Activity sharpens the map during the regeneration of the retinotectal projection in goldfish

In the regenerating retinotectal projection of goldfish, we have used intraocular injections of tetrodotoxin (TTX) to determine whether activity plays a role in organizing or refining the retinotopic map. Repeated injections produced a continuous 27-day block without producing extraocular effects or causing deleterious effects in the retinal ganglion cells⁸. The retinotectal maps regenerated in the TTX fish were normally organized but the multiunit receptive fields were grossly enlarged. In control regenerates, 1–3 units (arbors of retinal ganglion cell axons) were simultaneously recorded at each penetration and their combined receptive field averaged 11–12°, nearly the same as for single units. In TTX fish each penetration yielded at least 5–10 units whose receptive fields were clustered over a wider area averaging 27° across. Individual ganglion cell receptive fields were assessed both by tectal and by intraretinal recording and were not enlarged. Many fish were recorded up to 4 months after the release from TTX block, but no further refinement of the maps occurred. If the nerve was recrushed and regenerated a second time without TTX, a normal map was formed, ruling out any permanent changes in the retinal ganglion cells or in the tectum. Blocks during various portions of the regeneration process showed that lack of activity during the process of axonal elongation (first 2 weeks) does not cause enlargement of the multiunit receptive fields, but lack of activity during the period of synapse formation and maturation (14–34 days) does. The results are discussed in terms of an activity-dependent stabilization of synapses. Neighboring retinal ganglion cells are known to fire in a statistically correlated fashion and this could help in their elimination of incorrect branches following an early period of diffuse connections.     

RNA content of normal and axotomized retinal ganglion cells of rat and goldfish

The responses of rat and goldfish retinal ganglion cells to axotomy were examined by a quantitative cytochemical method for RNA and by morphometric measurement 1-60 (rat) and 3-90 (goldfish) days after interruption of one optic nerve or tract intracranially. Unoperated control animals were studied also. The RNA content of axotomized neurons of rat fell 7-60 days postoperatively. Additionally, atrophy of the axotomized somas occurred. Over time, neuronal atrophy approximately paralleled the loss of RNA, and mean cell area and RNA content were reduced by about 25% 60 days after axotomy. Incorporation of 3H-uridine by axotomized neurons declined also. Axotomized retinal ganglion cells of goldfish behaved differently from those of the rat and showed increases in RNA content, most conspicuously 14-60 days postoperatively. Enlargement of axotomized fish neurons occurred but was less proportionately than concomitant increases in RNA content. The nonaxotomized ganglion cells of goldfish displayed statistically significant increases in size and RNA content 14-49 days after unilateral optic nerve or tract lesions. In contrast, alterations in rat retinal ganglion cells contralateral to interruption of one optic nerve were of limited and questionable significance. The contrasting reactions to axotomy by the retinal ganglion cells of these two vertebrates, one of which regenerates optic axons and one of which does not, may support the proposition that the somal response to axon injury has an important bearing upon the success or failure of CNS regeneration.(ABSTRACT TRUNCATED AT 250 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.     

Map of retinal position onto the cross section of the optic pathway of goldfish

The position of a retinal cell is defined by the two polar coordinates: r, the distance from the optic disc, and theta, the angular (or clock-face) position. Axons of similar theta value were labeled by the punctate application of horseradish peroxidase (HRP) to optic axons in the retina, and axons of similar r-value were labeled by the application of this same marker to a tectal fascicle. Labeled axons were traced in serial transverse sections of the optic pathway from the retina to the tectum to learn the map of the retinal surface onto the cross section of the pathway. Retinas were flat-mounted and treated for HRP to show the retinal origins of the labeled axons. Axons of similar r were clustered together, and the fraction of the pathway's cross-sectional area occupied by the cluster was about the same as the fraction of the retinal area occupied by the group of labeled somata. Axons of similar theta were also clustered, but the fraction of the cross-sectional area they occupied was larger than the fraction of retinal area occupied by their somata. The geometry of the clusters of labeled axons depended on the proximodistal location in the pathway. Near the retina both were strip-shaped, but the location and orientation of the strip varied. Both an r-strip and a theta-strip were labeled in some pathways by dual applications of HRP; the two strips were mutually orthogonal at all levels. Each of r and theta mapped onto a separate axis. The axons from most peripheral retina (largest r) were everywhere adjacent to the pia, and axons of progressively more central retina (smaller r) were progressively more separated from the pia (except in the nerve, where the secondary fasciculation complicates the geometry by wrapping old axons in new pia). The map of the circular variable, theta, onto a line, required a discontinuity, the location of which differed, depending on the proximodistal level. From the retina to the chiasm, the discontinuity was at the ventral retinal radius (i.e., the right retinal clock-face positions were ordered 6-9-12-3-6 o'clock across the line); just central to the chiasm, the fibers reordered to put the discontinuity at the nasal radius (clock-face positions ordered 3-6-9-12-3); at the brachial bifurcation, the 3-6-9 half turned dorsally, the 9-12-3 half, ventrally.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Effect of nerve growth factor on regeneration of goldfish optic axons

Axonal outgrowth following a crush of the goldfish optic nerve was enhanced if nerve growth factor (NGF) was administered by intraocular injection or by local application to the lesion site. Various forms of NGF (β, 2.5S and 7S) were effective, producing a 20–40% decrease in the time required for recovery of the startle reaction to a bright light. A corresponding increase in axonal outgrowth was revealed by histological examination of the optic nerves. The effect produced by a single intraocular injection given at the time of the lesion was not further increased by subsequent injections. Up to 14 days after the lesion, the size of the retinal ganglion cell bodies and the incidence of nucleoli detectable by light microscopy were not affected by the NGF treatment.     

Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells

To characterize the fast component of axonal transport in regenerating goldfish optic axons, the incorporation of l-2,3-[³H]proline into newly-synthetized proteins in the cell bodies of the retinal ganglion cells and the amount of transported labeled protein were determined at 2–36 days after cutting the optic tract. Both the incorporation and the amount of transported protein had doubled by 10 days after the lesion and continued to increase to about 5 times normal at 15 days, a time when a large proportion of the regenerating axon population had reached the optic tectum. Near-normal levels were recovered by 36 days. In contralateral control neurons, the incorporation of l-2,3-[³H]proline was unchanged from normal throughout, whereas the amount of labeled transported protein entering control axons was decreased by 55% at 2 and 10 days after the testing lesion, returning to normal by 15 days. An increase in fast transport velocity was seen in the regenerating axons beginning at 10 days after the lesion. However, a similar velocity increase was also seen in the contralateral control axons and in undamaged axons following removal of the cerebral hemispheres. Therefore, the velocity increase was not a specific consequence of axotomy.     

Molecular events associated with increased regenerative capacity of the goldfish retinal ganglion cells following X-irradiation: Decreased level of axonal growth inhibitors

In our previous work we established conditions to study the contribution of non-neuronal cells to the process of goldfish optic nerve regeneration. This issue has been studied successfully by adapting the use of X-irradiation to manipulate division of non-neuronal cells associated with the injured nerve. The regenerative capacity of the goldfish retinal ganglion cells was determined subsequent to the X-ray treatment. In this work we present an analysis of the molecular events associated with regeneration and enhanced regenerative capacity which follows X-irradiation. Under normal conditions the non-neuronal cells surrounding an intact nerve released axonal growth inhibitors, the level of which was only slightly sensitive to X-irradiation. In contrast, regenerating nerves showed a marked decrease in substances having an inhibitory effect on sprouting in vitro. Moreover, their level was significantly reduced following X-irradiation which was accompanied by increased accumulation of fibrous collagen adjacent to astrocytes. The alteration in the reciprocal relationship between the axon and the surrounding non-neuronal cells was also manifested by changes in the profile of labeled proteins released by the non-neuronal cells. The results of this work, therefore, indicate the sensitivity of retinal ganglion cells to the environment provided by the cells surrounding the regenerating fibers and thus may open a new perspective relating to the capacity of neurons to regenerate.     

Effect of a conditioning lesion on optic nerve regeneration in goldfish

Following a ‘testing lesion’ (crush) of the optic nerve in goldfish, histological study of axons in silver-stained sections showed that outgrowth of the leading axons began after an initial delay of 4.3 days and proceeded at0.34 ± 0.03mm/day. When a ‘conditioning lesion’ (crush at the same site) preceded the testing lesion by 2 weeks, the initial delay was 2.5 days and the outgrowth rate was0.74 ± 0.13mm/day (P < 0.01).Two additional methods, utilizing intraocular injections of tritiated proline or fucose to label axonally transported proteins, were used to examine the outgrowth of leading optic axons. (a) Measurement of the distances reached by labeled axons in the nerve at 6 and 10 days after a testing lesion alone yielded an initial delay of 4.6 days and an outgrowth rate of0.41 ± 0.04mm/day. However, when a conditioning lesion preceded the testing lesion, labeled optic axons were already found to have reached the optic tectum by 10 days after the testing lesion, indicating an outgrowth rate in excess of 0.64 mm/day. (b) Determination of the times at which labeled axons arrived at the optic tectum showed that the outgrowth rate after a testing lesion alone was 0.40 mm/day whereas when the testing lesion was preceded by a conditioning lesion it was 0.74 mm/day.Thus, as a result of a conditioning lesion the initial delay was reduced bynearly half and the outgrowth rate was nearly doubled.     

Removal of optic tectum prolongs the cell body reaction to axotomy in goldfish retinal ganglion cells

Injury to the optic axons of goldfish elicits dramatic changes in the cell bodies of the neurons from which these axons arise, the retinal ganglion cells. The changes include a large increase in cell size and in synthesis and axonal transport of protein. The cells begin to return to normal about 3 weeks after the injury, when the axons invade the contralateral (homotopic) lobe of the optic tectum, and recovery is essentially complete by 8-10 weeks after the lesion. However, if the homotopic lobe of the tectum was removed at the time of nerve crush, we found that the cell body reaction was greatly prolonged. The cells remained enlarged, and [3H]proline incorporation and fast axonal transport of protein remained elevated, until at least 10-12 weeks after nerve crush, although by this time most of the regenerating axons had probably regained their normal length and many had entered the remaining ipsilateral (heterotopic) lobe of the tectum. The cells showed partial recovery by the latest time tested, 26 weeks after nerve crush, when the projections from the two eyes had segregated into separate bands in the heterotopic tectal lobe.     

Inhibition of non-neuronal cell proliferation in the goldfish visual pathway affects the regenerative capacity of the retina

Proliferating cells associated with the visual pathway were found in the present study to affect the regenerative capacity of the goldfish retina following optic nerve injury. The contribution of these cells to the process of regeneration was investigated in the goldfish visual system by reducing their proliferation in the optic tract and tecta, using X-irradiation. The regenerative ability of the retina was then evaluated by the following parameters: sprouting from retinal explants, protein synthesis in the retina and accumulation of radiolabeled transported components in the tectum. X-irridiation of the visual system at an early stage of the regeneration process had a promoting effect whereas irradiation at a later stage resulted in a reduced capacity to regenerate. The results are discussed with respect to the possibility that proliferating cells, possibly glia, exert two contradictory contributions: an inhibitory effect at the site of injury, whereas distal to it, a supportive, perhaps trophic effect.     

An electrophysiological study of early retinotectal projection patterns during regeneration following optic nerve crush inside the cranium in Hyla moorei

The sequence of regeneration following intracranial optic nerve crush has been studied using electrophysological visual mapping in the frog Hyla moorei. Compared to our earlier series⁹ with extracranial crush, the time course was slower and the intermediate projections more disorganized. It is suggested that the apparent discrepancy between the early patterns of regeneration in Xenopus⁴ and Rana^5,6 compared to Hyla⁹, fish⁸ and newt², is not due to species differences but to the location of the lesion site.     

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.     

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.     

An electrophysiological study of early retinotectal projection patterns during optic nerve regeneration inHyla moorei

The sequence of regeneration of the optic nerve into the tectum has been studied using electrophysiological visual mapping in the frog,Hyla moorei. Some individuals were remapped to confirm the sequential nature of the changes described. The projection was retinotopic throughout regeneration. Multiunit receptive field sizes were initially large but progressively decreased to normal over approximately 30 days. The differences between these results and earlier studies are discussed.     

Protein synthesis and axonal transport in goldfish retinal ganglion cells during regeneration accelerated by a conditioning lesion

Axonal outgrowth in goldfish retinal ganglion cells following a testing lesion of the optic axons is accelerated by a prior conditioning lesion. Changes in protein synthesis and axonal transport were examined during the accelerated regeneration. The conditioning lesion was an optic tract cut made 2 weeks prior to the testing lesion, which consisted of a tract cut at the chiasma, so that nerves subjected to either a conditioning lesion (‘conditioned nerves’) or a sham operation (‘sham-conditioned nerves’) could be examined in the same animal. In the retinal ganglion cells of conditioned nerves, the incorporation of [³H]proline into protein began to increase between 1 and 8 days after the testing lesion. The amount of fast-transported labeled protein was elevated to about 8 × normal by 1 day after the testing lesion but had decreased to about 3–5 × normal at 8 and 22 days. The 8 and 22 day values were not significantly different from those in sham-conditioned nerves or nerves that had received a testing lesion alone. For slow protein transport, the instantaneous amount transported was 15–16 × normal in the conditioned nerves at 1 and 8 days after the testing lesion, and the velocity of slow transport, which was already elevated above normal by 1 day after the testing lesion, was elevated still further by 8 days — to a value in excess of 1.5 mm/day (compared to 0.2–0.4 mm/day in normal animals). We believe that the enhanced outgrowth resulting from the conditioning lesion is due to a transient increase in the amount of fast transport (possibly responsible for a decreased delay in the initiation of sprouting), and a sustained increase in the amount and velocity of slow transport (which may account for an increased rate of elongation).