Aberrant optic axons in the retinal pigment epithelium during chick and quail visual pathway development

Examination of a large number of retinal pigment epithelia revealed that, in a small proportion, optic axons in chick and quail eyes aberrantly entered the pigment cell layer between embryonic day (E) 7 to E14. The aberrant retinal axons originated from the main stream of retinal fibers in the optic nerve and invaded the pigment layer from various positions of the optic nerve head or fissure by growing along the basal side of the pigment epithelium. The axon bundles grew several millimeters into the epithelial sheet and arborized at the margin of the eye. As shown by electron microscopy the nerve fibers occurred as bundles of three to several hundred axons. They always were located at the basal side of the epithelium, and were enveloped by processes of epithelial cells. Very large bundles of axons, however, displaced the epithelial cells from the basal matrix. These retinal axons contacted the pigment epithelial basal lamina. The basal extracellular matrix from the retinal pigment epithelium was isolated and used as substratum for in vitro cultures of various types of neural explants. The matrix preparations consisted of a sheet of a 50 nm thick basal lamina with a central lamina densa, two laminae rarae, and a 15 micron thick stroma. Axons from avian retina explants, as well as sensory ganglia, grew on the basal lamina side of the pigment cell matrix with the same growth rate and with the same fiber density as on similarly prepared basal laminae from the neural retina. These experiments show that the matrix from the pigment epithelium of the avian eye does not have negative effects on axonal growth and indicate that a basal lamina from a normally non-innervated tissue can provide a favorable matrix for axonal growth.     

Arrested development of myelin in chick optic tectum following deaferrentation

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The levels of both an enzyme associated with myelin (2′,3′-cyclic nucleotide 3′-phosphohydrolase) and of a lipid constituent of myelin (galactocerebrosides) rise markedly in the chick optic lobe during the maturation of the newly hatched bird.     

Cross-species collapse activity of polarized radial glia on retinal ganglion cell axons

Inhibition of incorrect axonal outgrowth has been shown to be a crucial guidance mechanism during the development of the nervous system. Within the visual system of chick and rat, extension of retinal ganglion cell axons is essentially restricted to distinct layers of the retina and distinct brain regions such as the tectum opticum. In addition, populations of ganglion cells from defined retina locations project topographically to defined tectal areas, their growth possibly being inhibited by radial glia in incorrect tectal regions. In the current study, we aimed to analyse potential inhibitory activity of retinal glia during outgrowth of ganglion cell axons of embryonic chick and rat. The response of ganglion cell axons originating from different retina locations when exposed to purified retinal radial glia cell membranes were monitored in collapse assays by time lapse video recording. The interaction of axons growing on purified glial somata or glial endfeet was analysed in outgrowth assays. Our results indicate that (1) nasal and temporal chick growth cones are equally induced to collapse by cell membranes from retinal radial glia: 75% nasal and 72% temporal. (2) The collapse inducing component of radial glia can be inactivated by defined heat treatment, reducing collapsing activity to 6% nasal and 5% temporal. (3) Rat growth cones respond in a similar way to chick radial glia. (4) Rat axons grow perfectly on endfeet but not on somata of radial glia of the chick. In summary, the data suggest that radial glia are functionally polarized with permissive endfeet and inhibitory somata based on heat-labile proteins. Glia polarization is likely to inhibit aberrant growth of ganglion cell axons into outer retina layers. However, retinal radial glia are unlikely to participate in preordering axons within the retina and therefore do not affect the topographic projection. Finally, the inhibitory function of radial glia is conserved between birds and mammals and represents possibly a fundamental mechanism for structuring the central nervous system. GLIA 25:143–153, 1999. © 1999 Wiley-Liss, Inc.     

Compartmentalization of anterogradely and retrogradely transported organelles in axons and growth cones from chick optic tectum

Previous work suggests that organelles contacting microtubules in axons are in fast transport. Here, we examine the distribution of organelles contacting microtubules in growing axons and growth cones from chick optic tectum. Five axon segments, each 10 microns long, and 4 entire growth cones were reconstructed from serial electron micrographs of quick-frozen, freeze-substituted chick optic tectum. Organelles contacting microtubules in axons are evenly distributed along all microtubules. Smaller organelles, presumably in anterograde transport, are enclosed in fascicles of microtubules, while larger organelles in retrograde transport lie outside the fascicles. In contrast, organelles contacting microtubules are prevalent only in the most proximal parts of the growth cone, before the microtubule fascicles splay out more distally. The distance between noncontacting organelles and microtubules also becomes progressively greater, reaching a maximum in the mid- and more distal region of the growth cone. Contacts with microtubules of both the smaller, presumably anterogradely transported organelles, as well as the larger, presumably retrogradely transported organelles, abruptly become less frequent in the proximal midregion of the growth cone. It is therefore of possible significance in stopping and starting microtubule-based organelle transport that microtubules change from a straight to an undulating configuration in the midregion of the growth cone. The decrease in organelle binding to microtubules at the demarcations between the straight and undulating microtubule segments may depend on proteins or other local factors as well as the splaying out of the microtubule bundles.     

Outgrowth and directional specificity of fibers from embryonic retinal transplants in the chick optic tectum

Retinal pieces taken from known positions of 6-day chick embryos were vitally labeled with the fluorescent dye Rhodamine-B-isothiocyanate (RITC). They were then transferred onto the surfaces of optic tecta following early bilateral removal of the embryo's optic vesicles. One to 5 days after transplantation the tecta were fixed and transplants that issued fibers were examined on tectal whole-mounts or were sectioned and viewed with a fluorescence microscope. Retinal fibers growing out from transplants on day E6 tecta showed a capacity for changing their initial outgrowth directions and for reorienting themselves towards their specific retinotopic projection area. Frequently, changes in growth direction appeared in a right-angled pattern. The capacity for turning was strongest for fibers of nasal retinal origin, less strong for fibers of temporal origin, and occurred rarely but unquantifiably in the case of fibers of ventral retinal origin. Fibers of all investigated retinal quadrants were found to reach their corresponding projection areas and to arborize there, that is, fibers of nasal retinal transplants in the posterior tectum, of temporal transplants in the anterior tectum, and of ventral transplants in the dorsal tectum. Furthermore, once in their target region, the fibers left the outer layer of the tectum and turned, again in right angles, to invade deeper layers. Capacity of fibers to turn towards their projection area was not observed for fibers issued from transplants placed on the tectum later than day E8. We suggest that there is a specific guidance of retinal axons on the tectum.     

Human fetal optic nerve: overproduction and elimination of retinal axons during development

We have estimated the number of axons in the optic nerves of human fetuses ranging in gestational age from approximately 10 to 33 weeks. At 10-12 weeks of gestation there were an estimated 1.9 million axons in the optic nerve. A peak count of 3.7 million axons was obtained from a specimen of 16-17 weeks gestation. The estimated number of axons then declined, stabilizing at an estimated 1.1 million axons by about week 29 of gestation. This figure is in close agreement with an estimate of 1.1-1.3 million optic axons in the human adult optic nerve. The results indicate that at least 70% of optic axons generated during development of the primary visual pathway are lost during fetal life. Part of this loss probably occurs as a result of the refinement of the terminal distribution of ganglion cell projections within their target nuclei. The significance of the relatively prolonged period of axonal loss is discussed.     

Cell death in the developing chick optic tectum

The degeneration and death of early developing neuroblasts in the optic tectum of chick embryo during normal development have been investigated by electron microscopy.     

Growth pattern of axons in the optic nerve of chick during myelogenesis

The purpose of this experiment was to study the diameter of axons at the time of the initiation of myelin and the pattern of growth of axons in the optic nerve of the chick. Embryos between 15 and 20 days and chicks 3, 5, 22 and 60 days of age were studied on the electron microscopic level. Based on axon diameter a unimodal distribution of unmyelinated axons is present through day 20 of incubation with a mean of approximately 0.35 micrometer. This population is represented through 22 days of age but from day 3 on, a second distinct population of unmyelinated axons is present which has a mean diameter that is approximately twice that of the smaller unmyelinated axons. All axons do not increase simultaneously in diameter but once growth starts, the unmyelinated axons apparently double in diameter at a relatively rapid rate prior to myelination. On incubation day 17 less than 1% of the axons in the optic nerve is myelimated. The number of axons in this group and their diameter (mean approximately 1.2 micrometer) remain relatively constant through day 3 but from days 5 through 22, two distinct populations of myelinated axons are present. By day 60, three distinct distributions of myelinated axons are present with mean diameters of 0.51 micrometer, 1.76 micrometer, and 3.90 micrometer. These populations represent approximately 20%, 67%, and 13% respectively of the total fiber population. As age increases the diameter of some myelinated axons is as small as or smaller than the unmyelinated axons at an earlier period in development. This suggests that factors other than axon diameter might be involved in the start of myelination. It appears that the increase in axon diameter does not occur in a continuous manner but in a saltatory manner from one size to another.     

Specific routing of retinal ganglion cell axons at the mammalian optic chiasm during embryonic development

During development of the mammalian CNS, axons encounter multiple pathway choices on their way to central target structures. A major pathway branch point in the visual system occurs at the optic chiasm, where retinal ganglion cell axons may either enter the ipsilateral or the contralateral optic tract. To investigate whether embryonic mouse retinal ganglion cell axons, upon reaching the optic chiasm, selectively grow into the correct pathway, developing retinal ganglion cells were retrogradely labeled using either 1,1'-dioctadecyl- 3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) or fluorescent microspheres placed into the optic tract on one side. The distribution of ipsilaterally and contralaterally projecting ganglion cells in the embryo was then examined and compared to that of the adult animal. Results show that axon routing at the chiasm is already extremely adult-like as early as embryonic day 15 (E15), shortly after retinal axons arrive at the chiasm. [Retinal ganglion cell neurogenesis = E11-E18 (Drager, 1985); birth = E21.] Throughout the development of this pathway, routing errors are infrequent and are on the order of only about 3-8/1000 retinal ganglion cells. Thus, embryonic retinal ganglion cell axons do not project randomly at the optic chiasm but instead appear to be highly specific in their choice of pathway. To learn how correct pathway choices are made, retinal axons were retrogradely labeled with Dil and their trajectories at the optic chiasm were reconstructed. Results show that ipsilaterally and contralaterally projecting axons are highly intermixed as they enter the chiasm region but selectively grow into the correct pathway. For example, a contralaterally projecting axon near the entrance of the ipsilateral optic tract will turn and bypass this pathway and grow towards the midline to head into the contralateral optic tract. Similarly, axons far away from the ipsilateral optic tract frequently turn abruptly at right angles to enter the ipsilateral tract, directly crossing over contralaterally projecting axons heading to the opposite side. The sorting out of intermixed ipsilaterally and contralaterally projecting retinal axons into the appropriate optic tracts strongly suggests the presence of specific guidance cues at the optic chiasm during embryonic development. Together, results from this study demonstrate that the pattern of axon projection at the adult mammalian optic chiasm is gradually built upon a highly specific pattern of axon routing laid down early during development.     

Expression of the AMPA-type glutamate receptor subunits in the chick optic tectum changes biphasically after retinal deafferentation

Effects of retinal lesions on the expression of AMPA-type glutamate receptor (GluR) subunits in the chick optic tectum were evaluated with immunohistochemistry and immunoblotting. Expression of GluR1 and GluR2/3 subunits decreased in the deafferented tectum after 2 days and increased after 7 days postlesion. These results suggest biphasic effects of retinal lesions upon the expression of GluR subunits, possibly due to removal of the glutamatergic input from the retina.     

Functional glutamate transport in rodent optic nerve axons and glia

Recent findings suggest that synaptic-type glutamate signaling operates between axons and their supporting glial cells. Glutamate reuptake will be a necessary component of such a system. Evidence for glutamate-mediated damage of oligodendroglia somata and processes in white matter suggests that glutamate regulation in white matter structures is also of clinical importance. The expression of glutamate transporters was examined in postnatal Day 14-17 (P14-17) mouse and in mature mouse and rat optic nerve using immuno-histochemistry and immuno-electron microscopy. EAAC1 was the major glutamate transporter detected in oligodendroglia cell membranes in both developing and mature optic nerve, while GLT-1 was the most heavily expressed transporter in the membranes of astrocytes. Both EAAC1 and GLAST were also seen in adult astrocytes, but there was little membrane expression of either at P14-17. GLAST, EAAC1, and GLT-1 were expressed in P14-17 axons with marked GLT-1 expression in the axolemma, while in mature axons EAAC1 was abundant at the node of Ranvier. Functional glutamate transport was probed in P14-17 mouse optic nerve revealing Na+-dependent, TBOA-blockable uptake of D-aspartate in astrocytes, axons, and oligodendrocytes. The data show that in addition to oligodendroglia and astrocytes, axons represent a potential source for extracellular glutamate in white matter during ischaemic conditions, and have the capacity for Na(+)-dependent glutamate uptake. The findings support the possibility of functional synaptic-type glutamate release from central axons, an event that will require axonal glutamate reuptake.     

Microscopical and ultrastructural investigations on the development of the blood-brain barrier in the chick embryo optic tectum

Summary The formation of a blood-brain barrier to horseradish peroxidase was microscopically and ultrastructurally investigated in the tectum opticum of the chick during development of the intraneural blood vessel network from the 6th incubation day to hatching, and in adult specimens.Extravasation of the circulating marker, apparently unimpeded during early stages of vasculogenesis, starts to diminish from the 14th incubation day (i.d.) and is prevented after the 18th i.d. The tracer seems to get out of the vessel lumina through the sites of reciprocal contact between adjacent endothelial cells, and the differentiation of tight junctions there hinders the passage of peroxidase particles. The formation of numerous endothelial vacuoles during early vasculogenesis and the setting of the blood-brain barrier are discussed in connection with the mechanisms of transendothelial transport, and respectively, the processes of moulding of the growing endothelia.     

Wnt signaling regulates proliferation and differentiation of radial glia in regenerative processes after stab injury in the optic tectum of adult zebrafish

Zebrafish have superior abilities to generate new neurons in the adult brain and to regenerate brain tissue after brain injury compared with mammals. There exist two types of neural stem cells (NSCs): neuroepithelial‐like stem cells (NE) and radial glia (RG) in the optic tectum. We established an optic tectum stab injury model to analyze the function of NSCs in the regenerative condition and confirmed that the injury induced the proliferation of RG, but not NE and that the proliferated RG differentiated into new neurons after the injury. We then analyzed the involvement of Wnt signaling after the injury, using a Wnt reporter line in which canonical Wnt signaling activation induced GFP expression and confirmed that GFP expression was induced specifically in RG after the injury. We also analyzed the expression level of genes related to Wnt signaling, and confirmed that endogenous Wnt antagonist dkk1b expression was significantly decreased after the injury. We observed that Wnt signal inhibitor IWR1 treatment suppressed the proliferation and differentiation of RG after the injury, suggesting that up‐regulation of Wnt signaling in RG after the stab injury was required for optic tectum regeneration. We also confirmed that Wnt activation by treatment with GSK3β inhibitor BIO in uninjured zebrafish induced proliferation of RG in the optic tectum. This optic tectum stab injury model is useful for the study of the molecular mechanisms of brain regeneration and analysis of the RG functions in physiological and regenerative conditions.     

Late developmental expression of DARPP-32 in the chick optic tectum

Immunohistochemical techniques reveal that the dopamine- and cAMP-regulated phosphoprotein DARPP-32 is detectable in neurons of the chick optic tectum starting on embryonic day 13. The expression levels then increase steadily from embryonic day 15 through the first posthatching day. After 15 days posthatching, expression of DARPP-32 reaches the adult pattern, with many labeled cells in tectal layers 11 and 12. These cells exhibit a bipolar shape, with long processes directed both to the deep and superficial layers. These results suggest that DARPP-32 is present in specific neuronal populations of the chick tectum and that this protein may not have a function in early ontogenetic processes.     

Light microscopic study of degenerating cobalt-filled optic axons in goldfish: role of microglia and radial glia in debris removal

Labeling severed axons with cobaltous-lysine ultimately leads to the degeneration of their distal segments. The present study was designed to determine whether microglia and radial glia have comparable roles in the elimination of degenerating axons. Another purpose was to determine whether the cobalt could escape from degenerating axons and enter intact neuronal cells. Optic axons were filled with cobaltous-lysine for 1 day and the retinal projections were examined from 1 to 106 days later. Optimal filling was obtained 1 day postlabeling. The number of filled axons in the optic tract was significantly reduced at the 2-day time point, indicating that many axons had disintegrated. Many axons contained large swellings that resembled cells. However, transneuronally labeled neuronal cells were never observed. Labeled, rounded microglia appeared among the degenerating axons at the 3-day time point, and the microglia changed shape at 5 days. They became elongated and manifested many processes. In addition, the microglia began to move toward, and entered, the ventricles and vasculature. Virtually all the labeled debris was removed between 17 and 28 days following the application of cobalt. The rapidity with which the axons were removed suggests that the cobalt accelerates the degenerative process either directly, or indirectly by accelerating the arrival of phagocytic cells. Radial glia appeared to play a smaller role in debris elimination. They took up labeled debris to a lesser degree than microglia and were briefly labeled. Interestingly, radial glia did not take up cobalt when it was injected intracranially and diffused through the brain. A previous claim of an axosomatic retinotectal projection to cells deep in the stratum periventriculare of a teleost fish is reinterpreted to represent cobalt within radial glia.     

Guidance of callosal axons by radial glia in the developing cerebral cortex

During development, columns of the mammalian cerebral cortex are formed by migration of neurons along fascicles of radial glia. Subsequently, axons of the corpus callosum connect reciprocal regions of each cerebral hemisphere. To determine whether the radial growth of callosal afferents through the developing cortex may be guided by particular cellular elements, we examined the ultrastructural relationship between callosal afferents and radial fibers in the early postnatal hamster sensorimotor cortex. Developing callosal axons and their growth cones were labeled with HRP injected into the cortex at 3 d postnatal when the growth cones have extended across the callosum and are just entering the contralateral cortex. An EM analysis of 30 HRP-labeled axons and their growth cones revealed that they extended upon fascicles of radial processes associated with migrating neurons. Reconstruction of seven of these growth cones, serially sectioned in their entirety, showed that growth cones were associated with the same radial fascicle as their axon. Growth cones also touched other cellular elements such as axons. However, the finding that callosal afferents, from the point at which they enter the cortex to their growth cones, were apposed to a continuous fascicle of radial fibers suggests that callosal axons are tracking along radial processes. We conclude that the majority of the radial processes within fascicles are likely to be glial, based on their relatively large diameters, electron-lucent cytoplasm with a regular array of microtubules, the presence of glycogen granules, occasional cytoplasmic protrusions lacking microtubules, and their consistent association with migrating neurons. We propose therefore that radial glia may serve a guidance function for growing callosal axons in their radial trajectory through the developing cerebral cortex.