close proximity to the ventral horn neuronal cell bodies (*). Scale bar = 50 μm.