Tests of the regenerative capacity of tectal efferent axons in the frog, Rana pipiens

Experiments were designed to determine if neurons of the ranid optic tectum, a major target of the optic nerve, possess the same regenerative potential as optic axons. Normal tectal efferent (TE) projections were reexamined by using the anterograde transport of 3H-proline and autoradiography (n = 18), bulk-filling damaged TE axons with horseradish peroxidase (HRP; n = 18) and anterogradely transporting wheat germ agglutinin-HRP (n = 8) to label TE axons. Results were similar to reports that used degeneration methods (Rubinson: Brain Behav. Evol. 1:529-561, '68; Lazar: Acta. Biol. Hung. 20:171-183, '69). Following a brainstem hemisection just caudal to the nucleus isthmi (1-20 weeks), the ipsilateral descending TE pathway was autoradiographically examined (n = 20). While all other TE projections appeared normal, there was no detectable ipsilateral descending projection beyond the lesion site. Ascending TE axons were cut at the anterior tectal border by hemisecting the left diencephalon (LDH)--a lesion that also cuts optic axons projecting to the left tectum. There was no indication of TE axonal regeneration with the aid of autoradiography or HRP histochemistry 1-30 weeks postlesion (n = 48) even when the medial diencephalon was intentionally left intact (n = 4). However, in all four cases examined, optic axons regenerated following the same LDH where TE axonal regeneration failed (also see Stelzner, Lyon, and Strauss: Anat. Rec. 205:191A-192A, '83). Local effects of LDH should be similar for both the cut optic and cut TE axons. Other factors were tested that may contribute to the lack of TE axonal regeneration. Our results indicate that optic regeneration itself (n = 8), postaxotomy retrograde cell death of TE neurons (n = 6), deafferentation of the tectum of optic axons, and potential sprouting within tectal targets by intact contralateral TE axons (n = 10) are not critical factors aborting TE axonal regeneration. TE axons filled with HRP at chronic periods after LDH (n = 4) terminate anomalously near the LDH border. Many of these endings are similar to reactive endings or terminal clubs seen after axonal injury in the mammalian CNS. Our results suggest that this disparity in regenerative ability of optic and TE axons may be related to a difference in the responsive ability of these cell types to initiate or maintain axonal elongation after axotomy within the amphibian CNS environment.     

Evidence for the stability of positional markers in the goldfish tectum

Positional markers in the tectum, which are thought to guide growing axons to their target sites, have been proposed to be induced by axons, to be only transiently associated with the tectal cells, and then lost after long-term denervation periods (Schmidt: J. Comp. Neurol. 177:279-300, '78). To further investigate this concept, retinal axons were induced to regenerate into ipsilateral tecta which had been deprived of their retinal afferents for shorter (0-4 months) and longer periods (4-8 months). The paths of HRP-labeled regenerating axons of known retinal origin were traced and used as an operational test to decide whether the axons might navigate under the influence of positional markers. Two different kinds of experiments were performed: 1. The axons from a subpopulation of all ganglion cells in the retina were labeled by applying a small crystal of HRP at defined retinal regions. Independent of the denervation period of the tectum, the labeled regenerating axons traveled in abnormal but nonrandom routes. In early regeneration stages, axons exhibited signs of exploratory growth. They extended branches equipped with growth cones and filopodia into various regions of the tectum. In late regeneration stages, the axons lost these branches, exhibited U-turns and bends, and ended in terminal arbors in the retinotopic target region. These findings suggest that the axons travel under the influence of tectal positional markers and that these markers are not transient. 2. Axons from a surgically created temporal hemiretina were labeled by application of HRP to the optic nerve to test whether the temporal axons might expand into the caudal tectum in long-term-denervated tecta. The HRP-labeled axons coursed over rostral and midtectal regions. Instead of invading the caudal tectum they bent and terminated in the rostral tectal half. These results add further support for the conclusion that the path of regenerating retinal axons is governed by long-lasting positional markers.     

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.     

Trajectories of regenerating retinal axons in the goldfish tectum: I. A comparison of normal and regenerated axons at late regeneration stages

To visualize and compare the intratectal path of normal and regenerated retinal axons, HRP was applied to localized sites in the dorsotemporal and dorsonasal retina in normal goldfish and in goldfish at 3-12 months after optic nerve section. The anterogradely labeled axons were traced in tectal whole mounts. In normal animals the axons were confined to the appropriate ventral hemitectum. Therein they ran in very orderly routes (Stuermer and Easter: J. Neurosci. 4:1045-1051, '84) and terminated in regions retinotopic to the labeled ganglion cells in the retina. The terminal arbors of dorsotemporal axons resided in the ventrorostral tectum and those of dorsonasal axons in the ventrocaudal tectum. In regenerating animals the terminal arbors also resided at retinotopic regions, where they sometimes formed two separate clusters. In contrast to normal axons, the regenerating ones traveled in abnormal routes through the appropriate and inappropriate hemitectum. From various ectopic positions, they underwent course corrections to redirect their routes toward the retinotopic target region. In their approach toward their target sites, dorsotemporal and dorsonasal axons behaved differently in that the vast majority of dorsotemporal axons coursed over the more rostral tectum whereas dorsonasal axons progressed into the caudal tectal half. This differential behavior of regenerating dorsonasal and dorsotemporal axons was substantiated by a quantitative evaluation of axon numbers and orientations.     

Histochemical localization of cytochrome oxidase in the retina and optic tectum of normal goldfish: a combined cytochrome oxidase-horseradish peroxidase study

Cytochrome oxidase (C.O.) was histochemically localized in the normal retina and optic tectum of goldfish in order to examine the laminar and cellular oxidative metabolic organization of these structures. In the optic tectum, C.O. exhibited a distinct laminar, regional, and cellular distribution. The laminae with highest C.O. levels were those that receive optic input, suggesting a dominant role for visual activity in tectal function. This was demonstrated by colocalizing C.O. and HRP-filled optic fibers in the same section. However, the distribution of C.O. within the optic laminae was not uniform. Within the main optic layers, the SFGS, four metabolically distinct sublaminae were distinguished and designated from superficial to deep as sublaminae a, b, c, and d. The most intense reactivity was localized within SFGSa and SFGSd, followed by SFGSb, then SFGSc. In SFGSd, intense reactivity was found to occur specifically within a class of large diameter axons and terminals that were apparently optic since these were also labeled with HRP and cobaltous lysine applied to the optic nerve. Regional C.O. differences across the tectum were also noted. Low levels were found in neurons and optic terminals along the growing immature medial, lateral, and posterior edges of tectum, but were higher at the more mature anterior pole and central regions of tectum. This suggests that the oxidative metabolic activity is initially low in newly formed tectal neurons and optic axons, but gradually increases with neuronal growth and functional axon terminal maturation. Most C.O. staining was localized within neuropil, whereas the perikarya of most tectal neurons were only lightly reactive. Only a few neuron classes, mostly the relatively larger projection neurons, had darkly reactive perikarya. In the retina, intense C.O. reactivity was localized within the inner segments of photoreceptors, the inner and outer plexiform layers, and within certain classes of bipolar and ganglion cells. The large ganglion cells in particular were intensely reactive. Like the large diameter optic terminals in SFGSd, the large ganglion cells were preferentially filled with HRP, suggesting that they may project to tectum and are the source of the darkly reactive large diameter axons and terminals in sublamina SFGSd. We propose a new scheme to describe tectal lamination that integrates laminar differences in C.O. reactivity with classical histological work.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Synaptic interrelationships between the optic tectum and the ipsilateral nucleus isthmi in Rana pipiens

The nucleus isthmi is reciprocally connected to the ipsilateral optic tectum. Ablation of the nucleus isthmi compromises visually guided behavior that is mediated by the tectum. In this paper, horseradish peroxidase (HRP) histochemistry and electron microscopy were used to explore the synaptic interrelationships between the optic tectum and the ipsilateral nucleus isthmi. After localized injections of HRP into the optic tectum, there are retrogradely labeled isthmotectal neurons and orthogradely labeled fibers and terminals in the ipsilateral nucleus isthmi. These terminals contain round. Clear vesicles of medium diameter (40–52 nm). These terminals make synaptic contact with dendrites of nucleus isthmi cells. Almost half of these postsynaptic dendrites are retrogradely labeled, indicating that there are monosynaptic tectoisthmotectal connections. Localized HRP injection into the nucleus isthmi labels terminals primarily in tectal layers B, E, F, and 8. The terminals contain medium-sized clear vesicles and they form synaptic contacts with tectal dendrites. There are no instances of labeled isthmotectal terminals contacting labeled dendrites. Retrogradely labeled tectoisthmal neurons are contacted by unlabeled terminals containing medium-sized and small clear vesicles. Fifty-four percent of the labeled fibers connecting the nucleus isthmi and ipsilateral tectum are myelinated fibers (average diameter approximately 0.6 μm). The remainder are unmyelinated fibers (average diameter approximately 0.4 μm). © 1994 Wiley-Liss, Inc.     

Tectal projection neurons to the retinopetal nucleus in the filefish

Following horseradish peroxidase (HRP) injections into the preoptic retinopetal nucleus (PRN), neurons in the ipsilateral optic tectum were labeled retrogradely. Labeled neurons exhibited a 'Golgi-like' appearance, somata of these neurons were pyriform or round, and most of them were located in the stratum album centrale (SAC) or the stratum periventriculare (SPV). These neurons had a long apical dendrite, which ramified in the upper-half of SGC into horizontally arborized dendritic fields. The main trunk of the apical dendrites also gave off several branches in the stratum fibrosum et griseum superficiale (SFGS) and reached the stratum opticum (SO). These neurons resemble the 'large pyriform neurons' of Vanegas et al. (Vanegas, H., Laufer, M. and Amat, J., The optic tectum of a perciform teleost. I. General configuration and cytoarchitecture, J. Comp. Neurol., 154 (1974) 43-60) except that in the tecto-PRN neurons the axons originates from the apical dendritic shaft at or near the level of the SAC. Judging from their dendritic patterns, the tectal neurons projecting indirectly to the retina may receive non-retinal inputs besides the retinal input.     

Rules of order in the retinotectal fascicles of goldfish

Individual fascicles of retinal axons were labeled in the goldfish tectum with horseradish peroxidase (HRP). The contralateral retina was later processed for HRP histochemistry to mark the cells that had axons in the fascicles. Labeled cells were found in a partial half anulus in ventral hemiretina, centered on the optic disk. The distance of the partial anulus from the disk depended on which tectal fascicle had been labeled; the more rostrocentral the fascicle, the smaller was the annular radius. The angular subtense of the partial anulus with respect to the disk depended on where (along its tectal course) the fascicle had been labeled; the more rostral the label site, the longer was the angular subtense. These results were interpreted in the context of retinotectal growth, and it was inferred that the axons followed two rules: (1) grow in along the edge of the tectum and (2) exit and terminate in order, axons from temporal retina first, nasal retina last. These rules would produce a retinotopic projection in peripheral tectum, but they require that some of the terminals already in place must shift as the tectum grows.     

Intraocular tetrodotoxin reduces axonal transport and transcellular transfer of adenosine and other nucleosides in the visual system of goldfish

Intraocular injection of tetrodotoxin (TTX) in goldfish, which abolishes physiological activity in the optic axons, decreased by up to about 30% the amount of radioactively labeled adenosine, uridine and guanosine (and their nucleotide derivatives) that was axonally transported in the optic nerve. The amount of labeled nucleoside that reached the optic tectum and became incorporated into RNA in the postsynaptic tectal neurons and glial cells was reduced by up to about 50%. There was no change, however, in the amount of transported nucleoside that became incorporated into RNA in the optic nerve glia. The TTX-induced changes were eliminated when axonal transport was blocked with vincristine, indicating that this change did not involve material moving along the nerve by diffusion. If the TTX injection was delayed until several hours after labeling of the transported materials, the transported labeled nucleoside in the nerve was reduced very little, but the RNA labeling in the tectum was reduced just as much as when TTX was given prior to labeling. This indicates that the labeling of the tectal cells was affected more by the level of activity in the pathway than by the amount of transported nucleoside reaching the optic nerve terminals. It appears likely, therefore, that the process most affected by the decrease in physiological activity is the release of nucleoside from the terminals of the presynaptic neurons or its uptake into postsynaptic tectal neurons and glia. The fact that physiological activity may modify the amount of axonally transported nucleosides made available for metabolism (including RNA synthesis) in postsynaptic neurons may provide an explanation for activity-linked neurotrophic effects.     

Retinotopic organization of the developing retinotectal projection in the zebrafish embryo

Developing retinal axons in the zebrafish embryo were stained with HRP or with the fluorescent dyes dil and diO to study the formation of the retinotectal projection. Retinal axons leave the eye at 34-36 hr postfertilization (PF), invade the tectum at 46-48 hr PF, and innervate the tectal neuropil at 70-72 hr PF. Dorsal and ventral axons occupy separate aspects of the optic nerve and tract and pass into their retinotopically appropriate ventral and dorsal hemitectum, respectively. Nasal and temporal axons are segregated in the nerve, mixed in the tract, and are coextensive over the rostral half of tectum until 56 hr PF. They then segregate again, due to the progression of nasal axons into the open caudal tectum. Thus, at 70-72 hr PF, dorsal and ventral as well as temporal and nasal axons occupy their retinotopically appropriate tectal quadrants. After ablation of the temporal retina prior to the time of axonal outgrowth, the nasal axons bypass the vacant rostral tectum to terminate in the caudal tectal half. Temporal axons in the absence of nasal axons remain restricted to their appropriate rostral tectal half, suggesting that nasal and temporal axons possess a preference for their retinotopically appropriate tectal domains. Measurements of individual terminal arbors and the tectal areas in embryos and in adult zebrafish showed that individual arbors are large with respect to the embryonic tectum but are about 14-15 times smaller than in the adult. However, the proportion of tectum covered by embryonic arbors is about 7 times larger than in the adult, suggesting that a higher precision of the adult projection is achieved as a result of a greater enlargement of the tectum than of the arbors.     

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.     

Expansion of the ipsilateral retinal projection in the frog brain during optic nerve regeneration: Sequence of reinnervation and retinotopic organization

The sequential reinnervation and distribution of optics axons in diencephalic and mesencephalic targets was studied after crushing the optic nerve in 37 adult Rana pipiens. Following intravitreal injection of ³H-proline at various times after nerve crush the distribution of regenerating optic axons was traced using autoradiographic methods. The maximal distribution of regenerating axons was reached between 6 and 8 weeks after optic nerve crush. On the contralateral side of the brain, the distribution of fibers was similar to the normal projection. Ipsilaterally, silver grain density was greater than normal in the optic tract and projections were expanded to all optic targets on this side of the brain. These abnormal projections were sustained for a least 6 months after nerve crush. The sequence of reinnervation of targets on both sides of the brain differed from normal development. Unlike development, regenerating optic axons were found in the ipsilateral optic tract prior to the time they were found on the contralateral side of the brain. Also unlike development, regenerating axons did not begin to reinnervate targets in the anterior thalamus until several weeks after reinnervation of the posterior thalamus and tectum had begun. The expanded distribution of regenerating axons within optic targets on the ipsilateral side of the brain became evident at the time optic axons first invaded each area. Most optic axons appeared to regenerate from the point of nerve crush. The retinal stump of the crushed nerve was filled with labeled axons in all six frogs given intravitreal ³H-proline injections between 1 and 7 days after nerve crush. In addition, using a modified Fink-Heimer method, few degenerating axons were found in the retinal stump of four frogs sacrificed between 2 and 8 days after optic nerve crush. In ten frogs studied between 3 and 6 months after nerve crush, horseradish peroxidase (HRP) was placed on different portions of the tectum ipsilateral to the crush. In each case HRP was retrogradely transported to ganglion cells in both retinae. The cells labeled in the ipsilateral retina corresponded in position to the same region of the contralateral retina although many fewer cells were labeled ipsilaterally. Cutting the optic tract on the side opposite the HRP placement did not affect the results. No ganglion cells were labeled in the ipsilateral retina of two frogs not receiving optic nerve crush. These results show that axons from all parts of the retina regenerate to the ipsilateral side of the brain during optic nerve regeneration and the distribution of these misrouted axons, at least to the tectum, overlaps the intact distribution from the other eye. Differences between development and regeneration in the patterns of growth of optic axons may be related to this anomaly.     

Connections of eye-saccade-related areas within mesencephalic reticular formation with the optic tectum in goldfish

Physiological studies demonstrate that separate sites within the mesencephalic reticular formation (MRF) can evoke eye saccades with different preferred directions. Furthermore, anatomical research suggests that a tectoreticulotectal circuit organized in accordance with the tectal eye movement map is present. However, whether the reticulotectal projection shifts with the gaze map present in the MRF is unknown. We explored this question in goldfish, by injecting biotin dextran amine within MRF sites that evoked upward, downward, oblique, and horizontal eye saccades. Then, we analyzed the labeling in the optic tectum. The main findings can be summarized as follows. 1) The MRF and the optic tectum were connected by separate axons of the tectobulbar tract. 2) The MRF was reciprocally connected mainly with the ipsilateral tectal lobe, but also with the contralateral one. 3) The MRF received projections chiefly from neurons located within intermediate and deep tectal layers. In addition, the MRF projections terminated primarily within the intermediate tectal layer. 4) The distribution of labeled neurons in the tectum shifted with the different MRF sites in a manner consistent with the tectal motor map. The area containing these cells was targeted by a high-density reticulotectal projection. In addition to this high-density topographic projection, there was a low-density one spread throughout the tectum. 5) Occasionally, boutons were observed adjacent to tectal labeled neurons. We conclude that the organization of the reticulotectal circuit is consistent with the functional topography of the MRF and that the MRF participates in a tectoreticulotectal feedback circuit.     

Morphology and location of tectal projection neurons in frogs: A study with hrp and cobalt-filling

Tectal projection neurons were labeled by retrograde transport of horseradish peroxidase (HRP) or cobaltic-lysine. The tracer substances were delivered iontophoretically or by pressure injection or diffusion into various regions of the brain or spinal cord. Histochemical procedures allowed identification of labeled cells projecting to the injected regions. Many neurons were filled with cobaltic-lysine, resulting in a Golgi-like staining. After cobalt injections in the diencephalon most of the labeled cells, identified as small piriform neurons, were located in layer 8 of the tectum. Two types of small piriform neurons were distinguished. Type 1 neurons have flat dendritic arborizations confined to lamina D, while the dendrites of type 2 cells may span all of the superficial tectal strata. In smaller numbers large piriform, pyramidal, and ganglionic cells of the periventricular tectal layers were labeled after diencephalic injections. Rhombencephalic cobalt and HRP injections labeled cells whose axons form the tectobulbospinal tract. The neurons most frequently labeled were large ganglionic cells. Ipsilaterally, the majority of their somata were located in layer 7, and their dendrites arborized mainly in lamina F. Con-tralaterally, labeled ganglionic cell somata occupied the top of layer 6, and most of their dendritic end-branches entered lamina B. The possible functional significance of this anatomical arrangement is discussed. After tectal cobalt injections the topography of the tectoisthmic projection and the terminals of tectal efferent fibers in the diencephalon and brainstem were observed. It is concluded that the organization of frog tec-tofugal pathways is very similar to that of mammals.     

The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study

Retrograde transport of horseradish peroxidase (HRP) from the region of retinal genglion cell axon terminals back to the cell bodies has been studied by light and electron microscopy. After injection of HRP into the chick optic tectum, it was taken up by axon terminals and unmyelinated axons as well as by other processes and cell bodies of the outer tectal layers. Subsequently the HRP was obseved in vesicles, multivesicular bodies, cup-shaped organelles and small tubules within axons in the stratum opticum, optic tract, optic nerve and optic fiber layer of the retina with accumulation in the retinal ganglion cell bodies. Pinocytosis of extracellular HRP along the axon shaft was rare. After a short postinjection interval, HRP was found in organelles within the axons of the optic nerve but not in the extracellular spaces. After larger injections or longer postinjection intervals, extracellular HRP diffused from the injection site to the back of the eye, but none was found in the extracellular spaces of the retina; ganglion cells were the only cells of the retina which contained HRP product. HRP disappeared from the cell bodies 3–4 days after transport. These findings suport the concept of intraaxonal retrograde movement of HRP. Within axons the vesicles carrying HRP frequently were partially or completely surrounded by a regualr array of microtubules. Doses of colchicine greater than 5–10 µ/eye administered 4 days before tectal injection of HRP interfered with the uptake and/or transport of HRP. HRP also moved in an anterograde direction in membrane-bound vesicles within the ganglion cell axons, although apparently more slowly and in smaller quantities than in the retrograde direction. The localization of HRP in neurons of the isthmo-optic nucleus following intravitreal injections has also been studied. The marker enzyme was found in neuronal cell bodies 4 hours after injection, indicating a rate of retrograde transport of at least 84 mm/day in these neurons.     

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.     

Widespread regeneration of central axons through the central nervous system of the goldfish

The ability of neurons in the brain of the goldfish to regenerate their axons was examined using anterograde and retrograde axonal tracing methods. It was found that all neurons in the thalamus and brainstem that project to the optic tectum can regenerate their axons after removal of the tectal lobe, and that their axons can often grow for considerably greater distances than they normally extend. In addition, they can penetrate (and, presumably, innervate) their normal target tissue, even if it is displaced into an ectopic location.     

Anomalous retrograde labeling of brain cells from the eye in the goldfish: evidence for long distance growth of sprouted neurites

We have used retrograde labeling with horseradish peroxidase (HRP) and a wheat germ agglutinin conjugate of HRP (WGA:HRP) to investigate the projections of the nucleus postglomerulosus (nPg) both in normal goldfish and in animals which had undergone retinal removal. In normal animals, our evidence indicates that nPg projects only to the optic tectum. Using small HRP and WGA:HRP application sites in the tectum, we have shown that nPg cells have broadly spread terminals in the tectal neuropil and that there is no obvious correspondence between the rostrocaudal axis of the nPg and the deployment of the terminal arbors of its cells along the rostrocaudal axis of the tectum. In addition, we found no evidence for an nPg projection to the eye in normal animals. After retinal removal we found that nPg cells were more readily backfilled from small tectal applications of HRP. However, our most interesting observation was that at 4-6 weeks and more after ocular surgery, we could retrogradely label the cells of the nPg with intraocular or retroocular injections of WGA:HRP. At the same postoperative times, we were also able to label neurites in the atrophied optic nerve by microinjecting WGA:HRP into the contralateral midbrain tegmentum. Finally, we found that the cells of the nPg undergo a hypertrophic response, similar to that seen in other neurons after axotomy, following retinal removal or section of the dorsomedial brachium of the optic tract. Thus, these cells respond to retinal denervation of the tectum with a response characteristic of axotomized cells although their axons have not been cut. Similar changes were also seen in the nucleus isthmi on both sides of the brain following retinal removal. We interpret our data to indicate that cells of the nPg can respond to optic (and thus heterotypic) denervation of their terminal field by sprouting processes which grow away from the terminal field, through denervated optic pathways, to the retinaless eye. This interpretation requires that the sprouted processes grow for several millimeters.     

Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation

The development of the optic tectum and the establishment of retinotectal projections were investigated in the quail embryo from day E2 to hatching day (E16) with Cresyl violet-thionine, silver staining and anterograde axonal tracing methods. Both tectal cytodifferentiation and retinotectal innervation occur according to a rostroventral-caudodorsal gradient. Radial migration of postmitotic neurons starts on day E4. At E14, the tectum is fully laminated. Optic fibers reach the tectum on day E5 and cover its surface on day E10. 'Golgi-like' staining of optic fibers with HRP injected in vitro on the surface of the tectum reveals that: growing fronts are formed exclusively by axons extending over the tectal surface; fibers penetrating the outer tectal layers are always observed behind the growing fronts; the penetrating fibers are either the tip of the optic axons or collateral branches; as they penetrate the tectum, optic fibers give off branches which may extend for long distances within their terminal domains; the optic fiber terminal arbors acquire their mature morphology by day E14. The temporal sequence of retinotectal development in the quail was compared to that already established for the chick, thus providing a basis for further investigation of the development of the retinotectal system in chimeric avian embryos obtained after xenoplastic transplantation of quail tectal primordia into the chick neural tube.