The Compound Myelinated Nerve Fiber

From a longitudinal point of view, the compound myelinated axon is organized into segments: the node, paranode, juxtaparanode and the unspecialized internodal segment. Each of these domains has a unique structure and carries unique molecules. A detailed account of the molecular

Figure II (A) Camera lucida drawing showing a Type III oligodendrocyte in the adult rat medullary velum. The oligodendrocyte was visualized by application of the antibody Rip to a velum whole mount. The diagram on the right side shows the relation L/D for the fibers in this unit. Scale bar = 50 |m. (With permission from Butt et al., 1998a.) (B) Type IV oligodendrocyte in fetal day 47 cat spinal cord white matter. The drawing to the left was made from an EM reconstruction based on serial thin sections. It shows a myeli-nating oligodendrocyte associated with a single axon. Note the close relation between the oligodendroglial perikaryon and the myelin sheath. Electron micrograph on the right side shows a transverse section through this unit at the level indicated in the drawing. Asterisk indicates axon. (x12,400). (With permission from Remahl and Hildebrand, 1990.)

Figure II (A) Camera lucida drawing showing a Type III oligodendrocyte in the adult rat medullary velum. The oligodendrocyte was visualized by application of the antibody Rip to a velum whole mount. The diagram on the right side shows the relation L/D for the fibers in this unit. Scale bar = 50 |m. (With permission from Butt et al., 1998a.) (B) Type IV oligodendrocyte in fetal day 47 cat spinal cord white matter. The drawing to the left was made from an EM reconstruction based on serial thin sections. It shows a myeli-nating oligodendrocyte associated with a single axon. Note the close relation between the oligodendroglial perikaryon and the myelin sheath. Electron micrograph on the right side shows a transverse section through this unit at the level indicated in the drawing. Asterisk indicates axon. (x12,400). (With permission from Remahl and Hildebrand, 1990.)

anatomy of the oligodendrocyte-axon interface is presented elsewhere in this book.

1. Nodal Features a. The Nodal Axon Successive myelin sheaths are separated by short gaps, the nodes of Ranvier, where the axolemma is exposed to the extracellular space over a length of about 1 |im (Hildebrand, 1971; Hildebrand and Waxman, 1984; Peters et al., 1991), as in the PNS (Berthold and Rydmark, 1995). Longer nodes have been observed in preterminal-terminal CNS axon domains (see Peters et al., 1991). As at PNS nodes (Reles and Friede, 1991; Berthold and Rydmark, 1995) the cross-cut CNS nodal axon usually has a circular outline and is barrel-shaped in longitudinal sections. The diameter of the nodal axon is less than the internodal diameter, particularly in the largest fibers, where the ratio between the nodal and the internodal diameter may be 1/3 (Fig. 12; Hildebrand, 1971).

A few spherical invaginations (75-85 nm) regularly occur in the nodal axolemma. In the nodal axoplasm microtubules and neurofilaments are relatively tightly packed, together with mitochondria, empty vesicles, some lamellated bodies, and small irregular membranous profiles (Fig. 12) (Hildebrand, 1971). In EM the nodal axolemma is very distinct because of the presence of a dense granular undercoat-ing, which is 25 to 35 nm thick and separated from the axolemma by a 10 -nm light zone (Fig. 12) (Hildebrand, 1971; Reles and Friede, 1991). The undercoating seems to

Figure 12 Electron micrographs from thin sections through nodes of Ranvier. (A) Transverse section through large node in cat spinal cord white matter. The nodal axon (AX) contains many vesicles and mitochondria (m) in addition to various other bodies. Note the close packing of microtubules and neurofilaments. The nodal axon membrane has a dense appearance. The axon is apposed by terminating myelin (MY) on the left side. The uncovered right side of the axon is surrounded by an extracellular material that separates the axon from perinodal astrocytic processes (PAP) containing filaments and gliosomes (G). The perinodal astrocytic processes emit microvilli-like processes (small arrows) and other processes (FP), which approach the nodal axolemma. Note presence of a myelinoid body (MB) in the upper right corner. (x17,400). Inset shows nodal axon membrane at a higher magnification (AX = axon). Arrows indicate a dense undercoating on the axoplasmic side of the axon membrane. (B, C) Later in the series of sections. (x13,800). (With permission from Hildebrand, 1971.) (D) Longitudinal section from node in a thin axon in a 30-day-old rat optic nerve. Asterisk indicates nodal axon. Note that the nodal axon is contacted by astro-cytic processes (A). (x16,700). (With permission from Hildebrand and Waxman, 1984.)

Figure 12 Electron micrographs from thin sections through nodes of Ranvier. (A) Transverse section through large node in cat spinal cord white matter. The nodal axon (AX) contains many vesicles and mitochondria (m) in addition to various other bodies. Note the close packing of microtubules and neurofilaments. The nodal axon membrane has a dense appearance. The axon is apposed by terminating myelin (MY) on the left side. The uncovered right side of the axon is surrounded by an extracellular material that separates the axon from perinodal astrocytic processes (PAP) containing filaments and gliosomes (G). The perinodal astrocytic processes emit microvilli-like processes (small arrows) and other processes (FP), which approach the nodal axolemma. Note presence of a myelinoid body (MB) in the upper right corner. (x17,400). Inset shows nodal axon membrane at a higher magnification (AX = axon). Arrows indicate a dense undercoating on the axoplasmic side of the axon membrane. (B, C) Later in the series of sections. (x13,800). (With permission from Hildebrand, 1971.) (D) Longitudinal section from node in a thin axon in a 30-day-old rat optic nerve. Asterisk indicates nodal axon. Note that the nodal axon is contacted by astro-cytic processes (A). (x16,700). (With permission from Hildebrand and Waxman, 1984.)

represent a cytoskeletal anchoring for protein molecules in the nodal axolemma (Ellisman, 1979; Bray et al., 1981; Koenig and Repasky, 1986). Freeze-fracture EM studies show a high density (1,000-1,500 per |im2) of large (>10 nm) particles in the E-face of the nodal axolemma, whereas the internodal axolemma (25-150 per |im2) and the axolemma of unmyelinated axons (25-250 per | m2) have a low density of such particles (Fig. 13) (Schnapp and Mugnaini, 1978; Rosenbluth, 1983; Waxman, 1984; Black et al., 1990; Waxman and Ritchie, 1993).

Figure 13 Freeze-fracture electron micrographs showing a node of Ranvier in the adult rat optic nerve. The fracture has exposed the E-face of the axon membrane, which exhibits numerous large particles at the node (eN), but not at the flanking paranodes (ePN). (A) Perinodal astrocytic process. (x70,000). Inset shows the E-face of the nodal axon membrane at a higher magnification. (x175,000). (With permission from Waxman and Black, 1995.) (B) Distribution of large intramembranous E-face particles (black dots) in the nodal-paranodal-juxtaparanodal region of a myelinated CNS axon. Note that such particles are enriched at the node. They also form linear strings in the paranodal region and occur in the juxtaparanode adjacent to the paranode. (Modified from Rosenbluth, 1989, with permission.)

Figure 13 Freeze-fracture electron micrographs showing a node of Ranvier in the adult rat optic nerve. The fracture has exposed the E-face of the axon membrane, which exhibits numerous large particles at the node (eN), but not at the flanking paranodes (ePN). (A) Perinodal astrocytic process. (x70,000). Inset shows the E-face of the nodal axon membrane at a higher magnification. (x175,000). (With permission from Waxman and Black, 1995.) (B) Distribution of large intramembranous E-face particles (black dots) in the nodal-paranodal-juxtaparanodal region of a myelinated CNS axon. Note that such particles are enriched at the node. They also form linear strings in the paranodal region and occur in the juxtaparanode adjacent to the paranode. (Modified from Rosenbluth, 1989, with permission.)

Since the nodal axolemma exhibits a distinct Na+ channel immunoreactivity and a high binding of the Na+ channel probe saxitoxin (see Rosenbluth, 1983; Waxman and Ritchie 1993), it has been suggested that these particles may represent sodium channels (Rosenbluth, 1976, 1984; Kristol et al., 1978).

b. Nodal Adnexa The perinodal astrocyte: When the diameter of a large CNS axon decreases as the node is approached, the emerging perinodal space becomes filled by fibrous astrocytic processes. Nodes of small myelinated CNS fibers decrease less in diameter and are less well shielded. EM analysis of CNS nodes shows that fingerlike or sheetlike astrocytic extensions course from the perinodal shield down to within 10 nm from the nodal axolemma (Fig. 12) (Hildebrand 1971; Hildebrand and Waxman, 1984). There are gap junctions between perinodal astrocytic processes and nodal oligodendrocytic components (Massa and Mugnaini, 1982; Waxman and Black, 1984; Waxman, 1986). Large nodes have more nodal processes than small nodes, but the system of nodal astrocytic processes always remains less impressive than the elaborate collar of Schwann cell processes seen at medium-size and large PNS nodes (Bjartmar et al., 1994b). The perinodal astrocytic extensions, from which the nodal extensions emerge, contain filament bundles and occasional gliosomes (Fig. 12). A single astrocyte may send processes to more than one node, and a single node may receive processes from more than one astrocyte. Similar observations have been made on CNS nodes in the cat (Hildebrand, 1971), the rat (Hildebrand and Waxman, 1984; Butt et al., 1994b), the guinea pig (Raine, 1984b), the chicken (Anderson et al., 2000), reptiles and amphibia (Bodega et al., 1987, Sims et al., 1991), and in fish (Maggs and Scholes, 1990). For a comprehensive discussion of the molecular anatomy of the nodal glial processes see Black et al. (1990, 1995). It has been suggested that the nodal astrocytic processes produce and maintain the node gap substance (see later) and/or influence the ionic composition of the node gap. It has also been speculated that node-contacting glial cells might transfer glial Na+ channels to the nodal axon membrane (Bevan et al., 1985; Gray and Ritchie, 1985). However, more recent evidence indicates that the clustering of sodium channels in the nodal axolemma is due to oligoden-droglial signals (Kaplan et al., 1997).

The nodal NG2 immunoreactive cell: Rat CNS nodes of Ranvier are contacted by processes from fibrous astrocytes and glial cells expressing the NG2 (neuron-glial antigen 2) integral membrane chondroitin sulfate proteoglycan (Fig. 14) (Butt et al., 1999, 2002). The NG2 molecule has a large extracellular domain and interacts with other membrane molecules and extracellular matrix molecules. The NG2 cells are not astrocytes since they are devoid of GFAP (glial fibrillary acidic protein). Their expression of PDGFa

(platelet-derived growth factor a) receptors and the O4 antigen indicates that they may be related to immature oligo-dendrocytes (Fig. 15). Thus, NG2 cells are distinct from neurons, astrocytes, microglia, and mature oligodendro-cytes, and they are at least as common as other glial types (Nishiyama et al., 1999; Nishiyama, 2001).

The CNS node of Ranvier is a meeting place for four different cell types: the neuron, the myelinating oligodendro-cyte, the perinodal astrocyte, and the nodal NG2 cell. It has been suggested that nodal NG2 cells might participate in sodium channel clustering at nodes of Ranvier (Butt et al., 1999, 2002). After rat brain injury, NG2 cells proliferate and their NG2 expression increases (Levine, 1994). NG2 cells occur in MS lesions, and these cells proliferate and differentiate into myelinating cells in experimentally demyeli-nated areas. NG2 cells undergo reactive changes in inflammatory CNS conditions and occur in oligoden-drogliomas (Nishiyama et al., 1999). Finally, NG2 cells in the hippocampus receive a glutamatergic synaptic input from CA3 pyramidal cells (Bergles et al., 2000; Nishiyama, 2001). The function of this neuronal-glial synapse remains an enigma (LoTurco, 2000).

The node gap substance: In EM images from CNS nodes of Ranvier, a granular extracellular material is present in the node gap (Figs. 12A-C) (Hildebrand, 1971; Hildebrand and Waxman, 1984; Raine, 1984b; Remahl and Hildebrand, 1985). Staining of sections from cat white matter according to a histochemical method for demonstration of polyanions (Langley and Landon, 1969) results in a reaction product at the nodes with a pattern that is very similar to that seen at rat PNS nodes (Fig. 16) (Langley and Landon, 1969; Hildebrand and Skoglund 1971). A similar pattern is seen in adult rat white matter nodes of Ranvier after application of antibodies against chondroitin sulfate or J1 glycoprotein molecules (Bjartmar et al., 1994b). These antibodies seem to label the node gap substance. According to Langley (1969) and Zagoren (1984), the node gap substance at PNS nodes acts as a cationic exchange resin, being strategically present where ion exchange takes place during impulse propagation. Since the PNS and CNS node gap substance can be visualized with the same reagents, they may be chemically similar and might play analogous roles. One function of this component of the CNS node could be to control the nodal ionic milieu (Hildebrand and Skoglund, 1971). It may also counteract sprouting from the nodal axon (Faissner and Schachner, 1995) and/or anchor nodal glial processes to the node (Davis et al., 1996).

2. Paranodal Features

Each node is flanked by two paranodes, where the myelin lamellae attach to the paranodal axolemma. Some previous investigators, including Ranvier himself, used the term paranode to describe a longer part of the myelin sheath, including both the paranodal myelin attachment

Figure 14 NG2+ glial cells visualized light microscopically by immunoperoxidase staining of a whole mount of the rat anterior medullary velum (A) and by preembedding EM immunocytochemistry (B, C). (A) Two NG2+ cells with a small soma and multiple branching primary processes, which either ramify along axon bundles (arrowheads) or terminate on individual axons (arrows). Some processes have spines that terminate on axons (curved arrows). (B) Electron micrograph showing an NG2+ cell (star = nucleus). Note the immunoreactive membrane. This cell sends a process (curved arrows) to a node of Ranvier nearby (asterisk, open arrows indicate paranodes). (C) Node of Ranvier (asterisk) contacted by NG2+ processes (curved arrows). x330, x10,600, and x7,600, respectively, for A, B, and C. (With permission from Butt et al., 1999.)

Figure 14 NG2+ glial cells visualized light microscopically by immunoperoxidase staining of a whole mount of the rat anterior medullary velum (A) and by preembedding EM immunocytochemistry (B, C). (A) Two NG2+ cells with a small soma and multiple branching primary processes, which either ramify along axon bundles (arrowheads) or terminate on individual axons (arrows). Some processes have spines that terminate on axons (curved arrows). (B) Electron micrograph showing an NG2+ cell (star = nucleus). Note the immunoreactive membrane. This cell sends a process (curved arrows) to a node of Ranvier nearby (asterisk, open arrows indicate paranodes). (C) Node of Ranvier (asterisk) contacted by NG2+ processes (curved arrows). x330, x10,600, and x7,600, respectively, for A, B, and C. (With permission from Butt et al., 1999.)

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