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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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2015 Anatomical Society.)

The autonomic nervous system (ANS) plays an essential role in the regulation of vascular tone. Sympathetic neurotransmitters epinephrine and norepinephrine are released from the terminals of perivascular nerves and suppress endothelial production of nitric oxide (NO), an important vasodilator. Sympathetic nerves also release neuropeptide Y, a co-transmitter that stimulates vasoconstriction and proliferation of vascular smooth muscle cells. Parasympathetic nerves release acetylcholine, which leads to vascular contraction when NO production is inhibited. The ANS produces a variety of other vasoactive substances including ATP, calcitonin gene-related peptide, dopamine, and serotonin. On the other hand, the vascular system can reciprocally influence ANS activity through the release of NO, reactive oxygen species (ROS), angiotensin II, and other mechanisms. In pathological conditions such as atherosclerosis, hyperactivation of sympathetic neural activity has pro-atherogenic effects on the vascular function by increasing vasoconstriction, accumulation of modified lipoproteins in the vascular wall, induction of endothelial dysfunction, and stimulation of oxidative stress and vascular remodeling. Indeed, suppression of the sympathetic ANS should be beneficial for the treatment of cardiovascular diseases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© 2015 Anatomical Society.)

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.

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.

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.

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., (Crown Copyright © 2014. Published by Elsevier GmbH. All rights reserved.)

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.

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., (© 2013 Anatomical Society.)

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.

The living monotremes (platypus and echidnas) are distinguished by the development of their young in a leathery-shelled egg, a low and variable body temperature and a primitive teat-less mammary gland. Their young are hatched in an immature state and must deal with the external environment, with all its challenges of hypothermia and stress, as well as sourcing nutrients from the maternal mammary gland. The Hill and Hubrecht embryological collections have been used to follow the structural development of the monotreme hypothalamus and its connections with the pituitary gland both in the period leading up to hatching and during the lactational phase of development, and to relate this structural maturation to behavioural development. In the incubation phase, development of the hypothalamus proceeds from closure of the anterior neuropore to formation of the lateral hypothalamic zone and putative medial forebrain bundle. Some medial zone hypothalamic nuclei are emerging at the time of hatching, but these are poorly differentiated and periventricular zone nuclei do not appear until the first week of post-hatching life. Differentiation of the pituitary is also incomplete at hatching, epithelial cords do not develop in the pars anterior until the first week, and the hypothalamo-neurohypophyseal tract does not appear until the second week of post-hatching life. In many respects, the structure of the hypothalamus and pituitary of the newly hatched monotreme is similar to that seen in newborn marsupials, suggesting that both groups rely solely on lateral hypothalamic zone nuclei for whatever homeostatic mechanisms they are capable of at birth/hatching., (© 2012 The Author. Journal of Anatomy © 2012 Anatomical Society.)

The living monotremes (platypus and echidnas) are distinguished from therians as well as each other in part by the unusual structure of the thalamus in each. In particular, the platypus has an enlarged ventral posterior (VP) nucleus reflecting the great behavioural importance of trigeminosensation and electroreception. The embryological collections of the Museum für Naturkunde in Berlin were used to analyse the development of the dorsal thalamus and ventral thalamus (prethalamus) in both species. Prosomeric organization of the forebrain emerged at 6 mm crown-rump length (CRL), but thalamic neurogenesis did not commence until about 8-9 mm CRL. Distinctive features of the dorsal thalamus in the two species began to emerge after hatching (about 14-15 mm CRL). During the first post-hatching week, dense clusters of granular cells aggregated to form the VP of the platypus, whereas the VP complex of the echidna remained smaller and divided into distinct medial and lateral divisions. At the end of the first post-hatching week, the thalamocortical tract was much larger in the platypus than the echidna. The dorsal thalamus of the platypus is essentially adult-like by the sixth week of post-hatching life. The similar appearance of the dorsal thalamus in the two species until the time of hatching, followed by the rapid expansion of the VP in the platypus, is most consistent with ancestral platypuses having undergone changes in the genetic control of thalamic neurogenesis to produce a large VP for trigeminal electroreception after the divergence of the two lineages of monotreme.

The modern monotremes (platypus and echidnas) are characterized by development of their young in a leathery egg that is laid into a nest or abdominal pouch. At hatching, the young are externally immature, with forelimbs capable of digitopalmar prehension, but hindlimbs little advanced beyond limb buds. The embryological collections at the Museum für Naturkunde in Berlin were used to examine the development of the spinal cord and early peripheral nervous system in developing monotremes and to correlate this with known behavioural development. Ventral root outgrowth to the bases of both the fore- and hindlimbs occurs at 6.0 mm crown-rump length (CRL), but invasion of both limbs does not happen until about 8.0-8.5 mm CRL. Differentiation of the ventral horn precedes the dorsal horn during incubation and separate medial and lateral motor columns can be distinguished before hatching. Rexed's laminae begin to appear in the dorsal horn in the first week after hatching, and gracile and cuneate fasciculi emerge during the first two post-hatching months. Qualitative and quantitative comparisons of the structure of the cervicothoracic junction spinal cord in the two monotremes with that in a diprotodont marsupial (the brush-tailed possum, Trichosurus vulpecula) of similar size at birth, did not reveal any significant structural differences between the monotremes and the marsupial. The precocious development of motor systems in the monotreme spinal cord is consistent with the behavioural requirements of the peri-hatching period, that is, rupture of embryonic membranes and egg, and digitopalmar prehension to grasp maternal hair or nest material.

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., (Copyright © 2012 S. Karger AG, Basel.)

The two groups of living monotremes (platypus and echidnas) have remarkably different olfactory structures in the adult. The layers of the main olfactory bulb of the short-beaked echidna are extensively folded, whereas those of the platypus are not. Similarly, the surface area of the piriform cortex of the echidna is large and its lamination complex, whereas in the platypus it is small and simple. It has been argued that the modern echidnas are derived from a platypus-like ancestor, in which case the extensive olfactory specializations of the modern echidnas would have developed relatively recently in monotreme evolution. In this study, the development of the constituent structures of the olfactory pathway was studied in sectioned platypus and echidna embryos and post-hatchlings at the Museum für Naturkunde, Berlin, Germany. The aim was to determine whether the olfactory structures follow a similar maturational path in the two monotremes during embryonic and early post-hatching ages or whether they show very different developmental paths from the outset. The findings indicate that anatomical differences in the central olfactory system between the short-beaked echidna and the platypus begin to develop immediately before hatching, although details of differences in nasal cavity architecture emerge progressively during late post-hatching life. These findings are most consistent with the proposition that the two modern monotreme lineages have followed independent evolutionary paths from a less olfaction-specialized ancestor. The monotreme olfactory pathway does not appear to be sufficiently structurally mature at birth to allow olfaction-mediated behaviour, because central components of both the main and accessory olfactory system have not differentiated at the time of hatching., (Copyright © 2011 S. Karger AG, Basel.)

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., (Copyright © 2011 S. Karger AG, Basel.)

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., (Copyright © 2012 S. Karger AG, Basel.)

Both lineages of the modern monotremes have distinctive features in the cerebral cortex, but the developmental mechanisms that produce such different adult cortical architecture remain unknown. Similarly, nothing is known about the differences and/or similarities between monotreme and therian cortical development. We have used material from the Hill embryological collection to try to answer key questions concerning cortical development in monotremes. Our findings indicate that gyrencephaly begins to emerge in the echidna brain shortly before birth (crown-rump length 12.5 mm), whereas the cortex of the platypus remains lissencephalic throughout development. The cortices of both monotremes are very immature at the time of hatching, much like that seen in marsupials, and both have a subventricular zone (SubV) within both the striatum and pallium during post-hatching development. It is particularly striking that in the platypus, this region has an extension from the palliostriatal angle beneath the developing trigeminoreceptive part of the somatosensory cortex of the lateral cortex. The putative SubV beneath the trigeminal part of S1 appears to accommodate at least two distinct types of cell and many mitotic figures and (particularly in the platypus) appears to be traversed by large numbers of thalamocortical axons as these grow in. The association with putative thalamocortical fibres suggests that this region may also serve functions similar to the subplate zone of Eutheria. These findings suggest that cortical development in each monotreme follows distinct paths from at least the time of birth, consistent with a long period of independent and divergent cortical evolution., (Copyright © 2011 S. Karger AG, Basel.)

CART (cocaine- and amphetamine-regulated transcript) is a neuromodulator involved in feeding, drug reward, stress and cardiovascular function. We have immunohistochemically studied the distribution of the CART peptide in the brains of two adult marsupial species: the brown antechinus (Antechinus stuartii) as a representative of polyprotodont marsupials and the tammar wallaby (Macropus eugenii) as a representative of diprotodont marsupials. We have also examined the distribution of CART during postnatal development in the tammar wallaby. There were similarities and differences both between the two marsupial species and between the marsupials and eutherians in CART distribution. Both marsupials showed immunoreactivity to CART in the olfactory bulb, piriform cortex, extended amygdala, the supraoptic, paraventricular and arcuate nuclei of the hypothalamus, somatosensory and auditory nuclei of the brainstem, vagal/solitary complex, raphe obscurus and raphe pallidus and presumptive presympathetic neurons of the ventrolateral medulla, as has been seen in eutherians. On the other hand, immunoreactivity to CART was weak in or absent from isocortical areas, and immunoreactivity to CART was poor or minimal in the ventral tegmental area and nucleus accumbens of both species; regions where immunoreactivity to CART is very strong in the brains of eutherians. During development, CART was present at birth (P0) in the lateral trigeminal ganglion, spinal trigeminal tract and the vagal sensorimotor complex, but did not appear in mid- or forebrain regions until much later (from P37). These anatomical findings indicate that although CART is likely to serve very similar functions in both eutherians and marsupials, there are potentially functionally significant differences between the two mammalian groups., (Copyright © 2010 S. Karger AG, Basel.)

The cyto- and chemoarchitecture of the cerebral cortex has been examined in three small (mouse-sized) polyprotodont marsupial carnivores from Australia (the stripe-faced dunnart, Sminthopsis macroura; the brown antechinus, Antechinus stuartii; and the red-tailed phascogale, Phascogale calura) in order to compare the cortical topography of these marsupials with that of diprotodontids, didelphids and eutherians. All three species studied had similar cortical cytoarchitecture. The isocortical surface was dominated by primary somatosensory (S1) and visual (V1) areas. Putative secondary sensory areas (S2, V2M, V2L) were also identified. The primary somatosensory cortex demonstrated clumps of granule cells in the presumptive mystacial field, whereas the primary visual area showed a distinctive chemical signature of intense calbindin immunoreactivity in layer IV. On the other hand, the primary auditory area was small and indistinct, but flanked by a temporal association area (TeA). A cytoarchitecturally distinct primary motor cortex (M1) with prominent pyramidal neurons in layer V and poor layer IV was identified medially to S1, and at rostral levels a putative secondary motor area was identified medial to M1. Transitional areas between isocortex and allocortical regions showed many cyto- and chemoarchitectural similarities to those reported for eutherian (and in particular rodent) cortex. Medially, two cingulate regions were found at rostral levels, with dysgranular and granular 'retrosplenial' areas identified caudally. Laterally, granular and agranular areas surrounded the rostral rhinal fissure, to be replaced by ectorhinal and perirhinal areas caudally. The findings indicate that the cyto- and chemoarchitectural features which characterize the iso- and allocortex in these small marsupial carnivores are similar to those reported in didelphids and eutherians and our findings suggest the existence of putative dedicated motor areas medial to the S1 field., (Copyright 2008 S. Karger AG, Basel.)

The topography and chemoarchitecture of the striatum and pallidum in a monotreme, the short-beaked echidna (Tachyglossus aculeatus) have been studied using Nissl staining in conjunction with myelin staining, enzyme reactivity to acetylcholinesterase and NADPH diaphorase, and immunoreactivity to parvalbumin, calbindin, calretinin, tyrosine hydroxylase, neuropeptide Y, and neurofilament protein (SMI-32 antibody). All those components of the striatum and pallidum found in eutherian mammals could also be identified in the echidna's brain, with broad chemoarchitectural similarities to those regions in eutherian brains also apparent. There was a clear chemoarchitectural gradient visible with parvalbumin immunoreactivity of neurons and fibers, suggesting a subdivision of the echidna caudatoputamen into weakly reactive rostrodorsomedial and strongly reactive caudoventrolateral components. This may, in turn, relate to subdivision into associative versus sensorimotor CPu and reflect homology to the caudate and putamen of primates. Moreover, the chemoarchitecture of the echidna striatum suggested the presence of striosome-matrix architecture. The morphology of identified neuronal groups (i.e., parvalbumin, calbindin, and neuropeptide Y immunoreactive) in the echidna striatum and pallidum showed many similarities to those seen in eutherians, although the pattern of distribution of calbindin immunoreactive neurons was more uniform in the caudatoputamen of the echidna than in therians. These observations indicate that the same broad features of striatal and pallidal organization apply across all mammals and suggest that these common features may have arisen before the divergence of the monotreme and therian lineages.

The dorsal nucleus of the vagus nerve plays an integral part in the control of visceral function. The aim of the present study was to correlate structural and chemical changes in the developing nucleus with available data concerning functional maturation of human viscera and reflexes. The fetal development (ages 9 to 26 weeks) of the human dorsal nucleus of the vagus nerve has been examined with the aid of Nissl staining and immunocytochemistry for calbindin and tyrosine hydroxylase. By 13 weeks, the dorsal vagal nucleus emerges as a distinct structure with at least two subnuclei visible in Nissl stained preparations. By 15 weeks, three subnuclei (dorsal intermediate, centrointermediate and ventrointermediate) were clearly discernible at the open medulla level with caudal and caudointermediate subnuclei visible at the level of the area postrema. All subnuclei known to exist in the adult were visible by 21 weeks and cytoarchitectonic differentiation of the nucleus was largely completed by 25 weeks. The adult distribution pattern of calbindin and tyrosine hydroxylase immunoreactive neurons was also largely completed by 21 weeks, although morphological differentiation of labeled neurons continued until the last age examined (26 weeks). The structural development of the dorsal nucleus of the vagus nerve appears to occur in parallel with functional maturation of the cardiovascular and gastric movements, which the nucleus controls.

Encephalization of Australian marsupials was analyzed using the endocranial volume (ECV) of 52 species of Dasyuromorphia and Notoryctemorphia, 14 species of Peramelemorphia and 116 species of Diprotodontia from Australia and New Guinea and compared with 16 species of Ameridelphian marsupials and 3 species of native and recently introduced Australian eutherian carnivores (dingo, feral cat and feral fox). Linear regression analysis of the relationship between ECV and body weight for marsupials revealed that allometric parameters for these groups are different from those previously derived for samples of (mainly eutherian) mammals, with higher slopes for Dasyuromorphia and Diprotodontia and lower slopes for Ameridelphians and Peramelemorphia. Absolute ECV for small Australian and New Guinea marsupial carnivores (Antechinus and Sminthopsis) were found to be comparable to eutherians of similar body weight, but large marsupial carnivores such as the Tasmanian devil and thylacine had substantially smaller ECVs than eutherian carnivores of similar body weight. Similarly, members of some superfamilies within Diprotodontia (Burramyoidea, Petauroidea, Tarsipedoidea) had ECVs comparable to prosimians, whereas bandicoots, bilbies and many macropods were found to be poorly encephalized. When both encephalization quotient (EQ) and residuals from regression analysis were used to compare relative ECV of extinct/threatened species with common species there were no significant differences for any of the orders of Australian marsupials, suggesting that encephalization is not a major factor in the current extinction crisis for Australian marsupials. Similarly there were no consistent differences in relative ECV between marsupials from New Guinea and associated islands compared to Australia or between arid and non-arid Australian regions for any of the marsupial orders. The results indicate that marsupials are not uniformly poorly encephalized and that small marsupial carnivores and some members of Diprotodontia are of comparable encephalization to eutherians of similar body weight. In particular, honey possums and some gliders show an encephalization level comparable to prosimians, perhaps reflecting convergence in adaptation to similar arboreal niches., (Copyright 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.)

The moa (Dinornithiformes: Aves) are an extinct group of ratites from the North and South Islands of New Zealand. The ancestors of both the moa and the kiwi were isolated from other Gondwanan fauna as much as 80 million years ago and evolved in the absence of large mammalian predators. As such they represent a natural experiment in the removal of mammalian predation pressure on the encephalization of these two groups of ratites. We have used endocranial and skull morphometry in conjunction with high resolution CT scanning of the skulls of 8 species of moa to assess encephalization and brain morphology in moa and compare these features with extant ratites. Absolute brain size among the moa ranged from 17.0 ml for Euryapteryx curtus to 60.0 ml for female Dinornis giganteus. Values for encephalization quotients (EQ) of moa ranged from 0.205 for Euryapteryx gravis of the southern North Island to a mean (+/- SD) of 0.475 (+/- 0.026) for Anomalopteryx didiformis, partially overlapping values for extant non-New Zealand ratites (emu: 0.402 +/- 0.042; rhea: 0.496 +/- 0.016; ostrich: 0.474 +/- 0.084). Nevertheless, mean +/- SD EQ for all moa examined (0.379 +/- 0.065) was significantly lower than EQ for extant non-New Zealand ratites (0.539 +/- 0.141). Bending of the endocranial axis was much less among moa than either the kiwi or non-New Zealand ratites, consistent with the caudal position of the foramen magnum and the horizontal carriage of the head and upper neck during life. Endocranial morphology of the moa species examined was similar to that for non-New Zealand ratites, with proportionally similar sizes of the olfactory bulb, Wulst, vagal and maxillomandibular foramina, suggesting that the moa occupied similar diurnal niches with comparable sensory specializations to the emu, rhea and ostrich. No evidence of olfactory specialization (i.e., enlarged olfactory bulbs and increased surface area of the olfactory nasal cavity or cribriform plate) was evident in any of the moa skulls, in contrast to the remarkable nasal and olfactory bulb specializations evident in the skull and brain of the little spotted kiwi (Apteryx owenii). We cannot exclude that isolation in the absence of highly encephalized mammalian predators might have contributed to the lower EQ among moa, but it certainly did not lead to any significant reduction in EQ for kiwi; rather the kiwi embarked on a remarkable path of neurological specialization, which allowed them to exploit a niche usually occupied elsewhere by mammals., (Copyright 2007 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 have examined the organization of the pretectal area in two monotremes (the short beaked echidna-Tachyglossus aculeatus, and the platypus-Ornithorhynchus anatinus) and compared it to that in the Wistar strain rat, using Nissl staining in conjunction with enzyme histochemistry (acetylcholinesterase and NADPH diaphorase) and immunohistochemistry for parvalbumin, calbindin, calretinin and non-phosphorylated neurofilament protein (SMI-32 antibody). We were able to identify distinct anterior, medial, posterior (now called tectal gray) and olivary pretectal nuclei as well as a nucleus of the optic tract, all with largely similar topographical and chemoarchitectonic features to the homologous regions in therian mammals. The positions of these pretectal nuclei correspond to the distributions of retinofugal terminals identified by other authors. The overall size of the pretectum in both monotremes was found to be at least comparable in size, if not larger than, the pretectum of representative therian mammals of similar brain and body size. Our findings suggest that the pretectum of these two monotreme species is comparable in both size and organization to that of eutherian mammals, and is more than just an undifferentiated area pretectalis. The presence of a differentiated pretectum with similar chemoarchitecture to therians in both living monotremes lends support to the idea that the stem mammal for both prototherian and therian lineages also had a differentiated pretectum. This in turn indicates that a differentiated pretectum appeared at least 125 million years ago in the mammalian lineage and that the stem mammal for proto- and eutherian lineages probably had similar pretectal nuclei to those identified in its descendants.

The monotremes are a unique group of living mammals, which diverged from the line leading to placental mammals at least 125 million years ago. We have examined the organization of pontine, inferior olivary, lateral reticular and vestibular nuclei in the brainstem of the short-beaked echidna (Tachyglossus aculeatus) to determine if the cyto- and chemoarchitecture of these nuclei are similar to that in placental mammals and marsupials. We have used Nissl staining in conjunction with enzyme-histochemistry for acetylcholinesterase, cytochrome oxidase and NADPH diaphorase as well as immunohistochemistry for non-phosphorylated neurofilament protein (SMI-32 antibody) and calcium binding proteins (parvalbumin, calbindin, calretinin). Homologies could be established between the arch shaped inferior olivary complex of the echidna and the principal, dorsal and medial accessory subdivisions of the therian inferior olivary complex. The pontine nuclei of the echidna included basilar and reticulotegmental components with similar cyto- and chemarchitectural features to therians and there were magnocellular and subtrigeminal components of the lateral reticular nucleus, also as seen in therians. Subdivisions and chemoarchitecture of the vestibular complex of the echidna were both similar to that region in rodents. In all three precerebellar nuclear groups studied and in the vestibular nucleus organization, the cyto- and chemoarchitecture of the echidna was very similar to that seen in therian mammals and no "primitive" or "reptilian" features were evident.

The monotremes (echidnas and platypus) have been claimed by some authors to show 'avian' or 'reptilian' features in the gross morphology and microscopic anatomy of the cerebellum. We have used Nissl staining in conjunction with enzyme histochemistry to acetylcholinesterase and cytochrome oxidase and immunohistochemistry to non-phosphorylated neurofilament protein (SMI-32 antibody), calcium binding proteins (parvalbumin, calbindin and calretinin) and tyrosine hydroxylase to examine the cyto- and chemoarchitecture of the cerebellar cortex and deep cerebellar nuclei in the short-beaked echidna. Immunoreactivity for non-phosphorylated neurofilament (SMI-32 antibody) was found in the deep cerebellar nuclei and in Purkinje cells of most regions except the nodule. Purkinje cells identified with SMI-32 immunoreactivity were clearly mammalian in morphology. Parvalbumin and calbindin immunoreactivity was found in Purkinje cells with some regional variation in staining intensity and in Purkinje cell axons traversing cerebellar white matter or terminating on Lugaro cells. Calbindin immunoreactivity was also present in inferior olivary complex neurons. Calretinin immunoreactivity was found in pontocerebellar fibers and small cells in the deep granule cell layer of the ansiform lobule. We found that, although the deep cerebellar nuclei were much less clearly demarcated than in the rodent cerebellum, it was possible to distinguish medial, interposed and lateral nuclear components in the echidna. As far as we can determine from our techniques, the cerebellum of the echidna shows all the gross and cytological features familiar from the cerebellum of therian mammals., (Copyright 2007 S. Karger AG, Basel.)

The present study investigated the prenatal development of the cyto- and chemoarchitecture of the human nucleus of the solitary tract from 9 to 35 weeks, by using Nissl staining and immunoreactivity to calbindin, calretinin, tyrosine hydroxylase and GAP-43. The nucleus began to gain heterogeneity and show different subnuclei as early as 13 weeks, and approached cytoarchitectural maturation from 21 to 25 weeks. The subnuclear division pattern observed in the fetal nucleus of the solitary tract at 25 weeks was very similar to that of the adult. Neurons immunoreactive to calbindin first appeared in the medial gastrointestinal area of the nucleus at 13 weeks, particularly within a putative gelatinosus subnucleus, while calretinin immunoreactivity during fetal life suggested the possible presence of a central subnucleus. Tyrosine hydroxylase immunoreactive neurons were seen in the medial subdivisions of the nucleus of the solitary tract as early as 13 weeks, but the population continued to increase until 25 weeks. Strong GAP-43 immunoreactivity was also present in the nucleus of the solitary tract at 13 weeks, especially in the dorsolateral and commissural subnuclei, while at 21 weeks there was a significant decline of GAP-43 expression. Results from the chemoarchitectural study showed that the human nucleus of the solitary tract expressed various neurochemical substances at an early developmental age (13 weeks), even before cellular and neuropil maturation was fully attained. Expression of these factors may play an important role in establishment and integration of viscerosensory function in the nucleus.

We have examined the cyto- and chemoarchitecture of the trigeminal nuclei of two monotremes using Nissl staining, enzyme reactivity for cytochrome oxidase, immunoreactivity for calcium binding proteins and non-phosphorylated neurofilament (SMI-32 antibody) and lectin histochemistry (Griffonia simplicifolia isolectin B4). The principal trigeminal nucleus and the oralis and interpolaris spinal trigeminal nuclei were substantially larger in the platypus than in either the echidna or rat, but the caudalis subnucleus was similar in size in both monotremes and the rat. The numerical density of Nissl stained neurons was higher in the principal, oralis and interpolaris nuclei of the platypus relative to the echidna, but similar to that in the rat. Neuropil immunoreactivity for parvalbumin was particularly intense in the principal trigeminal, oralis and interpolaris subnuclei of the platypus, but the numerical density of parvalbumin immunoreactive neurons was not particularly high in these nuclei of the platypus. Neuropil immunoreactivity for calbindin and calretinin was relatively weak in both monotremes, although calretinin immunoreactive somata made up a large proportion of neurons in the principal, oralis and interpolaris subnuclei of the echidna. Distribution of calretinin immunoreactivity and Griffonia simplicifolia B4 isolectin reactivity suggested that the caudalis subnucleus of the echidna does not have a clearly defined gelatinosus region. Our findings indicate that the trigeminal nuclei of the echidna do not appear to be highly specialized, but that the principal, oralis and interpolaris subnuclei of the platypus trigeminal complex are highly differentiated, presumably for processing of tactile and electrosensory information from the bill.

We have used carbocyanine dye tracing from the brachium of the superior colliculus in conjunction with Nissl staining and immunohistochemistry to GAP-43 and calretinin to study the development of retinal projections to the superior colliculus in 17 human embryos and fetuses aged from 8 to 28 weeks. Lamination of the superior colliculus begins to emerge by 11 weeks, and by 16 weeks all seven layers of the mature superior colliculus are visible. Fibres immunoreactive to GAP-43 were seen at 13 weeks in the most superficial layers. By 19 weeks, GAP-43 immunoreactivity was present in the stratum opticum as well as the deeper fibres layers, indicating the development of fibre pathways following those laminae. Carbocyanine dye tracing of retinocollicular projections showed extensive rostrocaudally running unbranched fibres in the superficial superior colliculus at 12 weeks. Shortly after this (13 weeks), retinocollicular fibres penetrate the deeper collicular layers and branching becomes apparent. We also saw occasional retrogradely labelled somata following DiI insertion into the superior brachium. Our findings indicate that development of the human superior colliculus and its connections is largely complete by 20 weeks. This would suggest that functional capacity of the human superior colliculus should also be mature by the middle of gestation.

The monotremes are an intriguing group of mammals that have major differences in their reproductive physiology and lactation from therian mammals. Monotreme young hatch from leathery skinned eggs and are nourished by milk secreted onto areolae rather than through nipples. Parturition and lactation are in part controlled through the paraventricular and supraoptic nuclei of the hypothalamus. We have used Nissl staining, enzyme histochemistry, immunohistochemistry for tyrosine hydroxylase, calbindin, oxytocin, neurophysin and non-phosphorylated neurofilament protein, and carbocyanine dye tracing techniques to examine the supraoptic and paraventricular nuclei and the course of the hypothalamo-neurohypophysial tract in two monotremes: the short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus). In both monotremes, the supraoptic nucleus consisted of loosely packed neurons, mainly in the retrochiasmatic position. In the echidna, the paraventricular nucleus was quite small, but had similar chemoarchitectural features to therians. In the platypus, the paraventricular nucleus was larger and appeared to be part of a stream of magnocellular neurons extending from the paraventricular nucleus to the retrochiasmatic supraoptic nucleus. Immunohistochemistry for non-phosphorylated neurofilament protein and carbocyanine dye tracing suggested that hypothalamo-neurohypophysial tract neurons in the echidna lie mainly in the retrochiasmatic supraoptic and lateral hypothalamic regions, but most neurophysin and oxytocin immunoreactive neurons in the echidna were found in the paraventricular, lateral hypothalamus and supraoptic nuclei and most oxytocinergic neurons in the platypus were distributed in a band from the paraventricular nucleus to the retrochiasmatic supraoptic nucleus. The small size of the supraoptic nucleus in the two monotremes might reflect functional aspects of monotreme lactation., (Copyright (c) 2006 S. Karger AG, Basel.)

The cyto- and chemoarchitecture of the anterior olfactory nucleus and piriform cortex of the short-beaked echidna and platypus were studied to determine: (1) if these areas contain chemically distinct subdivisions, and (2) if the chemoarchitecture of those cortical olfactory regions differs from therians. Nissl and myelin staining were applied in conjunction with enzyme reactivity for NADPH diaphorase and acetylcholinesterase, and immunoreactivity for calcium-binding proteins (parvalbumin, calbindin and calretinin) and tyrosine hydroxylase. Golgi impregnations were also available for the echidna. In the echidna, the anterior olfactory nucleus is negligible in extent and merges at very rostral levels with a four-layered piriform cortex. Several rostrocaudally running subregions of the echidna piriform lobe could be identified on the basis of Nissl staining and calcium-binding protein immunoreactivity. Laminar-specific differences in calcium-binding protein immunoreactivity and NADPH-d-reactive neuron distribution were also noted. Neuron types identified in echidna piriform cortex included pyramidal neurons predominating in layers II and III and non-pyramidal neurons (e.g., multipolar profusely spiny and neurogliaform cells) in deeper layers. Horizontal cells were identified in both superficial and deep layers. By contrast, the platypus had a distinct anterior olfactory nucleus and a three-layered piriform cortex with no evidence of chemically distinct subregions within the piriform cortex. Volume of the paleocortex of the echidna was comparable to prosimians of similar body weight and, in absolute volume, exceeded that for eutherian insectivores such as T. ecaudatus and E. europaeus. The piriform cortex of the echidna shows evidence of regional differentiation, which in turn suggests highly specialized olfactory function., (Copyright (c) 2006 S. Karger AG, Basel.)

This study was undertaken to determine whether the olfactory tubercles of two monotremes (platypus and echidna) showed cyto- or chemoarchitectural differences from the tubercles of therian mammals. Nissl staining was applied in conjunction with enzyme reactivity for NADPH diaphorase and acetylcholinesterase, and immunoreactivity for calcium binding proteins (parvalbumin, calbindin and calretinin) and tyrosine hydroxylase (echidna only). Golgi impregnations of the tubercle were also available for the echidna. The olfactory tubercle is a poorly laminated structure in the echidna, despite the pronounced development of other components of the echidna olfactory system, and the dense cell layer of the olfactory tubercle was found to be discontinuous and irregular. Granule cell clusters (islands of Calleja) were present, but were small, poorly defined and did not show the intense NADPH diaphorase activity seen in marsupial and placental mammals. A putative small island of Calleja magna was seen in only one echidna out of four. In Golgi impregnations of the echidna olfactory tubercle, the most abundant neuron type was a medium-sized densely spined neuron similar to that seen in the olfactory tubercle of some therians. Large spine-poor neurons were also seen in the polymorphic layer. In the platypus, the olfactory tubercle was very small but showed more pronounced lamination than the echidna, although no granule cell clusters were seen. In both monotremes, the development of the olfactory tubercle was poor relative to other components of the olfactory system (bulb and piriform cortex). The small olfactory tubercle region in the platypus is consistent with poor olfaction in that aquatic mammal, but the tubercle in the echidna is more like that of a microsmatic mammal than other placentals occupying a similar niche (e.g., insectivores)., (Copyright 2006 S. Karger AG, Basel.)

The cyto- and chemoarchitecture of the olfactory bulb of two monotremes (shortbeaked echidna and platypus) was studied to determine if there are any chemoarchitectural differences from therian mammals. Nissl staining in conjunction with enzyme reactivity for NADPH diaphorase, and immunoreactivity for calcium binding proteins (parvalbumin, calbindin and calretinin), neuropeptide Y, tyrosine hydroxylase and non-phosphorylated neurofilament protein (SMI-32 antibody) were applied to the echidna. Material from platypus bulb was Nissl stained, immunoreacted for calretinin, or stained for NADPH diaphorase. In contrast to eutherians, no immunoreactivity for either the SMI-32 antibody or calretinin was found in the mitral or dispersed tufted cells of the monotremes and very few parvalbumin or calbindin immunoreactive neurons were found in the bulb of the echidna. On the other hand, immunoreactivity for tyrosine hydroxylase in the echidna was similar in distribution to that seen in therians, and periglomerular and granule cells showed similar patterns of calretinin immunoreactivity to eutherians. Multipolar neuropeptide Y immunoreactive neurons were confined to the deep granule cell layer and underlying white matter of the echidna bulb and NADPH diaphorase reactivity was found in occasional granule cells, fusiform and multipolar cells of the inner plexiform and granule cell layers, as well as underlying white matter. Unlike eutherians, no NPY immunoreactive or NADPH diaphorase reactive neurons were seen in the glomerular layer. The bulb of the echidna was comparable in volume to prosimians of similar body weight, and its constituent layers were highly folded. In conclusion, the monotreme olfactory bulb does not show any significant chemoarchitectural dissimilarities from eutheria, despite differences in mitral/tufted cell distribution., (Copyright 2006 S. Karger AG, Basel.)

We have examined the cyto- and chemoarchitecture of the dorsal thalamus of the short beaked echidna (Tachyglossus aculeatus), using Nissl and myelin staining, immunoreactivity for parvalbumin, calbindin, calretinin and non-phosphorylated neurofilament protein (SMI-32 antibody), and histochemistry for acetylcholinesterase and NADPH diaphorase. Immunohistochemical methods revealed many nuclear boundaries, which were difficult to discern with Nissl staining. Parvalbumin immunoreactive somata were concentrated in the ventral posterior, reticular, posterior, lateral and medial geniculate nuclei, while parvalbumin immunoreactivity of the neuropil was present throughout all but the midline nuclei. Large numbers of calbindin immunoreactive somata were also found within the midline thalamic nuclei, and thalamic sensory relay nuclei. Immunoreactivity for calretinin was found in many small somata within the lateral geniculate "a" nucleus, with other labelled somata found in the lateral geniculate "b" nucleus, ventral posterior medial and ventral posterior lateral nuclei. Immunoreactivity with the SMI-32 antibody was largely confined to somata and neuropil within the thalamocortical relay nuclei (ventral posterior medial and lateral nuclei, lateral and medial geniculate nuclei and the posterior thalamic nucleus). In broad terms there were many similarities between the thalamus of this monotreme and that of eutheria (e.g. disposition of somatosensory thalamus, complementarity of parvalbumin and calbindin immunoreactive structures), but there were some unique features of the thalamus of the echidna. These include the relatively small size of the thalamic reticular nucleus and the preponderance of calbindin immunoreactive neurons over parvalbumin immunoreactive neurons in the ventral posterior nucleus.

We have examined the cyto- and chemoarchitecture of the temporal and extended amygdala in the brain of a monotreme (the short-beaked echidna Tachyglossus aculeatus) using Nissl and myelin staining, enzyme histochemistry for acetylcholine esterase and NADPH diaphorase, immunohistochemistry for calcium binding proteins (parvalbumin, calbindin and calretinin) and tyrosine hydroxylase. While the broad subdivisions of the eutherian temporal amygdala were present in the echidna brain, there were some noticeable differences. No immunoreactivity for parvalbumin or calretinin for somata was found in the temporal amygdala of the echidna. The nucleus of the lateral olfactory tract could not be definitively identified and the medial nucleus of amygdala appeared to be very small in the echidna. Calbindin immunoreactive neurons were most frequently found in the ventrolateral part of the lateral nucleus, intraamygdaloid parts of the bed nucleus of the stria terminalis and the lateral part of the central nucleus. Neurons strongly reactive for NADPH diaphorase with filling of the dendritic tree were found mainly scattered through the cortical, central and lateral subnuclei, while neurons showing only somata reactivity for NADPH diaphorase were concentrated in the basomedial and basolateral subnuclei. Most of the components of the extended amygdala of eutherians could also be identified in the echidna. Volumetric analysis indicated that the temporal amygdala in both the platypus and echidna is small compared to the same structure in both insectivores and primates, with the central and medial components of the temporal amygdala being particularly small.

We have examined the distribution and morphology of neurons immunoreactive for nonphosphorylated neurofilament protein (SMI-32 antibody), calcium-binding proteins (parvalbumin, calbindin, calretinin), and neuropeptide Y as well as neurons reactive for NADPH diaphorase in the cerebral cortex of the Australian short-beaked echidna (Tachyglossus aculeatus). We have also studied synaptic morphology and density in S1 somatosensory cortex and assessed parameters associated with metabolic activity of the cerebral cortex (vessel volume density, mitochondrial volume density, and mitochondrial numerical density) in semi- and ultrathin sections. SMI-32 immunoreactivity was found mostly in layer V pyramidal neurons in selected cortical regions (S1, PV, V1, A). These neurons often showed atypical morphology compared with therian cortex. Neurons immunoreactive for calcium-binding proteins were broadly similar in both morphology and distribution to those seen in therian cortex, although calretinin-immunoreactive neurons were rare. Both Gray type I and Gray type II synapses could be identified in echidna S1 cortex and were similar to those seen in therian cortex. Peak synaptic density was in upper layer IV, followed by layer I, lower layer II, and upper layer III. Most synapses were of type I (72%), although types I and II were encountered with similar frequency in lower layer II and upper layer III. The capillary volume fraction values obtained for the echidna (from 1.18% in V1 to 1.34% in S1 cortex) fall within the values for rodent cortex. Similarly, values for mitochondrial volume fraction in echidna somatosensory cortex (4.68% +/- 1.76%) were comparable to those in eutherian cortex., (2004 Wiley-Liss, Inc.)

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)

We have studied the distribution of the CD15 epitope in the developing brain of an Australian diprotodontid metatherian mammal, the tammar wallaby ( Macropus eugenii), using immunohistochemistry in conjunction with hematoxylin and eosin staining. At the time of birth (28 days after conception), CD15 immunoreactivity labeled somata in the primordial plexiform layer of the parietal cortex in a similar position to that seen in the early fetal eutherian brain. CD15 immunoreactivity in the brain of the developing pouch-young wallaby was found to be localized on the surface of radial glia at boundaries between developmentally significant forebrain compartments in a similar distribution to that seen in developing eutherian brain. These were best seen in the developing diencephalon, delineating epithalamus, ventral and dorsal thalamus and hypothalamic anlage, and in the striatum. Immunoreactivity for CD15 identified radial glia marking the lateral migratory stream at the striatopallial boundary, peaking in intensity at P19 to P25. From P37 to P54, CD15 immunoreactivity also demarcated patch compartments in the developing striatum. In contrast, CD15 immunoreactivity in hindbrain structures showed some differences from the temporospatial pattern seen in eutherian brain. These may reflect the relatively early brainstem maturation required for the newborn wallaby to be able to traverse the distance from the maternal genital tract to the pouch. The wallaby provides a convenient model for testing hypotheses concerning the role of CD15 in forebrain development because all events in which CD15 may play a critical role in forebrain morphogenesis occur during pouch life, when the young wallaby is accessible to experimental manipulation.

We have examined the topography of the cerebral cortex of the Australian echidna (Tachyglossus aculeatus), using Nissl and myelin staining, immunoreactivity for parvalbumin, calbindin, and nonphosphorylated neurofilament protein (SMI-32 antibody), and histochemistry for acetylcholinesterase (AChE) and NADPH diaphorase. Myelinated fibers terminating in layer IV of the cortex were abundant in the primary sensory cortical areas (areas S1, R, and PV of somatosensory cortex; primary visual cortex) as well as the frontal cortex. Parvalbumin immunoreactivity was particularly intense in the neuropil and somata of somatosensory regions (S1, R, and PV areas) but was poor in motor cortex. Immunoreactivity with the SMI-32 antibody was largely confined to a single sublayer of layer V pyramidal neurons in discrete subregions of the somatosensory, visual, and auditory cortices, as well as a large field in the frontal cortex (Fr1). Surprisingly, SMI-32 neurons were absent from the motor cortex. In AChE preparations, S1, R, V1, and A regions displayed intense reactivity in supragranular layers. Our findings indicate that there is substantial regional differentiation in the expanded frontal cortex of this monotreme. Although we agree with many of the boundaries identified by previous authors in this unusual mammal (Abbie [1940] J. Comp. Neurol. 72:429-467), we present an updated nomenclature for cortical areas that more accurately reflects findings from functional and chemoarchitectural studies., (Copyright 2004 Wiley-Liss, Inc.)

The embryonic and fetal development of the nuclear components and pathways of vagal sensorimotor circuits in the human has been studied using Nissl staining and carbocyanine dye tracing techniques. Eight fetal brains ranging from 8 to 28 weeks of development had DiI (1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate) inserted into either the thoracic vagus nerve at the level of the sternal angle (two specimens of 8 and 9 weeks of gestation) or into vagal rootlets at the surface of the medulla (at all other ages), while a further five were used for study of cytoarchitectural development. The first central labeling resulting from peripheral application of DiI to the thoracic vagus nerve was seen at 8 weeks. By 9 weeks, labeled bipolar cells at the ventricular surface around the sulcus limitans (sl) were seen after DiI application to the thoracic vagus nerve. Subnuclear organization as revealed by both Nissl staining and carbocyanine dye tracing was found to be advanced at a relatively early fetal age, with afferent segregation in the medial Sol apparent at 13 weeks and subnuclear organization of efferent magnocellular divisions of dorsal motor nucleus of vagus nerve noticeable at the same stage. The results of the present study also confirm that vagal afferents are distributed to the dorsomedial subnuclei of the human nucleus of the solitary tract, with particular concentrations of afferent axons in the gelatinosus subnucleus. These vagal afferents appeared to have a restricted zone of termination from quite early in development (13 weeks) suggesting that there is no initial exuberance in the termination field of vagal afferents in the developing human nucleus of the solitary tract. On the other hand, the first suggestion of afferents invading 10N from the medial Sol was not seen until 20 weeks and was not well developed until 24 weeks, suggesting that direct monosynaptic connections between the sensory and effector components of the vagal sensorimotor complex do not develop until this age.

This study used acetylcholinesterase (AChE) histochemistry to reveal the organization of the dorsomedial hypothalamic nucleus (DM) in the human. Topographically, the human DM is similar to DM in the monkey and rat. It is wedged between the paraventricular nucleus, dorsally, and the ventromedial nucleus, ventrally. Laterally, DM borders the lateral hypothalamic area while medially it approaches the 3rd ventricle. The AChE staining distinguished two subcompartments of the human DM: the larger diffuse and the smaller compact DM. The subcompartmental organization of the human DM appears homologous to that found in the monkey and less complex than that reported in rats. Understanding of the organization of DM creates meaningful anatomical reference for physiological and pharmacological studies in the human hypothalamus.

Many authors have reported that the claustrum, which comprises the insular claustrum and the endopiriform nucleus, is missing from the monotreme forebrain. We used Nissl and myelin staining in conjunction with enzyme histochemistry for acetylcholinesterase and immunohistochemistry for parvalbumin, calbindin, calretinin and tyrosine hydroxylase to examine the brains of two monotremes, the short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus). We found that although the insular claustrum is a small structure in the echidna brain, it is nevertheless clearly present as loosely clustered neurons embedded in the white matter ventrolateral to the putamen and deep to the piriform and entorhinal cortices. Neurons in this region share the chemical features of the adjacent cortex (presence of a similar proportion of parvalbumin immunoreactive neurons and minimal activity for acetylcholinesterase and tyrosine hydroxylase), unlike the adjacent putamen and ventral pallidum. A putative endopiriform nucleus can be identified in the interior of the piriform lobe of the echidna as calretinin immunoreactive neurons embedded within the white matter. The situation is much less clear in the platypus, but our data suggest that there may be an insular claustrum deep to frontal cortex, separated from layer VI by only a thin layer of white matter. We could not identify an endopiriform nucleus in our platypus material. Our findings indicate that presence of the claustrum cannot be considered a feature confined to therian mammals and lend weight to arguments that this structure was present in the ancestral mammalian brain.

The cerebral cortex of the echidna is notable for its extensive folding and the positioning of major functional areas towards its caudal extremity. The gyrification of the echidna cortex is comparable in magnitude to prosimians and cortical thickness and neuronal density are similar to that seen in rodents and carnivores. On the other hand, many pyramidal neurons in the cerebral cortex of the echidna are atypical with inverted somata and short or branching apical dendrites. All other broad classes of neurons noted in therian cortex are also present in the echidna, suggesting that the major classes of cortical neurons evolved prior to the divergence of proto- and eutherian lineages. Dendritic spine density on dendrites of echidna pyramidal neurons in somatosensory cortex and apical dendrites of motor cortex pyramidal neurons is lower than that found in eutheria. On the other hand, synaptic morphology, density and distribution in somatosensory cortex are similar to that in eutheria. In summary, although the echidna cerebral cortex displays some structural features, which may limit its functional capacities (e.g. lower spine density on pyramidal neurons), in most structural parameters (e.g. gyrification, cortical area and thickness, neuronal density and types, synaptic morphology and density), it is comparable to eutheria.

Monotremes, perhaps more than any other order of mammals, display an enormous behavioural reliance upon the tactile senses. In the platypus, Ornithorhynchus anatinus, this is manifest most strikingly in the special importance of the bill as a peripheral sensory organ, an importance confirmed by electrophysiological mapping that reveals a vast area of the cerebral cortex allocated to the processing of tactile inputs from the bill. Although behavioural evidence in the echidna, Tachyglossus aculeatus, suggests a similar prominence for tactile inputs from the snout, there is also a great reliance upon the distal limbs for digging and burrowing activity, pointing to the importance of tactile information from these regions for the echidna. In recent studies, we have investigated the peripheral tactile neural mechanisms in the forepaw of the echidna to establish the extent of correspondence or divergence that has emerged over the widely different evolutionary paths taken by monotreme and placental mammals. Electrophysiological recordings were made from single tactile sensory nerve fibres isolated in fine strands of the median or ulnar nerves of the forearm. Controlled tactile stimuli applied to the forepaw glabrous skin permitted an initial classification of tactile sensory fibres into two broad divisions, according to their responses to static skin displacement. One displayed slowly adapting (SA) response properties, while the other showed a selective sensitivity to the dynamic components of the skin displacement. These purely dynamically-sensitive tactile fibres could be subdivided according to vibrotactile sensitivity and receptive field characteristics into a rapidly adapting (RA) class, sensitive to low frequency (