Optic synapses are found in diencephalic neuropils before development of the tectum in Xenopus.

The position of the earliest optic synapses in Xenopus and the stage at which they developed were studied with the electron microscope after labelling of optic axons with horseradish peroxidase. In addition, tritiated thymidine autoradiography and bromodeoxyuridine immunohistology were used to identify the birth dates of cells in the regions where the synapses had been found. The earliest mature optic synapses were found in the mid-diencephalic region, where the major diencephalic optic neuropils were beginning to develop. These synapses were seen at stage 35/36, before cells in the tectal precursor region had become postmitotic. In other animals labelling with tritiated thymidine or bromodeoxyuridine showed that cells in the diencephalon, close to where the synapses had been seen, were becoming postmitotic at the time the earliest optic axons arrived. The first optic synapses to form in the developing Xenopus visual system thus appeared to do so in the neuropil of Bellonci and the rostral visual nucleus.

Regeneration in the Xenopus tadpole optic nerve is preceded by a massive macrophage/microglial response.

Changes in the optic nerve following a crush lesion and during axonal regeneration have been studied in Xenopus tadpoles, using ultrastructural and immunohistological methods. Degeneration of both unmyelinated and myelinated axons is very rapid and leads to the formation, within 5 days, of a nerve which consists largely of degeneration debris and cells. Immunohistological analysis with monoclonal antibody 5F4 shows that there is a rapid and extensive microglial/macrophage response to crush of the nerve. Regenerating axons have begun to enter the distal stump by 5 days and grow along the outer part of the nerve in close approximation to the astrocytic glia limitans. Between 5 and 10 days after nerve crush, regenerating axons reach and pass the chiasma. Macrophages are seen in the nerve at the site of the lesion within 1 h, and the response peaks between 3-5 days, just before axonal regeneration gets under way.

Development of the tectum and diencephalon in relation to the time of arrival of the earliest optic fibres in Xenopus.

The development of the tectum and diencephalon in Xenopus has been investigated in relation to recent descriptions of the establishment of the retinotectal projection. Tritiated thymidine autoradiography and bromodeoxyuridine immunohistology were used to identify the stages at which cells became postmitotic. Cells in the diencephalon were found to become postmitotic before cells in the tectum. At the time of arrival of the first optic fibres (stage 37/38) no postmitotic cells appeared to be present in the tectal precursor region. The first postmitotic cells which could be definitely assigned to the tectum appeared between stages 41 and 45. The results suggest that the initial retinotopic ordering of optic fibres observed from stage 37/38 relates to the position of fibres in the optic tract and not the tectum.

Spatio-temporal patterns of retinal ganglion cell death during Xenopus development.

During development of the retina in mammals and birds, most retinal ganglion cells (RGC) that are produced are eliminated later in development by cell death. In lower vertebrates, however, such massive cell death has not been observed; total ganglion cell number increases linearly during most of development. Using 3H-thymidine or 5-bromodeoxyuridine labeling of retinal cell nuclei, we have been able to identify postmitotic RGC populations in Xenopus central retina at different developmental stages and follow their fate during development to postmetamorphic stages. RGC populations that become postmitotic between embryonic stages 32 and 49, during the initial stages of retinal growth, lose 40-77% of their cells during metamorphosis (approximately 4,000-5,000 cells). Twenty percent of the RGC present at stage 54, which later disappear, represent the same population of dying RGC that were present at stage 49. This suggests that the ganglion cells that became postmitotic between stage 49 and 53/54 show no apparent decline in numbers during metamorphosis. Since thyroxine is known to stimulate an increase in RGC number as well as the extent of fiber projection on the tectum, we suggest that this reduction in RGC numbers is not due to thyroxine-induced neuronal cell death. After stage 54, however, binocular vision develops in Xenopus (Keating, '74) and ipsilateral fibers begin to grow into thalamic visual neuropils (Hoskins and Grobstein, '85). We suggest, therefore, that as in mammals, in which RGC elimination correlates with binocular segregation of contralateral and ipsilateral retinal axons in visual centers, a similar process may occur in the frog among those RGC projecting to thalamic visual neuropils.

Microglia in tadpoles of Xenopus laevis: normal distribution and the response to optic nerve injury.

We have studied the distribution of microglia in normal Xenopus tadpoles and after an optic nerve lesion, using a monoclonal antibody (5F4) raised against Xenopus retinas of which the optic nerves had been cut 10 days previously. The antibody 5F4 selectively recognizes macrophages and microglia in Xenopus. In normal animals microglia are sparsely but widely distributed throughout the retina, optic nerve, diencephalon and mesencephalon (other regions were not examined). After crush or cut of an optic nerve, or eye removal, there occurs an extensive microglial response along the affected optic pathway. Within 18 h an increase in the number of microglial cells in the optic tract and tectum can be detected. This response increases to peak at around 5 days after the lesion. At this time the nerve distal to the lesion contains many microglial cells; the entire optic tract is outlined by microglia, extended along the degenerating fibres; and the affected tectum shows a heavy concentration of microglia. This microglial response thereafter decreases and has mostly gone by 34 days. We conclude that the microglial response to optic nerve injury in Xenopus tadpoles starts early, peaks just before the regenerating optic nerve axons enter the brain, and is much diminished by the time the retinotectal projection is re-established. The timing is such that the microglial response could play a major role in facilitating regeneration.

Regeneration of optic fibres through the chiasma in Xenopus laevis tadpoles.

The path through the chiasma followed by regenerating optic nerve fibres in Xenopus tadpoles was studied at light- and electron-microscopic levels, and with horseradish peroxidase as a fibre label. Over the period (5-10 days) in which regenerating fibres reach and cross the chiasma, they did not follow residual deep fibres through the chiasma, nor were they associated with the trail of degeneration in the chiasma which represented the remains of the deeper (older) parts of the original projection. The regenerating optic fibres were always seen in close association with the inner surfaces of the ependymoglial endfeet, or in the extracellular spaces that lie close to the endfeet in the most superficial part of the chiasma, where newly-growing fibres from the retinal margin are normally to be found.

The induction of an anomalous ipsilateral retinotectal projection in Xenopus laevis.

The conditions necessary for extensive regeneration of fibres from one optic nerve to both tecta in Xenopus have been investigated. The effects of various types and positions of nerve lesion on the distribution of regenerated projections were examined by labelling the regenerated projections with either horseradish peroxidase or tritiated proline. The only types of nerve lesion which consistently gave rise to extensive regeneration to both the contralateral and the ipsilateral tectum were those close to the chiasma, liable to have caused damage to the nerve entry point. However, all other types of lesion studied (near the eye; near the skull; crush or cut) frequently led to regeneration of a very few fibres to the ipsilateral tectum. These fibres gained access to the ipsilateral tectum by various routes: in some cases via the optic tract; but more commonly either across the posterior commissure or by a complex pathway following the oculomotor nerve root. Over time periods of between one and seven months, the distribution of the regenerated fibres following each type of lesion showed little change. We conclude that the regeneration of bilateral retinotectal projections in Xenopus is caused by tissue damage to the region of the chiasma resulting in misrouting of fibres.

The course of regenerating retinal axons in the frog chiasma: the influence of axons from the other eye.

There is evidence, from a variety of species, that axons arising from the two eyes interact as they pass through the optic chiasma. In this paper we examine the effect of altering the composition of the normal chiasmatic environment on the course taken by regenerating axons in Xenopus. We show that the usual contralateral route of such axons in the optic chiasma is not affected by the absence of axons from the other eye. We have not observed any influence of embryonic enucleation, or eye removal before the onset of metamorphosis, on either the normal ipsilaterally projecting axons or the contralateral route taken by regenerating axons. Further, we have found no effect of the degeneration of the other projection, either for long or short periods of time, suggesting that axonal debris does not influence the course of regenerating axons. The simultaneous regeneration of the two projections also has no effect on the trajectory of regenerating axons. We conclude that in spite of the close association of axons from the two eyes in the chiasma, there is no interaction involved in Xenopus in the generation of the decussation pattern.

A developmental and ultrastructural study of the optic chiasma in Xenopus.

The structure of the optic chiasma in Xenopus tadpoles has been investigated by light and electron microscopy. Where the optic nerve approaches the chiasma, a tongue of cells protrudes from the periventricular cell mass into the dorsal part of the nerve. Glial processes from this tongue of cells ensheath fascicles of optic axons as they enter the brain. Coincident with this partitioning, the annular arrangement of axons in the optic nerve changes to the laminar organization of the optic tract. Beyond the site of this rearrangement, all newly growing axons accumulate in the ventral-most part of the nerve and pass into the region between the periventricular cells and pia which we have called the 'bridge'. This region is characterized by a loose meshwork of glial cell processes, intercellular spaces and the presence of both optic and nonoptic axons. In the bridge, putative growth cones of retinal ganglion cell axons are found in the intercellular spaces in contact with both the glia and with other axons. The newly growing axons from each eye cross in the bridge at the midline and pass into the superficial layers of the contralateral optic tracts. As the system continues to grow, previous generations of axon, which initially crossed in the existing bridge, are displaced dorsally and caudally, forming the deeper layers of the chiasma. At their point of crossing in the deeper layers, these fascicles of axons from each eye interweave in an intimate fashion. There is no glial segregation of the older axons as they interweave within the chiasma.

The discontinuous visual projections on the Xenopus optic tectum following regeneration after unilateral nerve section.

The establishment of retinotectal projections following transection of one optic nerve in developing Xenopus has been investigated. Between 3 weeks and 11 months after the operation, the nerve fibre tracer horseradish peroxidase (HRP) was applied to either the operated or the unoperated nerve, and the brains were prepared for examination as whole mounts. In most cases fibres from the operated nerve innervated both tecta, with the result that one tectum was doubly innervated and one tectum singly innervated. Two months after transection of the optic nerve in tadpole life, between stages 50 and 54, this nerve usually made a uniform projection on the contralateral tectum and a striped projection on the ipsilateral, doubly innervated, tectum. The projection made by the unoperated nerve on this tectum was a similar pattern of stripes, which ran generally rostrocaudally. Two months after transection of the optic nerve of newly metamorphosed animals, the projection formed by the operated nerve on the doubly innervated tectum was usually a pattern of spots or spots mixed together with stripes in no particular orientation superimposed on a roughly uniform background. In a small number of cases the projections made by the same nerve on the two tecta were approximately complementary; that is, the presence of label on one tectum corresponded with its absence on the other tectum. The results are examined in the context of the development of the retina and of the tectum. It is suggested that the consistently oriented stripes which result from nerve transection at a stage at which only a small proportion of the retinal fibres had reached the tectum are formed by the interaction of two equally matched sets of developing fibres, stripe orientation being determined by the mode of growth of the optic tectum. The formation of patterns of spots or spots mixed together with stripes following nerve transection after the end of the main phase of tectal histogenesis, and when 50% of the optic fibres had already reached the tectum, is attributed to an unequal competition between the two sets of fibres.

The effects of the fibre environment on the paths taken by regenerating optic nerve fibres in Xenopus.

The paths taken by fibres regenerating to the tectum from various parts of the Xenopus retina were investigated in whole-mount preparations, after localized retinal labelling with HRP. The effects of different environments on the fibres were studied by comparing contralateral with ipsilateral regeneration, in the presence of the other eye or after it had been removed in embryonic life. Under all conditions fibres from the various parts of the retina regenerated to the corresponding appropriate parts of the tectum, but they took a variety of pathways, some grossly abnormal, to get there. Contralaterally regenerating fibres tended to behave less abnormally than ipsilateral fibres; and regeneration in the absence of the other eye tended to be more abnormal than in its presence. In any one category of regeneration the most nearly normal pathways were those of fibres from temporal retina, followed by ventral, nasal and dorsal fibres. Fibres regenerating from all parts of the retina, in the presence of the other eye, tended to become gathered into the medial brachium as they approached the tectum. All regenerating fibres approached their tectal terminations by one or more of three main pathways: round one or both brachia, thus encircling the tectum to get to their terminal zone; directly across the tectum; or by passing on to the tectum before changing course. The changes of direction required to enable fibres wrongly positioned in the tract to reach their correct terminal zones were frequently sudden and considerable, and took place on the tectum or at the tectodiencephalic junction. The results are discussed in relation to the differing substrates over which the fibres regenerate.

The distribution of fibres in the optic tract after contralateral translocation of an eye in Xenopus.

In Xenopus embryos of stage 30 the right eye was translocated, without rotation, to a left host orbit. Shortly after metamorphosis the visuotectal projection through the operated eye was mapped electrophysiologically and shown to be normal dorsoventrally but reversed nasotemporally. Labelling of small groups of retinal axons with HRP showed that the fibre trajectories from dorsal and ventral retina were normal, whereas fibres from nasally placed retina had diencephalic pathways and tectal terminations typical and temporal fibres, and fibres from temporally placed retina had diencephalic pathways and tectal terminations typical of nasal fibres. Thus from just beyond the chiasma the fibres had already achieved the major uniaxial rearrangement necessary to establish a normal tract distribution despite the eye translocation. The fibre rearrangement required to permit the formation of a nasotemporally inverted visuotectal projection appears, therefore, to occur not on the tectum or in the optic tract, but either within the nerve or at the chiasma.

Fibre order in the normal Xenopus optic tract, near the chiasma.

In juvenile Xenopus retinotopic fibre order in the optic tract near the chiasma was investigated by labelling small groups of optic fibres from peripheral retina with HRP. This selective fibre labelling with HRP was combined with autoradiography following administration of tritiated thymidine to the eye, so that the HRP-labelled fibres could be located within the borders of the optic tract. Fibres arising from the periphery of all four retinal quadrants were superficially located in the optic tract near the chiasma, with dorsal retinal fibres showing the greatest tendency to travel deep in the diencephalon. Retinal lesions closer to the optic nerve head labelled fibres which ran deeper in the optic tract. Near the chiasma, fibres from ventral retina tended to group rostrally while fibres from dorsal retina tended to group caudally. However, no obvious localization of fibres arising in temporal or nasal retina was seen in the lower optic tract.

The visuotectal projection following translocation of grafts within an optic tectum in the goldfish.

Rectangular grafts were translocated between the rostral and caudal tectum in adult goldfish. In some animals the optic nerve was also cut. After periods ranging from 161 to 378 days the retinotectal projections were mapped. The results showed that translocation of tectal grafts is followed by translocation of the corresponding part of the visuotectal map. In many cases the visual field positions projecting to points in the region between the grafts were abnormal and in some cases intercalated field positions were found across the borders between grafted and normal tissue. The results are taken as strong evidence for the existence of affinity labels on the tectum and their importance in the restoration of the retinotectal projection.

The positional coding system in the early eye rudiment of Xenopus laevis, and its modification after grafting operations.

In a series of Xenopus embryos, 60-90 degrees sectors at various positions distributed around the eye rudiment were replaced with sectors grafted from the opposite position in the rudiment on the same side of the head of a donor. The majority of the operations were carried out before stage 28 (Nieuwkoop & Faber, 1956), and many before stage 26. The patterns of retinotectal connectivity which then developed were assayed electrophysiologically soon after metamorphosis. The visuotectal maps were frequently compound, giving evidence that many parts of the rudiment had already been equipped with distinct tissue positional codes by the time operations were performed (i.e. before neurogenesis). Although graft-derived sectors of retina connected to tectal sectors that were more nearly appropriate for their original positions in the rudiment than for their translocated ones, the 'handedness' of these ectopic components of the compound maps tended to bear a mirror-image relation to the major map, rather than the point-symmetrical one to be expected from a complete autonomy of mapping functions in grafted tissue. The results are discussed in relation to possible modes of organization of the developing eye, considered as a pattern-forming system.

The visuotectal projections made by Xenopus 'pie slice' compound eyes.

In xenopus embryos at stages 28-32 one quarter to one third of the left eye rudiment was replaced by a similarly sized piece from a different position in a right eye rudiment. Three groups of operations were performed: (1) temporal tissue was placed in a nasal position; (2) nasal tissue was placed temporally; (3) ventral tissue was placed dorsally. The visuotectal projections made by these 'pie-slice' compound eyes were assessed electrophysiologically at 1 week to 6 months after metamorphosis. Of 97 animals, 71 yielded interpretable projections. In most cases two projections could be identified in each map. One, ascribed to the host part of the retina, extended over the entire tectal surface mapped. The other, identified as that from tissue derived from the pie-slice graft, projected to the tectum in register with that part of the host retina which matched the pie-slice in origin. Both projections were well ordered, and in the orientation expected if the corresponding piece of retinal tissue had participated in a normal projection. Consistent differences in pie-slice size and tectal coverage between the three groups were found. Pie-slices of nasal origin gave maps showing that they came from a relatively large portion of the retina and projected to a relatively large amount of the tectum; those of temporal origin occupied relatively small amounts of field and tectum. It was concluded that these results are further evidence for the existence of positional markers in the retina which are used for the assembly of the retinotectal map.

Development of the orientation of the visuo-tectal map in Xenopus.

Eye rudiments from Xenopus embryos of stage 28 or younger were explanted on to the flank of similar embryos with normal or nasotemporally reversed orientation. After some hours the eyes were retransplanted to an orbit of a stage 32/34 embryo, with either normal or reversed nasotemporal orientation. Later the visuotectal projections through the operated eyes were mapped electrophysiologically. The maps obtained were oriented as if the mapping orientation of the eye had already been determined in the donor orbit before the first transplantation; i.e. if normally oriented in the final host orbit the eye gave a normal map, and if the eye was reversed in the final host orbit it gave a reversed map. In only one out of 25 cases did it seem that the orientation of the map could have been influenced by the orientation of the eye on the flank of the intermediate host, and here the evidence was weak. Two eyes gave reduplicated maps and 4 maps were uninterpretable. It was concluded that the orientation of the map is determined before stage 26 and is not altered by information derived from the flank during stages 26-34.

Pathways of Xenopus optic fibres regenerating from normal and compound eyes under various conditions.

We have used Horseradish peroxidase to investigate the pathways taken by Xenopus optic fibres regenerating from normal and electrophysiologically-confirmed compound eyes to the optic tectum. Optic fibres, when sectioned near the chiasma, regenerate up both sides of the diencephalon to both tecta. We have therefore been able, by using animals in which one eye had or had not been removed at early embryonic stages, to look at the behaviour of regenerating axons in three different situations: (1) regeneration to the contralateral tectum, previously innervated by the sectioned fibres; (2) regeneration to a "virgin' ipsilateral tectum, never before innervated by optic fibres; and (3) regeneration to an ipsilateral tectum already innervated by fibres from a normal eye. From the chiasma to the tectodiencephalic junction regenerating fibres behave similarly in all three situations, following roughly the course of the normal optic tract, but running in a rather disorganised way, with frequent crossing over of fibres. However fibres of nasal retinal origin (from an NN eye) spread to occupy a much larger area of the side of the diencephalon than those of temporal origin (from a TT eye). From the tectodiencephalic junction to the tectal termination of the fibres there are differences between the three situations investigated; fibres regenerating to a 'virgin' ipsilateral, or to a denervated contralateral tectum, tend to grow straight onto the tectum, instead of being channelled into lateral or medial brachium as uncut fibres tend to be. There is however, the remains of a brachial organisation, and of differential selection of these brachia by fibres from the different types of compound eye, this being well seen on "virgin' tecta. Fibres regenerating to an ipsilateral innervated tectum behave very differently. As they reach the tectodiencephalic junction they suddenly start to grow in a less disorganised way, and are channelled into well defined brachia. If from a compound eye, these fibres terminate on only that part of the tectum innervated by fibres from the corresponding part of the normal eye. Thus fibres from a VV eye and those from the ventral half of the normal eye all terminate on medial tectum; fibres from an NN eye, and those from the nasal half of the normal eye all terminate in caudal tectum; and temporal fibres from both normal and TT eyes terminate in rostral tectum.

Expansion and retinotopic order in the goldfish retinotectal map after large retinal lesions.

A peripheral annulus of neural retina and pigment epithelium was removed from the eye of adult goldfish. After survival times from 148 to 560 days, the retinotectal projection from the remaining central fragment was mapped. In most cases, the map was orderly and had expanded to fill the entire contralateral tectum, but when less than 10-15% of the original retina remained intact, the projection failed to fill the entire available tectal space and was abnormally disordered.

The retinotectal fibre pathways from normal and compound eyes in Xenopus.

Horseradish peroxidase was used to demonstrate the nature of the retinotectal fibre pathways from normal eyes and from compound double nasal (NN), double temporal (TT) and double ventral (VV) eyes in Xenopus. From normal eyes, nasal fibres were widespread in the optic tract and mostly entered the tectum through the medial and lateral brachia. Ventral retinal fibres approached the tectum via the medial brachium and dorsal retinal fibres passed through the lateral brachium, while temporal retinal fibres formed a narrow band in the centre of the tract and entered the tectum directly at its rostral border. Fibres from NN eyes formed a wide tract and strong medial and lateral brachia. Fibres from VV eyes all entered the tectum via the medial brachium and fibres from TT eyes formed a narrow tract and entered the tectum directly from its rostral extremity. Thus fibres from each type of compound eye followed pathways to the tectum that were appropriate to the embryonic origin of the retina forming the compound eye.