Development of the vestibular apparatus and central vestibular connections in a wallaby (Macropus eugenii).

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.

Development of the olfactory system in a wallaby (Macropus eugenii).

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.

Development of structural and functional connectivity in the thalamocortical somatosensory pathway in the wallaby.

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.

Retinocollicular synaptogenesis and synaptic transmission during formation of the visual map in the superior colliculus of the wallaby (Macropus eugenii).

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.

Cyto- and chemoarchitecture of the cortex of the tammar wallaby (Macropus eugenii): areal organization.

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.

Brain-derived neurotrophic factor is expressed in a gradient in the superior colliculus during development of the retinocollicular projection.

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.

Cyto- and chemoarchitecture of the hypothalamus of a wallaby ( Macropus eugenii) with special emphasis on oxytocin and vasopressinergic neurons.

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.

Developmental onset of functional activity in the wallaby whisker cortex in response to stimulation of the infraorbital nerve.

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.

Graded expression of EphA3 in the retina and ephrin-A2 in the superior colliculus during initial development of coarse topography in the wallaby retinocollicular projection.

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.

"Rodent-like" and "primate-like" types of astroglial architecture in the adult cerebral cortex of mammals: a comparative study.

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.

Neurogenesis and identification of developing layers in the visual cortex of the wallaby (Macropus eugenii).

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.

Serotonergic neurons in the brainstem of the wallaby, Macropus eugenii.

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.

Marsupial retinocollicular system shows differential expression of messenger RNA encoding EphA receptors and their ligands during development.

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.

Morphological development of afferent segregation and onset of synaptic transmission in the trigeminothalamic pathway of the wallaby (Macropus eugenii).

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.

Development of whisker-related patterns in marsupials: factors controlling timing.

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.

Development of commissural neurons in the wallaby (Macropus eugenii).

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.

Timecourse of development of the wallaby trigeminal pathway: III. Thalamocortical and corticothalamic projections.

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 in the optic nerve and superior colliculus during development of the retinocollicular projection in the wallaby (Macropus eugenii).

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 initial stages of development of the retinocollicular projection in the wallaby (Macropus eugenii): distribution of ganglion cells in the retina and their axons in the superior colliculus.

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.

Anterior commissure of the wallaby (Macropus eugenii): adult morphology and development.

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.