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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.