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Functional gap junctions in the schwann cell myelin sheath.

Balice-Gordon RJ, Bone LJ, Scherer SS - J. Cell Biol. (1998)

Bottom Line: Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32).The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32- mice, indicating that other connexins participate in forming gap junctions in these cells.Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6074, USA. rbaliceg@mail.med.upenn.edu

ABSTRACT
The Schwann cell myelin sheath is a multilamellar structure with distinct structural domains in which different proteins are localized. Intracellular dye injection and video microscopy were used to show that functional gap junctions are present within the myelin sheath that allow small molecules to diffuse between the adaxonal and perinuclear Schwann cell cytoplasm. Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32). The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32- mice, indicating that other connexins participate in forming gap junctions in these cells. Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

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Injected low molecular mass compounds fill the outer/ abaxonal and inner/adaxonal collars of Schwann cell cytoplasm.  A–D are from the same fiber after injection with 5,6-carboxyfluorescein and neurobiotin. Intensity profiles are illustrated for a  line perpendicular to the long axis of the fiber at the location indicated by the white arrowhead in each panel; the scale bar for  the intensity histogram (bottom right) is 0–255 intensity levels.  (A) Double line of 5,6-carboxyfluorescein observed immediately  after a perinuclear injection (left edge). Note doublet in the peak  of the intensity histogram representing each side of the fiber. (B)  Subsequent confocal analysis of the same fiber showed a similar  double train track pattern of 5,6-carboxyfluorescein within the  Schwann cell along the length of the fiber. A single confocal  plane, taken at near the resolution limit of the confocal microscope, midway through the depth of the cell adjacent to a node of  Ranvier (right edge) is shown; note that this region is brighter  than the double train track as there is more cytoplasm in this location. There is a doublet present in each intensity peak shown at  right of photomicrograph. (C) A single confocal plane of ∼5 μm  above the plane shown in B demonstrates the filling of fingers of  Schwann cell cytoplasm. Note absence of doublet in intensity  peaks. (D) After confocal imaging, the same fiber was processed  to visualize neurobiotin and was then reanalyzed by confocal microscopy. A single confocal plane is shown, midway through the  fiber, demonstrating that neurobiotin diffusion also results in a  double train track pattern, although the pattern is somewhat distorted by tissue processing. A doublet is apparent in each peak of  the intensity histogram on each side of the fiber. Arrow, location  of an incisure; visualization with polarized light confirmed this.  Bars, 10 μm.
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Figure 5: Injected low molecular mass compounds fill the outer/ abaxonal and inner/adaxonal collars of Schwann cell cytoplasm. A–D are from the same fiber after injection with 5,6-carboxyfluorescein and neurobiotin. Intensity profiles are illustrated for a line perpendicular to the long axis of the fiber at the location indicated by the white arrowhead in each panel; the scale bar for the intensity histogram (bottom right) is 0–255 intensity levels. (A) Double line of 5,6-carboxyfluorescein observed immediately after a perinuclear injection (left edge). Note doublet in the peak of the intensity histogram representing each side of the fiber. (B) Subsequent confocal analysis of the same fiber showed a similar double train track pattern of 5,6-carboxyfluorescein within the Schwann cell along the length of the fiber. A single confocal plane, taken at near the resolution limit of the confocal microscope, midway through the depth of the cell adjacent to a node of Ranvier (right edge) is shown; note that this region is brighter than the double train track as there is more cytoplasm in this location. There is a doublet present in each intensity peak shown at right of photomicrograph. (C) A single confocal plane of ∼5 μm above the plane shown in B demonstrates the filling of fingers of Schwann cell cytoplasm. Note absence of doublet in intensity peaks. (D) After confocal imaging, the same fiber was processed to visualize neurobiotin and was then reanalyzed by confocal microscopy. A single confocal plane is shown, midway through the fiber, demonstrating that neurobiotin diffusion also results in a double train track pattern, although the pattern is somewhat distorted by tissue processing. A doublet is apparent in each peak of the intensity histogram on each side of the fiber. Arrow, location of an incisure; visualization with polarized light confirmed this. Bars, 10 μm.

Mentions: We also evaluated whether other low molecular mass compounds known to cross gap junctions also filled the inner/adaxonal collar of Schwann cell cytoplasm. Fluorescence from other commonly used compounds, such as Lucifer yellow, propidium iodide, and ethidium bromide, was not as intense as that from 5,6-carboxyfluorescein, making it difficult to monitor dye movement by video microscopy. These dyes also have broad-spectrum emission that interfered with staining in other fluorescence channels. Moreover, since propidium iodide and ethidium bromide are impermeant to Cx32 channels in transfected cells (Elfgang et al., 1995), we selected Lucifer yellow and neurobiotin. Lucifer yellow injected into myelinating Schwann cells produced a double train track pattern of staining (n = 6 fibers from two mice; data not shown). Neurobiotin, a nonfluorescent, low-mass compound that crosses gap junctions, was coinjected iontophoretically with 5,6-carboxyfluorescein to monitor the injection (Fig. 5 A). The fibers were examined by confocal microscopy (Fig. 5 B). Evaluation of serial single confocal planes through the z axis confirmed the double train track pattern of labeling of 5,6-carboxyfluorescein, and demonstrated that this pattern was distinct from cytoplasmic labeling (Mugnaini et al., 1977; Gould and Mattingly, 1990). Fibers then were fixed, permeabilized, stained with rhodamine-conjugated strepavidin to visualize the neurobiotin, and then were then reexamined by confocal microscopy (Fig. 5 D; same fiber as in A–C), although the structure of the myelin sheath was distorted by this processing. In nine out of 12 fibers, both 5,6-carboxyfluorescein and neurobiotin stained in a double train track pattern, indicating that both compounds filled the inner collar of Schwann cell cytoplasm. Thus, several low molecular mass compounds known to pass through gap junctions appeared to diffuse across the myelin sheath.


Functional gap junctions in the schwann cell myelin sheath.

Balice-Gordon RJ, Bone LJ, Scherer SS - J. Cell Biol. (1998)

Injected low molecular mass compounds fill the outer/ abaxonal and inner/adaxonal collars of Schwann cell cytoplasm.  A–D are from the same fiber after injection with 5,6-carboxyfluorescein and neurobiotin. Intensity profiles are illustrated for a  line perpendicular to the long axis of the fiber at the location indicated by the white arrowhead in each panel; the scale bar for  the intensity histogram (bottom right) is 0–255 intensity levels.  (A) Double line of 5,6-carboxyfluorescein observed immediately  after a perinuclear injection (left edge). Note doublet in the peak  of the intensity histogram representing each side of the fiber. (B)  Subsequent confocal analysis of the same fiber showed a similar  double train track pattern of 5,6-carboxyfluorescein within the  Schwann cell along the length of the fiber. A single confocal  plane, taken at near the resolution limit of the confocal microscope, midway through the depth of the cell adjacent to a node of  Ranvier (right edge) is shown; note that this region is brighter  than the double train track as there is more cytoplasm in this location. There is a doublet present in each intensity peak shown at  right of photomicrograph. (C) A single confocal plane of ∼5 μm  above the plane shown in B demonstrates the filling of fingers of  Schwann cell cytoplasm. Note absence of doublet in intensity  peaks. (D) After confocal imaging, the same fiber was processed  to visualize neurobiotin and was then reanalyzed by confocal microscopy. A single confocal plane is shown, midway through the  fiber, demonstrating that neurobiotin diffusion also results in a  double train track pattern, although the pattern is somewhat distorted by tissue processing. A doublet is apparent in each peak of  the intensity histogram on each side of the fiber. Arrow, location  of an incisure; visualization with polarized light confirmed this.  Bars, 10 μm.
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Figure 5: Injected low molecular mass compounds fill the outer/ abaxonal and inner/adaxonal collars of Schwann cell cytoplasm. A–D are from the same fiber after injection with 5,6-carboxyfluorescein and neurobiotin. Intensity profiles are illustrated for a line perpendicular to the long axis of the fiber at the location indicated by the white arrowhead in each panel; the scale bar for the intensity histogram (bottom right) is 0–255 intensity levels. (A) Double line of 5,6-carboxyfluorescein observed immediately after a perinuclear injection (left edge). Note doublet in the peak of the intensity histogram representing each side of the fiber. (B) Subsequent confocal analysis of the same fiber showed a similar double train track pattern of 5,6-carboxyfluorescein within the Schwann cell along the length of the fiber. A single confocal plane, taken at near the resolution limit of the confocal microscope, midway through the depth of the cell adjacent to a node of Ranvier (right edge) is shown; note that this region is brighter than the double train track as there is more cytoplasm in this location. There is a doublet present in each intensity peak shown at right of photomicrograph. (C) A single confocal plane of ∼5 μm above the plane shown in B demonstrates the filling of fingers of Schwann cell cytoplasm. Note absence of doublet in intensity peaks. (D) After confocal imaging, the same fiber was processed to visualize neurobiotin and was then reanalyzed by confocal microscopy. A single confocal plane is shown, midway through the fiber, demonstrating that neurobiotin diffusion also results in a double train track pattern, although the pattern is somewhat distorted by tissue processing. A doublet is apparent in each peak of the intensity histogram on each side of the fiber. Arrow, location of an incisure; visualization with polarized light confirmed this. Bars, 10 μm.
Mentions: We also evaluated whether other low molecular mass compounds known to cross gap junctions also filled the inner/adaxonal collar of Schwann cell cytoplasm. Fluorescence from other commonly used compounds, such as Lucifer yellow, propidium iodide, and ethidium bromide, was not as intense as that from 5,6-carboxyfluorescein, making it difficult to monitor dye movement by video microscopy. These dyes also have broad-spectrum emission that interfered with staining in other fluorescence channels. Moreover, since propidium iodide and ethidium bromide are impermeant to Cx32 channels in transfected cells (Elfgang et al., 1995), we selected Lucifer yellow and neurobiotin. Lucifer yellow injected into myelinating Schwann cells produced a double train track pattern of staining (n = 6 fibers from two mice; data not shown). Neurobiotin, a nonfluorescent, low-mass compound that crosses gap junctions, was coinjected iontophoretically with 5,6-carboxyfluorescein to monitor the injection (Fig. 5 A). The fibers were examined by confocal microscopy (Fig. 5 B). Evaluation of serial single confocal planes through the z axis confirmed the double train track pattern of labeling of 5,6-carboxyfluorescein, and demonstrated that this pattern was distinct from cytoplasmic labeling (Mugnaini et al., 1977; Gould and Mattingly, 1990). Fibers then were fixed, permeabilized, stained with rhodamine-conjugated strepavidin to visualize the neurobiotin, and then were then reexamined by confocal microscopy (Fig. 5 D; same fiber as in A–C), although the structure of the myelin sheath was distorted by this processing. In nine out of 12 fibers, both 5,6-carboxyfluorescein and neurobiotin stained in a double train track pattern, indicating that both compounds filled the inner collar of Schwann cell cytoplasm. Thus, several low molecular mass compounds known to pass through gap junctions appeared to diffuse across the myelin sheath.

Bottom Line: Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32).The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32- mice, indicating that other connexins participate in forming gap junctions in these cells.Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6074, USA. rbaliceg@mail.med.upenn.edu

ABSTRACT
The Schwann cell myelin sheath is a multilamellar structure with distinct structural domains in which different proteins are localized. Intracellular dye injection and video microscopy were used to show that functional gap junctions are present within the myelin sheath that allow small molecules to diffuse between the adaxonal and perinuclear Schwann cell cytoplasm. Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32). The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32- mice, indicating that other connexins participate in forming gap junctions in these cells. Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

Show MeSH
Related in: MedlinePlus