Article Figures & Data
Figures
- Figure 1.
Oligodendrocytes express HCN2 ion channels. A, qPCR of immunopanned mouse oligodendroglia after 5 DIV showing the expression of Mbp and Hcn1-4 normalized to Gapdh; n = 3 independent cultures. B, C, qPCR of rat oligodendroglial cultures over 7 DIV showing the expression of Mbp and Hcn2 normalized to Gapdh; n = 3 independent cultures. D, Postnatal day 35 cortical gray matter CNPase-positive oligodendrocyte showing robust HCN2 expression within the cell soma and diffuse staining in myelin sheaths. E, G, HCN2 is expressed by some but not all CC1-positive oligodendrocytes in layer V of the mouse cortex (E) and spinal cord (G). White arrowheads depict HCN2-positive CC1-positive oligodendrocytes. Empty arrowheads depict HCN2-negative CC1-positive oligodendrocytes. F, Ratio of HCN2-positive CC1-positive oligodendrocytes in the medial prefrontal cortex layer V at P21, P35, P85, and P180 (mean ± SE; P21, 0.5643 ± 0.04; P35, 0.631 ± 0.045; P85, 0.698 ± 0.053; P180, 0.614 ± 0.057); n = 3 mice at each time point. H, Ratio of HCN2-positive CC1-positive oligodendrocytes in spinal cord gray matter at P21, P35, P85, and P180 (mean ± SE; P21, 0.646 ± 0.031; P35, 0.822 ± 0.028; P85, 0.915 ± 0.016; P180, 0.853 ± 0.037); n = 3 mice at each time point.
- Figure 2.
Functional properties of oligodendrocyte HCN channels. A, Whole-cell voltage-clamp currents recorded from an MBP-positive oligodendrocyte in response to an activation protocol (detailed in the bottom panel) in the absence (top) and presence (middle, blue) of HCN channel blocker ZD7288 (30 μm). Bottom, Leak-subtracted currents from the example. HCN currents were measured in the presence of Ba2+ (1 mm) in order to block the influence of inwardly rectifying potassium channel currents (see Materials and Methods for full details of the protocol). B, Whole-cell voltage-clamp currents recorded from an MBP-negative oligodendrocyte progenitor in response to an activation protocol (detailed in the bottom panel) in the absence (black) and presence (blue) of HCN channel blocker ZD7288 (30 μm). C, The black plot represents the mean ± SE activation curve of ZD7288-sensitive tail currents (n = 6). The mean RMP of oligodendrocytes (dashed line) predicts ∼15% channel opening. The plot in red represents the mean ± SE activation curve of ZD7288-sensitive tail currents in the presence of 8-bromo-cAMP [8-Br-cAMP] (n = 6). D, Current-clamp recording from an MBP-positive oligodendrocyte where ZD7288 application generates hyperpolarization of the RMP.
- Figure 3.
HCN channels regulate the length of myelin sheaths formed in oligodendrocyte-purified cultures. A, Representative MBP-positive oligodendrocytes after 14 d cultured on electrospun microfibers in the presence of either 0.1% DMSO or 10 μm ZD7288. Scale bars, 10 µm. B, Mean ± SE percentage of MBP-positive cells on microfibers: DMSO, 60.77 ± 2.972%; ZD7288, 61.76 ± 2.659%; n = 4 independent cultures; p = 0.8857, Mann–Whitney test. C, Mean myelin sheath length generated on microfibers. Values are reported as the mean ± SE; DMSO: 19.03 ± 0.6101µm; n = 342 sheaths from five independent cultures; ZD7288: 14.23 ± 0.9092 µm; n = 241 sheaths from four independent cultures; p = 0.0159, Mann–Whitney test. D, Histogram representation of the frequency of sheath lengths generated by oligodendrocytes as assessed by measuring complete MBP-positive sheaths surrounding microfibers in 5 µm bins. Pooled data from n = 4–5 independent cultures. E, Pooled data of the number of complete sheaths formed by individual oligodendrocytes in microfiber cultures. Values are reported as the mean ± SD; DMSO: 3.613 ± 2.4; n = 93 cells; ZD7288: 3.280 ± 1.935; n = 75 cells; pooled data from n = 4–5 independent cultures; p = 0.5695, Mann–Whitney test. F, qPCR of two-dimensional 3 DIV rat oligodendroglial cultures treated with ZD7288 for the expression of Mbp normalized to Gapdh. DMSO, 1.003 ± 0.03327; ZD7288, 0.9658 ± 0.03675; n = 4 independent cultures; p = 0.4857, Mann–Whitney test. G, Representative Western blot images for loading control β-actin, MBP, and CNPase from two-dimensional rat oligodendroglial cultures treated with ZD7288. H, MBP protein expression in arbitrary units (A.U.) normalized to β-actin levels. Values are reported as the mean ± SD; DMSO, 12.12 ± 2.084; ZD7288, 7.558 ± 2.540; n = 4 independent cultures; p = 0.0286, Mann–Whitney test.
- Figure 4.
HCN2 regulates the lengths of myelin sheaths formed in vivo. A, Postnatal day 21 gray matter oligodendrocyte showing no HCN2 expression in CC1-positive oligodendrocytes in PDGFRa-cre cKO mice. White arrowheads depict HCN2-positive CC1-positive oligodendrocytes. Empty arrowheads depict HCN2-negative CC1-positive oligodendrocytes. B, Representative images of P21 corpus callosum showing expression of CC1 and Sox10. Values are reported as the mean ± SE; wild type: 64,149 ± 3068 cells/mm3; Hcn2: 61,386 ± 2749 cells/mm3; n = 5–4. C, Mean number of CC1-positive oligodendrocytes in the corpus callosum. D, Mean ratio of CC1-positive cells over total Sox10-positive cells. Wild type: 0.7803 ± 0.025; Hcn2: cKO: 0.766 ± 0.003; n = 5–4. E, Representative postnatal day 21 gray matter CNPase-positive oligodendrocytes. CNPase staining labels the entire oligodendrocyte enabling the reconstruction of individual cells in the sparsely myelinated layer II/III of the cortex. Scale bars, 30 µm. F, Mean myelin sheath length per oligodendrocyte. Values are reported as the mean ± SD; wild type: 47.08 ± 10.07 µm; n = 35 cells from five mice; Hcn2 cKO: 39.34 ± 7.654 µm; n = 35 cells from five mice; p = 0.0002, Mann–Whitney. G, Mean myelin sheath length per oligodendrocyte per mouse. Wild type: 47.08 ± 2.157 µm; n = 1383 sheaths from five mice; Hcn2 cKO: 39.34 ± 1.775 µm; n = 1298 sheaths from five mice; p = 0.0317, Mann–Whitney test. H, Mean myelin sheath number per oligodendrocyte. Values are reported as the mean ± SD; wild type: 49.57 ± 9.992; n = 35 cells from five mice; Hcn2 cKO: 47.29 ± 10.30; n = 35 cells from five mice; p = 0.3493, unpaired t test. I, Mean myelin sheath number per oligodendrocyte per mouse. Values are reported as the mean ± SE: wild-type: 49.57 ± 2.746; n = 5; Hcn2 cKO: 47.29 ± 1.856; n = 5 mice; p = 0.6905, Mann–Whitney test. J, Postnatal day 21 spinal cord white matter teased fibers showing the expression of MAG-positive myelin sheaths and CASPR-positive paranodes. White arrowheads show complete myelin sheaths: two CASPR-positive paranodes connected by a continuous MAG-positive myelin sheath. Scale bars, 100 µm. K, Mean myelin sheath length from spinal cord teased fiber preparations. Values are reported as the mean ± SE; wild-type 442.2 ± 24.39 µm, n = 225 sheaths from four mice; Hcn2 cKO: 334.4 ± 24.99 µm; n = 230 sheaths from five mice; p = 0.0190, unpaired t test. L, Histogram representation of data from K showing the frequency of myelin sheath lengths in 50 µm bins. Pooled data are from n = 4–5 mice.
- Figure 5.
HCN2 regulates the length of myelin sheaths formed in vivo. A, Postnatal day 50 gray matter oligodendrocytes showing no HCN2 expression in CNP-positive oligodendrocytes in CNP-cre cKO mice. B, Representative postnatal day 50 gray matter CNPase-positive oligodendrocytes. C, Mean ± SD myelin sheath length per oligodendrocyte. Wild type: 50.12 ± 7.438 µm; n = 27 cells from four mice; Hcn2 cKO: 38.42 ± 5.89 µm; n = 28 cells from four mice; p = 0.0001 unpaired t test. D, Mean myelin sheath length per oligodendrocyte per mouse. Values are reported as the mean ± SE; wild-type: 50.27 ± 2.266 µm; n = 981 sheaths from four mice; Hcn2 cKO: 38.42 ± 1.711 µm; n = 1306 sheaths from four mice, p = 0.029, Mann–Whitney test. E, Mean ± SD myelin sheath number per oligodendrocyte. Wild-type 48.35 ± 5.621; n = 26 cells from four mice; Hcn2 cKO: 47.52 ± 4.552; n = 27 cells from four mice; p = 0.5577 unpaired t test. F, Mean ± SE myelin sheath number per oligodendrocyte per mouse. Wild-type: 48.35 ± 1.639; n = 5; Hcn2 cKO: 47.52 ± 0.36; n = 4 mice, p = 0.999, Mann–Whitney test. G, Mean ± SE myelin sheath length from spinal cord teased fiber preparations. Wild-type: 428.1 ± 24.52 µm; n = 281 sheaths from four mice; Hcn2 cKO: 318 ± 27.51 µm; n = 306 sheaths from four mice, p = 0.0244, unpaired t test. H, Histogram representation of data from G showing the frequency of myelin sheath lengths in 50 µm bins. Pooled data are from n = 4 mice.
