Faculty Bibliography

We have examined the ependymal astrocytes of the frog cerebellar cortex in thin sections and freeze-fracture replicas. The somata border the fourth ventricle and give rise to basal processes whose daughter branches cross the molecular layer and terminate as subpial endfeet. Irregular lamellar appendages arise from the basal processes and their branches. In the molecular layer the appendages selectively ensheath apposed parallel fiber boutons and Purkinje cell dendritic spines. Other appendages ramify throughout the neuropil, some contributing to extensive pericapillary sheaths. Freeze-fractured ependymal astrocyte plasma membrane consistently has a greater concentration of intramembranous particles (IMPs) and IMPs of larger mean size than neuronal plasma membrane in the same replicas. Like the astrocytes of the mammalian central nervous system, frog ependymal astrocytes form numerous gap junctions with each other. However, orthogonal arrays of IMPs ('assemblies') were not observed. Ependymal cells in the frog cerebellum combine the morphology, and probably the functions, of both ependymal cells and astrocytes.

(1) The principal result of freeze-fracture studies of myelinated axons is that the axolemma is clearly not uniform in its structure, but rather is highly differentiated in both paranodal and nodal regions. Thus, it is no longer correct to assume that the special physiological properties of myelinated nerve fibers derive only from the presence of the myelin sheath. The inhomogeneity of the axolemma must also be taken into account. (2) The nodal axolemma is characterized by a population of large intramembranous particles primarily in the E fracture face that may correspond to the voltage sensitive sodium channels known to be concentrated there. (3) Significant numbers of such particles also frequently occur in paranodal 'lakes' and in the internodal axolemma immediately adjacent to the paranodal region. These are probably accessible, albeit slowly, by way of the narrow extracellular cleft between the paranodal junctional membranes. (4) In the absence of ensheathment by myelinating cells, axons fail to develop normal nodal and paranodal membrane specializations. (5) When ensheathed by abnormal myelinating cells, corresponding abnormalities develop in both nodal and paranodal specializations of the axolemma. (6) Demyelination results in dedifferentiation of axolemmal specializations. (7) It is concluded that development and maintenance of normal axolemmal differentiation requires interaction of the axon with myelinating cells. These cells thus serve not only to produce myelin but also to regulate axolemmal differentiation. Alterations in axolemmal structure following demyelination may significantly affect the physiological properties of the axons. Specifically, ionophore redistribution may underlie the development of either continuous or nonuniform conduction in some demyelinated fibers.

The spinal cord, optic nerves, and cerebellum of the mouse mutant Shiverer were examined by electron microscopy of thin sections. Although central nervous system myelin is grossly deficient in amount, none of its basic structural elements are missing. Regions of compact myelin can be found composed of several layers of alternating major dense lines and intermediate lines repeating with normal periodicity. The 'radial component' consisting of periodic thickenings of the intermediate line aligned through several lamellae was also identified. Axoglial junctions characteristic of the type found in paranodal regions are present in greater than normal numbers but occur in aberrant locations. Myelin sheaths have marked reduced numbers of lamellae, which often contain cytoplasm, terminate in cytoplasmic 'loops' within and around myelin sheaths, and do not completely encircle axons. In addition, membranous debris appears within neuronal and glial profiles, suggesting some degree of myelin breakdown. Thus, the protein lacks in this mutant appear not to be associated with discrete deficiencies of specific structural components but rather with a variety of quantitative changes and irregularity of form.

Complementary freeze-fracture replicas of frog peripheral nerves have revealed new details of membrane structures at the node of Ranvier and paranodal axon-Schwann cell junction. At the node both E and P fracture faces of the axolemma have high particle concentrations (approximately 1350/sq. micron and 1600/sq. micron respectively) and these particles do not overlap when tracings from the respective fracture faces are superimposed. A high proportion of the E face particles are large (> 9.5 nm) and cast long shadows while the proportion of large particles in the P face is much lower. In the paranodal region the diagonal pattern of parallel rows in the junctional axolemma always has the same orientation within a given fracture face. In the E face, the parallel rows form a positive (+ 30 degrees) angle to the groove below and in the P face, a negative (-30 degrees) angle to the ridge above. This implies that the diagonal pattern derives from asymmetric subunits that are able to associate along only one axis and are unable to 'flip over' with respect to the junctional membranes.

The mouse mutant Shiverer has been shown previously to lack myelin basic protein and other myelin proteins in both the peripheral and central nervous systems. Examination by electron microscopy shows that the peripheral nervous system, in contrast to the markedly abnormal central nervous system, is grossly normal. Myelin sheaths are of the usual thickness and exhibit normal periodic structure consisting of alternating major dense and intermediate lines. Subtle abnormalities do occur, however, consisting of increased numbers of cytoplasm-containing lamellae, aberrant terminations of myelin lamellae in internodal regions, invagination of the axon by the inner tongue of the myelin sheath, myelin debris in both axon and Schwann cells, and disruption of outer myelin lamellae. Such changes have been seen previously in various types of neuropathy and are not pathognomonic of the Shiverer mutation. Despite the absence of myelin basic protein, the peripheral manifestations of this gene are relatively minor and probably not severe enough to compromise peripheral nerve function significantly.

We have examined the postsynaptic membrane of the synaptic junctions of frog cerebellar mossy fibers by electron microscopy of freeze-fracture replicas and thin sections. The intramembranous particles (imps) in the E fracture face of the postsynaptic membrane are approximately 10 nm in size and form conspicuous aggregates which we classified as macular, annular, or anastomotic in form, according to the occurrence and placement of imp-free 'windows' within the aggregate. The size and shape of the aggregates appear related in that the area of macular aggregates is consistently smaller than the area of annular or anastomotic aggregates. Measurements of aggregate area range from 0.06 to 0.75 micrometer2. The variable size and shape of the imp aggregate in the postsynaptic membrane sets it apart from other excitatory synapses in the central nervous system, where macular aggregates are usually described. Examination of serial thin sections suggests that the shape of the postsynaptic density is equivalent to that of the imp aggregate observed in the postsynaptic membrane by freeze-fracture. This supports the notion that the region of postsynaptic membrane associated with the postsynaptic density in thin sections corresponds to the particle-rich regions of E face membrane observed in freeze-fracture replicas.

'Amyelinated' axons in the spinal roots of dystrophic mouse nerves lack typical nodal and paranodal membrane specializations. However, at the periphery of the amyelinated bundles some of the naked axons form aberrant junctions with Schwann cells belonging to neighbouring myelinated axons. These junctions are characterized by a narrow intercellular cleft containing regularly-spaced densities that closely resemble the 'transverse bands' found at paranodal axoglial junctions with respect to both configuration and spacing. In addition, the Schwann cells sometimes extend fingerlike projections towards amyelinated axons in regions where the axolemma has a dense cytoplasmic undercoating. Such regions resemble nodes of Ranvier, where Schwann cell processes interlace over the axolemma. Freeze-fracture replicas show no typical nodal or paranodal membrane specializations in the amyelinated fibres where they are apposed to each other. However, isolated paracrystalline patches of membrane occur corresponding to the aberrant junctions between amyelinated axons and Schwann cells at the periphery of the bundles. The observations show that structural differentiation of the axolemma occurs only where axons are in intimate contact with myelinating cells and does not develop independently in the amyelinated regions. Sodium channels, which are normally concentrated in the specialized nodal membrane, are, therefore, probably distributed uniformly along the amyelinated axon segments that show no sign of such regional differentiation. In addition, it is shown that Schwann cells are capable of forming specialized junctions with more than one axon at the same time.

Previous freeze-fracture studies of central myelinated nerve fibres have demonstrated a distinctive junction in the paranodal region formed between the terminal loops of the glial cell and the axolemma. This unique junction is characterized by the presence of diagonally oriented rows of particles in the P face and to a lesser extent in the E face of the glial cell and an equivalent pattern in the axolemma. In both, the rows are spaced at 250--300 A intervals. Although this junction was originally thought to be peculiar to the paranodal region, examples of the same pattern have now been seen in extraparanodal regions in the central nervous system where they appear as circumscribed patches of membrane exhibiting a pattern identical to that in the paranodal glial loops. All examples found were in the immediate vicinity of myelinated nerve fibres and in one case the membrane containing the specialized patch was identified as a lamella of a myelin sheath. These observations constitute evidence that this distinctive membrane specialization is not limited to the paranodal axoglial junction but can also be found in glial membrane specialization is not limited to the paranodal axoglial junction but can also be found in glial membranes not in immediate contact with the specialized membrane of the paranodal axolemma.