Magnetic resonance imaging and diffusion tensor imaging reconstruction of connectomes in a macropod, the quokka (Setonix brachyurus).

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.

Numerical Analysis of the Cerebral Cortex in Diprotodontids (Marsupialia; Australidelphia) and Comparison with Eutherian Brains.

Diprotodontids are a diverse group of Australian metatherians, which occupy a range of ecological niches from nectar and pollen-feeders to grazers and folivores. The group encompasses small-brained nectar-feeding species (Tarsipes) and large-brained grazing and browsing species (macropods). This group of Australian metatherians therefore represents an opportunity to examine how the cerebral cortex has expanded in an adaptive radiation quite independent of that occurring among eutherians. We have used the Nelson Brain Collection and online resources to perform a quantitative analysis of the isocortex, hippocampal formation and olfactory structures in diprotodontids. We found that the scaling relationship between iso- and periallocortical grey matter and brain size, and between subcortical white matter and iso- and periallocortex grey matter, are both almost identical among diprotodontids and eutherians. By contrast, the relationship between gyrification and brain size is strikingly different between diprotodontids and eutherians, with gyrification being much lower for a given brain size among the diprotodontids, although gyrification is much more varied among macropods than other diprotodontids. The scaling of iso- and periallocortical volume with dorsal striatal and dorsal thalamic volume is almost identical among the diprotodontids and eutherians, but the claustrum is smaller, and amygdala larger, for a given brain size among diprotodontids than eutherians. The hippocampal formation and central olfactory areas (anterior olfactory region and piriform cortex) both scale more steeply with brain size among diprotodontids compared to eutherians. Our findings suggest that, although white matter scaling is identical among all therians, there are significant differences between diprotodontids and eutherians in the way that cortical folding and expansion of allocortical structures occurs with brain enlargement.

Quantitative analysis of cerebellar morphology in monotreme, metatherian and eutherian mammals.

To superficial inspection, the mammalian cerebellum appears to be a stereotypical structure that varies little in morphology across mammals. In the present study, the volumes of components of the corpus cerebelli, foliation of the cerebellar cortex and the volumes of the pontine and deep cerebellar nuclei have been measured and compared in three species of monotreme, 90 species of marsupial and 57 species of eutherian mammal. In all three mammalian groups, the volume of the corpus cerebelli scales isometrically with brain volume, and pontine nuclear volume also scales isometrically with cerebellar volume. The ratio of hemisphere to vermal cerebellar cortex is comparable in all mammals at small cerebellar volume, but elaboration of cerebellar hemispheres is largely confined to large cerebella of eutherian mammals. At small cerebellar volumes, diprotodontid metatherians have proportionally large cerebellar hemispheres compared to non-diprotodontid metatherians, and metatherian cerebella in general have a high volume of central white matter for a given cerebellar cortex volume compared to eutherians. The degree of foliation of the cerebellum scales similarly in therian mammals, but is relatively low in the monotremes for the volume of their corpus cerebelli. Among metatherians, cerebellar foliation is stronger among diprotodontid as compared to non-diprotodontids. Although the cerebellum has a similar structure in all mammals, there are subtle differences in structure between different mammal groups with possible functional implications.

Quantitative analysis of arterial supply to the developing brain in tetrapod vertebrates.

Understanding the metabolic cost of building developing tetrapod brains is critically important to explaining the more than 10-fold differences in encephalization of adult tetrapods that have emerged during evolution. The exact metabolic costs of developing the variety of tetrapod brains are impossible to determine, but one can compare cerebral artery caliber (internal radius raised to the fourth power-r4 ) across developing tetrapod vertebrate groups as a proxy of cerebral arterial flow, the delivery of nutrients during embryogenesis and early postnatal development, and hence the metabolic costs of brain development. In this study, r4 of aortic outflow and cerebral inflow arteries, as well as aortic wall thickness as a proxy of arterial pressure, were measured and compared between developing representatives of all four tetrapod classes (mammals, birds, reptiles, and amphibians). We found a clear endotherm/ectotherm dichotomy in aortic outflow and cerebral inflow between developing mammals and birds on the one hand, and developing reptiles and amphibians on the other. We did not find strong evidence for functionally significant differences in cerebral arterial caliber between groups at the order level (i.e., within birds, reptiles or amphibians). In particular, we did not find evidence in favor of increased blood supply to the brain for more behaviorally complex and encephalized avian species.

Quantitative Analysis of the Timing of Development of the Cerebellum and Precerebellar Nuclei in Monotremes, Metatherians, Rodents, and Humans.

We have used a quantitative statistical approach to compare the pace of development in the cerebellum and precerebellar systems relative to body size in monotremes and metatherians with that in eutherians (rodents and humans). Embryos, fetuses, and early postnatal mammals were scored on whether key structural events had been reached in the development of the cerebellum itself (CC-corpus cerebelli; 10 milestones), or the pontine and inferior olivary precerebellar nuclear groups (PC; 4 milestones). We found that many early cerebellar and precerebellar milestones (e.g., formation of Purkinje cell layer and deep cerebellar nuclei) were reached at a smaller absolute body length in both metatherians and eutherians together, compared to monotremes. Some later milestones (e.g., formation of the external granular layer and primary fissuration) were reached at a smaller body length in metatherians than eutherians. When the analysis was performed with proportional body length expressed as a natural log-transformed ratio of length at birth, milestones were reached at a much smaller proportional body length in rodents and humans than in the metatherians or monotremes. The findings are consistent with the slower pace of metabolic activity and embryonic development in monotremes. They also indicate slightly advanced maturation of some early features of the cerebellum in some metatherians (i.e., early cerebellar development in dasyurids relative to body size), but do not support the notion of an accelerated development of the cerebellum to cope with the demands of early birth. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1998-2013, 2020. © 2019 American Association for Anatomy.

Quantitative analysis of forebrain pallial morphology in monotremes and comparison with that in therians.

We have made quantitative volumetric analyses of cerebral cortical (pallial) structures in the brains of three species of monotreme (Ornithorhynchus anatinus, Tachyglossus aculeatus, Zaglossus bruijni) and compared the findings with similar measurements in a range of therian mammals (6 marsupials and 50 placentals). We have found that although the iso- and periallocortical grey matter volume of the monotremes is about what would be expected for their brain size, the proportion of iso- and periallocortical white matter in monotremes is substantially lower than that in the forebrains of therians. This suggests that the forebrains of the three monotremes have fewer association, commissural and/or projection connections than those of similarly sized forebrains of therian mammals. We also found that the iso- and periallocortex of the platypus is relatively smooth-surfaced compared to similarly sized brains of therian mammals, with a distinct caudal shift in the positioning of cortical white matter in the forebrain, consistent with expansion of the posterior thalamic radiation. Central laminated olfactory structures (anterior olfactory nucleus and piriform cortex) are large in the tachyglossid monotremes (Tachyglossus aculeatus and Zaglossus bruijni) and large in xenarthran placental mammals, suggesting convergence of the forebrain structure of monotreme formivores with that of similarly specialized therians like the xenarthrans Myrmecophaga tridactyla and Dasypus novemcinctus.

Magnetic Resonance Imaging of the Brain of a Monotreme, the Short-Beaked Echidna (Tachyglossus aculeatus).

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.

Reconstruction of the Cortical Maps of the Tasmanian Tiger and Comparison to the Tasmanian Devil.

The last known Tasmanian tiger (Thylacinus cynocephalus)-aka the thylacine-died in 1936. Because its natural behavior was never scientifically documented, we are left to infer aspects of its behavior from museum specimens and historical recollections of bushmen. Recent advances in brain imaging have made it possible to scan postmortem specimens of a wide range of animals, even more than a decade old. Any thylacine brain, however, would be more than 100 years old. Here, we show that it is possible to reconstruct white matter tracts in two thylacine brains. For functional interpretation, we compare to the white matter reconstructions of the brains of two Tasmanian devils (Sarcophilus harrisii). We reconstructed the cortical projection zones of the basal ganglia and major thalamic nuclei. The basal ganglia reconstruction showed a more modularized pattern in the cortex of the thylacine, while the devil cortex was dominated by the putamen. Similarly, the thalamic projections had a more orderly topography in the thylacine than the devil. These results are consistent with theories of brain evolution suggesting that larger brains are more modularized. Functionally, the thylacine's brain may have had relatively more cortex devoted to planning and decision-making, which would be consistent with a predatory ecological niche versus the scavenging niche of the devil.

Anterior commissure versus corpus callosum: A quantitative comparison across mammals.

Mammals rely on two major pathways to transfer information between the two hemispheres of the brain: the anterior commissure and the corpus callosum. Metatheria and monotremes rely exclusively on the anterior commissure for interhemispheric transfer between the isocortices and olfactory allocortices of each side, whereas Eutheria use a combination of the anterior commissure and an additional pathway exclusive to Eutheria, the corpus callosum. Midline cross-sectional area of the anterior commissure and corpus callosum were measured in a range of mammals from all three infraclasses and plotted against brain volume to determine how midline anterior commissure area and its size relative to the corpus callosum vary with brain size and taxon. In Metatheria, the square root of anterior commissure area rises in almost direct proportion with the cube root of brain volume (i.e. the ratio of the two is relatively constant), whereas among Eutheria the ratio of the square root of anterior commissure area to the cube root of brain volume declines slightly with increasing brain size. The total of isocortical and olfactory allocortical commissure area rises more rapidly with increasing brain volume among Eutheria than among Metatheria. This means that the midline isocortical and olfactory allocortical commissural area of metatherians with large brains (about 70 ml) is only about 50% of that among eutherians with similarly sized brains. On the other hand, isocortical and olfactory allocortical commissural area is similar in Metatheria and Eutheria at brain volumes around 1 ml. Among the Eutheria, some groups make less use of the anterior commissure pathway than do others: soricomorphs, rodents and cetaceans have smaller anterior commissures for their brain size than do afrosoricids, erinaceomorphs and proboscideans. The findings suggest that use of the anterior commissural route for isocortical commissural connections may have placed limitations on interhemispheric transfer of information among the metatherians, but only when brain size reaches 50 ml or more.

Quantitative comparison of cerebral artery development in metatherians and monotremes with non-human eutherians.

A quantitative comparison of the internal diameters of cerebral feeder arteries (internal carotid and vertebral) and the aorta in developing non-human eutherians, metatherians and monotremes has been made, with the aim of determining if there are differences in cerebral arterial flow between the three infraclasses of mammals such as might reflect differences in metabolism of the developing brain. There were no significant differences between eutherians and metatherians in the internal radius of the aorta or the thickness of the aortic wall, but aortic internal radius was significantly smaller in developing monotremes than therians at the < 10 mm body length range. Aortic thickness in the developing monotremes also rose at a slower rate relative to body length than in metatherians or eutherians. The sums of the internal calibres of the internal carotid and vertebral arteries were significantly lower in metatherians as a group and monotremes compared with non-human eutherians at body lengths up to 20 mm and in metatherians at > 20 mm body length. The internal calibre of the internal carotids relative to the sum of all cerebral feeder arteries was also significantly lower in monotremes at < 10 mm body length compared with eutherians. It was noted that dasyurids differed from other metatherians in several measures of cerebral arterial calibre and aortic internal calibre. The findings suggest that: (i) both aortic outflow and cerebral arterial inflow may be lower in developing monotremes than in therians, particularly at small body size (< 20 mm); (ii) cerebral inflow may be lower in some developing metatherians than non-human eutherians; and (iii) dasyurids have unusual features of cerebral arteries possibly related to the extreme immaturity and small size at which they are born. The findings have implications for nutritional sourcing of the developing brain in the three infraclasses of mammals.

Timing of mammalian peripheral trigeminal system development relative to body size: A comparison of metatherians with rodents and monotremes.

Specializations of the trigeminal sensory system are present in all three infraclasses of mammals (metatheria, eutheria, prototheria or monotremata). The trigeminal sensory system has been suggested as a critically important modality for sampling the path to the pouch and detecting the nipple or milk patch, but the degree to which that system may be required to function at birth varies significantly. Archived sections of the snout and brainstem of embryonic and postnatal mammals were used to test the relationship between structural maturity of the two ends of the trigeminal nerve pathway and the body size of mammalian young in metatherians, rodents and monotremes. A system for staging different levels of structural maturity of the vibrissae and trigeminal sensory was applied to embryos, pouch young and hatchlings and correlated with body length. Dasyurids are born at the most immature state with respect to vibrissal and trigeminal sensory nucleus development of any available metatherian, but these components of the trigeminal system are also developmentally advanced relative to body size when dasyurids are compared to other metatherians. Vibrissal and trigeminal sensory nucleus development is at a similar stage of development at birth and for a given body size in non-dasyurid metatherians; and trigeminal sensory nucleus development in monotremes is at a similar stage at birth to metatherians. Rodents reach a far more advanced stage of vibrissal and trigeminal sensory nucleus development at birth than do metatherians, and in the case of the mouse have a more developmentally advanced trigeminal system than all available metatherians at any given body length. Precocious development of the trigeminal sensory pathway relative to body size is evident in dasyurids, as might be expected given the small birth size of those metatherians. Nevertheless, the trigeminal sensory system in metatherians in general is not precocious relative to body size when these species are considered alongside the pace of trigeminal somatosensory development in rodents.

Quantitative comparison of cerebral artery development in human embryos with other eutherians.

The embryonic and early fetal human brain is known to undergo extraordinary expansion of its cellular population during embryonic and early fetal life, and is critically dependant on a steady supply of nutrients and oxygen for proper brain development. Quantitative analysis of the internal radius of the aorta and cerebral arteries in a range of eutherian mammals has been used to compare arterial flow to the developing human brain with that to the brains of non-human eutherians. Human embryos showed a much steeper rise of internal radius of the aorta with increasing body size than the embryos of non-human eutherians, but the thickness of the aorta rose at the same pace relative to body size in both humans and non-humans, suggesting that aortic pressure is similar in all eutherian embryos of a similar size. The sums of internal radii of both the internal carotids and vertebral arteries of human embryos raised to the fourth power were much lower at embryonic stages (less than 22 mm body length) than in non-human eutherians, were similar between humans and non-humans at 22-30 mm body length, and exceeded the non-humans at body lengths of more than 30 mm. The relative size of the internal calibre of the cerebral feeder arteries (internal carotid and vertebral) to the aorta did not change between embryonic and fetal sizes in either humans or non-humans. The findings suggest that the developing human brain may actually receive less blood flow at embryonic sizes (less than 22 mm body length) than do other mammalian embryos of a similar body size, but that internal carotid and vertebral flow is higher in human fetuses (body length greater than 30 mm) than in developing non-humans of the same body size. Increased flow to the developing human brain relative to non-humans is achieved by simultaneous increases in both aortic and cerebral feeder artery internal calibre.

Quantitative analysis of somatosensory cortex development in eutherians, with a comparison with metatherians and monotremes.

Extant eutherians exhibit a wide range of adult brain sizes and degree of cortical gyrification. Quantitative analysis of parietal isocortical sections held in museum collections was used to compare the pace of somatosensory cortex development relative to body size and pallial thickness among diverse eutherian embryos, foetuses, and neonates. Analysis indicated that, for most eutherians, cortical plate aggregation begins at about 6-18 mm greatest length or about 120-320 µm pallial thickness. Expansion of the proliferative compartment occurs at a similar pace in most eutherians, but exceptionally rapidly in hominoids. Involution of the pallial proliferative zones occurs over a wide range of body sizes (42 mm to over 500 mm greatest length) or when the cerebral cortex reaches a thickness of 1.2-9.8 mm depending on the eutherian group. Many of these values overlap with those for metatherians. The findings suggest that there is less evolutionary flexibility in the timing of cortical plate aggregation than in the rate of expansion of the pallial proliferative compartment and the duration of proliferative zone activity.

Quantitative analysis of somatosensory cortex development in metatherians and monotremes, with comparison to the laboratory rat.

Metatherians and monotremes are born in an immature state, followed by prolonged nurturing by maternal lactation. Quantitative analysis of isocortical sections held in the collections at the Museum für Naturkunde, Berlin was used to compare the pace of somatosensory cortex development relative to body size and pallial thickness between metatherian groups, monotremes, and the laboratory rat. Analysis indicated that the pace of pallial growth in the monotremes is much lower than that in the metatherians or laboratory rat, with an estimated 8.6-fold increase in parietal cortex thickness between 10 and 100 mm body length, compared to a 10- to 20-fold increase among the metatherians and the rat. It was found that aggregation of cortical plate neurons occurs at similar embryo size in the mammals studied (around 8-14 mm body length) and a similar pallial thickness (around 200 µm), but that proliferative zone involution occurs at a much higher body size and pallial thickness in the monotremes compared to the metatherians and the laboratory rat. The observations suggest that cortical development in the monotremes is slower and subject to different regulatory signals to the therians studied. The slow pace may be related to either generally slower metabolism in monotremes or less efficient nutrient supply to the offspring due to the lack of teats.

Brain and behaviour of living and extinct echidnas.

The Tachyglossidae (long- and short-beaked echidnas) are a family of monotremes, confined to Australia and New Guinea, that exhibit striking trigeminal, olfactory and cortical specialisations. Several species of long-beaked echidna (Zaglossus robusta, Zaglossus hacketti, Megalibgwilia ramsayi) were part of the large-bodied (10 kg or more) fauna of Pleistocene Australasia, but only the diminutive (2-7 kg) Tachyglossus aculeatus is widespread today on the Australian mainland. We used high-resolution CT scanning and other osteological techniques to determine whether the remarkable neurological specialisations of modern echidnas were also present in Pleistocene forms or have undergone modification as the Australian climate changed in the transition from the Pleistocene to the Holocene. All the living and extinct echidnas studied have a similar pattern of cortical gyrification that suggests comparable functional topography to the modern short-beaked form. Osteological features related to olfactory, trigeminal, auditory and vestibular specialisation (e.g., foramina and cribriform plate area, osseous labyrinth topography) are also similar in living and extinct species. Our findings indicate that despite differences in diet, habitat and body size, the suite of neurological specialisations in the Tachyglossidae has been remarkably constant: encephalisation, sensory anatomy and specialisation (olfactory, trigeminal, auditory and vestibular), hypoglossal nerve size and cortical topography have all been stable neurological features of the group for at least 300,000 years.

Spinal cord development in marsupials in relation to birthing strategies and in comparison with monotremes and the laboratory rat.

Marsupials are born in an immature state, followed by prolonged nurturing of pouch young by maternal lactation. Spinal cord sections held in the collections at the Museum für Naturkunde, Berlin were used to test the relationship between structural maturity of the spinal cord and the locomotor challenges that face young marsupials and monotremes. Analysis of variance indicated that body length is a much stronger determinant of variation in anatomical measures of spinal cord maturation than mammal type.

Vestibular development in marsupials and monotremes.

The young of marsupials and monotremes are all born in an immature state, followed by prolonged nurturing by maternal lactation in either a pouch or nest. Nevertheless, the level of locomotor ability required for newborn marsupials and monotremes to reach the safety of the pouch or nest varies considerably: some are transferred to the pouch or nest in an egg (monotremes); others are transferred passively by gravity (e.g. dasyurid marsupials); some have only a horizontal wriggle to make (e.g. peramelid and didelphid marsupials); and others must climb vertically for a long distance to reach the maternal pouch (e.g. diprotodontid marsupials). In the present study, archived sections of the inner ear and hindbrain held in the Bolk, Hill and Hubrecht collections at the Museum für Naturkunde, Berlin, were used to test the relationship between structural maturity of the vestibular apparatus and the locomotor challenges that face the young of these different mammalian groups. A system for staging different levels of structural maturity of the vestibular apparatus was applied to the embryos, pouch young and hatchlings, and correlated with somatic size as indicated by greatest body length. Dasyurids are born at the most immature state, with the vestibular apparatus at little more than the otocyst stage. Peramelids are born with the vestibular apparatus at a more mature state (fully developed semicircular ducts and a ductus reuniens forming between the cochlear duct and saccule, but no semicircular canals). Diprotodontids and monotremes are born with the vestibular apparatus at the most mature state for the non-eutherians (semicircular canals formed, maculae present, but vestibular nuclei in the brainstem not yet differentiated). Monotremes and marsupials reach the later stages of vestibular apparatus development at mean body lengths that lie within the range of those found for laboratory rodents (mouse and rat) reaching the same vestibular stage.

Clonogenic CD15 immunoreactive radial glial cells from the developing human lateral ganglionic eminence.

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.

Distinct development of the trigeminal sensory nuclei in platypus and echidna.

Both lineages of the modern monotremes have been reported to be capable of electroreception using the trigeminal pathways and it has been argued that electroreception arose in an aquatic platypus-like ancestor of both modern monotreme groups. On the other hand, the trigeminal sensory nuclear complex of the platypus is highly modified for processing tactile and electrosensory information from the bill, whereas the trigeminal sensory nuclear complex of the short-beaked echidna (Tachyglossus aculeatus) is not particularly specialized. If the common ancestor for both platypus and echidna were an electroreceptively and trigeminally specialized aquatic feeder, one would expect the early stages of development of the trigeminal sensory nuclei in both species to show evidence of structural specialization from the outset. To determine whether this is the case, we examined the development of the trigeminal sensory nuclei in the platypus and short-beaked echidna using the Hill and Hubrecht embryological collections. We found that the highly specialized features of the platypus trigeminal sensory nuclei (i.e. the large size of the principal nucleus and oral part of the spinal trigeminal nuclear complex, and the presence of a dorsolateral parvicellular segment in the principal nucleus) appear around the time of hatching in the platypus, but are never seen at any stage in the echidna. Our findings support the proposition that the modern echidna and platypus are derived from a common ancestor with only minimal trigeminal specialization and that the peculiar anatomy of the trigeminal sensory nuclei in the modern platypus emerged in the ornithorhynchids after divergence from the tachyglossids.

Development of the cerebellum in the platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus).

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.