Peculiar and typical oligodendrocytes are involved in an uneven myelination pattern during the ontogeny of the lizard visual pathway

We studied the myelination of the visual pathway during the ontogeny of the lizard Gallotia galloti using immunohistochemical methods to stain the myelin basic protein (MBP) and proteolipid protein (PLP/DM20), and electron microscopy. The staining pattern for the PLP/DM20 and MBP overlapped during the lizard ontogeny and was first observed at E39 in cell bodies and fibers located in the temporal optic nerve, optic chiasm, middle optic tract, and in the stratum album centrale of the optic tectum (OT). The expression of these proteins extended to the nerve fiber layer (NFL) of the temporal retina and to the outer strata of the OT at E40. From hatching onwards, the labeling became stronger and extended to the entire visual pathway. Our ultrastructural data in postnatal and adult animals revealed the presence of both myelinated and unmyelinated retinal ganglion cell axons in all visual areas, with a tendency for the larger axons to show the thicker myelin sheaths. Moreover, two kinds of oligodendrocytes were described: peculiar oligodendrocytes displaying loose myelin sheaths were only observed in the NFL, whereas typical medium electron-dense oligodendrocytes displaying compact myelin sheaths were observed in the rest of the visual areas. The weakest expression of the PLP/DM20 in the NFL of the retina appears to be linked to the loose appearance of its myelin sheaths. We conclude that typical and peculiar oligodendrocytes are involved in an uneven myelination process, which follows a temporo-nasal and rostro-caudal gradient in the retina and ON, and a ventro-dorsal gradient in the OT.     

The astrocytes in the retina and optic nerve head of mammals: A special glia for the ganglion cell axons

Summary The neuroglia in the retina and the intraocular portion of the optic nerve of the monkey and cat has been examined by light and electron microscopy. In the retina two types of macroglial cells can be distinguished: 1) Müller cells, and 2) astrocytes. The bipolar radial glial cells of Müller penetrate the entire thickness of the retina and their basal processes align in the nerve fibre layer to form septa that fasciculate the axons of the ganglion cells. In contrast to the Müller cells, the retinal astrocytes are not homogeneously distributed throughout the retina; their number correlates with the thickness of the nerve fibre layer. The processes of the astrocytes are confined to the ganglion cell layer and to the nerve fibre layer. In the latter, the astrocytic processes run parallel to and between the axons of a given nerve fibre bundle. According to cytological criteria, the retinal astrocytes are protoplasmic. In the intraocular portion of the optic nerve, however, the astrocytes are fibrous and their processes run perpendicular to the axon bundles of the prelaminar portion of the optic nerve. Thus, because of their intimate morphological relationship to axons of the nerve fibre layer and the intraocular portion of the optic nerve, the astrocytes in the eye of the monkey and the cat may be considered as a special glia for the axons of ganglion cells.     

Distribution of neurotrophin-3 during the ontogeny and regeneration of the lizard (Gallotia galloti) visual system

We have previously described the spontaneous regeneration of retinal ganglion cell axons after optic nerve (ON) transection in the adult Gallotia galloti. As neurotrophin-3 (NT-3) is involved in neuronal differentiation, survival and synaptic plasticity, we performed a comparative immunohistochemical study of NT-3 during the ontogeny and regeneration (after 0.5, 1, 3, 6, 9, and 12 months postlesion) of the lizard visual system to reveal its distribution and changes during these events. For characterization of NT-3(+) cells, we performed double labelings using the neuronal markers HuC-D, Pax6 and parvalbumin (Parv), the microglial marker tomato lectin or Lycopersicon esculentum agglutinin (LEA), and the astroglial markers vimentin (Vim) and glial fibrillary acidic protein (GFAP). Subpopulations of retinal and tectal neurons were NT-3(+) from early embryonic stages to adulthood. Nerve fibers within the retinal nerve fiber layer, both plexiform layers and the retinorecipient layers in the optic tectum (OT) were also stained. In addition, NT-3(+)/GFAP(+) and NT-3(+)/Vim(+) astrocytes were detected in the ON, chiasm and optic tract in postnatal and adult lizards. At 1 month postlesion, abundant NT-3(+)/GFAP(+) astrocytes and NT-3(-)/LEA(+) microglia/macrophages were stained in the lesioned ON, whereas NT-3 became downregulated in the experimental retina and OT. Interestingly, at 9 and 12 months postlesion, the staining in the experimental retina resembled that in control animals, whereas bundles of putative regrown fibers showed a disorganized staining pattern in the OT. Altogether, we demonstrate that NT-3 is widely distributed in the lizard visual system and its changes after ON transection might be permissive for the successful axonal regrowth.     

Neuroglia of the adult rat optic nerve in the course of wallerian degeneration

The neurological reactions in Wallerian degeneration have been studied by electron microscopy in the optic nerve of adult albino rats from 7 to 120 days after unilateral enucleation. Reactive astrocytes contained abundant dense bodies, numerous microtubules and hyperplastic glial filaments. These astrocytes also assisted phagocytosis of degenerated myelin sheaths and in glial scar formation. Oligodendrocytes disconnected their cytoplasmic extensions, which were phagocytosed by microglial cells and astrocytes, by increased production of lysosomes. Microglial cells consisted of crinkled, long, rough endoplasmic reticula, several highly-active Golgi complexes, laminar inclusions and globoid lipid droplets. Microglia engulfed and lysed the disintegrated axons and myelin sheaths.     

Accumulation of lipid inclusions in astrocytes of aging human optic nerve

We examined age-related changes in the human optic nerve (ON) from 10 postmortem donor eye samples (age: 21- to 94-year-old). In aged ON, many axons showed paucity of cytoskeleton, and possessed disorganized myelin that remained in the extracellular space. Lipid inclusions were detected in glia, as stained by oil red O, and these accumulated with aging. To identify and confirm which glial cell type possessed lipid inclusions, we performed immunohistochemistry (IHC) and transmission electron microscopy (TEM). Comparisons were made from TEM features and size of the glia immunolabeled with glial fibrillary acidic protein and glutamine synthetase (markers for astrocytes) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (a marker for oligodendrocytes). It was found that lipid inclusions were restricted to the astrocytes having larger perikarya than the oligodendrocytes (IHC) and possessing filaments in cytoplasm (TEM). These astrocytes also possessed myelin debris and it is thus likely that those inclusions originated from degenerated myelin of the ON axons. These data indicate that astrocytes play a role in phagocytosis and clearance of disorganized myelin in aging human ON.     

Activation of epidermal growth factor receptors directs astrocytes to organize in a network surrounding axons in the developing rat optic nerve

In postnatal developing optic nerves, astrocytes organize their processes in a cribriform network to group axons into bundles. In neonatal rat optic nerves in vivo, the active form of EGFR tyrosine kinase is abundantly present when the organization of astrocytes and axons is most actively occurring. Blocking activity of EGFR tyrosine kinase during the development of rat optic nerves in vivo inhibits astrocytes from extending fine processes to surround axons. In vitro, postnatal optic nerve astrocytes, stimulated by EGF, organize into cribriform structures which look remarkably like the in vivo structure of astrocytes in the optic nerve. In addition, when astrocytes are co-cultured with neonatal rat retinal explants in the presence of EGF, astrocytes that are adjacent to the retinal explants, re-organize to an astrocyte-free zone into which neurites grow out from the retinal tissue. We hypothesize that in the developing optic nerve, EGFR activity directs the formation of a histo-architectural structure of astrocytes which surrounds axons and provides a permissive environment for axon development.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Enriched Environment Protects the Optic Nerve from Early Diabetes-Induced Damage in Adult Rats

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Axoglial alterations of the distal (close to the chiasm) optic nerve (ON) could be the first structural change of the visual pathway in streptozotocin (STZ)-induced diabetes in rats. We analyzed the effect of environmental enrichment on axoglial alterations of the ON provoked by experimental diabetes. For this purpose, three days after vehicle or STZ injection, animals were housed in enriched environment (EE) or remained in a standard environment (SE) for 6 weeks. Anterograde transport, retinal morphology, optic nerve axons (toluidine blue staining and phosphorylated neurofilament heavy immunoreactivity), microglia/macrophages (ionized calcium binding adaptor molecule 1 (Iba-1) immunoreactivity), astrocyte reactivity (glial fibrillary acid protein-immunostaining), myelin (myelin basic protein immunoreactivity), ultrastructure, and brain derived neurotrophic factor (BDNF) levels were assessed in non-diabetic and diabetic animals housed in SE or EE. No differences in retinal morphology or retinal ganglion cell number were observed among groups. EE housing which did not affect the STZ-induced weight loss and hyperglycemia, prevented a decrease in the anterograde transport from the retina to the superior colliculus, ON axon number, and phosphorylated neurofilament heavy immunoreactivity. Moreover, EE housing prevented an increase in Iba-1 immunoreactivity, and astrocyte reactivity, as well as ultrastructural myelin alterations in the ON distal portion at early stages of diabetes. In addition, EE housing avoided a decrease in BDNF levels induced by experimental diabetes. These results suggest that EE induced neuroprotection in the diabetic visual pathway.     

Turnover of Axonally Transported Phospholipids in Nerve Endings of Retinal Ganglion Cells

We have investigated the metabolic turnover of axonally transported phospholipids in myelinated axons (optic tract) and nerve endings (superior colliculus) of retinal ganglion cells. One week following intraocular injection of [2-3H]glycerol, turnover rates for individual phospholipid classes in the retina (which contains a number of other cell types in addition to the ganglion cells) were all very similar to each other, with apparent half-lives of approximately 7 days. Apparent half-lives of labeled phospholipids in superior colliculus (presumably primarily in retinal ganglion cell nerve endings) were 10 days for both choline and inositol phosphoglycerides and 13 days for both serine and diacylethanolamine phosphoglycerides. Subcellular fractionation data obtained from superior colliculus at various times after injection suggested that apparent turnover rates determined for nerve ending phospholipids probably were not significantly affected by transfer of axonally transported 3H lipids into myelin. Apparent half-lives for phospholipids in optic tract were somewhat longer than in superior colliculus, ranging from 11 to 18 days. The slower turnover rates in optic tract may, in part, reflect the transfer of some axonal lipids to the more metabolically stable pool of lipids in the myelin ensheathing the retinal ganglion cell axons. In both optic tract and superior colliculus, apparent half-lives for axonally transported phospholipids labeled with [32P]phosphate were only slightly longer than for [2-3H]glycerol, while those for [14C]choline and [3H]acetate were markedly longer, indicating differing degrees of metabolic conservation or reutilization of these precursors relative to glycerol.     

Concentration of astrocytic filaments at the retinal optic nerve junction is coincident with the absence of intra-retinal myelination: comparative and developmental evidence

The structure of the lamina cribrosa (LC) and astrocytic density were examined in various species with and without intra-retinal myelination. Sections of optic nerve from various species were stained with Milligan's trichrome or antibodies to glial fibrillary acidic protein, myelin basic protein (MBP) and antibody O4. Marmoset, flying fox, cat, and sheep, which lack intraretinal myelination, were shown to possess a well-developed LC as well as a marked concentration of astrocytic filaments distal to the LC. Rat and mouse, which lack intraretinal myelination, lacked a well-developed LC but exhibited a marked concentration of astrocytic filaments in this region. Rabbit and chicken, which exhibit intraretinal myelination, lacked both a well-developed LC and a concentration of astrocytes at the retinal optic nerve junction (ROJ). A marked concentration of astrocytes at the ROJ of human fetuses was also apparent at 13 weeks of gestation, prior to myelination of the optic nerve; in contrast, the LC was not fully developed even at birth. This concentration of astrocytes was located distal to O4 and MBP immunoreactivity in human optic nerve, and coincided with the site of initial myelination of ganglion cell axons in marmoset and rat. Myelination proceeded from the chiasm towards the retinal end of the human optic nerve. Moreover, the outer limit of oligodendrocyte precursor cells (OPC) migration into the rabbit retina was restricted by the outer limit of astrocyte spread. These observations indicate that a concentration of astrocytic filaments at the ROJ is coincident with the absence of intraretinal myelination. Differential expression of tenascin-C by astrocytes at the ROJ appears to contribute to the molecular barrier to OPC migration (see Bartsch et al., 1994), while expression of the homedomain protein Vax 1 by glial cells at the optic nerve head appears to inhibit migration of retinal pigment epithelial cells into the optic nerve (see Bertuzzi et al., 1999). These observations combined with our present comparative and developmental data lead us to suggest that the astrocytes at the ROJ serve to regulate cellular traffic into and out of the retina.     

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.     

In Vitro Protein Synthesis in the Goldfish Retinotectal Pathway During Regeneration: Evidence for Specific Axonal Proteins of Retinal Origin in the Optic Nerve

Four proteins with molecular weights of 58,000 can be separated as a linear array by two-dimensional gel electrophoresis. They are highly concentrated in the goldfish optic nerve and are designated as ON1, ON2, ON3, and ON4. Proteins ON1 and ON2 are undetectable in the optic nerve after disconnection and their concentration is gradually restored during regeneration. In vitro incubations of retinas, optic nerves, or tecta in the presence of [35S]methionine indicate that proteins ON1 and ON2 are of retinal origin. The labeling rate of these proteins in the retina increases fourfold after optic nerve crush whereas the overall labeling rate in the retina remains largely constant. Their synthesis cannot be detected in tissues devoid of retinal ganglion cells. This is consistent with the view that ON1 and ON2 are synthesized by retinal ganglion cells and are consequently of neuronal origin in the optic nerve. In contrast, similar experiments indicate that ON3 and ON4 are of nonneuronal origin. They are synthesized in the optic nerve in the absence of retinal ganglion cells.     

Neuronal Intermediate Filament Expression During Neurite Outgrowth from Explanted Goldfish Retina: Effect of Retinoic Acid

Regulation of the goldfish neuronal intermediate filament proteins ON1 and ON2 was investigated in a retinal explant system. The synthesis of these proteins in explanted retina decreased with increasing time in culture, despite continuing neurite outgrowth. Thus, ON1/ON2 neurofilament expression is regulated independently from neurite outgrowth. During regeneration of the goldfish optic nerve in vivo, the expression of these proteins increased during the later phase of the process, when growing axons make contact with the optic tectum. The declining synthesis of ON1 and ON2 during neurite outgrowth in culture suggests that factors extrinsic to the retina are necessary to support synthesis of these proteins. Treating retinal explants with retinoic acid stimulated the synthesis of the ON1/ON2 proteins in a dose-dependent manner. This stimulation was effective during a period of declining synthesis of the ON1/ON2 proteins, restoring their synthesis towards initial levels of expression. These results show that retinoic acid serves as a modulator of neurofilament expression in this in vitro model of nerve regeneration.     

Astrocytes as gate-keepers in optic nerve regeneration--a mini-review

Animals that develop without extra-embryonic membranes (anamniotes--fish, amphibians) have impressive regenerative capacity, even to the extent of replacing entire limbs. In contrast, animals that develop within extra-embryonic membranes (amniotes--reptiles, birds, mammals) have limited capacity for regeneration as adults, particularly in the central nervous system (CNS). Much is known about the process of nerve development in fish and mammals and about regeneration after lesions in the CNS in fish and mammals. Because the retina of the eye and optic nerve are functionally part of the brain and are accessible in fish, frogs, and mice, optic nerve lesion and regeneration (ONR) has been extensively used as a model system for study of CNS nerve regeneration. When the optic nerve of a mouse is severed, the axons leading into the brain degenerate. Initially, the cut end of the axons on the proximal, eye-side of the injury sprout neurites which begin to grow into the lesion. Simultaneously, astrocytes of the optic nerve become activated to initiate wound repair as a first step in reestablishing the structural integrity of the optic nerve. This activation appears to initiate a cascade of molecular signals resulting in apoptotic cell death of the retinal ganglion cells axons of which make up the neural component of the optic nerve; regeneration fails and the injury is permanent. Evidence specifically implicating astrocytes comes from studies showing selective poisoning of astrocytes at the optic nerve lesion, along with activation of a gene whose product blocks apoptosis in retinal ganglion cells, creates conditions favorable to neurites sprouting from the cut proximal stump, growing through the lesion and into the distal portion of the injured nerve, eventually reaching appropriate targets in the brain. In anamniotes, astrocytes ostensibly present no such obstacle since optic nerve regeneration occurs without intervention; however, no systematic study of glial involvement has been done. In fish, vigorously growing neurites sprout from the cut axons and within a few days begin to re-enervate the brain. This review offers a new perspective on the role of glia, particularly astrocytes, as "gate-keepers;" i.e., as being permissive or inhibitory, by comparison between fish and mammals of glial function during ONR.     

Molecular cloning of gefiltin (ON1): serial expression of two new neurofilament mRNAs during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins of this pathway do not match the intermediate filament protein composition of adult higher vertebrate neurons, which lack the capacity for growth and development. Using a goldfish retina lambda gt10 library we isolated cDNA clones representing the predominant goldfish optic nerve neurofilament protein, ON1. The mRNA for this protein is abundant in retinal ganglion cells, and its level increases slowly during optic nerve regeneration. The rate of ON1 mRNA accumulation after optic nerve crush was compared with that of plasticin, a previously described novel type III neurofilament from goldfish retinal ganglion cells. Plasticin mRNA is normally expressed at low steady state levels, but accumulates dramatically and rapidly, preceding gefiltin mRNA, in response to optic nerve crush. The predicted amino acid sequence for ON1 indicates that it is a novel intermediate filament protein. We have named it gefiltin, for goldfish eye intermediate filament protein. The serial expression of plasticin and gefiltin is discussed with respect to the diversity of neurofilament proteins during neurogenesis.     

Remodeling and Sorting Process of Ethanolamine and Choline Glycerophospholipids During Their Axonal Transport in the Rabbit Optic Pathway

The existence of a mechanism by which the ester- and ether-linked aliphatic chains of the major phospholipids are retailored during their axonal transport and sorted to specific membrane systems along the optic nerve and tract was investigated. A mixture of [1-14C]hexadecanol and [3H]arachidonic acid was injected into the vitreous body of albino rabbits. At 24 h and 8 days later, the distribution (as measured by the 3H/14C ratio) and the positioning (as monitored by hydrolytic procedures) of radioactivity in the various phospholipid classes of retina, purified axons, and myelin of the optic nerve and tract were determined. At the two intervals after labeling, the 3H/14C ratios of each diradyl type of phosphatidylethanolamine and phosphatidylcholine were (a) substantially unchanged all along the axons within the optic nerve and tract and (b) markedly modified in comparison with those found in the retina and axons for molecular species selectively restricted to myelin sheath. Evidence is thus available that intraxonally moving ethanolamine and choline glycerophospholipids, among others, are added to axonal membranes most likely without extensive modifications. In contrast, they are transferred into myelin after retailoring. Through these two processes, the sorting and targeting of newly synthesized phospholipids to their correct membrane domains, such as axoplasmic organelles, axolemma, or periaxonal myelin, could be controlled.     

Expression of αB-Crystallin in the Peripapillary Glial Cells of the Developing Chick Retina

Peripapillary glial cells (PPGCs) are a peculiar macroglia in avian species, located in the central retina adjacent to the optic nerve head. PPGCs have a similar shape and orientation to Müller cells, which traverse the entire layer of the retina; however, there are differences in protein expression between the two cell types. In the present study, we first demonstrated that PPGCs expressed αB-crystallin, which is not expressed in Müller cells, during retinal development. αB-crystallin was first faintly expressed in PPGCs of the E5 retina, adjacent to the optic nerve head. Further, αB-crystallin was exclusively expressed in PPGCs up to E14. The shape of these cells was bipolar with vitread and ventricular processes. The vitread processes of αB-crystallin+ PPGCs became finer at E18. Double labeling analysis clearly demonstrated that only vimentin+ or GFAP+ astrocytes were located in the optic nerve head and were demarcated from the retina by αB-crystallin+ PPGCs. Furthermore, we determined that αB-crystallin+ PPGCs, with a number of processes, completely wrapped the optic nerve head and were densely located in the junction of the optic nerve head and the retina in a whole mount preparation and in vertical-sectioned retinae. The results of present study, together with reports that retinal astrocytes migrate from the optic nerve head, suggest that PPGCs prevent astrocytes from migrating into the retina in avian species.     

A developmental and ultrastructural study of the optic chiasma in Xenopus

The structure of the optic chiasma in Xenopus tadpoles has been investigated by light and electron microscopy. Where the optic nerve approaches the chiasma, a tongue of cells protrudes from the periventricular cell mass into the dorsal part of the nerve. Glial processes from this tongue of cells ensheath fascicles of optic axons as they enter the brain. Coincident with this partitioning, the annular arrangement of axons in the optic nerve changes to the laminar organization of the optic tract. Beyond the site of this rearrangement, all newly growing axons accumulate in the ventral-most part of the nerve and pass into the region between the periventricular cells and pia which we have called the 'bridge'. This region is characterized by a loose meshwork of glial cell processes, intercellular spaces and the presence of both optic and nonoptic axons. In the bridge, putative growth cones of retinal ganglion cell axons are found in the intercellular spaces in contact with both the glia and with other axons. The newly growing axons from each eye cross in the bridge at the midline and pass into the superficial layers of the contralateral optic tracts. As the system continues to grow, previous generations of axon, which initially crossed in the existing bridge, are displaced dorsally and caudally, forming the deeper layers of the chiasma. At their point of crossing in the deeper layers, these fascicles of axons from each eye interweave in an intimate fashion. There is no glial segregation of the older axons as they interweave within the chiasma.