Pathway choice by regenerating optic fibers following tectal lobectomy in the goldfish: Inferences from the study of gliosis in tectal efferent bundles

Cell counts in various fiber bundles of the goldfish brain have demonstrated a profound, but transient gliosis of tectal (and pretectal) efferent pathways following removal of a tectal lobe. In the majority of cases, the pathways which underwent gliosis were also those which were penetrated by regenerating optic fibers which had been sectioned by the tectal surgery. However, the dorsal trunk of the horizontal commissure on the intact side of the brain showed only a minimal gliotic response but was consistently innervated by the optic fibers. Conversely, the ansate commissure and the crossed tectobulbar tract invariably demonstrated a marked gliotic response but only rarely received more than minimal innervation by the regenerating fibers.These observations are discussed with regard to the modifications which they demand of the hypothesis that degenerating axon bundles in the goldfish brain are in some way attractive to regenerating axons.     

Time course and pattern of optic fiber regeneration following tectal lobe removal in the goldfish

Following single tectal lobe removal in the adult goldfish, Carassius auratus, the pattern of regeneration of the optic fibers which had previously projected to that tectum was examined at 1, 2, 4, 6, 8, 10, and 12 weeks postoperative using ³H-proline radioautography. We found that regenerating optic fibers grew across the midline through the transverse, minor, horizontal, and posterior commissures to innervate the remaining tectum. At early postoperative times innervation of the tectum was continuous, while later, the regenerating fibers segregated into discrete patches in the superficial layers of the tectum. In addition, regenerating fibers also grew into non-optic centers/pathways such as the habenula, the fasciculus retroflexus, the forebrain, the torus semicircularis, the valvula and corpus cerebelli, the hypothalamus, and the medulla. While optic fibers were no longer apparent in the habenula and the fasciculus retroflexus after 2 weeks postoperative, all other structures were still occupied by the fibers at 12 weeks postoperative. Since most of the innervated pathways were either tectal efferent pathways, which should contain degenerating debris and proliferating glial cells after the tectal removal, or pathways closely associated with traumatized areas, we suggest that degenerating axonal debris and proliferating glia may play an important role in guiding regenerating fibers in this system.     

Retinal projection to a rotated tectal reimplant following long-term tectal denervation in adult goldfish

Following optic nerve crush, the precise termination sites of regenerating goldfish optic axons may be influenced by the presence or abscence of degenerating axonal debris from the previous projection. We investigated whether tectal polarity reversal can be induced in the absence of axonal debris The right optic tectum was denervated by contralateral eye removal. One year later, when no debris was present, a piece of the right tectum was rotated and innervation by the right eye was induced by removal of the left tectum. The new ipsilateral projection to the rotated region was correspondingly rotated. It is concluded that retention of tectal polarity is not dependent upon degenerating axonal debris.     

Denervation of non-optic brain areas along the course of the optic tract does not affect the success of optic nerve regeneration in frogs

Unsuccessful axonal regeneration in frog or goldfish spinal cord is thought to be due in part to inappropriate denervated synaptic sites attracting regenerating axons as they pass by the lesion zone (Bernstein and Bernstein, '69; Bernstein and Gelderd, '73). The reported success of optic nerve regeneration in these same animals may result because there are no inappropriate synaptic sites available to the regenerating axons. To test for this we denervated areas within thalamus and mesencephalon which lie along the path of regenerating optic axons to determine whether or not abnormal connections would form in these areas and affect the success of optic nerve regeneration. After transection of the brainstem through the isthmal region, terminal degeneration was found in several zones adjacent to optic nerve targets or the optic tract (i.e., nucleus rotundus, corpus geniculatum laterale, and torus semicircularis). In adult frogs receiving left isthmal transection, we also crushed the right optic nerve and then examined the regenerated optic projection at intervals up to six months after nerve crush using anterograde transport of ³H-proline. In no instance did the regenerating optic axons alter their distribution within visual neuropil zones or invade those areas deafferented by the isthmal lesion. Histological study showed that the axons cut by the isthmal lesion did not regenerate back to their sites to prevent the invasion of optic axons into these zones. We then attempted to force optic axons into foreign territory by removing a major projection zone, the optic tectum. With tectal ablation and isthmal transection, regenerating optic axons were offered synaptic space made available by both lesions. However, we found abnormal optic projections only in the middle and posterior thalamic neuropil and in the remaining tectal hemisphere. Optic axons did not expand into any of the areas deafferented by the isthmal transection, even though some of them were further denervated by the tectal ablation. We conclude that optic axons will not invade non-optic areas deafferented by an isthmal lesion even if a large number of optic axons have no normal target to innervate.     

The miswired goldfish brain: Long-term persistence and transience of retinal projections following tectal lobe removal

Following tectal lobe removal in the goldfish, optic fibers, which are sectioned by the surgery, regenerate through various abnormal pathways to both the optic tectal lobe which remains and to various non-optic sites in the brain. In this communication we present anatomical evidence that regenerated optic fibers in many of these pathways atrophy or disappear within several months after surgery. By contrast, in some pathways the regenerated fibers persist for at least 1.5 years. We suggest that the majority of fibers which persist for long periods do so because they have reached the remaining tectal lobe and been able to make synapses there.The results from this system are briefly compared to those which have been obtained in studies of regeneration in the peripheral nervous system and parallels between the two are noted.     

Intraocular colchicine inhibits competition between resident and foreign optic axons for functional connections in the doubly innervated goldfish optic tectum

After unilateral optic tectum ablation in the goldfish, regenerating optic axons grow into the optic layers of the remaining ipsilateral tectal lobe and regain visual function. The terminal arbors of the foreign fibers are initially diffusely distributed among the resident optic axons, but within two months the axon terminals from each retina are seen to segregate into irregular ocular dominance patches. Visual recovery is delayed until after segregation. This suggests that the foreign fibers compete with the residents for tectal targets and that the segregation of axon terminations is an anatomical characteristic of the process. Here we investigate whether inhibiting axonal transport in the resident fibers inhibits competition with foreign fibers. The eye contralateral to the intact tectal lobe received a single injection of 0.1 μg colchicine, which does not block vision with the intact eye. We measured visual function using a classical conditioning technique. Segregation of axon terminations was examined shortly following visual recovery by autoradiography. The no-drug control fish showed reappearance of vision with the experimental eye at 9 weeks postoperatively and ocular dominance patches were well developed. Colchicine administered to the intact eye (resident fibers) several weeks postsurgery decreased the time to reappearance of vision with the experimental eye by several weeks. Autoradiography revealed some signs of axonal segregation but the labeled foreign axons were mainly continuously distributed. Administration of colchicine at the time of tectum ablation, or of lumicolchicine at two weeks postoperatively produced normal visual recovery times. Fast axonal transport of³H-labeled protein was inhibited by 1.0 and 0.5 μg but not by 0.1 μg of colchicine or by 1.0 μg of lumicolchicine. Previous studies showed that while 0.1 μg of colchicine does not block vision it is sufficient to inhibit axonal regeneration following optic nerve crush. We conclude that two retinas can functionally innervate one tectum without forming conspicuous ocular dominance columns, and that the ability of residents to compete with the in-growing foreign axons is very sensitive to inhibition of axoplasmic transport or other processes that are inhibited by intraocular colchicine.     

Visual recovery in goldfish following unilateral optic tectum ablation: Evidence of competition between optic axons for tectal targets

Anatomical studies suggest that regenerating optic axons which invade the ipsilateral lobe of the optic tectum following ablation of the contralateral lobe compete with resident optic axons for synaptic sites on tectal neurons. Invader optic axons are initially uniformly distributed over the entire tectal lobe. With time, the invader and resident optic axons progressively segregate so that the invaders are localized in bands or islands separated by areas that are innervated mainly by the residents. When the resident optic axons are destroyed by ablating the eye opposite to the experimental eye, the invader axons remain continuously distributed and the segregation process apparently does not occur. We investigated the relationship between the segregation process and the recovery of visual function by the invader axons. Visual recovery was measured with a behavioral method in which the index of vision was the occurrence of a branchial suppression response to a moving spot of red light that was classically conditioned to an electric shock stimulus. The minimum time to reappearance of vision following ablation of the contralateral lobe of the tectum in two-eye fish was similar to the reported time of onset of the segregation process. Visual recovery occurred sooner when the opposite eye was removed. The restored vision in both groups disappeared following subsequent ablation of the remaining lobe of the tectum. These results suggest that the goldfish optic tectum normally contains no free synaptic sites for anomalous optic afferents and that the invader axons must compete for targets with the resident optic afferents. The invader axons can apparently remain unconnected or non-functional for several weeks following their arrival in the ipsilateral tectal lobe.     

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.     

On the formation of eye dominance stripes and patches in the doubly-innervated optic tectum of the chick

By surgically dividing the region of the presumptive optic chiasm in chick embryos on the third day of incubation (around stage 15), we have been able to induce substantial numbers of optic nerve fibers to grow aberrantly into the ipsilateral optic tract. As a result, many of the visual centers that are normally innervated only by fibers from the contralateral retina received fibers from both eyes. The proportion of fibers going to each tectal lobe varied from case to case, but in about one-third of the animals the tectal lobes received approximately equal numbers of fibers from each eye. In animals that survived until embryonic days 17-19 (which is beyond the period of retinal ganglion cell death) labeling of the two eyes with WGA-HRP and [3H]proline respectively, revealed a pattern of sharply defined eye dominance stripes or patches in the stratum griseum et fibrosum superficiale (SGFS) of the optic tectum, and in the ventral lateral geniculate nucleus. Less clearly segregated eye dominance zones were seen in the ectomammillary nucleus and the nucleus externus. The size and distribution of the stripes varied depending on the number of fibers projecting from each eye to a given tectal lobe; the minimum size was about 75 micron, while the maximum was large enough to occupy almost the entire tectal lobe. In animals in which the tectal input from the two eyes was roughly equal, the stripes varied in width between 75 micron and about one-third of the surface of the tectal lobe. The orientation of the stripes was consistently orthogonal to the direction of fiber ingrowth from the optic tract. From the earliest stages of optic fiber ingrowth, the fibers from the two eyes are completely intermixed in the stratum opticum (SO). However, on embryonic day 12, shortly after they have begun to penetrate into the SGFS, they are already segregated into stripes, although the stripe borders are very fuzzy. This suggests that the fibers from the two eyes may overlap at this stage. The phase of stripe formation coincides with that of naturally occurring retinal ganglion cell death, and we suggest that the two processes are interlinked.     

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.     

Long-term degeneration renders central tracts refractory to penetration by regenerating optic fibers

We have examined time-dependent changes in the ability of degenerating central pathways in the goldfish to be penetrated by regenerative axons. We have found that when a tract has degenerated for 2–5 weeks it is readily penetrated by regenerating optic fibers. However, tracts which degenerated for any longer than 6 weeks, before being exposed to the regenerating fibers, were only sparsely penetrated by them. We conclude that over a period of no less than 6 weeks, degenerating central tracts in the goldfish change their character and become relatively refractory to penetration by regenerating axons.     

Anomalous retinal projection after removal of contralateral optic tectum in adult goldfish

Retinotectal projections were mapped in a series of adult goldfish at various intervals after complete ablation of one tectum, or after enucleation of one eye and removal of its ipsilateral tectum. In the first case, regenerating optic axons entered the ipsilateral tectum and innervated it retinotopically; the normal and ipsilateral projections overlapped each other. In the second case, the remaining eye projected over the remaining ipsilateral tectum in normal retinotopic order. Thus, the presence of already innervated optic tectum does not deter regenerating axons from innervating it. It is suggested that the visual projection of goldfish tends to retain its completeness in the absence of normal terminal spaces.     

Restoration of vision in genetically eyeless axolotls (Ambystoma mexicanum).

Eyes grafted into genetically eyeless axolotls at embryonic stages 26 or 27 (early tailbud stage) are capable of establishing retinotectal connections and restoring near normal vision. Normal vestibulo-ocular reflexes are also present in most of the eyeless mutants having grafted eyes. The animals are capable of accurately localizing objects in visual space and demonstrate following movements in an optokinetic drum. Evoked potentials can be recorded from the surfaces of the tectal lobes of eyeless mutants having a right eye graft which do not differ significantly from those recorded from a normal animal, except that recordings can still be obtained from the ipsilateral tectal lobe in the former following section of the intertectal fibers. This indication of direct retinotectal connections to the ipsilateral tectum was confirmed by histological examination which also showed that the optic fibers entering the diencephalon high on the lateral wall are initially directed toward the normal optic tract position before proceeding to be tectum.     

Visual orienting response in goldfish: a multidisciplinary study

The neural basis underlying the orienting response has been thoroughly studied in frontal-eyed mammals. However, in non-mammalian species, including fish, it remains almost unknown. Therefore, we studied the contribution of the optic tectum and the mesencephalic reticular formation to the performance of the orienting response in goldfish, using behavioural, physiological, and anatomical tracer techniques. The appearance of a visual stimulus (a pellet of food) in the environment of a goldfish evoked a turn of the body to reorient the line of sight. Left-tectal lobe ablation abolished the orienting turn response towards the contralateral hemifield. Electrical microstimulation of the optic tectum suggested the presence of a motor map, which is in correspondence with the overlying visual representation, as previously reported in other vertebrates. The tracer biotin-dextran amine was injected into different functionally identified tectal zones. The results showed that rostral and caudal poles of the mesencephalic reticular formation receive outflow mainly from the rostral and caudal tectal poles, respectively. This suggests that the tectal wiring with downstream structures is site-dependent. Furthermore, the electrical activation of rostral and caudal mesencephalic reticular formation revealed a different contribution to vertical and horizontal orienting eye movements. We conclude that the basic neural system coding the orienting response appears early in phylogenesis, although some specific characteristics are selected by adaptive pressure.     

Target selection by surgically misdirected optic fibers in the tectum of goldfish

This study tested the capacity of regenerating optic fibers to read tectal markers and thereby grow to their appropriate tectal loci when initial position, optic pathway, and interfiber interactions are eliminated as useful cues. The stability of these markers with long-term optic denervation of the tectum was also examined. In adult goldfish optic fibers innervating lateroposterior optic tectum were dissected free of tectum and inserted into the medial anterior region of the opposite "host" tectum. Normally, fibers at this position either innervate medial anterior tectum or follow the medial division of the optic pathway into medioposterior tectum. Host tectum was denervated of all other optic fibers by enucleating its contralateral eye either at the time of the deflection or at various times up to 18 months prior to deflection. The regeneration of these deflected fibers into host tectum was examined by autoradiography and electrophysiology at 1 to 11 months later. At the insertion site deflected fibers split into two groups of roughly equal size. One group directly entered the optic layers of medial tectum and grew posterolaterally across the medial half of tectum into the lateral half. The second group followed an almost direct path to the lateral tectum, sometimes traversing through the deep cell layers of tectum in which optic fibers are not usually found. These fibers subsequently entered the optic layers at the lateral edge of tectum and grew posteriorly. This second path was not seen in controls in which optic fibers from medioposterior tectum were similarly deflected. Instead growth was almost entirely posteriorly directed. On the average by 1.5 months deflected lateroposterior fibers were preferentially distributed in the lateral half of the tectum. Densitometric measurements indicated nearly a 4-fold difference in lateroposterior compared with medial posterior labeling. By contrast, controls in which medial posterior fibers were deflected had 4 times more grains medially than laterally. There was also a posterior over anterior preference, but this was weak. There was no suggestion that long periods of optic denervation prior to deflection or long postoperative periods after deflection of lateroposterior fibers diminished the lateral over medial preference. These findings support the idea that stable tectal markers exist which are differentially read by medial and lateral optic fibers. However, in no case was the innervation by deflected fibers as selective as in the normal projection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrophoretic analysis of specific proteins in the regenerating goldfish retinotectal pathway

Proteins in the goldfish retinotectal pathway were analyzed by 2-D gel electrophoresis, under conditions of optic nerve crush or eye removal. A specific cluster of proteins was detected, consisting of 4 components, all of which are highly concentrated in the intact optic nerve. Two components were not detectable in non-visual areas of the goldfish brain. The total cluster was diminished by about 80% in the denervated optic tectum, and its level was restored during optic nerve regeneration. These data were interpreted as evidence for visual system-specific proteins in the goldfish retinotectal pathway.     

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.     

Effect of target removal on goldfish optic nerve regeneration: analysis of fast axonally transported proteins.

How is axonal transport in regenerating neurons affected by contact with their synaptic target? We investigated whether removing the target (homotopic) lobe of the goldfish optic tectum altered the incorporation of 3H-proline into fast axonally transported proteins in the regenerating optic nerve. Regeneration was induced either by an optic tract lesion (to reveal the changes in the original axon segment that remained connected to the cell body) or by an optic nerve lesion (to reveal the changes in the newly formed axon segment). Of 26 proteins analyzed by 2-dimensional gel electrophoresis and fluorography, all but one showed increased labeling as a result of tectal lobe ablation. By 2 d after the lesion, significantly increased labeling of some proteins was seen with a 6-hr labeling interval, but not with a 24-hr labeling interval. This is probably indicative of an increased velocity of transport, which may have been a nonspecific consequence of the surgery. Otherwise, tectal lobe removal had relatively little effect until 3 weeks, when there was a transitory increase in labeling of transported proteins in the new axon segments of the tectum-ablated animals. Beginning at 5 weeks, tectal lobe ablation caused considerably higher labeling of many of the proteins in the original axon segments. Because this was seen with both 6-hr and 24-hr labeling intervals, it is probably indicative of increased protein synthesis. The increased synthesis lasted until at least 12 weeks, though some proteins were beginning to show a diminished effect at this time. In the late stages of regeneration (8-12 weeks), there was also increased labeling of proteins in the new axon segments as a result of the absence of the target tectal lobe. This included a disproportionately large increase in the relative contribution of cytoskeletal proteins and of protein 4, which is the goldfish equivalent of the growth-associated protein GAP-43 (neuromodulin). We conclude that, after the regenerating axons begin to innervate the tectum, the expression of most of the proteins in fast axonal transport is down-regulated by interaction between the axons and their target. However, the changes in expression may be preceded by a modulation of the turnover and/or deposition of proteins in the newly formed axon segment.     

Developing retinotectal projection in larval goldfish

The retinotectal projection in larval goldfish was studied with the aid of anterograde filling of optic fibers with HRP applied to the retina. The results show that optic fibers have already reached the tectum and begun to form terminal arbors in newly hatched fish. The projection is topographic in that fibers from local regions of the retina project to discrete patches of tectum, with the smallest patch covering 3.5% of the total surface area of tectal neuropil. Many fibers in young larvae have numerous short side branches along their length and only some of them show evidence of terminal sprouting. The arbors are approximately elliptical in shape and average about 1,500 microns 2. Growth cones are seen frequently. In older larvae, terminal arbors are larger and more highly branched, and they have begun to resemble those in adult fish. Fibers terminate in two strata; those in the upper layer are smaller (1,800 microns 2 on average) than those in the deeper stratum (4,000 microns 2 on average). The fraction of tectal surface area covered by individual arbors (the "tectal coverage") ranges from 1.5% to 3% of the total surface area of the tectal neuropil. In contrast, the tectal coverage of individual arbors in young adult goldfish is much smaller, ranging from 0.02% to 0.42% of tectal surface area (Stuermer, '84, and unpublished). This apparent increase in precision of the map in older animals is not due to retraction of arbors, which are slightly larger in adults, but is accounted for by overall tectal growth: the tectal neuropil in goldfish increases in area by about 250-fold during this period (Raymond, '86).