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The dorsal lateral geniculate nucleus (dLGN) contains a retinotopic map where input from the two eyes map in register to provide a substrate for binocular vision. Ten-m3, a transmembrane protein, mediates homophilic interactions and has been implicated in the patterning of ipsilateral visual projections. Ease of access to early developmental stages in a marsupial wallaby has been used to manipulate levels of Ten-m3 during the development of retinogeniculate projections. In situ hybridisation showed a high dorsomedial to low ventrolateral gradient of Ten-m3 in the developing dLGN, matching retinotopically with the previously reported high ventral to low dorsal retinal gradient. Overexpression of Ten-m3 in ventronasal but not dorsonasal retina resulted in an extension of ipsilateral projections beyond the normal binocular zone. These results demonstrate that Ten-m3 influences ipsilateral projections and support a role for it in binocular mapping., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Retinal projections to the superior colliculus are organised into retinotopic maps. Binocular vision requires that inputs from the two eyes map in register with each other. Studies in mice lacking Ten-m3, a homophilic transmembrane protein, indicate that it plays a key role in this process by influencing ipsilateral projections. The postnatal, ex utero development of the wallaby allows the targeted manipulation of molecules of interest during development. The distribution of mRNA for Ten-m3 in the retina and superior colliculus of the wallaby, and the effects of its spatiotemporally restricted retinal overexpression was investigated, in particular on the mapping of ipsilateral projections. Quantitative polymerase chain reaction found that Ten-m3 mRNA is expressed at relatively higher levels in the retina and colliculus early in development. Further, it is higher in ventral than dorsal retina, and increased in the retinotopically corresponding medial compared to lateral superior colliculus. In situ hybridisation demonstrated an increasing dorsoventral gradient in retinal ganglion cells was matched to an increasing lateromedial gradient in the superior colliculus. Overexpression of Ten-m3 by in vivo retinal electroporation produced an increase in ipsilateral projections to the binocular rostromedial colliculus, fitting with the proposal that Ten-m3 mediates mapping by attractant homophilic interactions. Retrograde labelling of the projection from this region suggested that overexpression produces a shift in the axons of existing ipsilaterally projecting ganglion cells rather than a rerouting of the axons of contralaterally projecting cells. Retinal manipulation of Ten-m3 levels produces changes in ipsilateral mapping, supporting a role for it in binocular mapping., (Copyright © 2013 ISDN. Published by Elsevier Ltd. All rights reserved.)

The visual system is characterized by precise retinotopic mapping of each eye, together with exquisitely matched binocular projections. In many species, the inputs that represent the eyes are segregated into ocular dominance columns in primary visual cortex (V1), whereas in rodents, this does not occur. Ten-m3, a member of the Ten-m/Odz/Teneurin family, regulates axonal guidance in the retinogeniculate pathway. Significantly, ipsilateral projections are expanded in the dorsal lateral geniculate nucleus and are not aligned with contralateral projections in Ten-m3 knockout (KO) mice. Here, we demonstrate the impact of altered retinogeniculate mapping on the organization and function of V1. Transneuronal tracing and c-fos immunohistochemistry demonstrate that the subcortical expansion of ipsilateral input is conveyed to V1 in Ten-m3 KOs: Ipsilateral inputs are widely distributed across V1 and are interdigitated with contralateral inputs into eye dominance domains. Segregation is confirmed by optical imaging of intrinsic signals. Single-unit recording shows ipsilateral, and contralateral inputs are mismatched at the level of single V1 neurons, and binocular stimulation leads to functional suppression of these cells. These findings indicate that the medial expansion of the binocular zone together with an interocular mismatch is sufficient to induce novel structural features, such as eye dominance domains in rodent visual cortex.

The mammalian cerebellar cortex is apparently uniform in composition, but a complex heterogeneous pattern can be revealed by using biochemical markers such as zebrin II/aldolase C, which is expressed by a subset of Purkinje cells that form a highly reproducible array of transverse zones and parasagittal stripes. The architecture revealed by zebrin II expression is conserved among many taxa of birds and mammals. In this report zebrin II immunohistochemistry has been used in both section and whole-mount preparations to analyze the cerebellar architecture of the Australian tammar wallaby (Macropus eugenii). The gross appearance of the wallaby cerebellum is remarkable, with unusually elaborate cerebellar lobules with multiple sublobules and fissures. However, despite the morphological complexity, the underlying zone and stripe architecture is conserved and the typical mammalian organization is present., (Copyright © 2012 S. Karger AG, Basel.)

We have studied the early development of the vestibular apparatus and its central connections in the tammar wallaby (Macropus eugenii) in order to determine whether the vestibular system anatomy is sufficiently mature at birth to assist in climbing to the pouch. Structural development was studied with the aid of hematoxylin and eosin stained sections and immunoreactivity for GAP-43, whereas the development of vestibular system connections was examined by carbocyanine dye tracing. At the time of birth, the otocyst has distinct utricle, saccule and semicircular canals with immature sensory regions receiving innervation by GAP-43 immunoreactive fibers. Vestibular nerve fibers can be traced into the brainstem to the developing vestibular nuclei, which are not yet cytoarchitectonically distinct. The vestibular nuclei do not contribute direct projections to the lower cervical spinal cord at birth; most bulbospinal projections in the newborn appear to be derived bilaterally from the gigantocellular, lateral paragigantocellular reticular and ventral medullary nuclei. A substantial bilateral projection to the vestibular ganglion and apparatus from the region of the gigantocellular and lateral paragigantocellular nuclei was seen at birth, but not in subsequent ages. This is similar to a projection seen in newborn Ameridelphians. By postnatal day (P) 5, the vestibular apparatus had extensive projections to all vestibular nuclei and neurons projecting in the lateral vestibulospinal tract could be identified in the lateral vestibular nucleus. Cytoarchitectonic differentiation of the vestibular nuclei proceeded over the next 3 to 4 weeks with the emergence of discrete parvicellular and magnocellular components of the medial vestibular nucleus by P19. GAP-43 immunoreactivity stayed high in the lateral vestibulospinal tract for several months after birth, suggesting that the development of this tract followed a prolonged timecourse. Our findings indicate that central and peripheral connections of the vestibular ganglion are present at birth, but that there is no direct projection from the vestibular nuclei to the cervical spinal cord until P5. Nevertheless, the possibility remains that an indirect projection between the vestibular nuclei and the medial reticular formation is present at birth and mediates control of the climb., ((c) 2008 S. Karger AG, Basel)

We used carbocyanine dye tracing techniques in conjunction with hematoxylin and eosin staining, immunohistochemistry for GAP-43, and tritiated thymidine autoradiography to examine the development of the olfactory pathways in early pouch young tammar wallabies (Macropus eugenii). The overarching aim was to test the hypothesis that the olfactory system of newborn tammars is sufficiently mature at birth to contribute to the guidance of the pouch young to the nipple. Although GAP-43 immunoreactive fibers emerge from the olfactory epithelium and enter the olfactory bulb at birth, all other components of the olfactory pathway in newborn tammars are very immature at birth, postnatal day (P0). In particular, maturation of the vomeronasal organ and its projections to the accessory olfactory bulb appears to be delayed until P5 and the olfactory bulb is poorly differentiated until P12, with glomerular formation delayed until P25. The lateral olfactory tract is also very immature at birth with pioneer axons having penetrated only the most rostral portion of the piriform lobe. Interestingly, there were some early (P0) projections from the olfactory epithelium to the medial septal region and lamina terminalis (by the terminal nerve) and to olfactory tubercle and basal forebrain. The former of these is presumably serving the transfer of LHRH(+) neurons to the forebrain, as seen in eutherians, but neither of these very early pathways is sufficiently robust or connected to the more caudal neuraxis to play a role in nipple finding. Tritiated thymidine autoradiography confirmed that most piriform cortex pyramidal neurons are generated in the first week of life and are unlikely to be able to contribute to circuitry guiding the climb to the pouch. Our findings lead us to reject the hypothesis that olfactory projections contribute to guidance of the newborn tammar to the pouch and nipple. It appears far more likely that the trigeminal pathways play a significant role in this behavior because the central projections of the trigeminal nerve are more mature at birth in this marsupial., (Copyright 2008 S. Karger AG, Basel.)

Neuronal activity is implicated as a driving force in the development of sensory systems. In order for it to play a developmental role, however, the pathways involved must be capable of transmitting this activity. The relationship between afferent arrival, synapse formation and the onset of chemical neurotransmission has been examined using the advantageous model of a marsupial mammal, the wallaby (Macropus eugenii), to determine at what stage activity has the capacity to influence cortical development. It is known that thalamocortical afferents arrive in the somatosensory cortex on postnatal day (P)15 and that their growth cones reach to the base of the compact cell zone of the cortical plate. However, electronmicroscopy showed that thalamocortical synapses were absent at this stage. Glutamatergic responses were recorded in the cortex following stimulation of the thalamus in slices at this time but only in magnesium-free conditions. The responses were mediated entirely by N-methyl-d-aspartate (NMDA) receptors. From P28, responses could be recorded in normal magnesium and comprised a dominant NMDA-mediated component and a non-NMDA mediated component. At this time thalamocortical synapses were first identified and they were in the cortical plate. By P63 the non-NMDA-mediated component had increased relative to the NMDA-mediated component, and by P70 layer IV began to emerge and contained thalamocortical synapses. By P76 a fast non-NMDA-mediated peak dominated the response. This coincides with the appearance of cortical whisker-related patches and the onset in vivo of responses to peripheral stimulation of the whiskers.

Spontaneous retinal activity has been implicated in the development of the topographic map in the superior colliculus (SC) but a direct demonstration that it reaches the colliculus is lacking. Here we investigate when the retinocollicular projection is capable of transmitting information from the retina in a marsupial mammal, the wallaby (Macropus eugenii). The projection develops postnatally, allowing in vivo analysis throughout development. Quantification of retinocollicular synaptogenesis has been combined with electrophysiology of the development and characteristics of retinocollicular transmission, including in vivo and in vitro recording in the same animals. Prior to postnatal day (P) 12-14 in vitro recording detected only presynaptic activity in retinal axons in the colliculus, in response to stimulation of the optic nerve. Postsynaptic responses, comprising both N-methyl-d-aspartate (NMDA) and non-NMDA responses, were first detected in vitro at P12-14 and retinal synapses were identified. In contrast, postsynaptic responses to optic nerve stimulation could not be detected in vivo until P39, around the time that retinal axons begin arborizing. Around this age density and numbers of total synapses began increasing in the retinorecipient layers of the colliculus. By P55-64, the numbers of retinal synapses had increased significantly and density and numbers of retinal and total synapses continued to increase up to P94-99. During this time the map is undergoing refinement and degenerating axons and synapses were present. The discrepancy between in vitro and in vivo onset of functional connections raises the question of when retinal activity reaches collicular cells in the intact, unanaesthetized animal and this will require investigation.

In the wallaby, whisker-related patterns develop over a protracted period of postnatal maturation in the pouch. Afferents arrive simultaneously in the thalamus and cortex from postnatal day (P) 15. Whisker-related patterns are first seen in the thalamus at P50 and are well formed by P73, before cortical patterns first appear (P75) or are well developed (P85). This study used the slow developmental sequence and accessibility of the pouch young to investigate the effect of nerve lesions before afferent arrival, or at times when thalamic patterns are obvious but cortical patterns not yet formed. The left infraorbital nerve supplying the whiskers was cut at P0-93 and animals were perfused at P112-123. Sections through the thalamus (horizontal plane) and cortex (tangential) were reacted for cytochrome oxidase to visualize whisker-related patterns. Lesions of the nerve at P2-5, before innervation of the thalamus or cortex, resulted in an absence of patterns at both levels. Lesions from P66-77 also disrupted thalamic and cortical patterns, despite the fact that thalamic patterns are normally well established by P73. Lesions from P82-93 resulted in normal thalamic and cortical patterns. Thus, despite the wallaby having clearly separated times for the development of patterns at different levels of the pathway, these results suggest a single critical period for the thalamus and cortex, coincident with the maturation of the cortical pattern. Possible mechanisms underpinning this critical period could include dependence of the thalamic pattern on corticothalamic activity or peripheral signals to allow consolidation of thalamic barreloids.

We have examined the cyto- and chemoarchitecture of the isocortex of a diprotodontid marsupial, the tammar wallaby (Macropus eugenii), using Nissl staining in combination with enzyme histochemical (acetylcholinesterase - AChE, NADPH-diaphorase - NADPHd, cytochrome oxidase) and immunohistochemical (non-phosphorylated neurofilament - SMI-32) markers. The primary sensory cortex showed distinctive patterns of reactivity in cytochrome oxidase, acetylcholinesterase and NADPH diaphorase. For example, in AChE material, S1 showed a heterogeneous appearance, with regions exhibiting a double layer of AChE activity (layers II and IV) adjacent to poorly reactive regions. In NADPHd preparations, activity in S1 was strongest in layers I to IV although, as in AChE material, there were consistent patches of reduced NADPHd activity which corresponded to poorly reactive regions in the AChE sections. Each of the primary sensory areas of the isocortex showed a different pattern of distribution of SMI-32+ neurons. In V1, SMI-32+ neurons were distributed in two layers (III and V) throughout the tangential extent of that region. In S1, SMI-32+ neurons were concentrated in layer V, but large and discrete patches within S1 had additional SMI-32+ neurons in layer III. In primary auditory cortex there was a dense band of SMI-32+ neurons in layer V, with only occasional labeled pyramidal neurons in layer III. In the secondary sensory areas (V2 and S2) SMI-32+ neurons were either distributed in layers III and V (V2) or solely within layer V (S2). The tangential and laminar distribution of Type I reactive NADPH diaphorase neurons in the tammar wallaby cortex was more like that seen in eutheria than in polyprotodontid metatheria., (Copyright (c) 2005 S. Karger AG, Basel)

Abstract Theoretical models of topographic map formation have postulated a gradient of attractant in addition to a gradient of repulsion in the target. In species where many axons grow past their correct positions initially, it has also been argued that a parallel gradient of attractant or branching signal is required to ensure collateral formation at the correct position (O'Leary et al., 1999). Brain-derived neurotrophic factor (BDNF) is a known attractant and promotes branching of retinal axons. We have examined its distribution in the superior colliculus and that of its receptor, trkB, in the retina, using immunohistochemistry and in situ hybridization, respectively, during the development of the topographic retinocollicular projection in the wallaby, a marsupial mammal. The number of glial endfeet expressing BDNF at the surface of the colliculus was found to be in a high caudal-to-low rostral gradient during the time when the retinocollicular projection was developing. When the projection was mature the rostrocaudal gradient had disappeared and the number of detectable endfeet expressing BDNF was very low. Messenger RNA for TrkB was expressed in the retinal ganglion cell layer throughout the time when the retinocollicular projection was developing, with no difference in expression across the nasotemporal axis of the retina. The low rostral to high caudal distribution of BDNF in glial endfeet supports the idea that it is providing a parallel gradient of attractant or branching signal in the colliculus.

We have studied the organization of the hypothalamus in an Australian diprotodontid metatherian mammal, the wallaby ( Macropus eugenii), using cytoarchitectural, histochemical and immunohistochemical techniques. Coronal sections of adult brains were processed for Nissl staining, histochemical reactivity (cytochrome oxidase, nicotinamide adenine dinucleotide phosphate diaphorase and acetylcholinesterase) and immunohistochemistry (antibodies to tyrosine hydroxylase, calbindin, calretinin, non-phosphorylated neurofilament protein, oxytocin and vasopressin). The distribution of immunoreactive neurons for these substances was mapped with the aid of a computer-linked microscope. In general, the wallaby hypothalamus showed a similar nuclear organization to that seen in rodents. The paraventricular nucleus could be divided into several subdivisions based on the different cellular parcellation, similar to that described in rodents. The ventromedial hypothalamic nucleus had cell-sparse dorsomedial and cell-dense ventrolateral subdivisions as seen in eutheria, suggesting a similar functional compartmentalization in all theria. The positions of tyrosine hydroxylase-positive neurons in the wallaby hypothalamus were also similar to those in eutheria. Oxytocin and vasopressinergic neurons were found in all the same major nuclear groups as seen in eutheria, although a nucleus circularis could not be identified. The general similarities between wallaby and eutherian hypothalamus indicate that the basic chemo- and cytoarchitectural features of the hypothalamus are common to eutheria and metatheria and validate the use of the wallaby as a mammalian model of wide applicability in investigations of hypothalamic functional development.

The expression of EphB1 and B2 receptors and ephrins-B1, -B2 and -B3 in the retina and superior colliculus of the wallaby (Macropus eugenii) was examined during the development of the retinocollicular projection, using reverse transcription-polymerase chain reaction and immunohistochemistry. There was an early transient differential expression of EphB2 that was higher in ventral retina and restricted to the outer neuroblast layer, whereas a high ventral to low dorsal gradient of ephrin-B2 expression occurred there throughout the study period. However, there was no dorsoventral gradient of receptors or ligands in retinal ganglion cells or a mediolateral gradient of ephrins in the colliculus. These findings suggest a limited role for these molecules in topographic mapping across the mediolateral colliculus in the wallaby. Early in retinal development there is a complementary pattern of expression of ephrin-B1 and -B2 in the outer neuroblast layer that overlaps with expression of EphB2. Ganglion and amacrine cells also express EphB2. As development proceeds subpopulations of putative horizontal and bipolar cells, also expressing EphB2, come to reside in the inner nuclear layer and ephrin-B1 is expressed throughout the outer nuclear layer. At the same time cells expressing ephrin-B2, and subpopulations of horizontal and bipolar cells come to reside in the inner nuclear layer and there is a corresponding decrease in ephrin-B2 expression in the outer nuclear layer. This pattern of coexpression of receptors and ligands suggests a role for them in cell migration and maintenance of laminar boundaries.

The time course of development and laminar distribution of thalamocortical synapses in the visual cortex of the marsupial mammal the wallaby (Macropus eugenii) has been studied by electron microscopy from the time of afferent ingrowth to the appearance of layer 4, the main target for thalamic axons. Axons were labeled from the thalamus by a fluorescent carbocyanine dye in fixed tissue or by transneuronal transport of horseradish peroxidase conjugated to wheat germ agglutinin from the eye. Thalamic axons first reached the cortex 2 weeks after birth and grew into the developing cortical plate without a waiting period in the subplate. The first thalamocortical synapses were detected 2 weeks later solely throughout the loosely packed zone of the cortical plate, where layer 6 cells previously have been shown to reside. As the thickness of the cortex increased with age, thalamocortical synapses were increasingly prevalent in the loosely packed zone of the cortical plate. With the appearance of layer 4, thalamocortical synapses were found there as well as in the marginal zone and layer 6. There was no evidence for an early population of thalamocortical synapses in the subplate. The first synapses made by thalamic axons were in a region containing layer 6 cells, one of their normal targets in the mature cortex., (Copyright 2003 Wiley-Liss, Inc.)

We have examined the distribution of immunoreactivity for GAP-43 in the developing and adult brain of a diprotodontid metatherian, the tammar wallaby ( Macropus eugenii). The distribution of GAP-43 immunoreactivity in the neonatal wallaby brain was strikingly heterogeneous, in contrast to that reported for the newborn polyprotodontid opossum. Immunoreactivity for GAP-43 in the developing wallaby brain showed a caudal-to-rostral spatiotemporal gradient, with the brainstem well in advance of the telencephalon throughout the first 100 days of postnatal life. In many regions examined, GAP-43 immunoreactivity passed through the following phases: 1. intense immunoreactivity in developing fiber tracts and occasional somata; 2. diffuse homogeneous immunoreactivity; 3. selective loss of immunoreactivity in particular nuclei or cortical regions. In the isocortex, selective loss of GAP-43 immunoreactivity in the somatosensory and visual cortex (at postnatal day 115) coincided with the maturation of the laminar distribution of terminal thalamocortical axonal fields. Within adult cortical regions, GAP-43 immunoreactivity was highest in layer I of all regions, lower layers (V and VI) of primary somatosensory and visual cortices, layers II/III of motor and cingulate cortex, and layer IV of entorhinal cortex. Our findings suggest that, while patterning of GAP-43 immunoreactivity in the mature brain is similar across meta- and eutheria, there may be early developmental differences in the distribution of GAP-43 immunoreactivity between poly- and diprotodontid metatheria.

We have studied the development of the hypothalamus of an Australian marsupial, the tammar wallaby (Macropus eugenii), to provide an initial anatomic framework for future research on the developing hypothalamus of diprotodontid metatheria. Cytoarchitectural (hematoxylin and eosin), immunohistochemical (CD 15 and growth associated protein, GAP-43), tritiated thymidine autoradiography, and carbocyanine dye tracing techniques were applied. Until 12 days after birth (P12), the developing hypothalamus consisted of mainly a ventricular germinal zone with a thin marginal layer, but by P25, most hypothalamic nuclei were well differentiated, indicating that the bulk of hypothalamic cytoarchitectural development occurs between P12 and P25. Strong CD 15 immunoreactivity was found in radial glial fibers in the rostral hypothalamus during early developmental ages, separating individual hypothalamic compartments. Immunoreactivity for GAP-43 was used to reveal developing fiber bundles. The medial forebrain bundle was apparent by P0, and the fornix appeared at P12. Tritiated thymidine autoradiography revealed lateral-to-medial and dorsal-to-ventral neurogenetic gradients similar to those seen in rodents. Dye tracing showed that projections to the posterior pituitary arose from the supraoptic nucleus at P5 and from the paraventricular nucleus at P10. Projections to the medulla were first found from the lateral hypothalamic area at P0 and paraventricular nucleus at P10. In conclusion, the pattern of development of the wallaby hypothalamus is broadly similar to that found in eutheria, with comparable neurogenetic compartments to those identified in rodents. Because most hypothalamic maturation takes place after birth, wallabies provide a useful model for experimentally manipulating the developing mammalian hypothalamus., (Copyright 2002 Wiley-Liss, Inc.)

Recordings from direction-selective neurons in the nucleus of the optic tract (NOT) of the marsupial wallaby, Macropus eugenii, show that 53% of cells are sensitive to visual stimulation of both eyes. Anatomical tracing studies using horseradish peroxidase reveal many retinal terminals in the contralateral NOT but very few in the ipsilateral nucleus. There was no convincing evidence of cortical inputs to the ipsilateral NOT despite large injections of tracer into the visual cortex. During visual stimulation in the visual field of the contralateral eye with moving patterns, the excitatory responses in the NOT generated by ipsiversive motion (right-to-left when recording from the left NOT) were usually larger than the inhibitory responses produced by contraversive motion. Conversely, during ipsilateral eye stimulation, the negative motion components to contraversive motion were usually larger than the positive components to ipsiversive motion. This response pattern resembles that observed in the NOT of the American opossum, Didelphis aurita, where binocularity appears to arise through a commissural subcortical pathway that connects the two nuclei and inverts the directional tuning of the transmitted signals. We propose that the lack of significant input from the ipsilateral eye and cortex in the wallaby suggests that binocularity must arise from another pathway, possibly a commissural route between the nuclei. As directional information appears not to be carried by the internucleus pathway in rats and cats, our results suggest that binocularity in the NOT arises from different sources in marsupials as compared to eutherians.

This study used the extrauterine development of a marsupial wallaby to investigate the onset of functional activity in the somatosensory pathway from the whiskers. In vivo recordings were made from the somatosensory cortex from postnatal day (P) 55 to P138, in response to electrical stimulation of the infraorbital nerve supplying the mystacial whiskers. Current source density analysis was used to localize the responses within the cortical depth. This was correlated with development of cortical lamination and the onset of whisker-related patches, as revealed by cytochrome oxidase. The earliest evoked activity occurred at P61, when layers 5 and 6 are present, but layer 4 has not yet developed. This activity showed no polarity reversal with depth, suggesting activity in thalamocortical afferents. By P72 synaptic responses were detected in developing layer 4 and cytochrome oxidase showed the first hint of segregation into whisker-related patches. These patches were clear by P86. The evoked response at this age showed synaptic activity first in layer 4 and then in deep layer 5/upper layer 6. With maturity, responses became longer lasting with a complex sequence of synaptic activity at different cortical depths. The onset of functional activity is coincident with development of layer 4 and the onset of whisker-related pattern formation. A similar coincidence is seen in the rat, despite the markedly different chronological timetable, suggesting similar developmental mechanisms may operate in both species.

We describe the expression of EphA3 and EphA7 receptors and ephrin-A2 ligand in the retina and the superior colliculus during the development of the retinocollicular projection in the marsupial wallaby (Macropus eugenii), using immunoblotting and immunohistochemistry. EphA3 in the retina was in a striking, low central to high peripheral gradient, superimposed on which was a high temporal to low nasal level of expression. This distribution was evident from postnatal day 30, when axons are growing into the colliculus and forming a coarsely organized topographic projection, to postnatal day 65, when axons have arborized in their correct retinotopic positions. EphA7 showed a shallow centroperipheral gradient with no nasotemporal differences in expression. In the superior colliculus no rostrocaudal differences in distribution were detected for either of these receptors. Ephrin-A2 was distributed in a gradient increasing from the rostral to the caudal pole in the superficial layers of the superior colliculus only up to postnatal day 30. Ephrin-A2 was evenly distributed in the retina throughout development of the projection. Expression of EphA3 in the retina increased, while the expression of ephrin-A2 in the colliculus was downregulated over time. The graded expression of EphA3 and ephrin-A2 early in the development of the projection suggests that they play a role in establishment of coarse topography of retinal axons along the rostrocaudal axis of the superior colliculus. However, the gradients were not complementary, meaning that EphA3 alone cannot mediate the repulsive interactions with ephrin-A2 that have been postulated to underlie formation of the topographic map.

Previous observations disclosed that astroglia with interlaminar processes were present in the cerebral cortex of adult New and Old World monkeys, but not in the rat, and scarcely in the prosimian Microcebus murinus. The present report is a more systematic and comprehensive comparative analysis of the occurrence of such processes in the cerebral cortex of several mammalian species. Brain samples were obtained from adult individuals from the following orders: Carnivora (canine), Rodentia (rat and mouse), Marsupialia (Macropus eugenii), Artiodactyl (bovine and ovine), Scandentia (Tupaia glis), Chiroptera (Cynopteris horsfieldii and C. brachyotis), and Primate: Prosimian (Eulemur fulvus), non-human primate species (Cebus apella, Saimiri boliviensis, Callithrix, Macaca mulatta, Papio hamadryas, Macaca fascicularis, Cercopithecus campbelli and C. ascanius) and from a human autopsy. Tissues were processed for immunocytochemistry using several antibodies directed against glial fibrillary acidic protein (GFAP), with or without additional procedures aimed at the retrieval of antigens and enhancement of their immunocytochemical expression. The cerebral cortex of non-primate species had an almost exclusive layout of stellate astrocytes, with only the occasional presence of long GFAP-IR processes in the dog that barely crossed the extent of lamina I, which in this species had comparatively increased thickness. Species of Insectivora and Chiroptera showed presence of astrocytes with long processes limited to the ventral basal cortex. Interlaminar GFAP-IR processes were absent in Eulemur fulvus, at variance with their limited presence and large within- and inter-individual variability as reported previously in Microcebus murinus. In New World monkeys such processes were absent in Callithrix samples, at variance with Cebus apella and Saimirí boliviensis. Overall, the expression of GFAP-IR interlaminar processes followed a progressive pattern: bulk of non-primate species (lack of interlaminar processes)--Chiroptera and Insectivora (processes restricted to allocortex) < strepsirhini < haplorhini (platirrhini < catarrhini). This trend is suggestive of the emergence of new evolutionary traits in the organization of the cerebral cortex, namely, the emergence of GFAP-IR long, interlaminar processes in the primate brain. Interlaminar processes may participate in a spatially restricted astroglial role, as compared to the one provided by the astroglial syncytium. It is proposed that the widely accepted concept of an exclusively astroglial syncytium is probably linked with a specific laboratory animal species ("rodent-type" or, rather, "general mammalian-type" model) that misrepresents the astroglial architecture present in the cerebral cortex of most anthropoid adult primates ("primate-type" model), including man.

Birthdates of the neurons that comprise the layers of the mature visual cortex in the wallaby (Macropus eugenii) have been determined with the aid of tritiated thymidine autoradiography. The laminar positions of cells, identified by their birthdates, have then been followed at early stages during development and compared with previously published data on the distribution of thalamocortical afferents and corticothalamic projecting cells (Sheng et al. [1991] J. Comp. Neurol. 307:17-38). Neurons are born in a deep to superficial sequence typical of other mammals. The loosely packed zone of cells, which develops at the base of the thin compact zone of cells at the superficial margin of the cortical plate early in development, was identified as being part of the cortical plate. Afferents did not wait below this zone but grew into the developing cortical layers immediately after the cells that form these layers began accumulating in the loosely packed zone, starting with layer 6 on postnatal day 22 (P22). The genesis of layer 4 did not begin until P32, and these cells reached the superficial cortical plate at P54 and entered the loosely packed zone by P65. Cells of layers 5 and 6 formed the initial projection to the thalamus. Despite the protracted development of the wallaby and the large discrepancy between the time of thalamic ingrowth and genesis of layer 4, there was no extended waiting period for afferents in the subplate., (Copyright 2000 Wiley-Liss, Inc.)

The organisation and cytoarchitecture of the serotonergic neurons in a diprotodont marsupial were examined by using serial sections of the brainstem processed for serotonin immunohistochemistry and routine histology. The topographic distribution of serotonergic neurons in the brainstem of the adult wallaby (Macropus eugenii) was similar to that of eutherian mammals. Serotonergic neurons were divided into rostral and caudal groups, separated by an oblique boundary through the pontomedullary junction. Approximately 52% of the serotonergic neurons in the wallaby brainstem were located in the rostral midline nuclei (caudal linear nucleus, dorsal, median, and pontine raphe nuclei and the interpeduncular nucleus), whereas 21% were found in the caudal midline region (nuclei raphe magnus, obscurus, and pallidus). The remaining serotonergic neurons (27%) were located in more lateral regions such as the pedunculopontine tegmental nuclei, the supralemniscal nuclei (B9 group), and the ventrolateral medulla. The largest serotonergic group, the dorsal raphe, contained one-third of the brainstem serotonergic neurons and showed five subdivisions, similar to that described in other species. In contrast, the median raphe did not show clear subdivisions. The internal complexity of the raphe nuclei and the degree of lateralisation of serotonergic neurons suggest that the wallaby serotonergic system is similar in organisation to that described for the cat and rabbit. This study supports the suggestion that the serotonergic system is evolutionally well conserved and provides baseline data for a quantitative study of serotonergic innervation of the developing cortex in the wallaby., (Copyright 1999 Wiley-Liss, Inc.)

The protracted development of the wallaby (Macropus eugenii) has allowed study of messenger RNAs encoding Eph receptors EphA3 and EphA7 and ligands ephrin-A2 and -A5 in the retina and superior colliculus at intervals throughout the development of the retinocollicular projection: from birth, before retinal innervation, to postnatal day 95, when the projection is mature. Reverse transcription-polymerase chain reaction showed messenger RNAs for both receptors and ligands were expressed at all ages. EphA7 was expressed more highly in the rostral superior colliculus. Ephrin-A2 and -A5 were expressed more highly in the caudal colliculus. EphA3 was expressed in a complementary manner, more highly in temporal than in nasal retina. There are higher levels of expression of the ligands when the projection is only coarsely topographically organised. This suggests a role for them and their receptor EphA3 in this stage, by repulsive interactions which restrict temporal axons to rostral superior colliculus. This is the first account in a marsupial mammal of the appearance of this molecular family, substantiating its ubiquitous role in topographically organised neuronal connections. Nevertheless, expression is not the same as in the mouse, suggesting differences in the details of topographic coding between species., (Copyright 1999 Wiley-Liss, Inc.)

A light and electron microscopic study has been made of the time of formation of whisker-related patterns in trigeminothalamic afferents and the onset of synapse formation between afferents and cells in the ventroposteromedial nucleus (VPM) of the marsupial mammal, the wallaby, by labelling afferents with a carbocyanine dye. A parallel in vitro study was made of the functional development of the trigeminothalamic pathway to the VPM. Evoked synaptic responses could be recorded in the VPM from the time that afferents first reached the VPM at postnatal day 15 (P15). At all stages, the excitatory response comprised both N-methyl-D-aspartate- and non-N-methyl-D-aspartate-mediated components. At P40, the response decreased markedly in duration, coinciding with the onset of synaptogenesis. This implies that transmission is occurring prior to synapse formation, probably through transmitter release from growth cones. At P50, synaptic responses became dominated by a fast, non-N-methyl-D-aspartate potential, and this coincided with the first appearance of whisker-related patterns in the VPM. A gamma-aminobutyric acid (subtype A)-mediated, inhibitory component was also present from the time of afferent arrival. These findings support the idea that functional interactions between afferents and their targets may play a role in pattern formation in the somatosensory thalamus.

In mature rodents, whisker-related patterns are known to be present in three levels of the brain: the brainstem trigeminal nuclei, the ventrobasal thalamus and the somatosensory cortex. These patterns have been demonstrated using neuroanatomical tracing techniques, histological and histochemical staining methods and electrophysiological recordings. The development and topography of these patterns are dependent on an intact periphery. But what governs when patterns form at the three levels? Possibilities include a controlling signal from the periphery or local mechanisms at each site, such as the arrival of afferent inputs or the maturation of target tissue. In this review, we report on the maturation of the whisker pathway in a marsupial, the wallaby, where the slow tempo of development is a feature. At each level, afferent fibres grow into the region of termination many weeks before the whisker-related pattern emerges. The results suggest that the maturity of the target tissue as well as signals from the periphery combine to trigger pattern formation at each level of the pathway.

We have examined the development of the laminar and areal distribution of cortical commissural neurons in a marsupial mammal, the wallaby Macropus eugenii. In this species, commissural axons approach the major cerebral commissure, the anterior commissure, via either the internal capsule or the external capsule and first cross the midline at postnatal day 14 (P14). By retrogradely labelling these axons with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) at P15, we show here that the cell bodies of these neurons are restricted to a region of cortex adjacent to the rhinal fissure. Most of these labelled neurons are located in the compact cell zone of the cortical plate, with only a few labelled cells found in the zone of loosely packed cells deep to this layer. Over the subsequent 66 days, commissural neurons are found progressively more dorsally, rostrally, and caudally, so that, by P80, they are present throughout the extent of the neocortex. At this age, they are mainly pyramidal in morphology and form a single band within the deeper part of layer 5 of the developing cortex. From P80 to adulthood, the distribution of commissural neurons has been assessed in the visual cortex by using retrograde transport of horseradish peroxidase. At P80, labelled neurons with immature pyramidal morphology are present throughout the occipital cortex; as in DiI material, somata are located in deep layer 5. At P165, previously shown to be the age when commissural axon numbers peak, widespread labelling is present in the occipital region, with labelled cells now found in two bands corresponding to layers 3 and 5. After this age, neurons become more restricted in distribution, so that, by adulthood, commissural neurons are no longer apparent throughout area 17 but are restricted to a localised region around the area 17/18 boundary. Within this region, labelling is still present in layers 3 and 5 but is more dense in layer 3. The gradual restriction of commissural fields seen here in the wallaby is similar to that reported in the neocortex in many eutherians. These findings also support studies in eutheria, suggesting that subplate neurons do not appear to play a major role in commissural development.

The development of trigeminal projections between the thalamus and cortex has been investigated in the marsupial mammal, the wallaby, by using a carbocyanine dye, horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP), Neurobiotin, and biocytin as pathway tracers. The appearance of whisker-related patterns in the cortex in relation to their appearance in the brainstem and thalamus was examined, as was the presence or absence of a waiting period for thalamocortical afferents and the identity of the first cortical cells to project to the thalamus. Thalamic afferents first reached the cortex at postnatal day (P) 15 and were distributed up to the deep edge of the compact cell zone in the superficial cortical plate throughout development, in both dye and WGA-HRP labelled material, with no evidence of a waiting period. The initial corticothalamic projection, detected by retrograde transport of WGA-HRP from the thalamus, occurred at P60 from layer 5 cells. This was confirmed by labelling of corticothalamic axons after cortical injections of Neurobiotin and biocytin. Scattered, labelled cells seen before P60 after dye labelling from the thalamus presumably resulted from transcellular labelling via thalamic afferents. Clustering of afferents in layer 4 and cell bodies and their dendrites in layers 5 and 6 first occurred simultaneously at P76. There is a sequential onset of pattern formation, first in brainstem, then in thalamus, and finally in cortex, with a long delay between afferent arrival and pattern formation at each level. Independent regulation at each level, likely depending on target maturation, is suggested.

Retinotopic order of optic axons in the optic nerve and superior colliculus of the marsupial mammal, the wallaby (Macropus eugenii), has been examined and compared during development of the retinocollicular projection to investigate the role of order in the nerve in map formation. Small groups of axons from different retinal quadrants were labelled in vivo with a carbocyanine dye from just after axons first reached the colliculus to when the projection was mature. The distribution and branching patterns of axons and their arbors on the colliculus were assessed quantitatively during this period, as was the degree of order in the nerve. Initially, axons accumulated in coarse retinotopic order in the colliculus, with little branching and no sign of arborization to form terminal zones. Axons labelled from deposits covering a mean of 2.2% of the retina reached a mean collicular coverage of around 30% at 41-47 days, at which time they began arborizing in their retinotopically correct positions. By 55 days axons from all retinal quadrants had formed terminal zones in their retinotopically correct positions. Axons did not arborise in incorrect positions as has been reported in the rat. By 61-68 days coverage had decreased to around 10%. By 90-95 days only axons supplying terminal zones were present and terminal zones were smaller. In the nerve, axons showed a coarse and consistent order throughout development. This order was retinotopic only immediately behind the eye. Temporal and nasal axons occupied corresponding halves of the nerve along its course. Axons from dorsal and ventral retina shifted from dorsal and ventral positions in the nerve, respectively, to opposite sides of the nerve just before the chiasm. This would assist in positioning them in the appropriate lateral and medial optic tracts, respectively, in the positions they occupied as they approached the colliculus. However, the position in the nerve was not related to the ability to arborize in the correct collicular position. In particular, the increase in retinotopic order in the colliculus late in development was not accompanied by an increase in order in the nerve. Since the final organization in the colliculus shows greater order than is ever seen in the nerve, additional mechanisms must be involved in the maturation of the collicular map.

The time course of ingrowth of retinal projections to the superior colliculus in the marsupial mammal, the wallaby (Macropus eugenii), was determined by anterograde labelling of axons from the eye with horseradish peroxidase, from birth to 46 days, when axons cover the colliculus contralaterally and ipsilaterally. The position of retinal ganglion cells giving rise to these projections over this period was determined in fixed tissue by retrograde labelling from the colliculus with a carbocyanine dye. Axons first reach the rostrolateral contralateral colliculus 4 days after birth and extend caudally and medially, reaching the caudal pole at 18 days and the far caudomedial pole at 46 days. The first contralaterally projecting cells are in the central dorsal and temporal retina, followed by cells in the nasal and finally the ventral retina. They are distributed closer to the periphery with increasing age. The first sign of a visual streak appears by 18 days. Axons reach the ipsilateral colliculus a day later than contralateral axons and come from a similar region of the retina. The sparser ipsilateral projection reaches the caudal and medial collicular margins by 46 days but by 16-18 days, ganglion cells giving rise to this transient projection are already concentrated in the temporoventral retina. The orderly recruitment of ganglion cells from retinotopically appropriate regions of the retina as axons advance across the contralateral colliculus suggests that the projection is topographically ordered from the beginning. The ipsilateral projection is less ordered as cells are located in the temporoventral crescent at a time when their axons are still transiently covering the colliculus prior to becoming restricted to the rostral colliculus. Features of mature retinal topography such as the visual streak and the location of ipsilaterally projecting cells begin to be established very early in development, before the period of ganglion cell loss and long before eye opening at 140 days.

In metatheria, all neo- and paleo-cortical commissural connections are made by the anterior commissure. We have examined the adult morphology of this commissure and its development in a diprotodontid metatherian, the wallaby (Macropus eugenii), at both the light and electron microscope level. The total number of axons in the adult anterior commissure was 21.7 million, of which 55-62% were myelinated. The dorsal two thirds of the commissure, containing neocortical commissural axons, showed a higher percentage of larger, myelinated axons than the ventral one third, which contains paleocortical commissural axons. The commissure also showed a topographical gradient, with cells in the dorsal cortex projecting through the dorsal region of the commissure, the fasciculus aberrans. In the rostrocaudal axis, axons from the frontal cortex tended to pass more anteriorly through the commissure and those from the occipital more posteriorly, but there was extensive overlap of projections from different areas. The gestation length of this wallaby is 28.3 days, and all commissural development occurs postnatally. The anterior commissure first appeared at P (postnatal day) 14, at which time commissural fibres were apparently derived from the external capsule exclusively. Commissural fibres passing through the internal capsule, and joining the anterior commissure via the fasciculus aberrans, were first noted at P18. By that age there were 94,000 to 161,000 axons. Peak axon counts of 50 to 63 million occurred between P100 and P150. The number of growth cones in a single midline section peaked at approximately P114 (480,000) and dropped to 0 by P170. The distribution of growth cones was analysed during the early stages of anterior commissure development (P18, P30, P82). At P18, growth cones were concentrated in the dorsal parts of the commissural bundle, suggesting a ventrodorsal sequence of addition of axons. There was no apparent preferential association of growth cones with the periphery of the commissure or glial structures at any of the three ages examined. The results show that axonal overproduction and regression in cortical commissural connections are features of development in diprotodontid metatheria, as in eutheria.

This paper is the second in a series which makes use of the protracted postnatal maturation of the wallaby to study the development of the trigeminal sensory system. Previous work has established similarities in the organisation of the trigeminal sensory system in the wallaby and in rodents. This study describes the structure and development of the ventroposteromedial nucleus in the wallaby in relationship to the arrival of afferents from the trigeminal nuclei, the formation of neuronal aggregations and naturally occurring cell death. Enzyme histochemistry, Nissl and myelin stains were used. Pathway development was followed using carbocyanine dyes. In the adult wallaby the nucleus demonstrates evidence of a parcellated organisation. Cells are arranged in dorsoventrally aligned bands resembling fingers. In the horizontal plane, these appear as circular clusters which are encircled by fine myelinated bundles. The clusters of cells are believed to correspond to the mystacial vibrissae. The first afferents from the principal trigeminal nucleus arrive between 10 and 15 days postnatal. This is more than two weeks prior to the time at which the borders of the nucleus can be discerned cytoarchitecturally. The first hints of segmentation are visible around day 50, and discrete aggregations form over the ensuing 3-4 weeks. Coincident with the aggregation of the neurons is an increase in their level of reactivity for acetylcholinesterase. A high level of acetylcholinesterase reactivity is maintained for at least 4 months, but has disappeared in adult animals. The peak of cell death occurs subsequent to the appearance of aggregations in the thalamus, but coincident with the appearance of vibrissae related patches in the cortex at day 85 (Waite et al. [1991] Dev. Brain Res. 58:35-41). The timing of the appearance of the neuronal aggregations supports the hypothesis that pattern formation occurs sequentially at successive levels of the pathway, and suggests the importance of target maturation in pattern formation.

1. Total optokinetic responses measured by electro-oculography and single unit recordings from the nucleus of the optic tract and dorsal terminal nucleus (NOT-DTN) of the accessory optic system were taken from young adult wallabies, whose one eye had been rotated about the optic axis at birth, and were compared with those from normal controls. 2. The velocities of the horizontal component of the slow phases of optokinetic nystagmus were measured in the horizontal plane as a function of the direction of stimulus motion. In normal animals the overall gain during monocular stimulation was greatest for horizontal temporonasal movement, with a lesser response to movement in the opposite, nasotemporal, direction. Upward or downward vertical stimulus motion did not elicit horizontal responses. In animals where one eye was removed at birth and the other eye was normal, the characteristic bidirectional response was retained; the response was identical with that elicited from one eye of a normal animal. 3. After surgical rotation (extorsion) of the left eye by approximately 90 degrees on or within a few days of birth, the animals were grown to adulthood. The visual streak of the retina of the operated eye was then found, in individual cases, to be between 30-100 degrees from horizontal with the head held in the standard resting position. This angle was taken as the definitive degree of cyclotorsion resulting from the operation in each animal. The extraocular muscles connected with regions of the eye adjacent to the location of their outgrowth in the orbit and not with the normal point of attachment on the globe.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of the vibrissae and their innervation and the maturation of the brainstem trigeminal sensory nuclei have been studied in the wallaby, Macropus eugenii, from birth to adulthood. At birth, developing vibrissal follicles consist of solid epidermal pegs surrounded by dermal condensations. The developing follicles and adjacent skin are innervated by trigeminal afferents. Ten days after birth the follicle contains a dermal papilla and the deep vibrissal nerve can be recognised. A hair cone is present at postnatal day (P) 30 and hairs are apparent on the skin surface by P35. By P63 the deep vibrissal nerve can be seen innervating Merkel cells in the outer root sheath; in addition, the first signs of the blood sinus can be recognised. Innervation of the inner conical body and lanceolate and lamellated receptors supplying the mesenchymal sheath and waist region are not seen until P119, when the follicle resembles that seen in the adult. At birth, central processes of the trigeminal ganglion cells have entered the trigeminal tract and extend from the rostral pons to the upper cervical cord. Labelling with a carbocyanine dye at P0 shows afferents extending medially from the tract into the trigeminal subnuclei at all levels. At this stage the trigeminal nuclei appear as areas of increased cell density in the lateral brainstem. By P30-40 the four subnuclei can be distinguished on the basis of shape, cytoarchitecture, and succinic dehydrogenase reactivity. Adult morphology is not fully established until P210. In mature animals, nucleus principalis contains closely packed, polymorphic cells, frequently aligned parallel to thick fibre bundles that traverse the nucleus obliquely. Subnuclei oralis and interpolaris contain sparsely distributed, medium to large cells, randomly oriented, as well as prominent rostrocaudally directed fibre bundles. Subnucleus caudalis consists of the marginal layer, substantia gelatinosa, and magnocellular layers as described in other species. Patches of increased succinic dehydrogenase or cytochrome oxidase reactivity, presumably corresponding to the vibrissae, are present in subnuclei principalis, interpolaris, and caudalis in developing and adult animals, although the pattern is less clear than in rats. The brainstem patches are first seen at P40, approximately 6 weeks before the corresponding vibrissal-related pattern develops in the cortex. This suggests that the onset of patch formation may be regulated independently at different levels of the pathway.

The retinocollicular projection in the marsupial mammal the wallaby Macropus eugenii, has been investigated anatomically to determine the order in the developing projection and electrophysiologically to determine the time of onset of synaptic transmission by recording evoked potentials in the colliculus in response to stimulation of the optic nerve. There are two clear stages: a protracted period when retinal axons grow into the colliculus in coarse retinotopic order with no recordable electrical activity followed by the formation of terminal zones in retinotopically correct positions, the loss of more widely distributed axons and the onset of evoked potentials. The two stages are not seen in non-mammalian vertebrates where the projection is functional from the beginning.

The position of ganglion cells contributing to the early imprecision in retinotopic order in the developing retinocollicular projection in the wallaby (Macropus eugenii) has been determined. Deposits of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) were made in the caudal pole of the superior colliculus (SC) at ages ranging from 22 days after birth, when sparse retinal axons have only just reached the caudal pole of the SC and are yet to cover its surface completely, to 96 days when the retinotopy of ganglion cell terminals in the SC is precise (Marotte, '90). From 30 days onwards, the deposit of WGA-HRP resulted in a dense patch of retrogradely labelled retinal ganglion cells that could be seen to be appropriately positioned in nasal retina. However, at all ages prior to 92 days, there were inappropriately positioned labelled cells between the densely labelled patch and the central retina and both dorsal and ventral to the patch. They were not found in far distant regions of retina and composed a relatively small proportion of labelled cells. They reached a peak at 45 days, had decreased to low levels by 63 days, were rare by 81 days, and by 92 days were absent. This latter age fits with the time when retinotopy was judged to be precise in a previous study (Marotte, '90). Inappropriately projecting cells never originate from the entire retina but only from regions adjacent to the appropriate region. Thus, during development there are no gross projection errors. Initially, ganglion cell axons are distributed on the colliculus in a coarse retinotopy. Refinement of the projection then follows, revealed by this technique as a loss of inappropriately projecting ganglion cells. This is complete by 92 days well before eye opening at around 140 days.

The morphology of the vibrissal follicles on the mystacial pad of the tammar wallaby is similar to that seen in other species except that the follicles lack a ringwulst or ring sinus. Instead, the mesenchymal sheath is thickened around the central region of the hair shaft. The follicle is innervated by both deep and superficial vibrissal nerves. The deep nerve enters as 4-11 fascicles which can be in close proximity or widely distributed around the hair. C1 follicles received more myelinated nerve fibres (252 +/- 31) than the smaller C4 follicles (174 +/- 43). The deep vibrissal nerve supplies the thickened mesenchymal sheath, the narrow 'waist' region above and the majority of endings in the inner conical body (ICB), while the superficial nerves provide a sparse innervation to the ICB and rete ridge. Receptors present in the follicle were of 4 types: (1) Merkel cells, especially numerous in the outer root sheath of the 'waist' region and occasionally in the ICB and rete ridge; (2) and (3) lanceolate and lamellated endings parallel to the hair shaft in both the mesenchymal thickening and the 'waist' region where they were particularly dense; (4) free nerve endings in the mesenchymal thickening, 'waist' region and ICB. No corpuscular, bulbous or Ruffini endings were seen. The innervation of the intervibrissal fur was similar to that described in other species.

This article makes two points. First, the diprotodont marsupials, including the kangaroos, wallabies and the Australian possum are not primitive mammals, and their brains make as good a general model of the higher mammals such as monkeys and humans as do those of the more common laboratory mammals such as cats and rats. Second, the peculiarities of marsupial reproduction, which comprises a very short period of intrauterine development, followed by a relatively protracted period of development in the pouch, provide unparalleled advantages for research into mammalian neuroembryology. Examples will be provided of how such research has made a contribution to our understanding of neural development, concentrating primarily on the visual system.

The distribution of afferents from the dorsal lateral geniculate nucleus (LGNd) and the lateral posterior nucleus (LP) and of cell bodies projecting to these nuclei has been studied in the visual cortex of the wallaby (Macropus eugenii) throughout development to determine how the characteristic laminar distribution of afferents and efferents of the mature cortex is achieved. Young are born after 26-28 days of gestation and do not open their eyes until around 140 days after birth. Horseradish peroxidase conjugated to wheatgerm agglutinin was injected in the visual thalamus in adults and in pouch young aged from 22 days after birth, just after thalamic axons first reach the visual cortex, to 118 days, when cortical lamination resembles the adult. From 22 to 65 days, the developing visual cortex consists of a marginal zone (MZ), cortical plate (CP), and intermediate zone (IZ) including the superficial subplate (SP), subventricular zone, and ventricular zone. There is a thin compact cell zone (CCZ) at the top of the CP and below it a less densely packed region that increases in thickness with age. Retrogradely labelled cells in two bands were first seen at 40 days, one in the CCZ and the other at the base of the CP. Two bands of cells were seen at all subsequent times if the injection covered both LGNd and LP, and by 76 days, these cells were located within cytoarchitectonically recognizable layers V and VI. Anterograde label prior to 45 days was distributed densely and evenly throughout the IZ and the CP up to the CCZ. Label in MZ was first seen at 25 days and was substantial by 54 days. Anterograde label than became gradually reduced in the IZ, whereas in the CP it remained evenly and densely distributed until 82 days. At this age, coincident with the emergence of layer IV, label within the CP first showed variations in density and by 99 days was concentrated over layer IV and, to a lesser extent, over layer VI. By 118 days label resembled the adult after injections covering both LGNd and LP, with label concentrated in layer I, IV, and VI with a less dense projection to lower layer III and upper layer V. There is a relatively earlier initial ingrowth of axons into the visual cortex in the wallaby and throughout development thalamocortical axons appear to be more widely distributed in the depth of the visual cortex than has been demonstrated for placental mammals.(ABSTRACT TRUNCATED AT 400 WORDS)

The somatosensory cortex associated with the whiskers has been studied in adult tammar wallabies (Macropus eugenii) and in pouch young from 60-120 days of pouch life. The time course of anatomical changes examined with succinic dehydrogenase (SDH) histochemistry and Nissl staining has been correlated with the maturation of electrically evoked cortical responses to stimulation of the whisker follicles. The earliest signs of aggregates of SDH reaction product in layer IV of the cortex were seen at 85 days, coincident with the first recordings of an immature cortical evoked potential. Aggregates, in a pattern corresponding to that of the facial whiskers, were most clearly seen from 90-140 days. At later stages, and in the adult, they were present but their arrangement was less clearly seen. By 186 days the electrical activity resembled the mature pattern. Patches of SDH activity in layer IV were not associated with changes in soma density characteristic of true barrels.

The time course of the development of connections between the visual cortex and the main subcortical visual structures, as well as intrahemispheric and interhemispheric connections, has been studied in the marsupial wallaby (Macropus eugenii) to compare its development with that of placental mammals. Pouch young are born prior to retinal innervation of the primary visual centers and spend a protracted period of development in the pouch, making them ideal for visual, developmental studies. Horseradish peroxidase conjugated to wheatgerm agglutinin was injected into either the presumptive visual cortex or the superior colliculus in young of varying ages. Thalamocortical projections from the dorsal lateral geniculate and lateral posterior nuclei reach the presumptive visual cortex between 12 and 15 days after birth. Descending cortical connections form later. Corticogeniculate axons are first detected in the geniculate and lateral posterior nucleus at 48 days after birth, while corticocollicular axons first reach the superior colliculus at 71 days and, by 81 days, have innervated the superficial layers. Intrahemispheric and interhemispheric connections form even later. By 99 days intrahemispheric axons from area 17 have accumulated in visual association areas but are yet to invade layers III and IV, their major termination zones in adult, while axons projecting back to area 17 have also reached their target area. At this time interhemispheric axons from area 17 have begun to accumulate in the opposite visual cortex, although they have not invaded the cortical layers. By 111 days cortical cells projecting to the opposite visual cortex are first labelled. These have a more widespread distribution in area 17 at 111 and 122 days compared to the adult, where they are confined to the 17/18 border. The results show that the marsupial wallaby has a timetable of similar sequence, but different relative timing, in the formation of cortical connections compared to that of placental mammals. In the first half of the period between conception and eye opening, the timing in the wallaby precedes considerably that in placental mammals. Ascending connections from the thalamus develop relatively earlier in the wallaby but descending collicular connections are delayed until the same relative time that they appear in placental mammals.

The development of retinotopy in projections from the eye to the dorsal lateral geniculate (dLGN) and superior colliculus (SC) has been studied in the marsupial wallaby. Discrete retinal lesions were made and the remaining retinal projections were traced with horseradish peroxidase in animals at stages ranging from just after optic innervation of the dLGN and SC to the time when the projections are mature. Topographically organised projections could be recognized a few weeks after axons first reached the dLGN and SC with a topographically discrete projection from nasoventral retinal recognized later than from dorsal, dorso-temporal, temporal, and temporoventral retina. Over time there was an increase in precision of the retinotopy as judged by an increase in sharpness of the borders of filling defects in the projection labelled with horseradish peroxidase. Refinement of the projection from temporal retina preceded that from nasal retina in both the dLGN and SC and in the former occurred concomitantly with the segregation of eye-specific terminal bands. Refinement was complete 16 weeks after birth, prior to eye opening at around 20 weeks after birth. Inequalities in retinal representations in both nuclei were present from the time retinotopy could first be detected. This was before the inequalities in retinal ganglion cell distribution, which underly these representations in the adult, were obvious. Retinotopy and inequalities in retinal representation characteristic of the adult are present from a very early stage in the protracted development of visual projections in the wallaby. Refinement may involve death of inappropriately projecting cells, pruning of inappropriately projecting axon arborizations or could be achieved by growth of the retinorecipient neuropil. Temporonasal differences in the time course of refinement may reflect gradients of maturation in the retina.

The topography of retinal projections to the superior colliculus and dorsal lateral geniculate nucleus of a wallaby, the tammar (Macropus eugenii), was investigated by an anatomical method. Small laser lesions were made in the retinas of experimental animals, and the remaining retinal projections were visualized by means of horseradish-peroxidase histochemistry. The position of each lesion was correlated with the position of the filling defects in the terminal label. The whole of the retina projects to the contralateral superior colliculus. The nasal retina is represented caudally, and the temporal retina rostrally. The ventral retina is represented medially, and the dorsal retina laterally. There is a projection to the ipsilateral superior colliculus, but it is patchy and its topography could not be determined by this method. The retinotopic map in the contralateral dorsal lateral geniculate nucleus has the nasal retina represented rostrally and the temporal retina caudally in the nucleus. The dorsal retina is represented ventrally, and the ventral retina is represented dorsally. It appears that the whole of the retina projects contralaterally, and in addition the temporal retina projects ipsilaterally. The maps of visual space through the two eyes were shown to be in topographic register in the binocular region by making a deposit of HRP in the visual cortex. This resulted in a column of retrogradely labeled cells in the nucleus. This column crossed the laminae, which are innervated by the ipsilateral and contralateral eye at right angles.

During compression of the entire retinotectal projection into the rostral half of the tectum after ablation of the caudal half there is widespread sprouting of ganglion cell axons, not only those cut during the operation but also those left intact. However, unlike cut axons those left intact sprout without their cell bodies showing chromatolysis or swelling. Chromatolysis and swelling of the cell bodies of cut axons are more prolonged than after optic nerve section and resolve in more central regions of retina first. The cut axons of cell bodies in these regions tend to be the first to form terminal arborizations during the compression process as judged electrophysiologically. However, there is no clear correlation in individual fish between these measures and the state of compression assessed electrophysiologically. Large areas of retina may contain chromatolysed cells even after compression has occurred. Electrophysiological mapping alone may give a misleading picture of the interactions occurring between retinal and tectal cells during reorganization.

Retinal differentiation in the pouch young of the wallaby Macropus eugenii was characterized microscopically and morphometrically. Mitosis occurs until early in the second month in the central retina, and until early in the fourth month, peripherally. Separation of the neuroblast layer by the outer plexiform layer did not immediately halt cell division. The retinal surface continued to expand well past the time of cessation of proliferation. Cell death in the ganglion cell layer continued through the fourth month centrally and to nearly five months in the periphery. The major period of cell death was coincident with the segregation of retinal afferents and the refinement of topography in the superior colliculus and dorsal lateral geniculate nucleus. Beginning in the third month retinal thickness, measured between the outer limiting membrane and nerve fiber layer, declined equally in peripheral and central regions. At all stages the combined thicknesses of the outer and inner nuclear layer in the retinal periphery was greater than that in the center. Together with a late thickening of the inner plexiform layer, the data are consistent with the suggestion that expansion of peripheral non-ganglion cell elements may play a role in development of center to periphery differences in ganglion cell distributions. Retinal differentiation of the wallaby follows the pattern of most mammals. The onset of development of key milestones for the acquisition of retinal function occurred in the sequence: conventional synapse formation prior to ribbon synapse formation in the inner plexiform layer, and photoreceptor outer segment differentiation prior to terminal triad synapse formation.