Generation and death of cells in the dorsal lateral geniculate nucleus and superior colliculus of the wallaby, Setonix brachyurus (quokka).

To study postnatal cell generation in primary visual centres of the quokka, tritiated thymidine was injected into pouch-young aged postnatal day (P)1-P85. Brains were examined at P100, just before eye-opening, when primary visual projections are essentially mature. Neurons in the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC) were generated at P1-P10 and P1-P18 respectively. Peak numbers of labelled cells were seen at P3 and P5 in the dLGN and SC. Cell death was assessed in the dLGN and SC of young aged P10-P150. Low numbers of dying cells were seen in the dLGN throughout this period, with a small peak at P85. A more substantial peak of cell death was seen in the SC, also at P85. In the quokka, the time interval between the peaks of cell generation and of cell death in the dLGN and SC is 70-80 days, considerably longer than the interval of 40 days between birth and death of retinal cells.

Distinctive pattern of organisation in the retinofugal pathway of a marsupial: I. Retina and optic nerve.

The nasotemporal division in the retina and the pattern of crossed and uncrossed axons in the optic nerve were determined in an Australian marsupial, a wallaby, Setonix brachyurus (the quokka), following unilateral horseradish peroxidase injections into primary visual centres. The gross morphology of the nerve was also examined. Ipsilaterally projecting ganglion cells were restricted to the temporal retina, whereas those that project contralaterally were located in all retinal regions. The morphological study of the nerve showed that fasciculation patterns, evident along much of the length of the nerve, became indistinct centrally and were replaced in the prechiasmatic region by dorsoventrally oriented fissures. In this prechiasmatic region, axons were oriented in two directions. Whereas the majority were aligned centroperipherally with the long axis of the nerve, a proportion were aligned dorsoventrally in the fissures. Labelling with HRP revealed that uncrossed axons were restricted to the lateral region of the optic nerve and possibly to discrete fascicles, whereas those destined to cross at the chiasm occupied all regions of the nerve but were less dense on the lateral side. This spatial distribution of crossed and uncrossed projections did not change along the length of the nerve. These results demonstrate that fibre organisation in the marsupial optic nerve is different than that found in eutherian mammals.

Distinctive pattern of organisation in the retinofugal pathway of a marsupial: II. Optic chiasm.

In the mammalian optic chiasm retinal axons from each eye divide into two populations, those that decussate and those that remain uncrossed. In eutherian (placental) mammals, the separation of these pathways is not reflected in the structure of the chiasm. The two populations from each eye are mixed through each hemichiasm, segregating only at the midline, where the uncrossed projection turns back. In this study the optic chiasm of a marsupial, the wallaby, Setonix brachyurus (quokka) has been investigated with staining and neuronal tracing techniques. The chiasm of this mammal is quite different from that of eutherian mammals. In coronal section it can be morphologically subdivided into three regions, a central body in which fasciculated groups of axons from each eye interdigitate across the midline, and two distinct lateral regions, one on each side, which contain the uncrossed retinal projections. In the rostral chiasm the lateral regions are separated from the main body of the chiasm by vertically oriented fibre-free regions. Caudally, the lateral regions increase in size and become less distinct as increasing numbers of contralaterally projecting axons that have crossed the midline project into them. However, the two populations remain predominantly segregated in this region. As the lateral regions develop, the central body of the chiasm becomes thinner and finally detaches at the midline to form the two optic tracts. The routes taken by retinal axons through the eutherian and marsupial chiasm appear to be fundamentally different. Therefore, the developmental factors that determine the laterality of retinal projections are likely to show significant differences in the two mammalian groups.

Development of the chiasm of a marsupial, the quokka wallaby.

We have previously shown that the mature optic chiasm of a marsupial is divided morphologically into three regions, two lateral regions in which ipsilaterally projecting axons are confined and a central region containing only contralaterally projecting axons. By contrast, in the chiasms of eutherian (placental) mammals studied to date, there is no tripartite configuration. Ipsilaterally and contralaterally projecting axons from each eye are mixed in the caudal nerve and in each hemichiasm and encounter axons from the opposite eye near the midline of the chiasm. Here, we show that, unlike eutherians, marsupials have astrocytic processes in high concentrations in lateral regions of the nerve and rostral chiasm. Early in development, during the period when optic axons are growing through the chiasm, many intrachiasmatic cells are seen with densities five to eight times higher in lateral than in central chiasmatic regions. Such cells continue to be added to all chiasmatic regions; later in development, considerably more are added centrally, as the chiasm increases in volume. In the mature chiasm, cell densities are similar in all regions. By contrast to the marsupial, cell addition in the chiasm of a placental mammal, the ferret, is almost entirely restricted to later developmental stages, after axons have grown through the chiasm, and there are no obvious spatial variations in the distribution of cells during the period examined. During development, similar to the adult marsupial, ipsilaterally projecting axons do not approach the chiasmatic midline but remain confined laterally. We propose that the cells generated early and seen in high densities in the lateral chiasmatic regions of the marsupial may play a role in guiding retinal axons through this region of pathway selection. These data suggest that there is not a common pattern of developmental mechanisms that control the path of axons through the chiasm of different mammals.

Morphology and birth dates of horizontal cells in the retina of a marsupial.

Most eutherian (placental) mammals have two horizontal cell types; however, one type only has been seen in rodents. In order to assess whether one type of horizontal cell or two is a basic mammalian feature, we have examined the morphology of horizontal cells in a marsupial, the quokka wallaby, by Golgi staining or horseradish peroxidase labelling. The birth dates of horizontal cells have also been determined by 3H-thymidine/autoradiography. There are two types of horizontal cell in the wallaby retina. One type has no axon and corresponds to the axonless cell in eutherian species; the other has shorter dendrites, an axon, and an axonal arbor, corresponding to the eutherian short-axon cell. As in eutherian mammals, the dendrites of each horizontal cell type lie in the outer plexiform layer (OPL) and contact cones and the axonal arbor of the short-axon cell contacts rods. The dendrites of the axonless cells are long, with an average length of 250 microns, and each cell has one, sometimes two, short, stubby processes, which branch off a dendrite, traverse the inner nuclear layer, and reach the inner plexiform layer. The dendritic field of these cells is elongated, and dendrites show a preferential orientation at right angles to the trajectory of overlying ganglion cell axons. Short-axon cells have a morphology similar to that seen in other species, although the axonal arbor is relatively small. Both types of horizontal cell are generated in the first phase of retinal cell generation.

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.

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.

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.

Generation of retinal cells in the wallaby, Setonix brachyurus (quokka).

We have examined the generation of retinal cells in the wallaby, Setonix brachyurus (quokka). Animals received a single injection of tritiated thymidine between postnatal days 1-85 and retinae were examined at postnatal day 100. Retinae were sectioned, processed for autoradiography and stained with Cresyl Violet. Ganglion cells were labelled by injection of horseradish peroxidase into the optic tracts and primary visual centres. Other cells were classified according to their morphology and location. Retinal cell generation takes place in two phases. During the first phase, which concludes by postnatal day 30, cells destined to lie in all three cellular layers of the retina are produced. In the second phase, which starts by postnatal day 50, cell generation is almost entirely restricted to the inner and outer nuclear layers. Cells produced in the first phase are orthotopic and displaced ganglion cells, displaced and orthotopic amacrine cells, horizontal cells and cones. Glia in the ganglion cell layer, orthotopic amacrine cells, bipolar and horizontal cells. Muller glia, and rods are generated in the second phase. Cells became heavily labelled with tritiated thymidine in the central retina before postnatal day 7, over the entire retina (panretinal) by postnatal day 7 and from postnatal day 18, only in the periphery. The second phase of cell generation is initiated at P50, in a region extending from the optic nerve head to mid-temporal retina. Subsequently, cells are generated in annuli, centred on mid-temporal retina, which are seen at progressively more peripheral locations. Therefore, cell addition to the inner and outer nuclear layers continues for longer in peripheral than in mid-temporal retina. We suggest that such later differential cell addition to the inner and outer nuclear layers contributes to an asymmetric increase in retinal area. This non-uniform growth presumably results in more expansion of the ganglion cell layer peripherally than in mid-temporal retina and may play a role in establishing density gradients of ganglion cells.

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.

Development and aging of cell topography in the human retinal pigment epithelium.

PURPOSE: To determine how regional cell density of this tissue changes with age, the authors examined the topography of the human retinal pigment epithelium (RPE) in wholemounted tissue obtained from eyes aged 12 to 89 years, donated for corneas. METHODS: The RPE, with choroid attached, was wholemounted and stained with cresyl violet. From these preparations, the authors analyzed retinal area, RPE cell number, and cell density. RESULTS: Retinal pigment epithelial cell number is highly variable between persons but does not appear to be age related. Retinal area increases until approximately 30 years of age, but beyond this age individual variation masks further enlargement. The distinctive topography of the RPE changes markedly with age. There is a modification from the relatively homogeneous cell density distribution in the youngest retinas examined toward a more heterogeneous pattern in older retinas. From mid-adolescence, a band of larger cells appears at the extreme periphery, adjacent to the ora serrata, which gradually widens so that by 90 years of age, it occupies the outermost 30% of the retinal area. Cell density is highest in the central temporal retina, adjacent to the macula in the neural retina, throughout life. Cell density values in this region increase slightly with age, and the difference between this and surrounding regions becomes more marked with age. CONCLUSIONS: With no marked change in total cell number, peripheral RPE in humans enlarges in area throughout life, but the RPE in more central regions decreases in area.

Visual acuity of the northern native cat (Dasyurus hallucatus)--behavioural and anatomical estimates.

Behavioural estimates of the visual acuity of the Northern Native Cat (or Northern Quoll)--Dasyurus hallucatus--were made using the Mitchell jumping stand technique. A value of 2.3-2.8 cycles per degree was obtained. This functional acuity compared well with predictions based on the peak ganglion cell density (2600 cells/mm2) determined from the retinal ganglion cell density map.

Development and cell generation in the hippocampus of a marsupial, the quokka wallaby (Setonix brachyurus).

Development and cell generation in the hippocampus of the marsupial, the quokka wallaby, has been examined. Cells in this brain region are similar in morphology to those in eutherian species, with predominantly pyramidal and granule cells. In the quokka, development of the hippocampus takes place postnatally; this region is first seen just after birth on postnatal day 1 (P1) as an out-pouching of the medial cortical wall into the lateral ventricle. The cornu ammonis (CA) region first appears at P20 as a line of denser cells and by P30, CA3 and the granule cell layer of the dentate gyrus (DG) can be defined. A specific region of the ventricle, near to the developing fimbria, produces the granule cells destined for the dentate gyrus. These cells initially migrate in a curved trajectory into the hilus, following the path of thick, vimentin-positive glial fibres. Cells are generated in the hippocampus from around P5 until at least P85 when some cells in the hilus and also glial cells are labelled with [3H]thymidine. In the cell sparse region around the hippocampal fissure there is a peak of neuron production before P20 followed by a decline and subsequent increase in the production of probably glial cells after P60. The peak of cell generation in the CA region and the granule cell layer of the DG is around P40. Cells continue to be produced in the hilus of the DG much later, with numbers still high at P85, presumably these cells are destined to reach the granule cell layer later in development.

Displaced retinal ganglion cells in the wallaby Setonix brachyurus.

Displaced ganglion cells have been examined in wholemounted and sectioned retinae following bilateral injection of horseradish peroxidase into optic tracts of the wallaby, Setonix brachyurus, "quokka". Such cells, which lie in the vitread part of the inner nuclear layer, are located mainly in superior retina as a streak-like band dorsal to the area centralis and visual streak of orthotopic ganglion cells. Only between 1 and 2% of the total ganglion cell population were displaced, but an analysis of cell morphology and soma diameter suggested that displaced ganglion cells represented several cell types.

Continued neurogenesis is not a pre-requisite for regeneration of a topographic retino-tectal projection.

Electrophysiological recording demonstrated that visuo-tectal projections are topographically organised after optic nerve regeneration in aged Xenopus laevis. 3H-thymidine autoradiography confirmed previous reports [Taylor, Lack, & Easter, Eur. Journal of Neuroscience 1 (1989) 626-638] that cell division had already ceased at the retinal ciliary margin. The results demonstrate that, contrary to a previous suggestion [Holder & Clarke, Trends in Neuroscience 11 (1988) 94-99], continued neurogenesis is not a pre-requisite for the re-establishment of appropriate connections with target cells.

Development of visual projections in the marsupial, Setonix brachyurus.

Retinal projections to the primary visual centres were studied following injection of tritiated proline into one eye in the Marsupial, Setonix brachyurus between 10 and 100 days postnatal and in adults. Initially, projections from the two eyes overlapped extensively, particularly between 20 and 50 days. There was a gradual refinement thereafter, including a segregation of inputs from the two eyes within both the lateral geniculate nucleus (LGN) and superior colliculus (SC) by 70 days. Such refinement in visual centres is discussed in relation to the concurrent emergence of retinal ganglion cell density gradients, a decrease in ganglion cell numbers, cell death in the ganglion cell layer and loss of optic axonal profiles.

Patterns of cytogenesis in the developing retina of the wallaby Setonix brachyurus.

Patterns of mitosis were examined during development from embryonic day (E) 19, 9 days before birth, for retinae of the wallaby Setonix brachyurus, using cresyl violet stained material. For neural retina, mitosis took place at the ventricular surface from the earliest stage of eye formation until postnatal day (P) 100. Numbers of mitotic figures reached a peak of approximately 12,000 by P43. Average densities ranged between 300/mm2 and 600/mm2 up to P12 and then fell to below 50/mm2 by P25 before reaching a second peak of over 400/mm2 at P43. Up to P50, mitoses were present across the entire retina. By P60, a 'cold spot' lacking mitotic activity had formed in temporal retina and progressively extended to reach peripheral regions by P100; no mitoses were seen at P150. The timing and location of the 'cold spot' coincided with our previous description of the appearance of an area centralis in the ganglion cell layer (Dunlop and Beazley 1985). Inner and outer plexiform layers (IPL and OPL) formed between P24-40 and P50-100 respectively and were seen first in temporal retina. Furthermore, the extent of the OPL matched the mitotic 'cold spot'. By contrast to neural retina, mitosis in the pigment epithelium was panretinal and was largely complete by P3. The data suggest that cell addition to the inner or outer nuclear layers contribute to differential retinal expansion and the establishment of cell density gradients in the ganglion cell layer.

Horse vision and an explanation for the visual behaviour originally explained by the 'ramp retina'.

Here we provide confirmation that the 'ramp retina' of the horse, once thought to result in head rotating visual behaviour, does not exist. We found a 9% variation in axial length of the eye between the streak region and the dorsal periphery. However, the difference was in the opposite direction to that proposed for the 'ramp retina'. Furthermore, acuity in the narrow, intense visual streak in the inferior retina is 16.5 cycles per degree compared with 2.7 cycles per degree in the periphery. Therefore, it is improbable that the horse rotates its head to focus onto the peripheral retina. Rather, the horse rotates the nose up high to observe distant objects because binocular overlap is oriented down the nose, with a blind area directly in front of the forehead.

Number of neurons in the retinal ganglion cell layer of the quokka wallaby do not change throughout life.

During adult life, the topography of the retinal pigment epithelium (RPE) of the quokka wallaby changes gradually. Cells in peripheral retina enlarge in surface area while those in mid-temporal retina, adjacent to the area centralis, a high density region in the ganglion cell layer, decrease in area, implying that the tissue in this area is drawing together. We speculated that high ganglion cell densities in temporal regions might be maintained, in the face of cell loss due to aging, by this apparent drawing together of the RPE sheet. Therefore, we examined the retinal ganglion cell layer of the quokka in cresyl violet stained wholemounts from animals aged from 0. 55 to 13.5 years. We found that total neuron number in the retinal ganglion cell layer of the quokka did not decrease significantly throughout life even though individuals in captivity live long lives (9-15 years). Ganglion and amacrine cells were counted separately and identified by strict morphological criteria. Nevertheless, the proportion of ganglion to amacrine cells appeared to decrease linearly throughout life, indicating that the morphology of a proportion of neurons became more amacrine-like during aging. Mean cell size did not change throughout life. In the quokka, retinal area increases slowly throughout life and may account for the small reduction in cell density seen in most retinal regions.