Faculty Bibliography

Chronically demyelinated lesions of cat dorsal columns were created by focal injection of the glial toxin ethidium bromide. Freeze-fracture studies show that the center of the lesion, which is devoid of glial cells and processes, contains axons having neither node-like nor paranodal-type membrane specializations. Near the margin of the lesion, however, where axons are in contact with glial cells, the axolemma sometimes displays focal accumulations of E- and P-face particles resembling those at nodes of Ranvier. In cases where the adjacent cell could be identified, it had the characteristics of an astrocyte. Linear indentations of the axolemma displaying a paracrystalline pattern like that of the paranodal axolemma also occur in the marginal region. Here, the adjacent cell had the characteristics of an oligodendrocyte. These specializations may be closely associated with each other or spatially separate. Normal nodal and paranodal specializations were absent throughout the lesion at all time periods examined. These findings support the view that both the formation and the maintenance of nodal and paranodal axon membrane specializations require contact with glial cells.

Focal accumulations of E-face intramembranous particles occur in spinal cord axons of myelin-deficient rat mutants. These resemble nodal particles and, like them, may represent voltage-sensitive sodium channels. It is proposed that axonal activity at these foci could increase extracellular potassium to the point of triggering activity in adjacent axons. Rapid spread of such potassium-induced activity among bare axons could underlie the seizures and other neurological abnormalities that develop in this mutant. A similar mechanism may account for the paroxysmal attacks sometimes seen in multiple sclerosis.

Analysis of the plasmalemma of frog dorsal root ganglion cells by freeze-fracture demonstrates regional differences in the distribution of intramembranous particles. Although P-face particles are distributed rather uniformly, the E-face particle concentration at the cell body (approximately 300 micron -2) is much lower than that at the axon hillock (approximately 900 micron -2), proximal initial segment (approximately 1000 micron -2), or intermediate portion of the initial segment (approximately 800 micron -2). The particle concentrations in the latter regions approach that at the node of Ranvier and, moreover, particle size analysis reveals that the E-face particles, like those at the node, include a large number that are 10 nm or more in diameter. Thin sections reveal patches of a dense undercoating on the cytoplasmic surface of the axolemma in some regions of the initial segment but not the axon hillock. It is concluded from these results that the axon hillock and the initial segment of dorsal root ganglion cells have some of the structural characteristics of the node of Ranvier.

Optic nerves of adult frogs were freeze-fractured with the proximal to distal orientation and distances from retina monitored throughout the process. E face particle accumulations are commonly found (approximately 90% of all examples) in the juxtaparanodal portion of the internode (JPI) immediately adjacent to the paranodal junction. The concentration of these particles is usually highest (200-700/micron 2) immediately adjacent to the last strip of the paranodal junction and then decreases over approximately 1-4 micron to the background level (approximately 100/micron 2) of the more remote portions of the internode. Accumulations with high particle concentrations generally extend further into the internode than those with low concentrations. JPI particle accumulations occur with equal frequency in proximal and distal JPIs, and no apparent difference was seen between optic nerve segments adjacent to or distant from the retina. The majority of the JPI particles are large (10 nm or more in diameter), and they resemble the large nodal particles in size and shape. Particle size analysis in different areas of the internode shows that the concentration of small particles does not change significantly along the internode (including the JPI), but the concentration of large particles is significantly higher in the immediate JPI (140-600/micron 2) than in internodal regions (30-55/micron 2). Thus, the high particle concentration at the JPI region is mainly due to the accumulation of large particles. Such accumulations also occur frequently in irregularly shaped 'lakes' between paranodal junctional strips. Here too the particles are primarily large, and the accumulations occur equally in segments adjacent to or distant from the retina and in both proximal and distal paranodal regions. Heminodes occur in all segments of the frog optic nerve. Most of these lack typical nodal specializations.

Axons in chronically demyelinated spinal cord lesions, induced by ethidium bromide injection, display patches of intramembranous particles and indentations resembling nodal and paranodal axolemmal specializations respectively. Both occur in the marginal region of the lesions where the demyelinated axons are intimately associated with astrocytic and oligodendrocytic processes, and probably correspond to the aberrant node-like and paranodal junctional complexes seen in thin sections of this region. Demyelinated axons in the center of the lesion, which are not in contact with glial processes, do not display these membrane specializations.

In developing rat peripheral fibers, nodal specialization appears early, prior to myelin compaction, and is first detected as a junction between the axon and the overhanging Schwann cell process characterized by a uniformly wide (approximately 18 nm) intercellular gap containing a patchy dense substance and a cytoplasmic undercoating subjacent to the axolemma. The gap width is rather consistent but the axolemmal undercoating is more variable and lower in density than that found at more mature nodes of Ranvier, and it is also highly variable in length, ranging from 0.5 to 3 micron. The outermost Schwann cell layer is usually prominent with a large volume of cytoplasm and many organelles. In freeze-fracture replicas, modal specializations are characterized by accumulations of large (approximately 10 nm) particles in the axolemma, especially the E face, but immature nodes generally have a lower particle concentration than mature nodes. No node-like particle aggregates have been found in axons not intimately associated with Schwann cells. Mature paranodal axon-Schwann cell junctions are usually formed first by the loops closest to the node and are characterized by a 2-3 nm gap between the apposed membranes, periodic intercellular densities (transverse bands) in the gap and cisternae flattened against the junctional Schwann cell membrane. The loops further removed from the node display a wider gap containing irregularly spaced or diffuse intercellular densities, or none. Mature junctions appear relatively late in the rat, and it is not unusual to find developing nodes with several Schwann cell loops present that do not indent the axolemma significantly and are not associated with the paracrystalline pattern characteristic of the mature junctional axolemma. In such instances, the nodal particle aggregates do not have sharply circumscribed boundaries. The majority of the developing nodes are asymmetric with one paranodal segment more mature than the other.

The binding of colloidal iron hydroxide (CI) and ruthenium red (RR) to the plasma membrane of frog ependymal astrocytes was examined by electron microscopy. Positively charged CI and RR bind to the external surface of the plasma membrane of all parts of the ependymal astrocyte. Prior treatment with neuraminidase markedly reduces the number of bound CI particles, suggesting that the sialic acid of carbohydrates associated with the cell surface is responsible for much of the CI binding. Comparable observations were made on mouse ependymal cells. These findings indicate that an anionic, carbohydrate-rich cell coat occurs on the plasma membrane of amphibian ependymal astrocytes and mammalian ependymal cells. This cell coat may be related to transport, barrier, or receptive functions of ependymal cells.

Peripheral nerves from the hind legs of frog tadpoles were examined in order to ascertain the pattern of development of nodal and paranodal specializations in myelinated fibers. In thin sections the earliest detectable node-related specializations resemble 'intermediate' junctions between axons and Schwann cell processes. These occur in individually ensheathed axons near the edges of the sheath segments and could represent early nodal or paranodal components or transient structures. The characteristic nodal 'undercoating' is indistinct and highly variable in thickness in immature fibers and its density is lower in developing nodes than in adult nodes. Corresponding freeze-fracture replicas of developing axons demonstrate aggregates of nodal E face particles whose concentration is lower than that in the adult. Such aggregates usually occur immediately adjacent to Schwann cell indentations, even though early in development the latter may not exhibit the paracrystalline pattern seen in the adult paranodal axolemma. On rare occasions, node-like particle aggregates and presumptive nodal undercoatings have been observed without recognizable paranodal junctions or indentations nearby. However, neither specialization has been found in axons not individually ensheathed by Schwann cells. Paranodal Schwann cell loops are widely separated and irregularly arranged in the developing nodes, and the paranodal regions flanking a node usually mature asymmetrically. Differentiated paranodal junctions appear early in axons ensheathed by only a few loose Schwann cell lamellae. However, such junctions are not formed by all paranodal loops; they consistently appear first in the loops close to the node and only later in those further removed. No junctional specialization has been observed in either the axolemma or the Schwann cell membrane without the close association of the other.

Analysis of Shiverer central nervous tissue by the freeze-fracture method shows that axoglial junctions of the type found normally in the paranodal region occur commonly despite the gross reduction in myelin. On a substructural level these junctions appear identical to those that form between paranodal oligodendroglial processes and axolemma. On a grosser level, however, they are bizarre in shape, arrangement and distribution. Isolated glial processes, or small sheaves of them, course among axons and form such junctions in an irregular patchy manner, usually without apparent relationship to paranodal regions. These aberrant junctions may be oriented transversely, obliquely or longitudinally with respect to the axonal axis. Axolemmal E face particle accumulations, which characterize normal nodes of Ranvier, are usually not found in the membrane adjacent to the aberrant junctional patches. Thus, axoglial junctional specializations of the paranodal type can form in this mutant in the absence of the myelin proteins that are deficient in Shiverer, and such junctions may appear in areas not related to other paranodal or nodal structures. The relevance of these findings to differentiation of the axolemma and to the neurological defects in this mutation is discussed.