Retrograde degeneration of myelinated axons and re-organization in the optic nerves of adult frogs (Xenopus laevis) following nerve injury or tectal ablation

Summary The optic nerve proximal to the lesion (toward the retina) was examined by light and electron microscopy in adultXenopus laevis after various types of injury to optic nerve fibres. Intraorbital resection, transection or crush of the optic nerve or ablation of the contralateral optic tectum all resulted in marked alterations in the myelinated axon population and in the overall appearance of the nerve proximal to the site of injury. Examination of the nerves from 3 days to 6 months postoperatively indicated that a progressive, retrograde degeneration of myelin and loss of large-diameter axons occurred throughout the retinal nerve stump regardless of the type of injury or distance of the injury from the retina. The retinal stump of nerves receiving resection or transection showed a nearly complete loss of myelin and large-diameter axons while the degree of degeneration was subtotal in nerves receiving crush injury or after lesions farther from the retina (i.e. tectal ablation). In addition, the entire retinal nerve stump after all types of injury was characterized by the appearance of an actively growing axon population situated circumferentially under the glia limitans. The latter fibres are believed to represent regrowing axons which are being added onto the nerve, external to the original axon population and are suspected to modify actively the glial terrain and glia limitans.     

A pattern of optic axons in the normal goldfish tectum consistent with the caudal migration of optic terminals during development

Optic axons were cut in the goldfish optic nerve or tectum, filled with horseradish peroxidase and traced in tectal wholemounts. Many of them ran in conspicuous fascicles which curved across the tectum. Axons from central nasal retina, which ran in the most rostral fascicles, turned abruptly as they left these fascicles; ran caudally in a diffuse, parallel array for up to half the tectal length; and passed beneath more caudal fascicles to innervate the caudal half-tectum. Axons from peripheral nasal retina ran in the most caudal fascicles and terminated near their turning-points. Axons from temporal retina entered the tectum at its rostral margin and ran caudally from their points of entry to innervate the rostral half-tectum. The resultant pattern was entirely consistent with the proposal that a slow caudal migration of optic terminals compensates during normal development for disparate modes of retinal and tectal growth.     

Changes in NADPH diaphorase expression in the fish visual system during optic nerve regeneration and retinal development

The various functions of nitric oxide (NO) in the nervous system are not fully understood, including its role in neuronal regeneration. The goldfish can regenerate its optic nerve after transection, making it a useful model for studying central nervous regeneration in response to injury. Therefore, we have studied the pattern of NO expression in the retina and optic tectum after optic nerve transection, using NADPH diaphorase histochemistry. NO synthesis was transiently up-regulated in the ganglion cell bodies, peaking during the period when retinal axons reach the tectum, between 20–45 days after optic nerve transection. Enzyme activity in the tectum was transiently down-regulated and then returned to control levels at 60 days after optic nerve transection, during synaptic refinement. To compare NO expression in the developing and regenerating retina, we have looked at NO expression in the developing zebrafish retina. In the developing zebrafish retina the pattern of staining roughly followed the pattern of development with the inner plexiform layer and horizontal cells having the strongest pattern of staining. These results suggest that NO may be involved in the survival of ganglion cells in the regenerating retina, and that it plays a different role in the developing retina. In the tectum, NO may be involved in synaptic refinement.     

Assessment of acetylcholine as an optic nerve neurotransmitter in Bufo marinus.

The optic nerve and optic tectum of the marine toad, Bufo marinus, contain significant levels of acetylcholine as measured by gas chromatography-mass spectroscopy and significant activities of the enzymes choline acetyltransferase and acelylcholinesterase. Following eye enucleation. acetylcholine levels and the activities of these two enzymes fall in the denervated tectum. The decrease in acetylcholinesterase activity was shown histochemically to occur in tectal layers containing class II and class IV retinal ganglion cell terminals.These studies, in conjunction with previous biochemical and physiological experiments, provide additional evidence for the role of acetylcholine as an optic nerve neurotransmitter in the toad.     

Changes in retinotectal projection in adult xenopus following partial retinal ablation

The retinotectal projection was mapped electrophysiologically and autoradiographically after surgical removal of the nasal or temporal retinal quadrants in Xenopus. The immediate result of retinal ablation was a corresponding temporal or nasal scotoma in the visual field and the absence of part of the optic fibre projection from the tectum. Thirty to fourty days after operation the visual field scotoma still persisted but the vacated tectal area became reinnervated by collateral sprouting of optic fibres from the fringe of the residual retinal projection. It is concluded that the retinotectal connection pattern can be altered by retinal ablations in adult toads.     

Transformations of the retinal topography along the visual pathway of the chicken

Summary It is still unclear how the retinotectal map of the chick is formed during development. In particular, it is not yet known whether or not the organization of fibres plays a role in the formation of this map. In order to contribute to the solution of this problem, we analysed the representation of the retinal topography at closely spaced intervals along the fibre pathway. We injected HRP into various sites of the tectal surface and traced the labelled fibre bundles back to the retina. The retinal topography was reconstructed at ten different levels, i.e. in the retina, the optic nerve head, the middle of the optic nerve, the chiasm (three levels), the optic tract (three levels), and the optic tectum. We obtained the following results: (1) The labelled fibre bundles as well as the fields of labelled retinal ganglion cells were always well delimited and coherent. (2) The reconstructions show that transformations of the retinal topography occur in the fibre pathway. The first and most important transformation is found in the optic nerve head where the retinal image is mirrored across an axis extending from dorsotemporal to ventronasal retina. In addition, the retinal representation is split in its temporal periphery. Thus, central and centrotemporal fibres are no longer in the centre of the image but close to the dorsal border of the nerve. Peripheral fibres are found along the medial, ventral and lateral circumference of the nerve. In the optic tract a second transformation occurs. The retinal topography is rotated clockwise by about 90 degrees and flattened to a band. The flattening is accompanied by a segregation of fibre bundles so that eventually central and centrotemporal retinal fibres are located centrally, ventral fibres dorsally and dorsal retinal fibres ventrally in the tract. By these two transformations an organization of fibres is produced in the optic tract which can be projected onto the tectal surface without major changes given that dorsal and ventral fibres remain in their relative positions, and that deep lying fibres project to the rostral and central tectum, superficial fibres to the caudal tectum.The transformations which we have observed follow specific rules and thus maintain order in the pathway although retinotopy is lost. In conjunction with our earlier studies on the development of the retinotectal system we conclude that fibres are laid down in a chronotopic order. The transformations take place under particular structural constraints. Thus, an organization of fibres is provided in the optic tract which results in a retinotopic map when projected onto the tectal surface. This is stated for the order of magnitude of fibre bundles as investigated in this study. At the level of individual fibres additional factors may play an important role.     

Current source density analysis of ON/OFF channels in the frog optic tectum

In the vertebrate retina, it is well known that an ON/OFF dichotomy is present. In other words, ON-center and OFF-center cells participate in segregated pathways morphologically and physiologically. However, there is no doubt that integration of both channels is necessary to generate the complicated response properties of visual neurons in higher optic centers. So far, functional organization of the ON and OFF channels in the optic centers has not been demonstrated at the level of neuronal populations. In this review article, we summarize our experimental approaches to demonstrate functional organization of the ON and OFF channels using current source density (CSD) analysis in the frog optic tectum. First, we show that one-dimensional CSD analysis, assuming constant conductivity, is applicable in the tectal laminated structure. The CSD depth profile of a response to electrical stimulation of the optic tract is composed of three current sinks (A, B, and D) in the retinorecipient layers and two current sinks (C and E) below those layers. This result is in agreement with previous morphological and physiological findings, and shows that CSD analysis is very useful to demonstrate the flow of visual information processing. Second, CSD analysis of tectal responses evoked by diffuse light ON and OFF stimuli reveals obviously different distributions of synaptic activity in the laminar structure. Two or three current sinks (I, II and III) are generated in response to ON stimulation only in the retinorecipient layers, while up to six current sinks (IV, V, VI, VII, VIII and IX) to OFF stimulation throughout the tectal layers. Based on well known properties of retinal ganglion cells of the frog, possible neuronal mechanisms underlying each current sinks and their functional roles in visually guided behavior are considered.     

Evidence that regenerating optic axons maintain long-term growth in the lizard Ctenophorus ornatus: growth-associated protein-43 and gefiltin expression

In the lizard, Ctenophorus ornatus, the optic nerve regenerates but animals remain blind via the experimental eye, presumably as a result of axons failing to consolidate a retinotopic map in the optic tectum. Here we have examined immunohistochemically the expression of the growth-associated protein GAP-43 and the low-molecular-weight intermediate filament protein gefiltin, up to one year after optic nerve crush. Both proteins were found to be permanently up-regulated, suggesting that regenerating axons are held in a permanent state of re-growth. We speculate that, in the lizard, the continued expression of GAP-43 and the failure to switch from the expression of low- to high-molecular-weight intermediate filament proteins are associated with the inability to consolidate a retinotopic projection.     

Aberrant axonal paths in regenerated goldfish retina and tectum opticum following intraocular injection of ouabain

Following ouabain-induced degeneration, the neural retina and the retinotectal axons regenerate. The pathways of regenerated retinal ganglion cell axons in retina and in tectum are visualized by labeling with horseradish peroxidase (HRP) applied to the optic nerve. In retina, the axons exhibit highly abnormal courses, including extensive fascicle crossing, hairpin loops and circular routes. In tectum, retinal axon fascicles are not neatly aligned in a normal fascicle fan. Instead, long and short fascicles are mixed, and take erratic routes, crossing each other and crossing the tectal equator.     

Roles of periventricular neurons in retinotectal transmission in the optic tectum

The midbrain roof is a retinorecipient region referred to as the optic tectum in lower vertebrates, and the superior colliculus in mammals. The retinal fibers projecting to the tectum transmit visual information to tectal retinorecipient neurons. Periventricular neurons are a subtype of these neurons that have their somata in the deepest layer of the teleostean tectum and apical dendrites ramifying at more superficial layers consisting of retinal fibers. The retinotectal synapses between the retinal fibers and periventricular neurons are glutamatergic, and ionotropic glutamate receptors mediate the transmission in these synapses. This transmission involves long-term potentiation, and is modulated by hormone action. Visual information processed in the periventricular neurons is transmitted to adjacent tectal cells and target nuclei of periventricular neuron axonal branches, some of which relay the visual information to other brain areas controlling behavior. We demonstrated that periventricular neurons play a principal role in visual information processing in the teleostean optic tectum; the effects of tectal output on behavior is discussed also in the present review.     

Impaired refinement of the regenerated retinotectal projection of the goldfish in stroboscopic light: a quantitative WGA-HRP study

Summary The retinotectal projection of the goldfish was studied after regeneration of a cut optic nerve in stroboscopic light, constant light or diurnal light, with the lens removed to blur the retinal image. Retrograde transport of wheatgerm agglutinin, conjugated to horseradish peroxidase, from a standard tectal injection site was used to measure the topographic precision of the projection. The dispersion of labelled retinal ganglion cells, which reflects this precision, was assessed by a method based on distance to nearest neighbour. In normal fish treated similarly, these cells are known to be clustered into about 1% of the retinal area. Early in regeneration, however, they are widely dispersed. The projection map then re-acquires its precision over two or three months.In diurnal light, lens ablation had no effect on refinement of the regenerated map. Constant light increased the number of labelled cells but also had no significant effect on the map. But in stroboscopic light with a continuous pseudorandom pattern of flash intervals (average rate 4.8 Hz), much less refinement was seen. Even after 70–98 days of regeneration, labelled cells remained scattered, on average, over 20% of the retinal area. These retinae were indistinguishable by several criteria from those obtained in diurnal light after only 32–39 days. Mislocated axon terminals, which are largely eliminated during the second and third months of regeneration in diurnal light, evidently persist much longer in stroboscopic light that synchronizes ganglion cell activity across the retina. These results, like previous ones obtained by blocking the transmission of activity to the tectum, support a model of map refinement based on correlation in the firing of neighbouring neurons, which may have wide application within the nervous system.     

Establishment of synaptic connections between explants of embryonic neural tissue in culture: experimental ultrastructural studies

Summary The development of synaptic interconnections between co-cultured explants of central and peripheral nervous tissue from chick embryos has been investigated by light and electron microscopy. Two sets of co-cultured explants were used: (a) dorsal root ganglion (DRG) and spinal cord and (b) retina and tectum. Both sets of co-cultured explants became linked by bundles of fibres but the most consistent results were obtained with the DRG-spinal cord explants. Thus axons from the DRG extended large distances across the culture substrate to reach and enter mainly the dorsal horn region of the spinal cord explants. In contrast retina-tectum links were less frequently established and were less extensive, possibly because there are fewer cells in retinal explants capable of establishing contacts in tectal explants than there are cells in DRG explants capable of establishing contacts in the spinal cord. In order to distinguish between synapses involving only neuronal elements within an expiant and those involving ingrowing fibres, fibre bundles linking adjacent explants were transected and the preparations fixed two to six hours later. Electron microscope study of such cultures revealed degenerating neurites and terminals in the spinal cord explants receiving DRG fibres but none in the corresponding DRG explants. Retinal explants contain numerous synapses of many types but degenerating terminals could not be found within the retinal explants after nerve fibre transections. Degenerating neurites and terminals were found within tectal explants but they were fewer and more difficult to locate than those found within spinal cord explants. The reasons for such differences are discussed.     

Readjustment of retinotectal projection following reimplantation of a rotated or inverted tectal tissue in adult goldfish.

1. The pattern of visual projection from the retina on to the optic tectum following reimplantation of a piece of the tectal tissue was studied with neurophysiological mapping methods in adult goldfish. 2. When a rectangular piece of the tectum was dissected, lifted free, and then reimplanted to the same tectum after rotation by 180 degrees around the dorsoventral axis, the re-established visual projection later showed a complete reversal of retinotopic order within the reimplanted area with reference to the normal projection on to the intact surrounding area of the same tectum. The localized reversal was observed as early as 65 days, and also as late as 721 days after the 180 degree rotated reimplantation. 3. If a square piece of the tectal tissue was reimplanted after rotation by 90 degrees anticlockwise around the dorsoventral axis, the restored visual projection later showed a corresponding localized 90 degrees rotation within the reimplanted ares. 4. When the entire laminar structure of a dissected tectal tissue was inverted, and the reimplanted upside-down along the same rostrocaudal axis of the tectum, the restored visual projection on to the inverted tectal reimplant was found to be organized in a reverse retinotopic order along only the mediolateral axis within the reimplanted area. The restored visual projection retained a correct retinotopic order along the rostrocaudal axis. The same trends were also observed after regeneration of the optic fibres following section of the contralateral optic nerve. 5. If the inverted tectal tissue was reimplanted along the same mediolateral axis of the tectum, the re-established visual projection showed a localized reversal of retinotopic order along only the rostrocaudal axis within the reimplanted area. Sectioning the contralateral optic nerve made no difference to the result. 6. These results suggest that a piece of adult tectal tissue retains its original topographic polarity regardless of the orientation of reimplantation after either a rotation or an inversion. Furthermore the retention is not a short-lived transitory phenomenon. It persisted as long as the reimplanted tissue survived. 7. Histological examination of the operated tecta revealed that the reimplanted tectal tissues underwent a severe derangement in their laminar structures. It was impossible to identify the main target zone of retinotectal projection (the stratum fibrosum et griseum superficiale) or the central cellular layer (the stratum griseum centrale) in the reimplants. The prominent feature of the deranged tectal tissue was irregular vortices of tangled fibre bundles. Sparse tectal neurones of bipolar and granular types were irregularly scattered in the deranged structure of the reimplant. 8. Thus, the retention of original topographic polarity did not require an integrity of the cytoarchitectonic structure of the reimplanted tectal tissue.     

Motor responses to localized electrical stimulation of the tectum in the freshwater perch (Perca fluviatilis)

The results of localized electrical stimulation of the teleostean tectum indicate the presence within each tectal lobe of separate motor areas mediating ipsilateral turning, contralateral turning, and rolling movements. Stimulation of caudal regions produced larger turning circles than stimulation of rostral sites. Both these sets of observations conflict with the retinotopic map. Stimulation sites connected with turning and rolling movements were mostly located in the upper layer of the tectum. Other kinds of movement including aggressive behaviour, escape movements, head dipping, and forward swimming were obtained by stimulating the deeper tectal sites and subtectal areas. These results suggest that the tectum may be differentiated into areas with specific motor functions and afferent connections. This has important consequences for studies on optic nerve regeneration and neuronal specificity.     

Foveal topography in the optic nerve and primary visual centers in Falconiforms

The topography of the retinal nasal and temporal foveal projections upon the optic nerve and primary visual centers was studied in diurnal bifoveate birds of prey by means of restricted tritiated proline intraocular injection. According to the degree of retinotopy, this study reveals that a single injection of tracer in the nasal or temporal fovea produces a well-defined and complementary pattern of projections in the following contralateral nuclei: lateral anterior thalamus, lateroventral geniculate nucleus (glv), superficial synencephalic (ss), tectal grey (gt), and optic tectum. In the thalamic nucleus dorsolateral anterior, the nasal foveal projections are seen mainly in the lateral and rostrolateral subdivision, while temproal projections are seen mainly in the magnocellular subdivision. In the external and ectomammillary nuclei there is some evidence of retinotopic innervation. Finally, a discrete field of projection from the nasal or temporal fovea is detected in lateral hypothalamus, ventrolateral thalamus, lateral geniculate intercalated nucleus, and pretectal optic area. The nasotemporal axis of the retina is ventrodorsally oriented in the optic nerve with ganglion cell axons of the temporal fovea more dorsally placed than the nasal ones. In the primary visual centers this retinal axis is mediolaterally rpresented in the nuclei glv, ss, and gt, and dorsoventrally oriented in the optic tectum. © 1993 Wiley-Liss, Inc.     

Ingrowth and ramification of retinal fibers in the developing optic tectum of the chick embryo

Summary Onset, temporal sequence, and pattern of ingrowth of retinal fibers into the developing optic tectum of the chicken were investigated with histological procedures including the Golgi technique. Invading fibers could first be detected by stage 34 (eight days of incubation) at a specific locus which is the central area of the optic tectum. Compared to other tectal regions the central area is distinguished at this time by its advanced cytoarchitectural development and by the maturation of dendrites of radial cells located within superficial laminae. Immediately after their arrival at the central area some fibers can be observed invading the outer tectal layers and forming side branches. These observations permit the conclusion that fibers do not wait at their termination site for several days, as has been suggested earlier. Retinal axons start to invade the tectum at the site which is most advanced in its structural development. This early maturation of neurons in a specific tectal region might be a sufficient explanation for the central retinal fibers connecting to neurons of this area, which, propter hoc, is called the central tectal area.     

Nucleus isthmi: its contribution to tectal acetylcholinesterase and choline acetyltransferase in the frog Rana pipiens

The distribution of acetylcholinesterase and the activity of choline acetyltransferase was studied in the tecta of normal frogs and frogs without retinal and/or nucleus (n.) isthmi inputs. In normal animals acetylcholinesterase activity is found primarily in three bands in the outer layers of the tectum-lamina A, laminae C-F, and lamina G. After retinal and contralateral n. isthmi deafferentation three distinct bands of tectal acetylcholinesterase activity are still present. After bilateral n. isthmi deafferentation there is loss of activity in lamina G and reduced activity in lamina A. With retinal and ipsilateral n. isthmi deafferentation, activity is seen only in lamina A. With retinal and bilateral n. isthmi deafferentation there is virtually no acetylcholinesterase activity in the outer tectal layers. Following unilateral retinal deafferentation there is no statistically significant difference in choline acetyltransferase specific activity between intact and deafferented tectal lobes after two, four and nine weeks. With unilateral nucleus isthmi lesions and survival times of between 10 and 40 days, choline acetyltransferase specific activity in the tectal lobe ipsilateral to the ablation is approximately 38% of the specific activity of the contralateral lobe. With bilateral n. isthmi lesions there is a strong correlation between amount of n. isthmi ablated and reduction of choline acetyltransferase activity. In vitro tectal acetylcholine synthesis was also determined in animals with unilateral n. isthmi ablation. On average, tectal lobes ipsilateral to the ablated n. isthmi synthesize acetylcholine at a rate which is approximately 58% of that of contralateral tecta. Collectively, these results imply that n. isthmi is the sole cholinergic input to the frog optic tectum, with ipsilaterally projecting isthmotectal fibers accounting for the greater share.     

Some aspects of the organization of the optic tectum of the skate Raja

The cellular organization of the optic tectum of the skate Raja was studied by anatomical and physiological methods. Injection of [³H]proline into one eye resulted in labelling of the dorsal one-third to one-half of the contralateral tectum. Additional label was found in a tegmental locus thought to be the nucleus of the basal optic root. Cellular profiles of tectal neurons were defined by Golgi methods. A variety of approximately horizontally arborizing neurons were found primarily in superficial and central tectal zones. Neurons with one or more radial dendrites were confined either to central and periventricular zones or to the layer innervated by optic afferents. Evoked potentials as a function of tectal depth were elicited by shocks applied to the contralateral optic nerve. A current-source-density analysis of the evoked potentials revealed that their current sources and sinks were confined to the dorsal tectal layers. This suggests that the radially oriented neurons found in dorsal tectum are the primary generators of the optic-evoked response.     

Neuronal organization underlying visually elicited prey orienting in the frog--I. Effects of various unilateral lesions

We have studied the effects on frog orienting behavior of three lesions: unilateral optic nerve section, unilateral tectal lobe ablation, and unilateral transverse hemisection of the neuraxis at a level just caudal to the optic tectum. Unilateral optic nerve section and unilateral tectal lobe ablation produce very similar deficits in visually elicited responses to prey items, an absence of responses for stimuli at locations within the monocular field of one eye. Unilateral hemisection, in contrast, results in abnormalities in visually elicited responses over a wider area, encompassing the entire ipsilateral visual hemifield. The hemisection deficit also differs in character from that following optic nerve section or tectal lesion. Within the affected hemifield, frogs do not fail to respond to stimuli but rather respond with abnormally directed movements. The movements, regardless of stimulus eccentricity on the horizontal, are always forwardly directed. While not varying with horizontal eccentricity, the movements do vary with stimulus elevation and distance. The variation with stimulus distance in the affected hemifield is somewhat different from that in the opposite hemifield. We conclude from the behavior that remains after hemisection lesions that there must exist bilateral descending tectofugal paths capable of triggering movements which vary with stimulus elevation and distance, and a crossed descending tectofugal path capable of triggering turns into one visual hemifield. That the deficit area is larger following a hemisection than following tectal lobe ablation indicates that the hemisection has affected the ability of both tectal lobes to trigger turns in one direction. A possible interpretation of this finding is that the lesion has interrupted not only the crossed descending tectofugal path from one tectal lobe but an uncrossed descending tectofugal path from the other. This hypothetical pathway as well as the others mentioned is incorporated in a model of the organization of the post-tectal circuitry involved in orienting.     

The conditioning effect of optic nerve injury upon axonal regrowth from adult rat retinal ganglion cells explanted in vitro

An in vitro assay was used to determine the effects of conditioning nerve lesions on the regeneration of adult rat retinal ganglion cell (RGC) axons from retinal explants. Following the conditioning lesion (CL) of unilateral optic nerve transection, maximal regrowth was seen from RGC explanted from ipsilateral retinae 10 days post-CL. Explants from this group initiated axonal regrowth earlier and a greater percentage regrew axons when compared with explants from normal rats. Axonal regrowth from explants of retinae contralateral to CL was also seen earlier than normal. In further experiments, the effects of both exposure of the optic nerve sheath in the orbit and the incision of the dura without injury to optic nerve axons were studied. The conditioning effect of a dural incision was found to be the same as that of optic nerve transection, whilst exposure of the optic nerve sheath had no conditioning effect on RGC axonal regrowth in vitro.