Regenerating optic axons restore topography after incomplete optic nerve injury.

Following complete optic nerve injury in a lizard, Ctenophorus ornatus, retinal ganglion cell (RGC) axons regenerate but fail to restore retinotectal topography unless animals are trained on a visual task (Beazley et al. [ 1997] J Comp Neurol 370:105-120, [2003] J Neurotrauma 20:1263-1270). Here we show that incomplete injury, which leaves some RGC axons intact, restores normal topography. Strict RGC axon topography allowed us to preserve RGC axons on one side of the nerve (projecting to medial tectum) while lesioning those on the other side (projecting to lateral tectum). Topography and response properties for both RGC axon populations were assessed electrophysiologically. The majority of intact RGC axons retained appropriate topography in medial tectum and had normal, consistently brisk, reliable responses. Regenerate RGC axons fell into two classes: those that projected topographically to lateral tectum with responses that tended to habituate and those that lacked topography, responded weakly, and habituated rapidly. Axon tracing by localized retinal application of carbocyanine dyes supported the electrophysiological data. RGC soma counts were normal in both intact and axotomized RGC populations, contrasting with the 30% RGC loss after complete injury. Unlike incomplete optic nerve injury in mammals, where RGC axon regeneration fails and secondary cell death removes many intact RGC somata, lizards experience a "win-win" situation: intact RGC axons favorably influence the functional outcome for regenerating ones and RGCs do not succumb to either primary or secondary cell death.

Changing Pax6 expression correlates with axon outgrowth and restoration of topography during optic nerve regeneration.

Pax6, a member of the highly conserved developmental Pax gene family, plays a crucial role in early eye development and continues to be expressed in adult retinal ganglion cells (RGCs). Here we have used Western blots and immunohistochemistry to investigate the expression of Pax6 in the formation and refinement of topographic projections during optic nerve regeneration in zebrafish and lizard. In zebrafish with natural (12-h light/dark cycle) illumination, Pax6 expression in RGCs was decreased during axon outgrowth and increased during the restoration of the retinotectal map. Rearing fish in stroboscopic illumination to prevent retinotopic refinement resulted in a prolonged decrease in Pax6 levels; return to natural light conditions resulted in map refinement and restoration of normal Pax6 levels. In lizard, RGC axons spontaneously regenerate but remain in a persistent state of regrowth and do not restore topography; visual training during regeneration, however, allows a stabilization of connections and return of topography. Pax6 was persistently decreased in untrained animals but remained increased in trained ones. In both species, changes in expression were not due to cell division or cell death. The results suggest that decreased Pax6 expression is permissive for axon regeneration and extensive searching, while higher levels of Pax6 are associated with restoration of topography.

Generation of transgenic mice with mild and severe retinal neovascularisation.

AIM: To generate a mouse model for slow progressive retinal neovascularisation through vascular endothelial growth factor (VEGF) upregulation. METHODS: Transgenic mice were generated via microinjection of a DNA construct containing the human VEGF165 (hVEGF) gene driven by a truncated mouse rhodopsin promoter. Mouse eyes were characterised clinically and histologically and ocular hVEGF levels assayed by ELISA. RESULTS: One transgenic line expressing low hVEGF levels showed mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation, which remained stable up to 3 months postnatal. Histologically, there were some disturbance and thinning of inner and outer nuclear layers, with occasional focal areas of neovascularisation. By contrast, three other lines expressing high hVEGF levels presented with concomitantly severe phenotypes. In addition to the above, clinical features included extensive neovascularisation, haemorrhage, and retinal detachment; histologically, focal to extensive areas of neovascularisation associated with retinal folds, cell loss in the inner and outer nuclear layers, and partial retinal detachment were common. CONCLUSIONS: The authors generated four hVEGF overexpressing transgenic mouse lines with phenotypes ranging from mild to severe neovascularisation. These models are a valuable research tool to study excess VEGF related molecular and cellular changes and provide additional opportunities to test anti-angiogenic therapies.

The accessory optic system of the wallaby, Setonix brachyurus: anatomy in normal animals and after early unilateral eye removal.

We have traced primary visual projections to nuclei of the accessory optic system in the mature wallaby, Setonix brachyurus, the "quokka," following unilateral intraocular injections of horseradish peroxidase. The organization of pathways and nuclei is similar to that of other marsupials and to that of eutherian mammals. The dorsal, lateral and medial terminal nuclei receive bilateral input, though nuclei ipsilateral to the injected eye are weakly labelled in comparison with their contralateral counterparts. We also report on the accessory optic system in animals which were unilaterally enucleated neonatally or at postnatal day 35. At maturity in enucleated animals, ipsilateral projections to all nuclei of the accessory optic system are more densely labelled than normal. This exuberance is more pronounced in neonatally enucleated animals than in those enucleated at the later stage.

Cell death in the developing retinal ganglion cell layer of the wallaby Setonix brachyurus.

The distribution and number of dying cells in the developing retinal ganglion cell layer of the wallaby Setonix brachyurus were assessed by using cresyl violet stained tissue. The density of dying cells has been expressed per 100 live cells for the entire retinal surface, data being presented as a grid of 500 micron squares. For statistical analysis, retinae were divided into 8 regions; dorsal, ventral, nasal, and temporal quadrants, each further divided into center and periphery. This method allowed comparison of the extent of cell death at different retinal locations as the high density area centralis of live cells developed temporal to the optic disk from 60 days onward. Between 30 and 70 days, dying cells were seen across the entire retina; beyond 100 days very few were seen. Initially, there was a significantly higher incidence of dying cells in the central retina compared to the periphery, whereas from 50 days this situation was reversed. Analysis of the central retina before and during area centralis formation consistently indicated a significantly lower number of dying cells per 100 live cells in temporal compared to other retinal quadrants. This differential pattern suggests that cell death lowers live cell densities less in the emerging area centralis than elsewhere, and therefore must play a part in establishing live cell density gradients. However, we cannot exclude the possibility that other factors are also instrumental. Indeed, factors such as areal growth (Beazley et al., in press) presumably operate at later stages since live cell density gradients continue to be accentuated even after cell death is complete. Numbers of dying cells peaked by 50 days, reaching approximately 1% of the live cell population. At this stage, counts were also maximal for live cells with values up to 30% above the adult range.

Neurogenesis and cell death in the isthmic nuclei of the frog Limnodynastes dorsalis.

In Limnodynastes dorsalis, neurogenesis of the isthmic nuclei was determined by 3H-thymidine autoradiography. The nuclei developed in a rostroventral to caudodorsal direction. Peak neurogenesis occurred in midlarval life, resulting in the formation of a substantial portion of each nucleus. Cell death was evidenced by the presence of pyknotic cells within the nucleus in all except the earliest stages. Peak cell death occurred around metamorphic climax. It was during this period that a rim neuropil appeared that segregates within the nucleus the contralateral and ipsilateral components of the tecto-isthmo-tectal pathways underlying binocular vision. It is also a time just prior to the onset of electrophysiologically detectable ipsilateral visual responses. However, no systematic cell death was found. Therefore the role of cell death in the formation of the isthmic nuclei remains obscure, although it may be involved in the establishment of tecto-isthmo-tectal connections.

Retinal ganglion cell death during optic nerve regeneration in the frog Hyla moorei.

In the frog Hyla moorei we have estimated there to be between approximately 450,000 and 750,000 cells in the retinal ganglion cell layer. Optic axon counts and retrograde transport of horseradish peroxidase (HRP) indicated that 72-76% of these were ganglion cells. Cells of this type were distributed as a temporally situated area centralis within a horizontal visual streak. Cell and optic axon counts showed that there was an approximately 40% loss of ganglion cells during optic nerve regeneration. Ganglion cells appeared chromatolysed by 6-8 days after an extracranial nerve crush but there was no indication of cell death until 15 days. By this stage anterograde transport of HRP indicated that axons had reached the chiasma. Death was first seen in the area centralis, extended along the streak, and finally was observed in the periphery by 65 days; cell counts demonstrated that at this time the wave of death was almost complete. We have previously shown by electrophysiological visual mapping (Humphrey and Beazley, '82) and confirmed in this study that visuotectal projections were retinotopically organized during regeneration. Multiunit receptive fields were initially large but progressively refined starting in nasal field (temporal retina) to restore a normal projection. The similar sequences whereby the visuotectal projection became refined and death took place in the retinal ganglion cell layer suggested that death may be related to a process of organization within the regenerating projection. In normal animals primary visual pathways revealed by anterograde transport of HRP were essentially similar to those of Rana pipiens and R. esculenta. Regenerating axons generally remained within optic pathways. Exceptions were a retinoretinal projection which was not completely withdrawn even after 1,028 days and a direct projection to the ipsilateral tectum via an inappropriate part of the optic tract.

The evolution of an area centralis and visual streak in the marsupial Setonix brachyurus.

The distribution, morphology, size, and number of cells in the retinal ganglion layer of the marsupial Setonix brachyurus, "quokka," was studied from 25 days postnatal to adulthood using Nissl-stained wholemounts. The total cell population was evenly distributed up to 50 days, but by 75 days highest densities were generally observed in a broad band extending across the nasotemporal axis. At 87 days, a temporally situated area centralis was seen for the first time. This was embedded in a horizontally aligned visual streak, the nasal arm of which contained areas of high density. By 106 days, densities in the area centralis had stabilized while peripheral values were higher than adult levels even at 180 days. In the adult, the area centralis was surrounded by a weak visual streak. Retinal area increased steadily during development to reach 168 mm2 at 180 days, the adult range being 225-250 mm2. All cells in the ganglion layer appeared undifferentiated and rounded at 33 days with soma diameters of 3-6 micrometers; by 70 days diameters had increased to 4-12 micrometers and some cells had axon hillocks containing Nissl substance. From 87 days we distinguished ganglion cells, which constituted 54-63% of the total. These were identified by deeply stained Nissl substance and had diameters of 7-18 micrometers, compared to 7-23 micrometers at 143 days and 7-24 micrometers in the adult; the remaining cells, termed glia/interneurons, were 5-8 micrometers throughout. Only ganglion cells were organized into an area centralis and visual streak. Glia/interneurons were evenly distributed except at the extreme periphery, where their density increased. In sectioned material, the ganglion layer was distinct from 25 days while the neuroblastic layer separated only between 48 and 85 days. From 25 to 250 days the total number of cells in the ganglion layer remained similar to the adult range of 336,000-393,000. At both 87 days and in adults optic axon counts fell between 180,000 and 224,000, close to ganglionic cell estimates. At 25 and 34 days, respectively, optic axon numbers were 75,000 and 172,000. Myelination was absent at 25 and 34 days, 3% at 87 days, and almost 100% in adults. Mechanisms are discussed whereby the area centralis and visual streak may evolve from an even distribution of cells while their number remains constant; migration is considered likely to be important.

Transient neovascularisation of the frog retina during optic nerve regeneration.

In conditions such as diabetic retinopathy, degenerative events in the retina are associated with neovascularisation. It is well established that a proportion of retinal ganglion cells die during optic nerve regeneration in the frog. The present study has determined whether neovascularisation takes place during this regenerative process. To do so, the pattern of blood vessels overlying the retinal ganglion cell layer was analysed in the frog Litoria (Hyla) moorei. We examined normal animals and those undergoing optic nerve regeneration following nerve crush. Blood vessels were visualised by perfusion with Indian ink and retinae were prepared as wholeamounts. In normal animals, the vascular tree was found to lie superficial to the nerve fibre layer and was more complex in regions overlying the area centralis and visual streak. After nerve crush, abnormal blood vessels transiently formed between the existing branches of the vascular tree. The new vessels were concentrated as an annulus centred on the optic nerve head and over the area centralis in midtemporal retina. The neovascularisation became most extensive between 6 and 10 weeks postcrush and disappeared by 12 weeks. The spatiotemporal sequence of neovascularisation suggests that it is triggered by accumulations of degenerating material formed as a proportion of the ganglion cells die during optic nerve regeneration.

Biphasic retinal neurogenesis in the brush-tailed possum, Trichosurus vulpecula: further evidence for the mechanisms involved in formation of ganglion cell density gradients.

We investigated cell generation in the retina of the brush-tailed possum (Trichosurus vulpecula) by using tritiated (3H)-thymidine labelling of newly generated cells. Animals aged between postnatal day (P) 5 and 85 each received a single injection of 3H-thymidine. Following autoradiographic processing, maps of labelled cells were constructed from retinal sections. Retinal cell generation takes place in two phases, the first is concluding in the retinal periphery at P53 as the second is seen to commence in midtemporal retina. In the first phase, cells in central retina are generated earlier than those in peripheral regions. In the second phase, cells complete their final division in midtemporal retina first and in the periphery last. Cells generated in the first phase comprise virtually all cells in the ganglion cell layer, amacrine cells, horizontal cells, and cones. Ganglion cells are produced at a slightly earlier stage than displaced amacrine cells, horizontal cells, or cones. Amacrine cells in the inner nuclear layer are the final cells produced in the first phase. When ganglion cells and amacrine cells are pooled, their combined rate of production matches that of the other cell types. These data indicate that the ratio of displaced amacrine cells: horizontal cells: cones: combined ganglion cells and amacrine cells does not change throughout development. However, the ratio of ganglion cells:macrines changes steadily as development proceeds to favour amacrine cells. In the second phase, sparse numbers of nonganglion cells in the ganglion cell layer and large numbers of bipolar and Müller cells are produced along with all rods. The two phases in the possum are similar to those seen in the wallaby, the quokka. However, fewer cells are added in central retina in the possum than in the quokka and cell addition continues for a more extended period in the periphery in the possum. We suggest that this difference in cell addition could account for the development of a more pronounced visual streak of retinal ganglion cells in the possum than in the quokka. A comparison of possum retinal cell generation with that of other marsupials adds support for the "homochrony theory."

Cell death in the inner and outer nuclear layers of the developing retina in the wallaby Setonix brachyurus (quokka).

We have examined the number and distribution of dying cells in the developing inner (INL) and outer (ONL) nuclear layers of sectioned quokka retinae (N = 31) from embryonic day (E)24 to postnatal day (P)192. Before birth, dying cells were seen in the optic fissure. Thereafter two major phases of cell death took place in the INL. The first phase was more pronounced within the vitread part with peak numbers of dying cells at P50. By contrast, during the second phase, cell death was more extensive in the sclerad portion; peak numbers of dying cells were recorded at P85 and P100 for the vitread and sclerad parts respectively. At these stages, photoreceptors were seen in the INL suggesting that these ectopic cells contribute to the pool of dying cells. The pattern of cell death broadly followed a central to peripheral sequence in the first phase but, in the second, was seen initially in mid-temporal retina and then became panretinal. Dying cells were seen in the ONL but in smaller numbers than in the INL. There was a peak of cell death at P26 which may represent death of mitotic cells at the ventricular surface. In the quokka, retinal cell genesis takes place in two phases (Harman and Beazley: Neuroscience 28:219-232, '89). The two major phases of cell death described here peak approximately 40 days after episodes of maximal cell genesis. These findings, together with data for the mouse, suggest that a biphasic pattern of cell genesis and cell death may be a feature of eutherian as well as marsupial retinal development.

Prevention of optic nerve regeneration in the frog Hyla moorei transiently delays the death of some ganglion cells.

We have previously reported that a proportion of ganglion cells die during optic nerve regeneration in the adult frog Hyla moorei (Humphrey and Beazley, '85). Here we assess the effect of preventing optic nerve regeneration on this cell loss. The optic nerve was crushed unilaterally and regeneration was allowed to progress unimpeded in one experimental series but was prevented by ligating or capping the nerve in another. We estimated total cell numbers in the ganglion cell layer from cresyl-stained wholemounts, comparing each experimental retina with its unoperated partner. At 70-78 days postcrush, mean cell numbers had fallen by 31.5% for frogs with unimpeded regeneration (N = 9), a significantly greater reduction than the 21.5% (N = 8) loss for the impeded regeneration series (p less than 0.001). Thereafter, cell numbers were stable for frogs with unimpeded regeneration. Cell death continued in the series with impeded regeneration, and losses exceeded those of frogs with unimpeded regeneration from 110 days postcrush. When regeneration was impeded, ganglion cell somas underwent an intense cell soma reaction and became arranged in rows radiating from the optic nerve head. Our findings indicate that some ganglion cells are transiently spared when regeneration of their axons is prevented. The abnormally extensive contacts formed between somas may delay ganglion cell loss. However, the eventual death of most ganglion cells shows them to be target-independent in the long term.

Topographic order of retinofugal axons in a marsupial: implications for map formation in visual nuclei.

We studied axon order in the primary visual pathway and in nine retinorecipient nuclei of a small marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata) using animals at postnatal day (P) 40 and P80. Dorsal, ventral, nasal, and temporal axons enter the optic nerve true to their retinal origin being respectively dorsal, ventral, medial, and lateral; the arrangement is retained to the chiasm. Dorsal and ventral axons maintain their respective locations within the chiasm but at the base of the contralateral optic tract undergo a 180 degrees axial rotation, thus reversing the dorsoventral axis with respect to the retina. The alignment is conserved along the optic tract with dorsal and ventral axons mapping directly into appropriate quadrants of each retinorecipient nucleus. Nasal and temporal axons remain segregated as they decussate and lie respectively superficially and deep along the optic tract but with some intermingling. Within each retinorecipient nucleus, the nasotemporal axis is clearly demarcated, being represented in either a rostrocaudal (ventral and dorsal lateral geniculate nuclei; lateral posterior, dorsal terminal, and pretectal nuclei) or caudorostral (medial terminal and caudal pretectal nuclei, intergeniculate nucleus and superior colliculus) direction. The results imply that the dorsoventral axis in the retinorecipient nuclei could be due to preordering within the pathway, whereas the nasotemporal axis is determined by target-based cues. Moreover, cues for the orientation of the nasotemporal axis within retinorecipient nuclei must be localised within individual nuclei rather than as a single organiser, as previously envisaged (Chung and Cooke [1978] Proc. R. Soc. Lond. B. 210:335-373).

Axon order in the visual pathway of the quokka wallaby.

Axon order throughout the visual pathway of the quokka wallaby (Setonix brachyurus) was determined after localised retinal applications of the tracers DiI and/or DiASP. Postnatal days (P) 22-90 were studied to encompass the development and refinement of retinal projections. Order was essentially similar at all stages. Axons entered the optic nerve head true to their sector of retinal origin. In the optic nerve, nasal and temporal axons continued to reflect their retinal origin, dominating, respectively, the medial and lateral halves. By contrast, dorsal and ventral axons exchanged locations between the retrobulbar level and one-third the distance along the nerve; thus, the inversion of the dorsoventral retinal axis, imposed by the lens, was corrected. Decussating axons maintained their relative locations through the chiasm. At the base of the optic tract, nasal and temporal axons underwent an axial rotation to lie on the medial and lateral sides, respectively; thus nasal overlapped with ventral axons and temporal with dorsal axons. Axons maintained their alignments throughout the tract, and as a result, nasal and ventral axons invaded the superior colliculus medially, whereas temporal and dorsal axons invaded laterally. Each retinal quadrant terminated preferentially in its retinotopically appropriate sector of the colliculus. The arrangement of axons in the quokka visual pathway displays several novel features. Axon order is distinct throughout, involving a well-demarcated exchange of dorsal and ventral axons in the nerve and an axial rotation of nasal and temporal axons at the base of the tract; these relocations suggest decision regions for growing axons. The organisation presumably underlies the less extensive searching within the developing superior colliculus to generate retinotopic maps in the quokka and also in tammar wallaby [Marotte, J. Comp Neurol. 293:524-539, 1990] than in the rat [Simon and O'Leary, J. Neurosci. 12:1212-1232, 1992].

Retinal pigment epithelium and photoreceptor maturation in a wallaby, the quokka.

Cell generation and the early stages of maturation of the retinal pigment epithelium (RPE) and photoreceptors were examined in a marsupial, the quokka, Setonix brachyurus. Results are presented for animals aged up to postnatal day (P)250. RPE cell generation was studied by analysis of cell number from wholemounted retinae and by tritiated thymidine (3HThy) autoradiography in sectioned material. For 3HThy autoradiography, quokkas aged P1-P200 were injected with 3HThy and killed either 6-20 hours later (pulse-kill) or at P100 or P250 (pulse-leave). The extent of pigmentation of the RPE sheet was examined from sections of embryonic and early postnatal stages. Retinae from animals aged P5 to P160 were also examined at the electron microscope. By P100, RPE cell number is within the range found in adults. New RPE cells are generated in a peripheral band which moves outwards as cells leave the cell cycle in more central locations. RPE cells thus complete their last cell division in a centre-to-periphery wave centred about the optic nerve head. At any given retinal location, RPE cells complete their last cell division earlier than the overlying layers of the neural retina. Cells of the RPE rapidly develop a mature morphology. For example, melanin granules are observed at P5 and Verhoeff's membrane (the terminal bar complex) is evident by P25. By contrast, photoreceptor development in this species is protracted; cone inner segments are observed by P40, whilst the first rod inner segments are observed at P60. Despite being generated earlier, morphological maturation of the cones appears retarded and prolonged compared with that of the rods. The last stages of RPE cell maturation occur late in development, in synchrony with the generation of rods.

Development of primary visual projections occurs entirely postnatally in the fat-tailed dunnart, a marsupial mouse, Sminthopsis crassicaudata.

We have examined the development of retinal projections in a diminutive polyprotodont marsupial, the fat-tailed dunnart, Sminthopsis crassicaudata. Here, we document the most immature mammalian visual system at birth described to date. At postnatal day (P) 0, the retinal ganglion cell layer has yet to form, and axons have not entered the optic stalk. By P4, the retinal ganglion cell layer could be distinguished at the posterior pole, and the front of growing axons extended one-third the length of the optic stalk, a distance of approximately 150 microm; a few pioneer growth cones had grown beyond the main axon group but had still to reach the midline. Axons had decussated at the optic chiasm by P10 to penetrate the base of the contralateral optic tract and, by P15, had reached the dorsal lateral geniculate nucleus (dLGN), superior colliculus (SC), and accessory optic system (AOS); ipsilaterally projecting axons matured slightly later. From P20, axons had reached the caudal SC both contralaterally and ipsilaterally and terminated throughout the depth of the retinorecipient layers. After P30, the projections gradually refined. Within the rostral dLGN, segregation into four contralateral and four ipsilateral bands occurred by P50, approximately 5 days after eye opening. The projection to the ipsilateral SC underwent refinement by P50, becoming restricted to its rostral pole, and presented as discrete patches within the stratum opticum. At birth, the dunnart visual system is comparable to early to midembryonic stages [embryonic day (E) 12, E14, E19, E24, and E30, respectively] in the mouse, rat, ferret, cat, and monkey. The extreme immaturity of the neonatal dunnart together with the observation that the entire development of the primary optic pathway occurs postnatally over a protracted period make this marsupial especially valuable for investigating factors that control pathway formation in the early developing mammalian primary visual system.

Optic nerve regenerates but does not restore topographic projections in the lizard Ctenophorus ornatus.

In adult fish and amphibians, the severed optic nerve regenerates and visual behaviour is restored. By contrast, optic axons do not regenerate in the more recently evolved birds and mammals. Here we have investigated optic nerve regeneration in a member of the class Reptilia, phylogenetically intermediate between the fish and amphibians and the birds and mammals. We assessed visual recovery anatomically and behaviourally one year after unilateral optic nerve crush in the adult ornate dragon lizard. Ctenophorus ornatus. Ganglion cell densities and numbers of axons in the optic nerve on either side of the crush site indicated that two-thirds of ganglion cells survived axotomy and regrew their axons. However, myelination fell from a mean of 21% in normals to 5.5% and 3%, proximal and distal to the crush, respectively. Anterograde labelling of the entire optic nerve showed that axons regenerated along essentially normal pathways and that the major projection, as in normals, was to the superficial one-third of the contralateral optic tectum. However, localised retinal injections indicated that regenerated projections lacked retinotopic order. Any one retinal region projected to the entire tectum. This feature presumably explains why the experimental lizards consistently appeared blind to stimuli via the regenerated nerve. Our findings indicate that although axons regenerate along essentially normal pathways in adult lizards, conditions within the visual centres do not allow regenerating optic axons to select appropriate central connections. In a wider context, the result suggests that the ability for regenerating central axons to form topographic maps may also have been lost in the more recently evolved vertebrate classes.

Retinal projections throughout optic nerve regeneration in the ornate dragon lizard, Ctenophorus ornatus.

In goldfish and frog, optic nerve regeneration is successful, with restoration of retinotopic projections in visual brain centres and the return of functional vision within 1-2 months. By contrast, at 1 year after unilateral optic nerve crush in the ornate dragon lizard (Ctenophorus ornatus), the regenerated retinotectal projections lack topographic order, presumably explaining why the lizards are blind via the experimental eye (Beazley et al. [1997] J. Comp. Neurol. 377:105-120). To determine whether other abnormalities are associated with the inability to restore topographic projections in the lizard, we charted anatomically the time course, accuracy, and stability of optic nerve regeneration by examining visual projections with the lipophillic dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) applied to the optic disk at intervals up to 1 year after optic nerve crush; in addition, DiI tracing of small groups of axons was used to examine the topicity of axons projecting to the tectum. Axons re-innervated visual centres from between 1 and 2 months, a time frame comparable with that in goldfish and frog. However, the projections in lizard were found to differ from those in goldfish and frog in three major ways. First, there was considerable variability within the projection patterns both between individual lizards at any one stage and with time. Second, the projections were inaccurate. As in normal lizards, the major projection was to the contralateral optic tectum, although it lacked detectable retinotopic axon order throughout. Furthermore, misrouting occurred such that regenerating axons formed a persistent projection to the ipsilateral side of the brain that was considerably stronger and more widespread than normal. Minor visual centres also became re-innervated but, in addition, regenerating axons formed persistent projections into the opposite optic nerve and to non-retino-recipient regions such as the nucleus rotundus, hypothalamus, and olfactory nerve, as well as the posterior and tectal commissures. Third, the projections appeared unstable. Projections to both tecta were strongest between 3 and 5 months, but they diminished thereafter. The results suggest that, compared with goldfish and frog, in lizards both pathway and target cues are degraded and/or cannot be read adequately; as a consequence, regenerating axons are unable to navigate exclusively to visual centres and cannot re-form stable connections.

Substance P, bombesin, and leucine-enkephalin immunoreactivities are restored in the frog tectum after optic nerve regeneration.

Extensive regeneration of the optic nerve takes place in adult Amphibia. In this study, we have determined whether one aspect of retinotectal organisation, namely immunoreactive laminae in the retinorecipient layers of the optic tectum, is restored after optic nerve regeneration. To do so, the distributions of substance-P, bombesin, and leucine-enkephalin immunoreactivities were examined in the optic tectum of the frog Litoria (Hyla) moorei. Results of a normal series were compared with those at intervals up to 84 days and at 196 days after either unilateral deafferentation or optic nerve crush. In the normal series, distinct neuropeptide immunoreactive laminae were located within the retinorecipient tectal layers. There were two major laminae with substance-P, two with bombesin, and one with leucine-enkephalin immunoreactivities. Additional faint laminae of both substance-P and bombesin immunoreactivity were present in the tectal region that receives input from the visual streak. In addition, labelling of cell bodies and dendrites was seen elsewhere in the tectum. All except one immunoreactive lamina changed after deafferentation. The deeper of those with substance-P immunoreactivity, along with both bombesin laminae, were eventually lost; the lamina with leucine-enkephalin immunoreactivity was halved in intensity. We assume that these laminae are wholely or, in the case of the leucine-enkephalin lamina, partially associated with primary optic input. By contrast, the more superficial lamina with substance-P immunoreactivity remained unchanged and is presumably not directly related to visual input. During nerve regeneration, the intensity of all laminae associated with optic input initially fell as in the deafferentation series but, in the long term, recovered to approximately 80% of normal intensities. We conclude that ganglion cells associated with each of the immunoreactivities tested had successfully regenerated. The reduced intensity of immunoreactivities after regeneration is due presumably in part to the cell loss from the ganglion cell population. Furthermore, we discuss the findings of similar studies for Rana pipiens (Kuljis and Karten [1983] J. Comp. Neurol. 217:239-251 and [1985] 240:1-15) in light of the present findings. We argue that some of the previous observations can be reinterpreted to indicate that regeneration was not limited to ganglion cells associated with substance-P immunoreactivity as first thought.

Anatomical comparison of the macaque and marsupial visual cortex: common features that may reflect retention of essential cortical elements.

This study identifies fundamental anatomical features of primary visual cortex, area V1 of macaque monkey cerebral cortex, i.e., features that are present in area V1 of phylogenetically distant mammals of quite different lifestyle and features that are common to other regions of cortex. We compared anatomical constituents of macaque V1 with V1 of members of the two principal marsupial lines, the dunnart and the quokka, that diverged from the eutherian mammalian line over 135 million years ago. Features of V1 common to both macaque and marsupials were then compared with anatomical features we have previously described for macaque prefrontal cortex. Despite large differences in overall area and thickness of V1 cortex between these animals, the absolute size of pyramidal neurons is remarkably similar, as are their specific dendritic branch patterns and patterns of distribution of intrinsic axons. Pyramidal neuron patchy connections exist in the supragranular V1 in both the marsupial quokka and macaque as well as in macaque prefrontal cortex. Several specific types of aspinous interneurons are common to area V1 in both marsupial and macaque and are also present in macaque prefrontal cortex. Spiny stellate cells are a common feature of the thalamic-recipient, mid-depth lamina 4 of V1 in all three species. Because these similarities exist despite the very different lifestyles and evolutionary histories of the animals compared, this finding argues for a highly conserved framework of cellular detail in macaque primary visual cortex rather than convergent evolution of these features.