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The diversity of the diprotodontids provides an excellent opportunity to study how a basic marsupial cortical plan has been modified for the needs of the mammals living in different habitats. Very little is known about the connections of the cerebral cortex with the deep brain structures (basal ganglia and thalamus) in this evolutionarily significant group of mammals. In this study, we performed mapping of brain regions and connections in a diprotodontid marsupial from data obtained from an excised brain scanned in high-field (9.4 T) microstructural magnetic resonance imaging (MRI) instrument. The analysis was based on two MRI methodologies. First, high-resolution structural scans were used to map MRI visible brain regions from T1w and T2w images. Second, extensive diffusion tensor imaging (DTI) data were obtained to elucidate connectivity between brain areas using deterministic diffusion tracking of neuronal brain fibers. From the data, we were able to identify corticostriate connections between the frontal association and dorsomedial isocortex and the head of the caudate, and between the lateral somatosensory cortex and the putamen. We were also able to follow the olfactory and limbic connections by tracing fibers in the fornix, cingulum, intrabulbar part of the anterior commissure, and lateral olfactory tract. There was segregation of fibers in the anterior commissure such that olfactory connections passed through the rostroventral part and successively more dorsal cortical areas connected through more dorsal parts of the commissure. Our findings confirm a common pattern of cortical connectivity in therian mammals, even where brain expansion has occurred independently in diverse groups.

We used magnetic resonance imaging to study the anatomy of cortical regions, nuclear groups, and major tracts in the brain of a monotreme, i.e., the short-beaked echidna (Tachyglossus aculeatus). Our specimens were from a collection held at the Australian Museum in Sydney and had been stored in formaldehyde solution for at least 70 years. Despite this, we were able to detect fine detail in the nuclear divisions of structures as well as in fiber tracts. In particular, we could detect the medial lemniscus as it approached the ventral posterior thalamic nucleus, subdivisions within the ventral posterior thalamic nucleus, lamination and subdivisions within the hippocampal formation, components of the olfactory pathways, and nuclei within the temporal amygdala. We were able to map the topography of subcortical white matter and relate it to cortical regions determined on the basis of physiology, as well as chemical and cytoarchitecture. As expected, dense aggregations of fibers were noted in association with the primary sensory areas of the isocortex (somatosensory, visual, and auditory) and connecting primary olfactory regions (intrabulbar anterior commissure and associated fibers). We found longitudinal fibers in the basal forebrain (medial forebrain bundle) and brainstem (corticopontine and corticospinal tracts), as well as a dense array of fibers associated with the vermal and paravermal zones of the anterior lobe of the cerebellum. We also observed previously unrecognized fiber systems, i.e., commissural connections between the paired frontal isocortical fields (dorsal Fr1), dense fibers to the retrosplenial association cortex, and prominent, paired longitudinal fiber bundles in the dorsal forebrain (longitudinal fasciculus) that intersected the dorsal anterior commissure. The connectome results are consistent with the known neuroanatomy of this monotreme and they extend our knowledge of the fiber topography within this unusual brain. Our findings demonstrate the feasibility of using this sort of imaging of archived brains to analyze the neuroanatomy of rare, endangered, and evolutionarily significant species.

The monotremes are a unique group of mammals whose young are incubated in a leathery-shelled egg and fed with milk from teatless areolae after hatching. As soon as they hatch, monotreme young must be able to maneuver around the nest or maternal pouch to locate the areolae and stimulate milk ejection. In the present study, the embryological collections at the Museum für Naturkunde, Berlin, have been used to follow the development of the monotreme cerebellum through incubation and lactational phases, to determine whether cerebellar circuitry is able to contribute to the coordination of locomotion in the monotreme hatchling, and to correlate cerebellar development with behavioral maturation. The structure of the developing monotreme cerebellum and the arrangement of transitory neuronal populations are similar to those reported for fetal and neonatal eutherians, but the time course of the key events of later cerebellar development is spread over a much longer period. Expansion of the rostral rhombic lip and formation of the nuclear and cortical transitory zones occurs by the time of hatching, but it is not until after the end of the first post-hatching week that deep cerebellar neurons begin to settle in their definitive positions and the Purkinje cell layer can be distinguished. Granule cell formation is also prolonged over many post-hatching months and the external granular layer persists for more than 20 weeks after hatching. The findings indicate that cerebellar circuitry is unlikely to contribute to the coordination of movements in the monotreme peri-hatching period. Those activities are most likely controlled by the spinal cord and medullary reticular formation circuitry.

The extant monotremes (platypus and echidnas) are believed to all be capable of electroreception in the trigeminal pathways, although they differ significantly in the number and distribution of electroreceptors. It has been argued by some authors that electroreception was first developed in an aquatic environment and that echidnas are descended from a platypus-like ancestor that invaded an available terrestrial habitat. If this were the case, one would expect the developmental trajectories of the trigeminal pathways to be similar in the early stages of platypus and short-beaked echidna development, with structural divergence occurring later. We examined the development of the peripheral trigeminal pathway from snout skin to trigeminal ganglion in sectioned material in the Hill and Hubrecht collections to test for similarities and differences between the two during the development from egg to adulthood. Each monotreme showed a characteristic and different pattern of distribution of developing epidermal sensory gland specializations (electroreceptor primordia) from the time of hatching. The cross-sectional areas of the trigeminal divisions and the volume of the trigeminal ganglion itself were also very different between the two species at embryonic ages, and remained consistently different throughout post-hatching development. Our findings indicate that the trigeminal pathways in the short-beaked echidna and the platypus follow very different developmental trajectories from the earliest ages. These findings are more consistent with the notion that the platypus and echidna have both diverged from an ancestor with rudimentary electroreception and/or trigeminal specialization, rather than the contention that the echidna is derived from a platypus-like ancestor.

Radial glial cells represent a subpopulation of secondary neural precursor cells that differentiate from neuroepithelial progenitors and are transiently found in the developing CNS of mammals. There is ample evidence for a temporal and spatial arrangement of increasingly committed radial glial cells that is of critical importance for the organisation and specification of different brain regions. For the human ganglionic eminence, recent findings have shown an early molecular specification of this cell type by the CD15 carbohydrate epitope, beginning already at the end of the first trimester. Here we further characterise the CD15+ radial glia cells as bFGF/EGF responsive progenitors allowing its propagation in vitro. By magnet activated cell sorting, its trilineage differentiation potential can be shown by differentiation into (PSA-NCAM ß3 tubulin immunoreactive) neurones, GFAP expressing cells of astrocytic morphology, and O4 positive oligodendrocytes. Subcloning experiments under proliferation conditions reveal ongoing CD15 expression by dividing cells. Although the relative number of CD15+ progenitor cells is found to decrease in favour of CD15- precursor cells during continuous passaging, cell sorting experiments allow the repetitive purification of high numbers of positively selected precursor cells for up to 12 weeks. In conclusion, expression of the cell adhesion molecule CD15 by a subpopulation of proliferative cells from the lateral ganglionic eminence allows easy and reproducible purification of progenitor cells by cell sorting, enabling the generation of a compartment-specific cell pool as a prerequisite for a safe and standardised therapy of neurodegenerative basal forebrain diseases.