Aberrant growth of regenerating retinotectal axons subsequent to optic tract ablation in goldfish

This study examined the effect of optic tract ablation on retinotectal fiber regeneration in goldfish. Approximately two-thirds of the left optic tract was removed, and, at various times post lesion (10–75 days), the course of regenerating retinotectal fibers was traced using horseradish peroxidase. In all experimental animals, axons were observed regenerating through the visual pathway but at the brachia most of the fibers were channeled through the ventral brachium. We present evidence that fibers in the ventral brachium originated from ganglion cells in all regions of retina and that these fibers grew almost exclusively into ventral half tectum even though some of these fibers would normally synapse in dorsal half tectum. These observations suggest that optic tract ablation does not prevent retinal fiber regeneration but results in aberrant growth through the brachia and significant inhibition of exploratory fiber growth within the tectum.     

Optic nerve regeneration with return of vision through an autologous peripheral nerve graft

The optic fiber termination layer in the contralateral optic tectum was reinnervated and useful vision was recovered in the adult frog, after successful optic nerve regeneration through an autologous peripheral nerve-bridge used to replace the optic nerve and optic chiasma. During their course through the nerve-bridge, the optic fibers were associated with Schwann cells in the usual relationship observed in peripheral nerve.     

Axons added to the regenerated visual pathway of goldfish establish a normal fiber topography along the age-axis

Throughout a goldfish's life, new generations of ganglion cells are added on the retinal margin and their axons extend centrally to occupy predictable positions in the retinotectal pathway, adjacent to their predecessors and subjacent to the pia. The stacking of successive generations of axons defines the age-axis of the pathway. This study examined whether an ordered array of predecessor axons is a prerequisite for the patterned growth of new axons. One optic nerve was crushed intraorbitally and the fish was injected with 3H-thymidine to label the proliferating cells on the retinal margin. The ring of 3H-thymidine-labeled cells separated retina that was present at the time of nerve crush (inside the ring) from new retina added afterward (outside). After a period of 14-16 months postcrush, both tectal lobes received two punctate applications of horseradish peroxidase (HRP), one in the central and the other in peripheral tectum, to retrogradely label contralateral retinal ganglion cell bodies and their axons. The pattern of HRP labeling from the control tectum confirmed earlier work: axons on the central tectum had somata in the central retina, and axons on the peripheral tectum had somata in the peripheral retina. The labeled cells and axons were both in predictable patterns. The somata that were backfilled from applications to the center of the experimental tectum lay inside the radioactive ring and had therefore regenerated their axons. The patterns of their labeled axons in the optic pathway and of their somata in the retina were typical of the regenerated condition as described in earlier studies. The somata backfilled from the periphery of the experimental tectum were outside the radioactive ring and had been added after the optic nerve crush. The patterns of their labeled axons and somata were comparable to the normal pattern. These observations indicate that new axons do not depend on an ordered array of predecessors to reestablish normal order along the age-axis of the pathway.     

Permanent disorganization of the regenerating optic tract in the frog

The organization of the optic tract as it regenerates following optic nerve transection in adult frogs was studied using microelectrode recordings from optic nerve arborizations in the tectum. In normal frogs, a cut extending from the midline partway across the rostromedial margin of the tectum severs optic axons with receptive fields in the temporosuperior quadrant of the visual field. During regeneration, however, a similar cut spares many axons with temporosuperior fields. This result implies that some fibers which normally enter the tectum via the most medial parts of the optic tract regenerate through other parts of the tract. Despite their anomalous routes, many of these fibers eventually terminate at the appropriate locations in the tectum.     

Effect of different optic nerve lesions on retinal ganglion cell death in the frog Rana pipiens

Following optic nerve crush in various species of frog, a proportion of the retinal ganglion cells re-establishes functional contact with the optic tectum. However, as much as 50% of the retinal ganglion cells die during this process. The determinants of an individual ganglion cell's fate have not been established. In this study of Rana pipiens, cell survival after optic nerve crush was compared with that after nerve cut followed by stump separation, a procedure that considerably delayed entry of optic axons to the brain. It was also ascertained, in the case of delayed ingrowth, whether application of nerve growth factor immediately after lesion influenced the cell death process. This study confirmed that retinal ganglion cell death is a relatively late event in regeneration, because in several animals where anterograde HRP labeling demonstrated regenerating axons within the tectum, no cell death had occurred. There was no statistically significant difference in cell death at 75 days after lesion between animals receiving nerve crush and those receiving nerve cut with stump separation, even though most crush animals had regenerated a complete visual projection, whereas most nerve cut animals had not. The application of NGF did not influence the level of cell death at 75 days after lesion. These results suggest that contact of optic axons with the optic tract or tectum is not necessary for retinal ganglion cell death to occur. However, this does not necessarily mean that contact with the brain is not involved with cell death during regeneration following nerve crush because it is possible that the mechanisms of cell death are different when axons are prevented from regenerating. Further investigations are therefore required to establish the reasons for this cell death.     

Ultrastructural evidence of the formation of synapses by retinal ganglion cell axons in two nonstandard targets

After unilateral ablation of the optic tectum in the frog (Rana pipiens), retinal ganglion cell axons enter the lateral thalamic neuropil in large numbers. This area is normally a target of the tectal efferent projection but is not innervated directly from the retina in normal frogs nor in frogs undergoing optic nerve regeneration in the presence of an intact tectum. The ability of retinal axons to form synaptic contacts in this nonstandard target, previously suspected only from light microscope studies, has been ultrastructurally verified in the present investigation. Retinal axon terminals were selectively labeled for light and electon microscope study by introducing horseradish peroxidase (HRP) into the optic nerve 73-413 days after unilateral ablation of the contralateral optic tectum. In some of the frogs, the optic nerve had also been crushed to test the ability of retinal axons regenerating over a long distance to form this connection. The HRP-labeled retinal axon terminals had the same untrastructural morphology whether located in the lateral thalamic neuropil or in the correct regions of projection, e.g., the lateral geniculate complex. They contained clear, spherical synaptic vesicles and made Gray type I synapses on the unlabeled postsynaptic dendrites. The magnitude of the projection was disproportionately greater in animals having complete or nearly complete tectal ablation than in a specimen in which the lesion was significantly incomplete. An aberrant projection was also observed in the nucleus isthmi in some of the specimens. These findings have significance for chemoaffinity theories of the specification of synaptic connections in that the ability of retinal axons to synapse in nonstandard targets in this experimental context may be considered evidence for the expression of appropriate cell-surface recognition-molecules by the abnormally targeted postsynaptic neurons. The likelihood that the expression of these postsynaptic labels is normally repressed transynaptically by molecular signals from the intact tectal input is discussed.     

Retinogeniculate projection fibers in the monkey optic nerve: a demonstration of the fiber pathways by retrograde axonal transport of WGA-HRP

Seven Japanese monkeys (Macaca fuscata) were used to investigate the fiber pathways of the optic nerve. Optic nerve fibers and retinal ganglion cells were retrogradely labeled by iontophoretic injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into electrophysiologically defined positions of the lateral geniculate nucleus (LGN). By gross anatomical observation, the optic nerve usually had one distinct bend, which flexed dorsally 3-4 mm from the eyeball, and occasionally another ventrally directed bend was found just behind the eyeball. In the optic nerve head, fibers from the various retinal areas were arranged in a wedge according to the fiber trajectory on the retinal surface. For about a 3 mm distance from the disc, fibers rapidly spread out radially. Subsequently, rather than scattering dorsoventrally, they progressed to the chiasm with a gradual increase in the degree of mediolateral (nasotemporal) scatter. The degree of the scatter was different depending on the retinal site from which the axons originated. Fibers from the peripheral retina spread out widely for a few millimeters behind the eyeball. Thereafter the scatter was rather limited until the chiasm. On the other hand, the scatter of fibers from the foveal and parafoveal areas progressed gradually through the nerve. The present study also suggests that the difference in scatter depends on the types of cells of origin. Fibers from large ganglion cells displayed more extensive scatter than fibers from medium-sized cells. In spite of the extensive scatter of fibers, two clear segregations were found; one was a dorsoventral segregation, which was displayed by both central and peripheral retinal fibers, and the other was a center-peripheral segregation in which the fibers from the nasal central (papillomacular) retina were located almost exclusively in the central part of the optic nerve surrounded by peripheral retinal fibers. However, the temporal central retinal fibers were located in the lateral periphery of the nerve, and they overlapped significantly with fibers from the temporal peripheral retina. Furthermore, a broad intermingling was found between nasal and temporal peripheral retinal fibers owing to their mediolateral scatter. Thus, the present findings based on more precise anatomical techniques indicate that the classical notion of the retinal quadrant topography in the monkey optic nerve probably is suspect. In addition, the "rotation" of the fiber arrangement was not demonstrated.     

A comparison of the normal and regenerated retinotectal pathways of goldfish

This is a light and electron microscopic study of the retinotectal pathway: intact and after regeneration of the optic nerve. The spatiotemporal pattern of axonal outgrowth and termination was studied with the methods of proline autoradiography, horseradish peroxidase (HRP) labeling, and fiber degeneration. The spatial order of optic fibers in the normal and regenerated pathways was assessed by labeling small groups intraretinally with HRP and then tracing them to the tectum. The labeled fibers occupied a greater fraction of the cross section of the regenerated than the normal optic tract. At the brachial bifurcation, roughly 20% of the regenerated fibers chose the incorrect brachium vs. less than 1% of the normals. In tectum, the regenerated optic fibers reestablished fascicles in stratum opticum, but they were less orderly than in the normals. The retinal origins of the fibers in the fascicles were established by labeling individual fascicles with HRP and then, following retrograde transport, finding labeled ganglion cells in whole-mounted retinas. Labeled cells were more widely scattered over the previously axotomized retinas than over the normal ones. A similar result was obtained when HRP was applied in the tectal synaptic layer. All of these results indicate that the pathway of the regenerated optic fibers is less well ordered than the intact pathway. Both autoradiography and HRP showed that the regenerating optic fibers invaded the tectum from the rostral end, and advanced from rostral to caudal and from peripheral to central tectum, along a front roughly perpendicular to the tectal fascicles. Synapses of retinal origin were noted electron microscopically in the tectum at the same sites where autoradiography indicated that the fibers had arrived. No retinal terminals were seen where grain densities were at background levels. Fiber ingrowth and synaptogenesis apparently occurred simultaneously. The synapses were initially smaller and sparser than in normals, but were in the normal tectal strata and contacted the same classes of post synaptic elements as in normals.     

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.     

Regenerating optic axons restore topography after incomplete optic nerve injury

Following complete optic nerve injury in a lizard, Ctenophorus ornatus, retinal ganglion cell (RGC) axons regenerate but fail to restore retinotectal topography unless animals are trained on a visual task (Beazley et al. [ 1997] J Comp Neurol 370:105-120, [2003] J Neurotrauma 20:1263-1270). Here we show that incomplete injury, which leaves some RGC axons intact, restores normal topography. Strict RGC axon topography allowed us to preserve RGC axons on one side of the nerve (projecting to medial tectum) while lesioning those on the other side (projecting to lateral tectum). Topography and response properties for both RGC axon populations were assessed electrophysiologically. The majority of intact RGC axons retained appropriate topography in medial tectum and had normal, consistently brisk, reliable responses. Regenerate RGC axons fell into two classes: those that projected topographically to lateral tectum with responses that tended to habituate and those that lacked topography, responded weakly, and habituated rapidly. Axon tracing by localized retinal application of carbocyanine dyes supported the electrophysiological data. RGC soma counts were normal in both intact and axotomized RGC populations, contrasting with the 30% RGC loss after complete injury. Unlike incomplete optic nerve injury in mammals, where RGC axon regeneration fails and secondary cell death removes many intact RGC somata, lizards experience a "win-win" situation: intact RGC axons favorably influence the functional outcome for regenerating ones and RGCs do not succumb to either primary or secondary cell death.     

The re-establishment of synaptic transmission by regenerating optic axons in goldfish: Time course and effects of blocking activity by intraocular injection of tetrodotoxin

Intraocular injections of tetrodotoxin were used to block activity for 27 days in normal fish and for the first 27 or 31 days of regeneration in fish with one optic nerve crushed. Synaptic activity was then assessed by a current source-density analysis of field potentials evoked by optic nerve shock at different times following the TTX treatment. In normal fish, the lack of activity for 4 weeks had no significant effect on the maintenance of synaptic strength. Likewise, in fish with nerve crush, lack of activity did not prevent the regenerating optic fibers from forming synapses that were nearly as effective as those formed in controls injected with the citrate buffer vehicle. The earliest synapses were formed at the rostromedial corner of the tectum (where the tract enters) at 20 days after nerve crush, when fibers had not yet reached the caudal areas. By 28 days synaptic potentials could be recorded everywhere on the surface of the tectum in both controls and TTX injected fish. However, the latency of the responses with TTX were longer, suggesting a smaller caliber of fiber, which is consistent with an earlier finding of decreased axonal transport in TTX fish. Maturation of the regenerating fibers proceeded slowly in both TTX and control fish. After more than 5 months, the projections were nearly normal but still not completely normal.     

Normal and regenerating optic fibers in goldfish tectum: HRP-EM evidence for rapid synaptogenesis and optic fiber-fiber affinity

The distribution of normal and regenerating retinal fibers and synapses was studied on tectum in goldfish by light (LM) and electron microscopy (EM). Since labeling of the early regenerating fibers was previously reported to be difficult, a new 'cold-fill' HRP labeling protocol was developed, which labeled regenerating optic fibers and terminals on tectum as early as 14 days after nerve crush when they first arrive on tectum. In order to characterize the laminar distribution of optic afferents in normal fish and in fish regenerating for 14-240 days, EM photomontages of areas 14 microns wide by 160 microns deep through the HRP-labeled primary optic innervation layer (S-SO-SFGS) were constructed. The time points in regeneration that were examined spanned the period in which others have shown that an initially diffuse retinotopic map becomes spatially restricted. At the LM level regenerating optic fibers were restricted to the optic lamina. They reinnervated tectum in an anterior to posterior sequence as previously seen with autoradiography. In addition, at 14 days, some "pioneer" optic fascicles were found to have already grown to posterior tectum where they gave rise to branches with boutonlike terminations and growth-cone-like processes. Form the ultrastructural analysis it was clear that optic fibers and terminals observed strict laminar boundaries as they partitioned themselves in the optic laminae (S, SO and SFGS) in both normal and regenerating fish. The behavior of optic fibers was lamina specific with respect to synapse formation and the orientation of fiber outgrowth. As early as 14 days regeneration, optic fibers made synapses onto the four types of postsynaptic profiles observed in normal fish. Numerous optic terminals were labeled at 14 days, and there appeared to be no waiting period between fiber ingrowth to the SO and synapse formation in the S and SFGS. At 14-60 days, atypical synaptic contacts which appear to be nascent synapses were made by labeled optic fibers in fascicles and by growth-cone-like processes. By 21-30 days, the density of optic terminals was high and there were many more fasciculated optic fibers in the SFGS than normal as late as 350 days. These findings suggest that optic fiber lamination is highly constrained by tectal cues, that fibers rapidly regenerate many synaptic terminals before retinotopic map refinement is complete, and that fibers have a strong affinity for each other.     

Growth dynamics and morphology of regenerating optic fibers in tectum are altered by injury conditions: an in vivo imaging study in goldfish

The dynamic behavior of axons in systems that normally regenerate may provide clues for promoting regeneration in humans. When the optic nerve is severed in adult goldfish, all axons regenerate back to the tectum to reestablish accurate connections. In adult mammals, regeneration can be induced in optic and other axons but typically few fibers regrow and only for short distances. These conditions were mimicked in the adult goldfish by surgically deflecting 10-20% of optic fibers from one tectum into the opposite tectum which was denervated of all other optic fibers by removing its corresponding eye. At 21-63 days, DiI was microinjected into retina to label a few fibers and the fibers were visualized in the living fish for up to 5-7 h. The dynamic behavior and morphology of these regenerating deflected fibers were analyzed and compared to those regenerating following optic nerve crush. At 3-4 weeks, deflected fibers were found to form more branches and to maintain many more branches than crushed fibers. Although both deflected and crushed fibers exhibited stochastic growth and retraction, deflected fibers spent more time growing but grew for less distance. At 2 months, both deflected and crushed fibers became much more stable. These results show that the morphology and behavior of fibers regenerating into the same target tissue can be substantially altered by the injury conditions, that is, they show state-dependent plasticity. The morphology and behavior of the deflected fibers suggest they were impaired in their capacity to grow to their correct targets.     

On the asymmetry of the primary branching of vagal sensory axons: possible role of the supporting tissue

Central and peripheral nonmedullated processes of vagal nodosal neurons of the cat were studied in normal nerves and after regeneration along their anatomical course and along the hypoglossal nerve. Nonmedullated fibers above the ganglion and in the root had comparable sizes (approximately 0.37 micron2) and caliber distribution. Below the ganglion, the cross-sectional area increased to 1.0 micron2. In axons of equal caliber, supranodosal and radicular fibers had similar microtubular densities while infranodosal fibers had two- to threefold that of the former. Regenerated fibers were studied after a recovery period of 6-9 months. Regrown axons were smaller than their parent axons; in turn, these were smaller than normal axons. This holds for central and peripheral nodosal branches, for homologous and heterologous regeneration. Regrown peripheral branches, either along their anatomical pathway or along the hypoglossal nerve, showed no change in microtubular density. Central branches exhibited their characteristic microtubular content when they regenerated along their anatomical course, but when regrowth took place along the hypoglossal nerve, the original low microtubular content of these branches increased to match the high content of peripheral fibers; parent central axons also shifted their microtubular content toward the pattern of peripheral fibers. We propose that the supporting tissue participates in specifying the organization of axonal microtubules.     

Retinotopic and temporal organization of the optic nerve and tracts in the adult goldfish

In order to investigate the role of the different factors controlling the pathways and termination sites of growing axons, selected optic fibers were traced from the eye to the tectum in adult goldfish either by filling them with HRP, or by severing a group of fibers and tracing their degeneration in 2μm plastic sections stained with toluidine blue. Some fish received more than one lesion and others received both lesions and HRP applications. Two major rearrangements of the optic fibers were identified, one at the exit from the eye, the other within the optic tracts. Near the eye the optic fibers appear to be guided by the conformation of the underlying tissue planes that they encounter. The most recently added fibers, from the peripheral retina, grow over the vitread surface of the older fibers toward the blood vessel in the center of the optic nerve head. Behind the eye the fibers follow this blood vessel until it leaves the side of the optic nerve, and the fibers from peripheral retina are left as a single group on the ventral edge of the optic nerve cross section. As a consequence of this pattern of fiber growth the fibers form an orderly temporal sequence in the optic nerve, with the oldest fibers from the central retina on one side of the nerve and the youngest from peripheral retina on the other. In addition, the fibers are ordered topographically at right angles to this central-to-peripheral axis, with fibers from ventral retina on each edge of the nerve, dorsal fibers in the center, and nasal and temporal fibers in between. This arrangement of the optic fibers continues with only a little loss of precision up to the optic tracts. A more radical fiber rearrangement, seemingly incompatibe with the fibers simply following tissue planes occurs within the optic tracts. Each newly arriving set of fibers grows over the surface of the optic tracts so that the older fibers come to lie deepest in the tracts. This segregation of fibers of different ages ensures that the rearrangement is limited to each layer of fibers. The abrupt reorganization of the fibers occurs as the tracts split around the nucleus rotundus to form the brachia of the optic tracts. The fibers are then arranged with temporal fibers nearest the nucleus rotundus and nasal fibers on the opposite edges of the brachia. From this point the fibers grow out over the tectal surface to their termination sites with only minimal rearrangements. Therefore the optic fiber rearrangements show evidence of several different sorts of constraints acting on the fibers at separate points in the optic pathway, each contributing to the final orderly arrangement of the fibers on the optic tectum.     

PERIPHERAL NERVE REGENERATION THROUGH GRAFTS OF LIVING AND FREEZE-DRIED CNS TISSUE

The ability of peripheral nerve fibres to regenerate through the central nervous system (CNS) extracellular matrix in the presence of CNS myelin debris was examined using living and freeze-dried optic nerve grafts. The grafts were placed end-to-end with the proximal stumps of severed common peroneal nerves of inbred mice. Within a 4 week period, regenerating peripheral nervous system fibres were found in only two of 14 living grafts. However axons always grew into freeze-dried grafts within one week, despite the presence of CNS myelin debris. The regenerating axons in freeze-dried grafts were accompanied by Schwann cells and were initially found associated with the inner aspect of the glial basal lamina. Although the extracellular matrix of the freeze-dried CNS tissue was subsequently reorganized by invading cells, it seems likely that neither the nature of the CNS extracellular matrix nor the presence of CNS myelin debris had a major inhibitory influence on peripheral nerve regeneration. It is suggested that the presence of living astrocytes covered by a basal lamina at the proximal end of the living optic nerve grafts may inhibit their penetration by regenerating axons.     

Axonal pathfinding during the regeneration of the goldfish optic pathway

Retinal ganglion cells in fish and amphibians regenerate their axons after transection of the optic nerve. Fiber tracing studies during the third month of regeneration show that the axons have reestablished a basically normal fiber order in the two brachia of the optic tract; axons originating in the ventral hemiretina are concentrated in the dorsal brachium, axons from the dorsal hemiretina in the ventral brachium. Attardi and Sperry (Exp. Neurol. 7:46-64, 1963) first suggested that the reestablishment of the fiber order reflects path-finding by the regenerating axons. Recently, however, Becker and Cook (Development 101:323-337, 1987) have claimed that the fiber order observed at later stages of regeneration is due to secondary axonal rearrangements and that the initial brachial choice is random. In order to evaluate whether regenerating axons are capable of navigating in the optic tract and brachia and on the tectum, the present study examined the pathway choices and the morphology of regenerating axons en route to their tectal targets in goldfish. Subsets of axons were labeled at various time intervals (2 to 30 days) following an optic nerve crush, by intraretinal application of the lipophilic fluorescent tracer 1,1-dioctadecyl-3-3-3'-3'-tetramethylcarbocyanine (DiI). After a survival time of 18 to 72 hours (to allow for diffusion of DiI along the axons), the experimental animals were perfused with fixative and their right and left optic pathways (nerve, tract, and tectum) were dissected free and separated at the chiasm. Fluorescently labeled axons were traced in whole-mounted pathways. Pathway choices were examined at the brachial bifurcation where axons from ventral and dorsal hemiretinae normally segregate. DiI was found to label axons reliably up to their growth cones, even at the earliest stages of regrowth. The pathway choices of the axons were nonrandom. The majority of the ventral axons reached the appropriate, dorsal hemitectum through the appropriate dorsal brachium of the tract. Dorsal axons reached the ventral hemitectum mainly through the ventral brachium. This suggests the presence of specific guidance cues, accessible to the regenerating axons. Differences in the complexity of the growth cones of the regenerating axons (simple in the nerve and tectal fiber layer, complex in the tract and the synaptic layer of the tectum) provide further evidence for specific interactions between the regenerating axons and their substrates along the pathway. These results argue that regenerating retinal axons in fish are capable of axonal path-finding.     

The course of axons of retinal ganglion cells within the optic nerve and tract of the chick (Gallus gallus)

Small laser lesions placed in the posthatch chicken retina resulted in axotomy and then death of all ganglion cells located in a sector peripheral to the primary damage. With the use of silver techniques, the patterns of degenerating retinal fibers in the optic nerve, chiasm, and optic tract were examined. In the proximal part of the optic nerve, radial retinal lesions resulted in a sheet of degenerating axons along the rostrocaudal extent of the nerve. The position of degenerating axons was related to the site of their entry in the optic nerve head with an overlapping distribution of degenerating fibers entering the optic nerve head from equivalent points from the temporal and nasal sides. In the optic chiasm, the distribution of fibers was similar to that seen in the proximal part of the optic nerve. In the optic tract there was a similar mixing of fibers from opposite sides of the retina. The ventral, nasal and temporal retinal fibers lay in the superficial part of the tract whereas the fibers from the nasal and temporal dorsal retina ran in the deeper, medial aspect of the tract. The central-to-peripheral axes of the retina were mapped along the rostrocaudal axis of the tract. As the tract approached the tectum degenerating fibers from single retinal lesions did not always remain together. In the case of a lesion in the ventral nasal retina, degenerating fibers split into two bundles located at opposite ends of the tract only to reunite at their terminal regional at the caudal pole of the tectum.(ABSTRACT TRUNCATED AT 250 WORDS)     

The organization of the fibers in the optic nerve of normal and tectum-less Rana pipiens

We have examined the detailed order of retinal ganglion cell (RGC) axons in the optic nerve and tract of the frog, Ranapipiens. By using horseradish peroxidase (HRP) injections into small regions of theretina, the tectum, and at various points along the visual pathway, it hasbeen possible to follow labelled fibers throughout their course in the nerve and tract. Several surprising features in the order of fibers in the visual pathway were discovered in our investigation. The fascicular pattern of RGC axons in che retina is similar to that described in other vertebrates; however, immediately central to their entry into the optic nerve head, approximately half of the fibers from the nasal or temporal retina cross over to the opposite side of the nerve. Although the axons from the dorsal and ventral regions of the retina generally remain in the dorsal and ventral regions of the nerve, some fiber crossing occurs in those axons as well. The result of this seemingly complex rearrangement is that the optic nerve of Rana pipiens contains mirror symmetric representations of the retinal surface on either side of the dorsal ventral midline of the nerve. The fibers in each of these representation are arranged as semicircles representing the full circumference of the retina. This precise fiber order is preserved in the nerve until immediately periphearal to the optic chiasm, at which point age-related axon from both side of the nerve bundle together. Consequently, when a small pellet of HRP is placed in the chiasmic region of the nerve, an annualus of retinal ganglion cells and a corresponding annulus of RGC terminals in the tectum are la belled. As the age-related bundles of fibers emerge from the chiasm they split to form a medial bundle and a lateral bundle, which grow in the medial and lateral branches of the optic tract, respectively. Although the course followed by RGC axons in the visual pathv/ay is complex, we propose a model in which the organization of fibers in the nerve and tract can arise from a few rules of axon guidance. To determine whether the optic tecta, the primary retinal targets, play a role in the development and organization of the optic nerve and tract, we removed the tectal primordia in Rana embryos and examined the order in the nerve when the animals had reached larval stages. We found that the order in the nerve and tract was well preserved in tectumless frogs. Therefore, we propose that guidance factors independent of the target direct axon growth in the frog visual system.