Retinotectal map formation in dually innervated tecta: A regeneration study in Xenopus with one compound eye following bilateral optic nerve section

Retinotectal map formation was studied during regeneration in young adult Xenopus. Right compound double-temporal eyes (TT) were formed in tailbud stage embryos by the fusion of two temporal halves of the eye blastema in the same orbit. In other animals right compound double-nasal eyes (NN) were prepared. In both combinations the left eye was kept intact. After metamorphosis the right and left optic nerves were sectioned to induce optic fiber regeneration from each eye to both tecta. The patterns of retinotectal projections from the compound and normal eyes were studied from 37 to 364 days after optic nerve section, using electrophysiological recording of the visuotectal projections and ³H-proline autoradiographic assay from one of the two eyes. The left normal eyes projected in a retinotopic fashion, across the entire extent of the right and left dually innervated tecta. In contrast, the right compound eye projections were confined to the rostrolateral or to the caudomedial part of the right and left tecta in TT and NN animals, respectively. These tectal areas corresponded to the termination of temporal and nasal hemiretinal fibers of the normal eye. Discontinuous, interdigitating projection patterns from the right and left eyes were found in parts of the tecta where the compound and the normal eye projections overlapped. These results indicate that the normal optic fiber projections caused the originally expanded compound eye projections to be restricted to the corresponding part of the dually innervated tecta. It is suggested that the orderliness and the extent of the retinotectal map are established by the competition and interaction of optic fibers based on stable positional programming of the retinal ganglion cells.     

Retinotectal synapses formed by ipsilaterally projecting fibers in the doubly innervated goldfish tectum

When one tectum of an adult goldfish is removed, the severed retinal fibers regenerate ipsilaterally into the remaining tectal lobe. Initially fibers from the two eyes overlap in the tectum but EM-HRP data suggest that few mature retinal synapses are formed between the ipsilateral eye and tectum at this time. At longer time periods, when some fibers appear to segregate into eye-specific termination bands, our data suggest that a significant number of synapses from the ipsilateral eye are present. These findings have important implications for how eye-specific termination bands are formed in doubly innervated tecta.     

Projections of growth-cone-bearing fibers of retinal ganglion cells within co-cultured tectal explants: early branching depends on age of target tissue

The projections of retinal ganglion cell axons within co-cultured tectal explants were analyzed in order to investigate some of the factors that determine the earliest responses of retinal axons to cues present in an isolated target tissue. Half retinas and superior colliculi (tecta) from the embryonal mouse were explanted, separated by a 0.5 mm gap. After 5 days in vitro retinal ganglion cells were labeled by extracellular ionophoresis of HRP into the optic nerve head region. Cleared co-cultures were studied as whole mounts. Growth-cone-bearing retinal fibers were studied in standard tectal co-cultures, and in cases where tectum had been explanted 2 weeks prior to retina. The heterochronously prepared cultures had a higher proportion of fibers with complex branching patterns than the synchronous explants. Cultures in which retinas were explanted 1 week after tecta exhibited intermediate proportions of such fibers. These observations suggest that older tecta facilitate branching of ingrowing retinal fibers, although other alterations during in vitro development must be evaluated. The growth patterns of axons originating in nasal and temporal hemi-retinas were analyzed in terms of possible positional cues provided by the target tecta. Axons originating in temporal hemi-retinas did not show evidence of preferential branching in, or growth toward, appropriate anterior regions of co-cultured tectal explants. In contrast, the majority of nasal retinal axons showed enhanced terminations and complex branching in, and bending towards, the posterior tectum.     

Target regulation of synaptic number in the expanded retinotectal projection of goldfish: the half-retinal preparation

The nature of the expansion of the visual field projection was studied in goldfish in which size disparities were created between the retina and the tectum. After removal of one-half of the retina, the remaining retinal ganglion cells expand their projections so that the entire contralateral optic tectum is encompassed (Schmidt et al.1978). We wished to determine whether this expansion is accompanied by increased arborization including proliferation of synaptic terminals by the spared retinal ganglion axons or whether field expansion is accomplished by increased arborization without changes in synaptic number. Portions of the retina were ablated and the animals were allowed to survive for at least 5 months, the time at which expansion can be demonstrated, before sacrifice. We mapped retinotectal projections to determine the extent of the expanded visual fields and used stereological and morphometric analyses of synaptic contacts in the retinal target lamina, the stratum fibrosum et griseum superficialis (SFGS), in the optic tectum to estimate synaptic number. Numbers of synaptic terminals in the SFGS contralateral to the lesioned retina were not different from numbers in the comparable portion of control tecta. These observations indicate that the surviving retinal axons increased the number of synaptic contacts on tectal target cells in response to removal of other retinal ganglion cells.     

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.     

Retinal fibers alter tectal positional markers during the expansion of the retinal projection in goldfish

Although widely accepted, the theory, that neurones carry immutable cytochemical markers which specify their synaptic connections, is not consistent with plastic reorganizations. Half retinal fish were therefore tested for changed markers following expansion. Optic nerve crush at the time of the half retinal ablation resulted in regeneration of a normal, restricted projection; but nerve crush following expansion (many months later) resulted in reestablishment of the expanded projection, assessed both by electrophysiological mapping and by radioautography. Since this implied changed markers, the half retina and tectum were tested independently using the ipsilateral tectum and eye as controls. In normal fish, removal of one tectum and deflection of the corresponding optic tract toward the remaining tectum resulted in regeneration of a positionally normal but ipsilateral map. In experimental fish, after the half retina had expanded its projection to the contralateral tectum, its optic tract was deflected to the control tectum. After 40 days it had regenerated a normal, restricted map indicating that the retinal markers had not changed. Such restricted projections did not expand in the presence of the normal projection even after a year or more. Similarly, the optic tract from the normal eye was deflected to cause innervation of the tectum containing the expanded half retinal projection. After 40 days, the projection regenerated from the normal eye was similar to the expanded half retinal projection. Areas of the normal retina corresponding to the missing areas of the half retina were not represented. Tectal markers had been altered by the half retinal fibers. In a final group, tecta were denervated and tested at various intervals by innervation from ipsilateral half retinal eyes. After five months of denervation, the regenerating fibers were no longer restricted to the rostral tectum but formed an expanded projection initially. Apparently tectal markers are induced by the retinal fibers, changed during expansion, and disappear during long-term denervation.     

Intraretinal RGMa is involved in retino-tectal mapping

The repulsive guidance molecule (RGMa) is involved in controlling the topography of retinal ganglion cell axons along the anterioposterior axis of the tectum. Here, we generated a new RGMa-monoclonal antibody and show that it is expressed in the developing retina, suggesting that it may regulate retinal axon pathfinding. We tested this hypothesis by using in ovo electroporation to either overexpress or downregulate RGMa in the eye. Anterograde labeling of retinal axons entering the optic tecta revealed abnormal phenotypes when RGMa expression is perturbed. These included the absence of terminal zone, the premature stalling of arborization of fibers, overshooting of terminal zone, aberrant axonal turns in the optic tectum and abnormal projections into deeper tectal layers. Moreover, RGMa overexpression frequently leads to intraretinal pathfinding errors. Thus, these data suggest that RGMa expression on retinal axons is a major determinant of topographic targeting in the retino-tectal projection and in the retina.     

Pre- and postsynaptic correlates of interocular competition and segregation in the frog

Segregated zones of termination between converging inputs that arise from different presynaptic populations are a common property of topographically organized zones within the vertebrate central nervous system. Increasing evidence suggests that such segregation is at least in part established on the basis of competitive interactions that depend upon the activity patterns within each afferent population. However, the cellular mechanisms of these interactions are poorly understood. We have used a preparation in which a stereotyped interdigitating pattern of retina-specific termination stripes are produced in frog tecta innervated by two retinas as a result of embryonic implantation of a third eye primordia. In these animals it has been possible to examine the relationship between the number of retinal ganglion cells in each of the retinas innervating a striped tectum, the volumetric changes in the tectum as a result of this double innervation, and the pattern of eye-specific segregation that is produced. Counts of retinal ganglion cells in the retinas of the three-eyed frogs with one completely striped tectal lobe revealed no significant differences between cell numbers in the doubly innervating retinas and the normal retinas of the same animals. The average increase in retinal ganglion cell innervation to the striped tecta of these animals was 100%. However the tecta only increased in total volume by 26%. This later increase consisted of a 25% increase in the volume of the deep lying and predominantly cellular tectal laminae and a 37% increase in the superficial retinotectal synaptic zone. In many of these same animals HRP and 3H-proline were used to differentially label the set of stripes from each retina and measurements of the extent of each projection were performed. We found that the volume of tectal neuropil occupied by a striped projection is relatively unrelated to the number of ganglion cells making up that projection. Observations of the striping pattern after HRP processing to visualize stripes in whole unsectioned tecta indicate that the periodicities and rostrocaudal orientation of stripes are robust over a wide range of relative innervation densities. When one projection is much smaller than the other, stripes appear to break down into a series of "puffs" or islands of retina-specific termination zones. Nevertheless, these puffs still have a rostrocaudal alignment and the spacing of fully formed stripes. These observations suggest that the formation of exclusive termination zones may be a threshold phenomenon: so after a certain innervation density is reached one input can take over a unit of target neuropil in an all-or-none manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and development of nicotinic acetylcholine receptor subtypes in the optic tectum of Rana pipiens

Acetylcholine allows the elicitation of visually evoked behaviors mediated by the frog optic tectum, but the mechanisms behind its effects are unknown. Although nicotinic acetylcholine receptors (nAChRs) exist in the tectum, their subtype has not been assessed. By using quantitative autoradiography, we examined the binding of [(3)H]cytisine and [(125)I]alpha-bungarotoxin in the laminated tectum. In mammalian systems, these radioligands bind with high affinity to alpha4 nAChR subunits and alpha7 nAChR subunits, respectively. [(3)H]Cytisine demonstrated high specific binding in adult frogs in retinorecipient layer 9, intermediate densities in layer 8, and low binding in layers 1-7 of the tectum. [(3)H]Cytisine binding was significantly higher in the tecta of adults than in those of tadpoles. Lesioning the optic nerve for 6 weeks decreased [(3)H]cytisine binding in layers 8/9 by 70+/-1%, whereas 6-month lesions decreased binding by 76+/-3%. Specific binding of [(125)I]alpha-bungarotoxin in adults was present only at intermediate levels in tectal layers 8 and 9, and undetectable in the deeper tectal layers. However, the nucleus isthmi, a midbrain structure reciprocally connected to the tectum, exhibited high levels of binding. There were no significant differences in tectal [(125)I]alpha-bungarotoxin binding between tadpoles and adults. Six-week lesions of the optic nerve decreased tectal [(125)I]alpha-bungarotoxin binding by 33+/-10%, but 6-month lesions had no effect. The pharmacokinetic characteristics of [(3)H]cytisine and [(125)I]alpha-bungarotoxin binding in the frog brain were similar to those demonstrated in several mammalian species. These results indicate that [(3)H]cytisine and [(125)I]alpha-bungarotoxin identify distinct nAChR subtypes in the tectum that likely contain non-alpha7 and alpha7 subunits, respectively. The majority of non-alpha7 receptors are likely associated with retinal ganglion cell terminals, whereas alpha7-containing receptors appear to have a different localization.     

The response of optic tract glia during regeneration of the goldfish visual system. II. Tectal factors stimulate optic tract glia

After transection, retinal ganglion cell axons of the goldfish will regenerate by growing into a primary target tissue, the optic tectum. To determine what role the target tissue may play in regulating glial cell growth, we measured biosynthetic activity of optic tract glia following excision of the optic tectum and compared it to activity of glia found in the regenerating visual system. Ablation of the tectum reduced glial incorporation of both [³H]thymidine and [³⁵S]methionine. Tectal ablation also led to nearly 80% reduction of amino acids incorporated by oligodendroglia as well as a decrease in the amount of newly synthetized protein found within multipotential glia and within cytoplasmic projections of astroglia. Since the tectal influence upon optic tract glia was detected at a time when tract and tectum are physically separated, we sought to determine if the optic tectum contained soluble glia-promoting factors. A soluble fraction recovered from tecta of the regenerating visual system increased amino acid incorporation within optic tract glia at 2–3-fold above preparations incubated with fractions from control, intact tecta. Comparisons of radiolabeled proteins separated by sodium dodecyl polyacrylamide gel electrophoresis from regenerating and factor-stimulated optic tract were similar and indicated that a soluble tectal fraction promoted biosynthesis of specific glial proteins. Our findings suggest that during regeneration of the goldfish visual system glia are influenced by humoral factor(s) released from the synaptic target site.     

Rapid onset of neuronal death induced by blockade of either axoplasmic transport or action potentials in afferent fibers during brain development.

We have investigated how neurons in the optic tecta of embryonic day 16 chick embryos depend for survival on their afferents from the retina. To distinguish between activity-mediated effects and other, "trophic," ones, we compared the effects on the tectal neurons of blocking intraocular axoplasmic transport (with colchicine) or action potentials (by means of TTX). Both interventions rapidly induced the appearance of dying (pyknotic) neurons in the tectum, with major increases in their number occurring within 13 hr post-colchicine and within 9 hr post-TTX. Following both drugs, the dying neurons were morphologically similar, and in both cases the cell death depended on protein synthesis. However, the effects of colchicine and of TTX could be dissociated, since the most superficial tectal neurons became pyknotic only in response to colchicine, and, with a sufficiently short survival time (9 hr), the deep cells of the stratum griseum centrale became pyknotic only in response to TTX. We hence argue that the survival of the tectal neurons depends on their ongoing maintenance by substances released from retinotectal axon terminals, the release being activity dependent in the case of the deep neurons but independent of activity in the case of the superficial ones.     

Tectotectal connectivity in goldfish.

The vertebrate optic tectum is a functionally coupled bilateral structure which plays a major role in the generation of motor commands for orienting responses. However, the characteristics of the tectotectal connectivity are unknown in fish, and have been reported only to a limited extent in other vertebrates. The purpose of the present study was to determine the anatomical basis underlying the functional coupling between tecta in goldfish, and to identify both similarities and differences to those features reported in other vertebrate species. The present experiments used the bidirectional tracer biotinylated dextran amine to map the distribution of labeled cells and synaptic boutons in the contralateral tectum following injections into identified tectal sites. Fibers that interconnect both tecta coursed through the tectal commissure. The cells of origin of these fibers, the tectotectal cells, and their synaptic endings were located in the deep layers, mainly in the strata periventricular and griseum central, respectively. Corresponding sites throughout the two tecta were interconnected in a symmetrical point-to-point fashion. The tectal commissure was composed of at least two distinct bundles of axons, which differed in their dorsoventral location, fiber diameter, and projection targets. The dorsal axons were tectotectal axons, they were thinner in diameter and profusely branched, and gave off en passant and terminal boutons in the deep layers of the contralateral tectum. The ventral axons were thicker in diameter, and formed the contralateral tectofugal-descending tract. Such fibers had few axon collaterals and boutons in the contralateral tectum. Boutons adjacent to retrogradely labeled tectotectal cells were very scarce. The data are discussed in terms of the coupling between tecta generating the motor commands required for orienting movements.     

Retinal projections in the freshwater butterfly fish, Pantodon buchholzi (Osteoglossoidei). II. Differential projections of the dorsal and ventral hemiretinas.

Pantodon buchholzi, the freshwater butterfly fish, is a member of the Osteoglossomorpha, the most primitive of the four major teleost radiations. The projections of fibers originating in the dorsal and ventral hemiretinas in Pantodon, as determined with autoradiography, are reported here. Fibers originating in the ventral hemiretina reach their targets through the axial, medial and dorsal optic tracts. Fibers that originate in the dorsal hemiretina reach their points of termination by way of the axial, medial and ventral optic tracts. Projections of the various tracts to preoptic, thalamic, tubercular, pretectal and tectal regions, as described in the previous study of total retinal projections, were verified. The retinal projections to the preoptic, thalamic and tubercular nuclei do not map topographically. Ventral hemiretinal fibers are mapped, however, onto the dorsal part of the nucleus pretectalis superficialis pars parvocellularis, the rostral part of the dorsal accessory optic nucleus, the entire nucleus pretectalis periventricularis pars ventralis and the dorsomedial portion of the optic tectum. Ventral hemiretinal fibers also supply most if not all the retinal innervation to the central pretectal nucleus. In contrast, dorsal hemiretinal fibers are mapped onto the ventral part of nucleus pretectalis superficialis pars parvocellularis, the entire dorsal accessory optic nucleus and the ventrolateral portion of the optic tectum. The dorsal and ventral hemiretinal projections to the tectum about at a cytoarchitectonically recognizable point, indicating that no discontinuity is present in the retinal connectivity with the tectum. The pars parvocellularis of nucleus pretectalis superficialis is a simple, unfolded, and nonlaminar structure in Pantodon. This structure contrasts markedly with the more complex, folded structure of the nucleus in the majority of other examined teleosts. The orientation of the projections from the dorsal and ventral hemiretinas onto this nucleus in Pantodon is congruent with that seen in other fishes only after a schematic unfolding of the nucleus in these fishes.     

Topographic projections between the nucleus isthmi and the optic tectum in a teleost,Navodon Modestus

Following horseradish peroxidase injections into the optic tectum of a teleost,Navodon modestus, reciprocal and topographic projections between the nucleus isthmi and the ipsilateral optic tectum were determined. The isthmo-tectal fibers diverge to the optic tectum while maintaining the spatial arrangements of the isthmic cells from which the fibers originate. The tecto-isthmic projections also keep the spatial arrangements in the optic tectum. The tectal fibers converge near the nucleus isthmi and terminate in the non-cellular portion of the nucleus. The reciprocal topography is apparent in the combined results of 9 experiments with one tectal injection in each region. No labeled cells and fibers were found in the contralateral nucleus isthmi.     

Optic synapse number but not density is constrained during regeneration onto surgically halved tectum in goldfish: HRP-EM evidence that optic fibers compete for fixed numbers of postsynaptic sites on the tectum

The number of optic synapses in the half tectum of goldfish was counted by using an improved HRP-labeling protocol and a columnar sampling method that spanned the entire optic innervation layer, S-SO-SFGS. It was previously found by using this procedure in intact tectum that the normal number of optic synapses was regenerated by 30 days and maintained thereafter even in the absence of impulse activity. This suggested that the number of synapses in this system was intrinsically fixed. In order to examine whether this limit was imposed by optic fibers or by target cells, optic synapses were counted in surgically halved tecta which received compressed optic projections consisting of regenerating optic fibers from the entire retina. We reasoned that if synapse number is a function of the number of afferents, then there should be twice the normal number of optic synapses per column; on the other hand, if their number is fixed by target, then their number per column should be normal. We found that the number of optic (labeled) synapses was normal in sample columns from fish at 70 days and 160 days after optic nerve crush. Thus, retinal ganglion cells, on average, formed half as many synapses on the half tectum compared to intact tectum, indicating the number of optic synapses was limited by the tectum. The number of nonoptic (unlabeled) synapses was also found to be normal. By contrast, the S-SO-SFGS was found to be 88-103% thicker compared to normal fish, apparently because of a 20-fold increase in the number of optic fibers. As a result, the density of synapses was about half normal in half tecta, and so, in contrast to synapse number, synaptic density is not constrained during regeneration. We infer from these data that optic fibers compete for limited numbers of postsynaptic sites during regeneration and suggest that this competition promotes neural map refinement and the various plasticities described for this projection.     

Plasticity in the chick ventral lateral geniculate nucleus

The chick ventral lateral geniculate nucleus (GLv) receives topographically corresponding projections from the retina and optic tectum. Tectal lesions produced on the day of hatching removed the tectogeniculate input to the GLv region corresponding to the tectal lesion and also severed some retinotectal axons. Following a survival period of 3 to 10 weeks, a patch of augmented retinogeniculate projection was noted in the GLv segment that corresponds topographically to the damaged area of the tectum. Changing the site of the tectal lesion led to changes in the locus of heavy retinal projection to the GLv predictable from topographic maps. Nuclei which received retinal but not tectal projections did not appear to have regions of augmented retinal termination nor did nuclei which received tectal but not retinal innervation. It is unlikely that the increased retinogeniculate termination is due to rerouting of growing retinotectal axons since the chick retinofugal pathway is well established by the time of hatching. Furthermore, there was no evidence of a projection from the ipsilateral eye to the affected GLv. On the basis of these light microscopic studies, it would appear that retinogeniculate terminals have sprouted in the GLv and that competition for terminal space, conservation of terminal space, proximity, and perhaps other factors are necessary for the augmented projection to occur.     

Chronic application of NMDA decreases the NMDA sensitivity of the evoked tectal potential in the frog.

The activity-dependent mechanism that refines the topography of the retinotectal projection in frogs is mediated by the NMDA receptor. Earlier studies found that chronic treatment of the optic tectum with the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (DL-AP5) desegregated eye-specific stripes in three-eyed frogs, while chronic treatment with NMDA sharpened stripe borders (Cline et al., 1987; Cline and Constantine-Paton, 1990). We now report that this same chronic treatment with NMDA decreases the electro-physiologically measured sensitivity of the optic tectum to applied NMDA: acute application of a given concentration of NMDA decreased the evoked tectal potential to a lesser extent in animals chronically treated with NMDA than it did in normal and sham-treated animals. This is observed as a shift to the right in the NMDA dose-response curves for both the positive and negative postsynaptic components of the evoked tectal response. We believe that this decreased NMDA receptor effectiveness further restricts the intermingling of axon branches from the two eyes by limiting synapse stabilization to areas where afferent activity is most correlated. This would account for the anatomical sharpening of stripe borders (i.e., increased afferent segregation). Quantitative autoradiographic analysis of 3H-glutamate binding to NMDA receptors indicated that binding densities within the tectum did not differ between control groups and NMDA chronically treated groups. We suggest that in the experimental animals the response to NMDA may be decreased by a change in the effectiveness of individual NMDA receptors rather than by decreases in receptor number. This experimentally induced change may be analogous to naturally occurring decreases in receptor function that correlate with the end of some periods of visual plasticity in mammals.     

Retinal recipient nuclei in the painted turtle,Chrysemys picta: An autoradiographic and HRP study

Retinofugal pathways in the painted turtle were examined with autoradiographic and HRP methods. The majority of the retinal fibers decussate at the optic chiasm and course caudally to terminate in 12 regions of the diencephalon and mesencephalon. The pars dorsalis of the lateral geniculate nucleus is the densest target in the thalamus. Two nuclei dorsal to pars dorsalis—the dorsal optic and dorsal central nuclei—receive optic input. Three nuclei ventral to pars dorsalis are retinal targets—the ventral geniculate nucleus, nucleus ventrolateralis pars dorsalis, and nucleus ventrolateralis pars ventralis. Contralateral fibers course through the pretectum where they terminate in nucleus geniculatis pretectalis, nucleus lentiformis mesencephali, nucleus posterodorsalis, and the external pretectal nucleus. Retinal fibers also terminate within the superficial zone of the optic tectum. HRP material demonstrates three optic fiber layers—laminae 9, 12, and 14. Optic fibers leave the main optic tract as a distinct accessory tegmental optic pathway and terminate in the basal optic nucleus. Ipsilateral retinal terminals occur in a pars dorsalis and a pars ventralis of the lateral geniculate nucleus, the dorsal optic nucleus, nucleus posterodorsalis, the basal optic nucleus, and in laminae 9 and 12 of the optic tectum. Rostrally, the ipsilateral tectal fibers occupy two zones along the medial and lateral tectal roof; these zones converge caudally and are continuous along the caudal wall of the tectum.     

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.