Increased NCAM-180 immunoreactivity and maintenance of L1 immunoreactivity in injured optic fibers of adult mice

The injury related expression of two axon-growth promoting cell adhesion molecules (CAMs), NCAM-180 which is developmentally downregulated and L1 which is regionally restricted, were compared in optic fibers in the adult mouse. The neuron-specific isoform of NCAM (NCAM-180) is present at very low levels in unlesioned adult optic axons. At 7 days after nerve crush, immunoreactivity was strongly and uniformly increased in optic axons within the nerve and throughout retina. Reactivity in surviving axons had returned to control levels at 4 weeks. To induce regrowth of adult retinal ganglion cell axons retinal explants were placed in culture. Strong NCAM-180 staining was observed on these regenerating optic axons. The neuronal cell adhesion molecule L1 is restricted to retina and to the unmyelinated segment of the optic nerve near the optic nerve head in unlesioned adult animals. Following nerve crush, L1 immunoreactivity was retained within retina and proximal nerve and novel staining was detected in the more distal segment of the optic nerve up to the lesion site where it persisted for at least eight months. The capacity of optic fibers to show increased NCAM-180 immunoreactivity and maintain L1 expression after a lesion may explain why these fibers exhibit relatively good potential for regeneration.     

Persistence of graded EphA/Ephrin-A expression in the adult frog visual system

Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the adult or, if downregulated after metamorphosis, to be reexpressed after optic nerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic frogs, including mature adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nerve injury.     

Immunohistological localization of tenascin-c in the developing and regenerating retinotectal system of two amphibian species

The expression pattern of the extracellular matrix molecule tenascin-C was investigated in the retinotectal system of the frog Discoglossus pictus and the salamander Pleurodeles waltl during development and optic nerve regeneration in the adult. In both species, the retina was devoid of tenascin-C immunoreactivity at all ages studied. During development, tenascin-C was distributed in a gradient in the optic nerve, with the highest immunoreactivity in the eye near part of the optic nerve. The myelin-associated glycoprotein was distributed in a gradient with opposite polarity. In Discoglossus, but not Pleurodeles, tenascin-C was detected in the anterior chiasm. In the tectum of both species, tenascin-C was observed in deep cellular and fiber layers but not in the layers receiving optic fibers or proliferative zones. The distribution patterns of tenascin-C were the same during development and in the adult, except for a disappearance of the molecule from the intraocular part of the optic nerve. After lesioning the optic nerve of adult animals, tenascin-C was strongly reexpressed in the intraocular part of the optic nerve but was only weakly upregulated in the distal optic nerve stump. In contrast, a chondroitin sulfate epitope was strongly upregulated in the distal optic nerve stump. These observations suggest that during development, tenascin-C serves as an attenuating barrier for myelinating cells in the optic nerve and contributes to the guidance of growing retinal ganglion cell axons. Due to its sustained expression in the adult, tenascin-C may have similar functions during regeneration of the lesioned adult retinotectal system. © 1995 Wiley-Liss, Inc.     

Eph/ephrin gradients in the retinotectal system of Rana pipiens: Developmental and adult expression patterns

Eph/ephrin‐receptor/ligand A and B families play a variety of roles during CNS development, including patterning the retinotectal projection. However, the alignment of their expression gradients with developing retinotectal maps and gradients of cellular development is not well understood in species whose midbrain tecta undergo a protracted anterior to posterior development. By using anatomical tracing methods and ³H‐thymidine neuronography, we have mapped the retinotectal projection and the spatiotemporal progression of tectal cellular development onto Eph/ephrin expression patterns in the tectum of larval Rana pipiens, as studied by means of in situ affinity analysis with fusion proteins. EphA expression is maximal in anterior tectum (and temporal retina); ephrin‐A expression is maximal at the posterior pole (and nasal retina). EphB expression is graded in the early larva, where it is maximal in the posterior tectum just anterior to the posterior pole (and in the ventral retina). Tectal EphB expression becomes uniform at later stages and remains so in the adult, although its retinal expression remains maximal ventrally. In the early larva, EphA, EphB, and ephrin‐A protein gradients are parallel to each other and align with the temporonasal axis of the retinal projection. The early EphB expression maximum overlaps the boundary between the mantle layer of newly postmitotic cells and the posterior, epithelial region of cell proliferation, suggesting that the expression maximum is associated with the initial migrations of the postmitotic cells. Ephrin‐B expression was detected in the olfactory bulb and dorsal retina at all ages, but not in the tectum. J. Comp. Neurol. 514:30–48, 2009. © 2009 Wiley‐Liss, Inc.     

Retinal distribution of ganglion cells which project to the ipsilateral optic tectum in Bufo matinus

The retinotectal projection in anura is mainly crossed, although a small proportion of optic axons projects to the ipsilateral tectum. Using the fluorescent carbocyanide dye, DiI, we mapped the retinal topography of ganglion cells which project to the ipsilateral tectum in adult Bufo marinus. DiI was injected into particular locations in the right tectum. After 10 days survival both the right and the left retinas were wholemounted and the number and retinal position of retrogradely filled ganglion cells were determined. The contralateral and ipsilateral cells were visuotopically distributed in the retina in the majority of experiments. However, in two cases cells were located in visuotopically disparate parts of the retina. The ipsilateral cells represented 3.7% of contralaterally projecting cells in the temporal retina. 0.1% in the nasal and dorsal retina and 0.6% of the ventral retina. The density of ipsilaterally projecting ganglion cells varied from a top of 25 cells/mm² in the temporal retina, 9 cells/mm² in the nasal, 3 cells/mm² in the dorsal to 11 cells/mm² in the ventral retina. The diversity of size and shape of retrogradely filled ganglion cells indicated that the ipsilateral population corresponded to a heterogeneous class of ganglion cell types. The functional significance of the direct ipsilateral retinotectal projection of the anuran visual system has yet to be elucidated. However, in light of the involvement of the indirect ipsilateral retinotectal projection in binocular vision, the direct pathway is likely to be associated with a retino-tecto-spinal circuit subserving postural adjustment to visually derived stimulation.     

Neural cell adhesion molecule and its polysialic acid moiety exhibit opposing and linked effects on neuropathic hyperalgesia

Spinal lamina II, where nociceptive C-fibers terminate, expresses high amounts of the polysialylated form of neural cell adhesion molecule (PSA-NCAM). While enzymatic removal of the PSA moiety from NCAM did not affect normal sensitivity to thermal stimuli, it exacerbated nerve injury-induced neuropathic hyperalgesia. The genetic removal of the NCAM core protein also did not alter thermal sensitivity. However in the presence of a peripheral nerve injury, NCAM-null mutants exhibited a complete suppression of thermal hyperalgesia. This strong NCAM mutant phenotype appears to involve the long form of NCAM's cytoplasmic domain, in that it is duplicated by selective genetic deletion of the NCAM-180 isoform. PSA appears therefore to provide a mechanism for modulation of chronic sensory overload, by means of attenuation of the activity of the NCAM-180 isoform, which reduces nociceptive transmission.     

Visual projections to the optic tecta in Xenopus after partial extirpation of the embryonic eye

The temporal, nasal or dorsal half (40–60%) of the eye primordium was excised in Xenopus frog embryos stage 25/26 (optic vesicle), 31/32 (optic cup) or 38 ± 1 (early larval eye). The residual nasal (NF) or temporal (TF) or ventral (VF) fragment gradually rounded-up to form a grossly normal eye of normal or reduced size. Electrophysiologic analysis of the visuotectal projection after metamorphosis showed that (independent of stage of surgery and final eye size) most eyes of each type generated normal retinotectal maps (orderly and continuous retinotopic projection across the entire tectum). A minority of NF eyes generated double-nasal compound maps, in which nasal and temporal halves of the adult retina projected as mirror images (of the nasal pattern) over the whole tectum. A minority of TF eyes generated double-temporal compound maps, while a minority of VF eyes generated double-ventral compound maps. Respectively, these compound maps simulated those generated by NN, TT or VV “compound-eyes” prepared by fusing two nasal half eyes, or two temporal half eyes, or two ventral half eyes. The incidence of double-maps was lowest in TF eyes at all stages, and after stage 25/26 surgery in all eye types. Lure tests and post-synaptic recording confirmed that these eyes formed functional retinotectal synapses, and that the (presynaptic) map accurately mirrored the connectivity pattern. The results are considered in terms of (1) the patterning of locus specificities in the retinal ganglion cell population, (2) embryonic “regulation” and “reduplication” in non-neural systems, and (3) alternative mechanisms by which partial extirpation might modify the deployment or expression of ganglion cell locus specificities to produce the retinotectal connectivity patterns we observed.     

Development and maintenance of connectivity in the visual system of the frog. II. The effects of eye removal

The interdependence of retinotectal and intertectal connections in the ontogeny of the visual system of the frog (Rana pipiens) was studied. To remove the direct afferent input from retina to one side of the tectum one eye was taken out at different embryonic and larval stages of development. Animals were allowed to pass through metamorphosis, at which time intertectal connections linking the visual input to the two sides of the tectum normally develop. Responses to visual stimulation were then recorded with extracellular microelectrodes from both sides of the tectum.In all animals eye removal resulted in size reduction of the tectum on the side of the remaining eye. Eye removal prior to the onset of metamorphosis (stage XIX) resulted in gross abnormalities of visually evoked responses in the tectum ipsilateral to the remaining eye; receptive fields were much larger than normal, localization was poor, and responses fatigued rapidly upon repeated stimulation. Eye removal after the onset of metamorphosis, on the other hand, did not result in any detectable abnormalities of the ipsilateral visual responses; receptive fields were small and well localized. This normal ipsilateral visuotectal projection persisted indefinitely (up to 273 days) in the absence of one eye.We conclude that there is a Necessary interaction between the developing intertectal pathways and the afferent fibers linking retina and tectum. This interaction appears to be completed during metamorphosis. The data presented in this and the preceding paper¹² argue against any subsequent interactions between the retinotectal and intertectal connections. Furthermore, the two papers together suggest that the interactions taking place during metamorphosis are the result of intrinsic developmental mechanisms and are not altered by sensory stimulation.     

The lens has a specific influence on optic nerve and tectum development in the blind cavefish Astyanax

We used the teleost Astyanaxmexicanus to examine the role of the lens in optic nerve and tectum development. This speciesis unusually suited for studies of nervous system development and evolution because of its two extant forms: an eyed surface dwelling (surface fish) and several blind cave dwelling (cavefish) forms. Cavefish embryos initially form eye primordia, but the lens eventually dies by apoptosis, then the retina ceases to grow, and finally the degenerating eyes sink into the orbits. Transplantation of an embryonic surface fish lens into a cavefish optic cup restores eye development. We show here that retinal nerve fibers are formed and project to the optic tectum in cavefish embryos. In adult cavefish that have completed lens degeneration, however, the number of retinal axons in the optic nerve is substantially reduced compared to surface fish. The presumptive brain domains of embryonic cavefish are not altered relative to surface fish based on expression of the regional marker genes Pax6, Pax2.1, and engrailed2. In contrast, the adult cavefish brain is elongated, the optic tectum is diminished in volume, and the number of tectal neurons is reduced relative to surface fish. Unilateral transplantation of an embryonic surface fish lens into a cavefish optic cup increases the size of the optic nerve, the number of retinotectal projections from the restored eye, and the volume and neuronal content of the contralateral optic tectum. The results suggest that the lens has a specific influence on optic nerve and tectum development during eye growth in Astyanax.     

The development of NADPH-diaphorase and nitric oxide synthase in the visual system of the cichlid fish, Tilapia mariae

The pattern of NADPH-diaphorase expression was studied in the retina and optic tectum of the cichlid fish Tilapia mariae during the first developmental stages. NADPH-diaphorase activity was seen early, at hatching. In the retina a few cell bodies of the retinal inner nuclear layer showed a faint labeling. Scattered labeled cells were found in the stratum periventriculare of the optic tectum, while the optic nerve was unlabeled. Two days after hatching, the number of labeled neurons increased in the inner nuclear layer and a few stained cell bodies were also scattered in the ganglion cell layer. Both the inner and outer plexiform layers showed a diffuse staining and the optic nerve was devoid of labeling. In the optic tectum several positive cells in the periventricular layer, with their dendritic trees extending in the superficial fibrous layer, were found. In 1-month-old Tilapia, NADPH-diaphorase staining and nitric oxide synthase immunoreactivity were found to overlap in both the retina and optic tectum. The density of NADPH-diaphorase labeled neurons in the inner nuclear layer of the retina and in the stratum periventriculare of the optic tectum was largely reduced in comparison with 2 days posthatching embryos. These findings indicated an early and transient production of nitric oxide in the retina and optic tectum of Tilapia, suggesting a functional role for nitric oxide in the development of visual structures in aquatic vertebrates.     

Rearrangements of the retinotectal projection in Rana pipiens after unilateral caudal half-tectum ablation.

When the caudal half-tectum is ablated and the optic nerve is cut in adult Rana pipiens, recordings from optic nerve terminals show that the entire visual field comes to be represented in retinotopic order on the remaining rostral half-tectum (compression). In contrast, if the caudal half-tectum is ablated but the optic nerve is left intact, no compression of the retinotectal projection results. Instead, the terminals displaced from the ablated tissue form a permanently disorganized projection to the rostral half-tectum superimposed on the unaltered representation of those parts of the retina which normally project there. The receptive field locations of tectal neurons in both groups of animals show that the altered retinotectal projections make functional synapses. This conclusion is further supported by behavioral data, which show that the accuracy of prey-catching movements is altered in both groups of frogs. The anomalous retinotectal maps can be interpreted by means of a model which includes polarity cues derived from the tectum and ordering cues derived from repelling interactions among fiber terminals.     

The central projections in the retina in Necturus maculosus

The projections of the retina in Necturus maculosus were studied by injecting radioactive proline into one eye. Labeling was seen in both the contralateral and ipsilateral diencephalon and tectum. The contralateral fibers are divided into three major tracts: the marginal, axial, and basal. The ipsilateral fibers separate into a marginal and an axial optic tract. The contralateral and ipsilateral axial optic tracts have a similar distribution. The contralateral and ipsilateral marginal optic tracts projecting to the diencephalon also have a similar distribution. However, in the tectum the ipsilateral marginal optic tract ends in the anterior third while the contralateral extends almost the entire length of the tectum. The retinotectal ipsilateral projection ends in clumps as has been described in other vertebrates. A direct ipsilateral retinotectal projection has not been described in any other amphibian.     

Expression of neural cell adhesion molecule (NCAM) characterizes a subpopulation of type 1 astrocytes in human optic nerve head

The human optic nerve contains a heterogeneous population of astrocytes. In situ, a specialized subpopulation of astrocytes was distinguished in the adult optic nerve head by expression of neural cell adhesion molecule (NCAM). To further study the biology of astrocytes, we have developed and characterized cells grown from explanted optic nerve heads and myelinated optic nerves as in vitro model systems. Second or third passage cells were processed for immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP) and cell surface epitopes: CD56/NCAM, HNK-1/NCAM, A2B5, and O4. Synthesis and gene expression of NCAM were characterized by Western blot analysis and RNase protection assay. Cells grown from myelinated optic nerves expressing GFAP, but not NCAM or A2B5, were identified as type 1A astrocytes, and cells expressing GFAP and A2B5, but not NCAM, were identified as type 2 astrocytes. Cells grown from explanted optic nerve head expressing GFAP, NCAM, and O4 were identified as type 1B astrocytes. Expression of NCAM by type 1B astrocytes may provide these cells with adhesion properties that allow them specialized responses in their microenvironment. Astrocytes from the lamina cribrosa may form a functional barrier to prevent myelination of the retina. In glaucoma, these astrocytes may be exposed to stresses due to fluctuation in intraocular pressure and therefore participate in the optic nerve changes associated with glaucomatous optic neuropathy. GLIA 20:262–273, 1997. © 1997 Wiley-Liss, Inc.     

Immunocytochemical and Biochemical Evidence for Aromatase in Neurons of the Retina, Optic Tectum and Retinotectal Pathways in Goldfish

Using an animal model in which neural aromatase is apparently overexpressed (the goldfish, Carassius auratus) and an antihuman placental antibody which specifically crossreacts with goldfish brain aromatase, aromatase-immunoreactive neuronal cell bodies and fibers have been localized within the retina. These include a subset of horizontal cells, bipolar cells, and amacrine cells of the inner nuclear layer, some fibers of the outer and inner synaptic layers and certain cells of the ganglion cell layer; photoreceptors were never labeled. Some ganglion cell projections to the brain via the optic nerve and optic tract were aromatase-positive, as were small neurons of the stratum periventriculare (SPV) and fibers of two other strata of the optic tectum. Aromatase activity, as measured by [³H]estrogen formation from [³H]androgen by tissue homogenates and cell cultures, confirmed the presence of aromatase in retina and in brain regions containing the optic tectum. This localization of the rate-limiting enzyme in estrogen biosynthesis suggests that neuroestrogen derived from circulating androgen may modulate transmission and integration of visual information important for reproduction in this species.     

Reorganization of retinotectal projection following surgical operations on the optic tectum in goldfish

The pattern of neural reconnection between the retina and surgically operated tectum was studied in juvenile goldfish (8–11 cm long) with electrophysiological methods. The results confirm that the remaining rostral half-tectum reacquires a complete visual projection from the whole retina about 90 days after excision of the caudal half. The same reorganization of visual projection from the whole retina onto the rostral half-tectum was found to occur in the presence of the caudal half of the tectum, if the two halves were separated by a transverse surgical incision down to the level of the optic ventricle regardless of whether the contralateral optic nerve was left intact or crushed to regenerate. The reorganization of retinotectal projection was also found to occur biaxially along the mediolateral as well as the rostrocaudal axis of the tectum following excision of a caudomedial sector of the tectum. It is suggested that the reorganization of retinotectal projection is due to synaptic respecifications of individual tectal neurons in correct retinotopic order, and that the cellular discontinuity between the rostral and the caudal parts of the tectum is sufficient to induce the orderly synaptic respecifications in juvenile goldfish.     

Topographic restriction of TAG-1 expression in the developing retinotectal pathway and target dependent reexpression during axon regeneration

TAG-1, a glycosylphosphatidyl inositol (GPI)-anchored protein of the immunoglobulin (Ig) superfamily, exhibits an unusual spatiotemporal expression pattern in the fish visual pathway. Using in situ hybridization and new antibodies (Abs) against fish TAG-1 we show that TAG-1 mRNA and anti-TAG-1 staining is restricted to nasal retinal ganglion cells (RGCs) in 24- to 72-h-old zebrafish embryos and in the adult, continuously growing goldfish retina. Anti-TAG-1 Abs selectively label nasal RGC axons in the nerve, optic tract, and tectum. Axotomized RGCs reexpress TAG-1, which occurs as late as 12 days after optic nerve lesion, when regenerating RGC axons arrive in the tectum, suggesting TAG-1 reexpression is target contact-dependent. Accordingly, TAG-1 reexpression ceases upon interruption of the regenerating projection by a second lesion. The topographic restriction of TAG-1 expression and its target dependency during regeneration suggests that TAG-1 might play a role in the retinotopic organization and restoration of the retinotectal pathway.     

Topographic projections of the retina and optic tectum upon the ventral lateral geniculate nucleus in the chick

The topographic projections of the retina upon the optic tectum and ventral lateral geniculate nucleus (GLv) of the chick were investigated by making small intraretinal injections of ³H-proline. The retinotectal projection pattern was similar to that described for the pigeon. The retinal projection to the GLv was also topographic and was restricted to the outermost lamina of the nucleus. The anteroposterior retinal axis was reversed in the GLv relative to its orientation in the tectum but the superoinferior axis was oriented identically in both. Furthermore, the posterior retina had an enlarged area of projection in the GLv similar to the enlarged area of retinotectal projection for the “red field” found in pigeons. The tectogeniculate projection was topographic and was confined to the outermost geniculate lamina. The secondorder retionotopic map made by the tectogeniculate projections was in register with the retinogeniculate projection. Although the retinal and tectal projection areas were coextensive in the outermost geniculate lamina, the grain density distributions peaked at different points along a radial path through the geniculate laminae. Injections of HRP into the optic tectum led to very light retrograde labeling of a small population of GLv cells topographically corresponding to the tectogeniculate projection zone of the injection site. The data suggest that the chick GLv is comparable to the GLv of other non-primate mammals.     

Tests for neuroplasticity in the anuran retinotectal system

Mapping of retinotectal projections in the tree frog Hyla regilla was carried out by both behavioral and electrophysiological recording techniques following tectal ablations, with and without optic nerve regeneration. Scotomata produced by unilateral and bilateral half tectum ablations and by unilateral rectangular midtectal excisions were found to remain essentially unaltered in all cases through recovery periods up to 334 days. Similarly, electrophysiological mapping of the rostral half tectum separated by Gelfilm implants from the caudal tectum for up to 191 days yielded a normal rostral visual field. The stability of the retinotectal projection in adult Hylidae observed in these experiments contrasts with the plastic readjustments obtained in young goldfish in which the retinotectal system is still probably growing and presumably capable of field regulation. The results are taken to support the original explanatory model for developmental patterning of retinotectal connections in terms of selective cytochemical affinities between retinal and tectal neurons.