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.     

Anatomical evidence for the influence of degenerating pathways on regenerating optic fibers following surgical manipulations in the visual system of the goldfish

We have used [³H]proline radioautography to trace regenerating optic fibers in the goldfish following: (1) the removal of the right tectal lobe and the right eye, and (2) the removal of both tectal lobes. Our results indicate that following the removal of the right tectal lobe and the right eye, both the denervated tectal efferent pathways, and the denervated visual pathways and terminal zones of the enucleated eye were penetrated by the regenerating optic fibers. In addition, following bilateral lobectomy, the denervated tectal efferent pathways were bilaterally penetrated by the regenerating fibers. Since, in both types of operations, these denervated pathways and terminal zones should undergo degeneration, our results support the suggestion that the presence of degenerating axonal debris and proliferating glia may play an important role in guiding regenerating optic fibers in the visual system of the goldfish.     

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.     

Selective retinal reinnervation of a surgically created tectal island in goldfish. II. Electron microscopic analysis

In the preceding study (Edwards et al., '85), we showed that regenerating optic axons reestablish a topographically restricted projection to a caudal tectal island created by surgical removal of a 1-mm-wide strip of caudal tectum in goldfish. In the present ultrastructural study, we evaluated the dependence of this axonal outgrowth on the presence of tectal target tissue caudal to the gap. Axon counts in the lesion zone were compared between cases with complete caudal tectal ablation and cases with ablation sparing a caudal tectal island (with and without optic nerve crush). During the postoperative interval of 20-50 days (early period), up to about 1,000 unmyelinated axons with features characteristic of optic axons were present in numerous small subpial bundles in both preparations. In the subsequent interval of 50-110 days (middle period), less than 200 axons were counted caudal to simple half-tecta, whereas 4,000-14,000 myelinated and unmyelinated axons were present in a few large bundles which crossed the lesion zone of tectal island cases. In this period, optic terminals could be demonstrated in the tectal island using the anterograde horseradish peroxidase method. At 170-300 days after surgery (late period), bridging bundles contained between 2,000 and 6,000 largely myelinated axons. We conclude that caudal tectal tissue is not necessary for the initial outgrowth of a small number of axons beyond a rostral half-tectum. The target is essential, however, for the maintenance of these axon fascicles and for the subsequent massive outgrowth of axons to the island. The contributions of glial guidance, diffuse exploratory outgrowth, and target-produced trophic factors to the formation of an initially exuberant projection to the island are discussed. A process of selective axon collateral withdrawal is proposed to account for the decrease in axon numbers within bridging bundles in the late period and for the late restriction in the retinal origin of the island projection indicated by results in the preceding study (Edwards et al., '85).     

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.     

Vision in goldfish following bilateral tectal ablation

Visual sensitivity of optic tectum-ablated goldfish was investigated using a classical conditioning technique. Intact fish were screened to obtain individuals which showed suppression of breathing movements in response to the visual conditioned stimulus (CS) in the presence or absence of adapting illumination. Following bilateral optic tectum ablation, responding was blocked in light-adapted but not dark-adapted fish. Response threshold testing revealed no significant postoperative changes in visual sensitivity. Small remnants of tectal tissue containing cellular elements of the periventricular gray zone and optic axon terminals were detected in some ablates but there was no evident relationship to response threshold. Optic nerve crush blocked responding in ablates and recovery occurred within 2-3 weeks postaxotomy confirming that the response was mediated by retinal as opposed to extraretinal photostimulation. The experiments support the findings by others that tectum ablation results in decreased visual sensitivity and that conditioned visual responding can be obtained. However, we find no support for the suggestion that visual sensitivity in the ablates depends on functional recovery of regenerating optic axons which innervate non-tectal visual nuclei. Instead, the results indicate that the normal retinal projections to the non-tectal nuclei can mediate visual responding and, in addition, that postoperative conditioning experience facilitates recovery of response in ablates which initially appear to be blind to the CS.     

The optic tectum regulates the transport of specific proteins in regenerating optic fibers of goldfish

The pattern of rapidly-transported proteins in regenerating optic fibers of the adult goldfish is regulated by interactions between these fibers and their main target, the optic tectum. When the optic fibers are allowed to interact with the tectum, the transport of proteins with molecular weights in the range of 110-145 kilodaltons (kDa) increases, whereas the transport of proteins in the 24-27 kDa range declines from the previously high level which has been induced by axotomy. If the optic fibers are prevented from interacting with the tectum, the transport of the 24-27 kDa proteins remains elevated for months. Amounts of other rapidly-transported retinal proteins (e.g. the acidic 43-49 kDa proteins that increase in regenerating optic fibers after axotomy) are relatively unaffected by tectal ablation.     

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.     

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.     

The organization of CRF neuronal pathways in toads: Evidence that retinal afferents do not contribute significantly to tectal CRF content

Previous work has suggested that the peptide corticotropin-releasing factor (CRF) acts to inhibit visually guided feeding in anurans, but little is known about potential targets for CRF within the subcortical visuomotor circuitry. Here we investigated the relationship between CRF neuronal organization and visual pathways in toads. CRF-immunoreactive (ir) neurons and fibers were widely distributed throughout the ventral subpallial telencephalon and hypothalamus, although few fibers were found in telencephalic areas, such as the striatum, that are known to project to the tectum in anurans. Large populations of CRF-ir cells were observed in the bed nucleus of the stria terminalis and preoptic area as well as in the ventral infundibular hypothalamus. CRF-ir neurons and fibers also were observed in several midbrain and brain stem areas. Colchicine treatment significantly enhanced CRF-ir neurons and fibers throughout the brain, and revealed CRF-ir cell groups in several brain areas (including the dorsal hypothalamus) that were not observed in untreated animals. Intrinsic CRF-immunoreactive neurons were routinely observed in cell layer 8 and sometimes in layer 6 of the optic tectum in both untreated and colchicine-treated animals. CRF was detected in toad optic tectum by radioimmunoassay, although tectal CRF content was less than that of the hypothalamus and forebrain. Unilateral eye ablation did not affect CRF content of the contralateral optic tectum. We conclude that CRF-producing neurons are widely distributed in several areas of the toad brain known to be involved in regulating the behavioral, autonomic and endocrine response to stressors, including the optic tectum and several brain areas known to project to the optic tectum. Furthermore, retinal afferents do not contribute significantly to tectal CRF content.     

In vitro proliferation of radial glia within isolated tectal tissue explanted from adult goldfish

Radial glia located in tectal tissue isolated from adult goldfish retain the ability to incorporate exogenous thymidine into DNA up to 5 days in culture. The rate of their proliferation is maximally enhanced during reinnervation of the tectum by optic fibers about 5 weeks after unilateral optic nerve crush.     

Gliosis during optic fiber regeneration in the goldfish: an immunohistochemical study.

Antisera directed against the 48 kDa and 50 kDa cytoskeletal antigens were used to examine changes in the astroglial fabric of the goldfish visual pathways following optic nerve crush. Several major observations are described. First, an optic nerve crush lesion in these animals appears to be devoid of glial cells for at least the first month after surgery. As a corollary, regenerating axons that grow across the lesion may do so over an aglial substrate. Once the axons cross the lesion, their growth is confined to the astroglial domains of the proximal nerve stump. In the optic nerve, gliosis comprises hypertrophy of astrocytic processes such that the open framework characterizing the normal nerve is obscured. In addition, during regeneration, optic nerve glia express large amounts of the 50 kDa cytoskeletal protein, which they ordinarily express at only minimal levels. In the optic tract, gliosis is reflected in a markedly increased expression of the 50 kDa protein as well as an apparent increase in the number and complexity of glial processes. In addition, optic tract glia begin to express the 48 kDa antigen during regeneration. This protein is ordinarily confined for the most part to the optic nerve and is not seen in the tract glia. Finally, no obvious changes were seen in the glia of the optic tectum. These results demonstrate many points of similarity between gliosis in the goldfish and in mammals. However, in some particulars the two responses differ, and it is possible that these differences are related to the differing ability of central axons to regenerate in the two groups of organisms.     

Selective retinal reinnervation of a surgically created tectal island in goldfish. I. Light microscopic analysis

Through anatomical and physiological studies of the regenerating retinotectal projection of goldfish, we sought to determine whether the establishment of a topographic projection is attained through a refinement of an initially less precise pattern of innervation. A 1-mm-wide mediolateral strip of caudal tectum was removed so that a small island of tectal tissue was spared at the caudal pole, and the contralateral nerve was either crushed (TIX) or left intact (TI). The presence of regenerated axons in the ablated zone and the reinnervation of the caudal island were assessed with anterograde and retrograde labeling methods in the following postoperative intervals: early, 20-50 days; middle, 50-110 days; and late, more than 170 days. The anterograde radioautographic method revealed that the appropriate layers of the tectal island became reinnervated by optic axons during the early period. During the middle and late periods, one to several large, discrete bundles bridging the lesion zone along the surface of exposed subtectal structures were readily identified both by radioautography and by anterograde or retrograde labeling following application of horseradish peroxidase to the transected optic nerve or tectal island, respectively. In contrast, the anterograde horseradish peroxidase method did not reveal axon bundles extending caudal to the half-tectum in the absence of a tectal island. Among TIX cases, retrograde horseradish peroxidase labeling of the contralateral nasal retina was more widespread in the middle period than in the late period, a result we interpret as reflecting an improvement in topographical precision with time. The area of retinal labeling among TIX cases in the late period was similar to that following caudal tectal injection in cases with simple nerve crush, although it was still elevated above normal control values. Physiological maps indicated a focal representation of the nasal retina in the tectal island in both periods and did not reveal a transient extreme convergence of retinal input. These findings are discussed in relation to Sperry's chemoaffinity theory.     

Afferent and efferent fiber connections of the carp torus longitudinalis

The efferent and afferent pathways of the carp torus longitudinalis were studied by means of degeneration and retrograde HRP methods. Efferent projections were only seen in the most superficial layer of the ipsilateral optic tectum (stratum fibrosum marginale). Afferent pathways to the torus longitudinalis were found to originate mainly in the valvula cerebelli. Degenerating fibers course in the tractus mesencephalocerebellaris posterior within the valvula, and join the tractus mesencephalocerebellaris anterior in the tegmentum. The fibers which ascend in the tract gradually invade the optic tectum through which they are distributed to the torus longitudinalis. The remaining fibers pass through the posterior commissure and terminate in the torus longitudinalis at the rostral end of the tract. Degenerating terminals were also seen in the torus longitudinalis when lesions were made in the optic tectum, tectal commissure, torus semicircularis, and in the area between the valvula and the corpus cerebelli. The possibility of projection from these areas is discussed depending upon the results of the retrograde HRP method.     

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.     

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).     

Mesencephalic and diencephalic commissures and interocular transfer in the goldfish

Interocular transfer of a differential, classically-conditioned, cardiac response has recently been shown to occur in tectal commissure-sectioned goldfish. In this report, posterior commissure-sectioned goldfish are shown to transfer this paradigm, but fish with section of the postoptic commissure group do not. Of this group, the horizontal commissure is show to be important for the transfer of the response to the reinforced stimulus.     

Restoration of the visual projection following tectal lesions in goldfish

The retinotectal projection was mapped in a series of adult goldfish at various intervals after either a transverse surgical lesion extending down to the ventricle on the dorsal tectum or a three-sided lesion separating the rostral, medial, and lateral tectum. In both cases optic nerve fibers regenerated into the affected region thus restoring the normal visual projection. The cellular discontinuity created by surgical lesion did not induce synaptic respecification. It was concluded that regenerating optic axons reroute to innervate the affected areas of the tectum.     

Mapping the normal and regenerating retinotectal projection of goldfish with autoradiographic methods

In normal goldfish, lesions of various size were made in nasal or temporal retina immediately prior to retinal labeling with tritiated proline. The resulting gaps in retinal innervation of tectum indicated that the projection is retinotopographically ordered to a precision of about 50 μm. Similarly, acute tectal incisions transecting the optic pathways were combined with immediate retinal labeling. The resulting tectal denervation confirmed that most fibers follow highly ordered paths through the stratum opticum of tectum; but a few fibers were found to follow unusual paths to their appropriate tectal positions. In other fish, the optic nerve was crushed. At various times afterwards, retinotopography and pathway order were similarly analyzed by making retinal lesions or tectal incisions just prior to labeling. For up to 40 days after crush, the projection lacked any refined retinotopic order. Only a gross topography could be demonstrated. Over several months, retinotopography gradually improved eventually approaching that of normals. Correlated with this was an initial stereotypic growth through the pathways of the stratum opticum followed by a long period of highly anomalous growth through the innervation layer. Evidently, many regenerated fibers grew in through inappropriate routes to the wrong region of tectum but subsequently arrived at their appropriate locus by circuitous routes within the innervation layer.