Is There Evidence for Myelin Modeling by Astrocytes in the Normal Adult Brain?

Abstract

A set of astrocytic process associated with altered myelinated axons is described in the forebrain of normal adult rodents with confocal, electron microscopy, and 3D reconstructions. Each process consists of a protuberance that contains secretory organelles including numerous lysosomes which polarize and open next to disrupted myelinated axons. Because of the distinctive asymmetric organelle distribution and ubiquity throughout the forebrain neuropil, this enlargement is named paraxial process (PAP). The myelin envelope contiguous to the PAP displays focal disruption or disintegration. In routine electron microscopy clusters of large, confluent, lysosomes proved to be an effective landmark for PAP identification. In 3D assemblies lysosomes organize a series of interconnected saccules that open up to the plasmalemma next to the disrupted myelin envelope(s). Activity for acid hydrolases was visualized in lysosomes, and extracellularly at the PAP-myelin interface and/or between the glial and neuronal outer aspects. Organelles in astrocytic processes involved in digesting pyknotic cells and debris resemble those encountered in PAPs supporting a likewise lytic function of the later. Conversely, processes entangling tripartite synapses and glomeruli were devoid of lysosomes. Both oligodendrocytic and microglial processes were not associated with altered myelin envelopes. The possible roles of the PAP in myelin remodeling in the context of the oligodendrocyte-astrocyte interactions and in the astrocyte's secretory pathways are discussed.

Keywords: astrocytic process; lysosome; myelin remodeling; neuropil; secretion.

Figure 1

Distribution of acid phosphatase positive cells and lysosomes in the neuropil and white matter. (A) Ilium of the dentate gyrus. Presumptive microglial cells (arrows) containing numerous lysosomes. Note that cell processes course between granule cells (asterisks). (B) Microglia with abundant lysosomes at either pole of the nucleus (arrow) that is also positive to the histochemical reaction. (C) A presumptive pyramidal cell in layer VI of the frontal cortex. Note that numerous lysosomes are scattered in the perikaryon. (D) Two presumptive microglial cells in the neuropil of the deep bulbar white matter of the olfactory bulb. To highlight is the linear arrangement of lysosomes in the cell's processes (arrows) that appose unstained, myelinated axons (arrowheads). (E) Cell in the glomerular layer of the olfactory bulb displaying an overall diffuse positivity to the reaction products. Note the varicose appearance of processes due to rows of lysosomes. As processes bend repeatedly they bound squared- or polygonal-shaped (asterisks) areas of the neuropil. Arrowheads, root of a proximal process; N, cell nucleus. (F) Granule cell layer of the olfactory bulb. Isolated lysosomes can be seen among the bundles of myelinated fibers (circles) and between the adjacent clusters of granule cells (arrows). (G) Radial glial cell whose soma (arrowhead) lies in the Purkinje cell layer of the cerebellum; in the upper part, a microglial cell can also be seen. Mol, molecular layer; Pk, Purkinje cell layer; Gr, granule cell layer of the cerebellar cortex. (H) High magnification micrograph from the Bergman radial glial cell shown in “G.” To note is the pale cell nucleus (arrowhead) and the rows (arrowheads) of lysosomes along the ascending process. (I) A presumptive astrocyte whose pale nucleus (arrowhead) is surrounded by numerous lysosomes that penetrate proximal processes (double arrowheads). (J) A probable microglia having a dark nucleus, as well as tightly packaged lysosomes (double arrowhead) positive to the reaction. Adult rat brain. Normarski optics Scale bars = 100 μm in (A,G), 10 in (B–D) and (H–J).

Figure 2

Confocal microscopy of specimens from a mouse expressing green fluorescence protein (eGFP) in astrocytes, immuno-stained to myelin basic protein (MBP), lysosomes positive to Lyso-Tracker (LyTr-positive), and counterstained with DAPI in the olfactory bulb medulla (A,B,D) and layer VI of the frontal isocortex (C). (A) Low magnification view. Note that some lysosomes LyTr-positive throughout the neuropil over impose sites of eGFP and MBP (Merged) fluorescence. (B) Higher magnification view to the area framed in “A.” 3D video is avialable as Video 1 in “Supplemental Material.” Co-labeling to eGFP, MBP, and LyTr the astrocyte distal processes. (C) Note the discreet sites of co-labeling of the astrocyte processes, myelinated axons, and lysosomes. (D) Cluster of LyTr-positive lysosomes (red) co-labeled with eGFP fluorescence surrounding a shrunken nucleus (DAPI) of a presumptive dead cell. Note the numerous astrocytic processes eGFP-positive and LyTR-positive lysosomes. 3D video is avialable as Video 2 in “Supplemental Material.” Scale bars = 10 μm.

Figure 3

Electron micrographs from astrocytic paraxial processes in various areas of the adult rat forebrain. (A) Olfactory bulb medulla. Proximal process from that part of the astrocyte boxed in “i” (low magnification of the astrocyte whose process is shown in “A”). A thick, descending bundle of intermediate filaments (f) penetrates a paraxial process (PAP) that contains a large mitochondrion (m), Golgi apparatus (G), and several large electron-dense granules (db). Note the myelinated fiber (asterisk) next to the process. (B) A higher magnification view of a successive section to that shown in “A.” To note are the large lipid-like inclusions (arrowheads) in a dense-bodies (db) and the thin connecting tubule (arrow) with an adjacent, oval-shaped granule in the upper part of the micrograph. It is noticeable that the same axon shown in “A,” here (asterisk) reaches contiguity with the astrocytes plasma membrane. (C) A PAP contiguous to an axon (ax) whose myelin envelope protrudes (asterisk) to the surface of the former. To note are the disruption of that part of the myelin next to the dense-body in the upper part of the process (arrow). db, dense bodies; if, fascicle of intermediate filaments; m, mitochondria; rer, rough endoplasmic reticulum (rer). Specimen from the medial preoptic nucleus. (D) An example of a PAP bulging-out from an end foot to the neuropil of the ventro-medial hypothalamic nucleus. f, fascicles of intermediate filaments; G, Golgi apparatus; L, capillary lumen m, mitochondria. (E) A para-vascular astrocyte (AC) in the deep parietal cortex (i.e., Layer VIb). Note a proximal process that originates a PAP next to two myelinated axons (asterisk). (F) A large PAP (dotted) about a myelinated axon (boxed) in the olfactory bulb medulla. To be highlighted is the proximity between the process containing presumptive lysosomes and the myelinated fiber contiguous to it. (G) PAP (dotted) in the neuropil of the CA4-dentate intersection. Note the protrusion (arrow) and splitting of the myelin sheath encircling the axon next to the process. mf = mossy fiber. Calibration bars = 0.5 μm in (A–D,F,G), and 2 μm in “E.” Adult rat brain.

Figure 4

Series throughout paraxial processes. (A) Presumptive secondary lysosomes in the core of a paraxial process (PAP). To note are the side-to-side confluence (circle, section 3) or via thin communicating tubules (sections 3 and 4, arrowheads and sections 4–6, arrows). Deep frontal isocortex. (B) Series paralleling the myelinated fiber (arrows) in sections 3 and 4. To note is the progressive increase in both size and number of dense bodies overlaying the myelinated fiber. Asterisks label an axon reconstructed in “C” and coursing orthogonally throughout the series. Olfactory bulb medulla. (C) Reconstruction from 34 sections including those shown in “B.” (a,b) lateral views, showing the lysosomal (light blue) clustering and anastomoses within the PAP cytoplasm (blue). Next to the PAP myelinated axons course horizontally (green, arrows) and the orthogonally (turquois). Note the alignment of lysosomes with the myelin envelope and the short tubules (hollow arrows) anchoring in the plasma membrane (out-lined in blue) contiguous to the myelin covering. Gray = level of sectioning of micrographs shown in “B.” (c) Orthogonal view showing the group of lysosomes in apposition with the cross-sectioned axon (outlined in turquois). 3D figures were obtained with the “Reconstruct” software that is access-free (http://synapses.clm.utexas.edu/tools/reconstruct/reconstruct.stm). Adult rat, deep granule cell layer of the main olfactory bulb. Scale bars = 0.3 μm.

Figure 5

Secondary lysosomes (SL) in astrocytic processes. (A) A long, slender tubule running from the SL toward the process periphery. (B) A short tubule (arrow heads) bridging a SL to a distorted myelin envelope (double asterisk). (C) A SL fussed with the limiting plasma membrane of a paraxial process and opening to the altered myelin envelope (double arrow-head). To note is the continuity between the SL unitary membrane (arrow) and that of the process proper. Scale bars = 300 nm in (A); 100 in (B,C).

Figure 6

Series through the part of a paraxial process (PAP) in direct contact with a myelinated axon (upper right side). (A) As the sectioning proceeds throughout a lysosome, it opens (+4, black square and +4i, circle) to the PAP's surface, next to the myelin envelope. Observe that the myelin envelope collapses (+4i, circle) and embeds bubble-like formations (+4i, arrow heads) next to the lysosome opening. The myelin periodicity is restored in sections distal to the site of lysosome fusion (−3, arrows). Asterisks, inter-lysosomal loops. Adult rat, deep (i.e., layer VI) frontal isocortex. ax, axoplasm. (B) An electron-dense granule issuing a finger-like channel toward a distorted myelin envelope (my). (C) Alternate section, one section apart from that shown in “B,” showing the opening of the channel leading to an axon (ax) with a disrupted myelin envelope. Note the membranous appendage (arrow) attached to the upper aspect of the granule. (D) 3D reconstruction of the granule (green) sampled in “B” and “C.” It is evident that the “loop” shown in “C” is part of a funnel-like structure (arrow) apposing to the PAP's cell membrane (out-lined in blue). Circle, tubules shown in “B” and “C” opening to the process plasma membrane. (i). Serial out-line of the granule in 21 successive sections. (E) Electron micrograph of a cluster of presumptive lysosomes in a PAP opening (arrow) to a myelinated fiber (asterisk) in the neuropil of the deep frontal cortex. The side-to-side communication (white circle) between them is evident. Black circle, bundle of intermediate filaments. (F) 3D from a series through the PAP shown in “E.” Note the lysosome clustering (pale blue) toward the myelinated fiber (red) and the narrow ducts (arrows) opening to the process cell membrane (pale green) contiguous to the myelin sheath. Circles, anastomoses between lysosomes. Scale bars = 0.25 μm in (A) +1 to (A) +5, (B–F), and 200 nm in (A) −3 and (A) +4i.

Figure 7

Interaction of lysosomes with an altered myelin envelope in the hippocampus. (A) Alternating (1, 5, and 7) and successive (8–11) micrographs through a series at the interaction of a paraxial process and the paranodal region of a myelinated axon. Dissolution of the myelin sheath is deeper in micrograph 10 (squared). Asterisk, presumptive primary lysosome. Arrow = oligodendrocyte tongue protruding to the axoplasm. (i) At higher magnification the focal excavation of the myelin envelope and the lysosome's membrane opening (right side) are evident. Also note the flocculent, electron-opaque, material embedding the myelin stumps. (ii) Myelin envelope just apart from the lysosome-myelin interaction displaying a regular, alternating, pattern. (B) 3D reconstruction showing the cavitation (arrow) of the myelin envelope shown in series depicted in “A.” (C) Note that the site of myelin (outlined in pale blue) indentation matches with that of the lysosome-(red) cell membrane fusion. (D) Ibid. Scale bars = 0.2 μm in (A–D); 200 nm in inserts.

Figure 8

Cytochemistry to acid phosphatase in the neuropil of various forebrain areas. (A) A section through a paraxial process (PAP) (asterisk) counter-stained with uranium and lead. Note the electron-dense reaction product associated with lysosomes (ly) and a transverse interruption of the myelin envelope (arrow) at the PAP-axon intersection. Uranium-lead contrast. (B) Unstained specimen showing the site of membrane apposition of two astrocytes and a neuron (bottom-right). Note the inter-membrane reinforcement due to electron-dense reaction product. My, tangential section though the myelin envelope. (i) High magnification view at the site of convergence of the cell membranes and myelin sheath. Note the lead precipitate in the intercellular space (single arrowheads), myelin envelope (asterisk), and primary lysosome (double arrowhead). (C) Tangential section through a myelin envelope displaying dark precipitates to acid phosphatase. (D) A presumptive apoptotic cell surrounded by astrocytic processes with converging lysosomes. (E) Successive sections from an astrocytic process. Note the continuity of the reaction product to acid phosphatase from the core of the lysosome (arrow) to the adjacent myelin envelope (asterisk) in sections 7, 11, and 15. Scale bars = 1 μm in (A–D); 200 nm in (i), and (E).

Figure 9

Astrocytic perikaryon and a paraxial process in the main olfactory bulb medulla following cytochemistry to acid phosphatase. (A) Survey view of a section through the perikaryon and a thick, paraxial process both containing lysosomes. (B) High magnification micrograph of the area framed in “A.” The electron-dense reaction product is associated with the lysosome matrix (asterisk) and focally with the myelin envelope (arrowheads). Note the narrow ducts (arrow) between the lysosome and the myelin envelope. (C) Reconstruction from all lysosomes found throughout the series. Note that lysosomes in the perikaryon and paraxial process are grouped in that part of the cytoplasm next to myelinated fibers (blue and light blue-colored cylinders). (i) Rotation of the model showing lysosomal polarization toward the plasma membrane (out-lined in blue). Cell nucleus, outlined in light blue. (ii) Myelinated fibers have been added to that image in “i.” From a series of 62 sections. Scale bars = 1 μm.

Figure 10

Interactions between the astrocyte and unusual axons with severe myelin disruption. (A) A process in the neuropil of the cerebral cortex is in opposition to a myelin sheath displaying marked disruption as evidenced for the uneven, beaded, profile, and focal substitution by an amorphous, electron-opaque material (asterisk). Ax, axoplasm. (B) Transverse section through an astrocytic process (AP) interacting with a disrupted myelin sheath due to infiltration by amorphous electron-dense material (asterisk). Arrows, presumptive phagosomes containing fragments of the myelin envelope. AP, Golgi apparatus; Lf, lipofuscin granule. Specimen from the olfactory bulb medulla. (C,D) 3-D views of the astrocyte shown in “B” (gray). All lysosomes (bright red), and a lipofuscin granule (Lf) have been reconstructed. To note is the cluster of lysosomes in the part of the karyoplasm next to an uneven axon with a distorted myelin sheath (pale blue). Deep blue, astrocytic cell membrane; deep red, cell nucleus. (E) Electron micrograph of a specimen from the olfactory bulb medulla incubated for acid phosphatase visualization. The high magnification micrograph allows visualization of the site of intersection between a paraxial process (bottom) and the altered myelin envelope (My) of the axon (Ax). Note that the electron-opaque reaction product concentrates at sites where the myelin envelope losses its periodicity (arrow-heads) and is substituted by amorphous material, as well as in the matrix of a lysosome (ly). My = myelin envelope with preserved membrane periodicity. Scale Bars 0.5 μm in (A–D); 0.2 in (E).

Figure 11

Two and three dimensional views of the astrocyte and its processes in the neuropil. (A) Micrograph at the site of ramification of two capillary blood vessels that arbors two astrocytes (green and blue). Between the astrocyte's cell body (light blue) and the endothelial cell (e), a pyknotic cell (double asterisk) can be seen. Another dark cell (single asterisk) is identified between the two astrocytes. Note the clustering of electron-opaque granules (Ly) in the cytoplasm adjacent to the latter dark cell. (B) High magnification view from a successive section. The presumptive apoptotic cell (light purple) is surrounded by the outer aspect and a thin process of the astrocyte. To note are the frequent electron-dense bodies (light yellow) in the part of the cytoplasm next to the apoptotic cell. (C) 3D view of all the electron-dense granules (red and yellow) contained by both astrocytes within the series. Note that all of them are clustered in the area of the cytoplasm adjacent to pyknotic cells (brown and purple). (D) Survey 3D view depicting the levels of sectioning. (E) Electron micrograph of the capillary wall and neuropil in the ventro-medial hypothalamic nucleus. A “tripartite” axo (red)-spinous (blue) synapse is shown. Note is that the synaptic terminal is covered by a glial laminae from the end-foot (green) devoid of electron-dense granules. (F) 3D views of the end-foot and synapse shown in “E.” (F1) End-foot (green) and endothelium (turquois). (F2) 3D model of the axo-spinous terminal. (F3) Position of the synaptic terminal encased by a glial envelope. (G) Electron micrograph of a glomerulus in the granule cell layer of the olfactory bulb. Note the concentric arrangement of the astrocytic lamella with a paucity of organelles. (H1) Dendritic spines (solid colors) receiving synaptic contacts from the single bouton at the center (transparent blue). (H2) Rotation of the 3D model to depict the six dendritic spines tributary to a centrally located bouton (outlined in blue). (H3) External appearance of the glial covering of the glomerulus. Note that glial processes (red) encase the glomerular neural elements. Scale bars = 1 μm in (A–D); 0.5 in (E–H).

Figure 12

Oligodendrocyte and its processes in the neuropil. (A) Oligodendrocyte (Ol) with divergent processes piercing the neighboring neuropil and numerous myelinated axons. To note is the drum-stick appearance of the descending process that is crowned by a round, terminal (circle) (RTK) knob containing a large, solitary dense-body. Olfactory bulb medulla. (B) A lysosome in an Ol process displaying activity to acid phosphatase (dark, electronopaque deposits). (C) An RTK containing a putative lysosome in apposition with the process plasma membrane and, the later with a normal-appearing myelinated fiber. Cerebral cortex. (D) Higher magnification view of an RTK following incubation with a substratum containing B-glycerol phosphate. To note are the electron opaque products of the reaction and the continuity of the lysosomal membrane next to an adjacent normal-appearing myelin envelope. Specimen from the ventral lateral nucleus of the hypothalamus. (E) A thinner. i.e., 60 nm, section displaying the continuity of the RT plasma membrane, paralleling the outer aspect of the normal-appearing myelin sheath next to it. D and E specimen the CA4 sector of the hippocampus. Scale bars = 1 μm in (A), 200 nm in (B,C), 100 nm in (D,E).

Figure 13

Microglia (MG) and its interactions with the neuropil in the normal neuropil. (A) Paravascular microglia (MG) laying between the capillary basal lamina and neuropil (right bottom). To note are the heterochromatic clumps outlying the nuclear envelope and the concentration of organelles within the perinuclear domain, thereby leaving an organellefree, peripheral cytoplasm (asterisks). L, capillary lumen. Deep temporal cerebral isocortex. (B) A slender MG interacting with the neighboring neuropil. To note is the distribution of electron-dense granules and other organelles within the core of the perikaryon and the ascending process. (C) Higher magnification view depicting the organelle types and distribution in the juxtanuclear area. Db, electron-dense granules; G, Golgi apparatus; m, mitochondria. Ibid. (D) Spindle-shaped MG lying next to a neuron (upper half) and neuropil (bottom). Deep frontal isocortex. (E) MG process, presumptively proximal, harboring similar organelles than those in the perikaryal domain (see “A,” “C,” and “D”) leaving an organelle-free halo (asterisks). db, electron-dense granules; G, Golgi apparatus. (F) MG process in the neuropil of the medial preoptic nucleus. Ibid; asterisks, organelle-free halo (asterisks). Scale bars = 1 μm in (A–D); 0.3 in (E,F).

Figure 14

3D reconstructions showing organelles contained by a typical paraxial process (PAP) (out-lined in turquois). Progression (from left to right) of the reconstruction adding the PAP organelles and an adjacent myelinated axon to the succession.