Distribution of CART (cocaine- and amphetamine-regulated transcript) peptide in mature and developing marsupial brain.

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.

Cortical cyto- and chemoarchitecture in three small Australian marsupial carnivores: Sminthopsis macroura, Antechinus stuartii and Phascogale calura.

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.

Topography and chemoarchitecture of the striatum and pallidum in a monotreme, the short-beaked echidna (Tachyglossus aculeatus).

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.

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

We have studied the early development of the vestibular apparatus and its central connections in the tammar wallaby (Macropus eugenii) in order to determine whether the vestibular system anatomy is sufficiently mature at birth to assist in climbing to the pouch. Structural development was studied with the aid of hematoxylin and eosin stained sections and immunoreactivity for GAP-43, whereas the development of vestibular system connections was examined by carbocyanine dye tracing. At the time of birth, the otocyst has distinct utricle, saccule and semicircular canals with immature sensory regions receiving innervation by GAP-43 immunoreactive fibers. Vestibular nerve fibers can be traced into the brainstem to the developing vestibular nuclei, which are not yet cytoarchitectonically distinct. The vestibular nuclei do not contribute direct projections to the lower cervical spinal cord at birth; most bulbospinal projections in the newborn appear to be derived bilaterally from the gigantocellular, lateral paragigantocellular reticular and ventral medullary nuclei. A substantial bilateral projection to the vestibular ganglion and apparatus from the region of the gigantocellular and lateral paragigantocellular nuclei was seen at birth, but not in subsequent ages. This is similar to a projection seen in newborn Ameridelphians. By postnatal day (P) 5, the vestibular apparatus had extensive projections to all vestibular nuclei and neurons projecting in the lateral vestibulospinal tract could be identified in the lateral vestibular nucleus. Cytoarchitectonic differentiation of the vestibular nuclei proceeded over the next 3 to 4 weeks with the emergence of discrete parvicellular and magnocellular components of the medial vestibular nucleus by P19. GAP-43 immunoreactivity stayed high in the lateral vestibulospinal tract for several months after birth, suggesting that the development of this tract followed a prolonged timecourse. Our findings indicate that central and peripheral connections of the vestibular ganglion are present at birth, but that there is no direct projection from the vestibular nuclei to the cervical spinal cord until P5. Nevertheless, the possibility remains that an indirect projection between the vestibular nuclei and the medial reticular formation is present at birth and mediates control of the climb.

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

We used carbocyanine dye tracing techniques in conjunction with hematoxylin and eosin staining, immunohistochemistry for GAP-43, and tritiated thymidine autoradiography to examine the development of the olfactory pathways in early pouch young tammar wallabies (Macropus eugenii). The overarching aim was to test the hypothesis that the olfactory system of newborn tammars is sufficiently mature at birth to contribute to the guidance of the pouch young to the nipple. Although GAP-43 immunoreactive fibers emerge from the olfactory epithelium and enter the olfactory bulb at birth, all other components of the olfactory pathway in newborn tammars are very immature at birth, postnatal day (P0). In particular, maturation of the vomeronasal organ and its projections to the accessory olfactory bulb appears to be delayed until P5 and the olfactory bulb is poorly differentiated until P12, with glomerular formation delayed until P25. The lateral olfactory tract is also very immature at birth with pioneer axons having penetrated only the most rostral portion of the piriform lobe. Interestingly, there were some early (P0) projections from the olfactory epithelium to the medial septal region and lamina terminalis (by the terminal nerve) and to olfactory tubercle and basal forebrain. The former of these is presumably serving the transfer of LHRH(+) neurons to the forebrain, as seen in eutherians, but neither of these very early pathways is sufficiently robust or connected to the more caudal neuraxis to play a role in nipple finding. Tritiated thymidine autoradiography confirmed that most piriform cortex pyramidal neurons are generated in the first week of life and are unlikely to be able to contribute to circuitry guiding the climb to the pouch. Our findings lead us to reject the hypothesis that olfactory projections contribute to guidance of the newborn tammar to the pouch and nipple. It appears far more likely that the trigeminal pathways play a significant role in this behavior because the central projections of the trigeminal nerve are more mature at birth in this marsupial.

Encephalization of Australian and New Guinean marsupials.

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.

Big birds and their brains: paleoneurology of the New Zealand moa.

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.

Development of the human dorsal nucleus of the vagus.

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.

The pretectal nuclei in two monotremes: the short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus).

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.

Precerebellar and vestibular nuclei of the short-beaked echidna (Tachyglossus aculeatus).

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.

Cyto- and chemoarchitecture of the cerebellum of the short-beaked echidna (Tachyglossus aculeatus).

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.

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

We have examined the cyto- and chemoarchitecture of the isocortex of a diprotodontid marsupial, the tammar wallaby (Macropus eugenii), using Nissl staining in combination with enzyme histochemical (acetylcholinesterase - AChE, NADPH-diaphorase - NADPHd, cytochrome oxidase) and immunohistochemical (non-phosphorylated neurofilament - SMI-32) markers. The primary sensory cortex showed distinctive patterns of reactivity in cytochrome oxidase, acetylcholinesterase and NADPH diaphorase. For example, in AChE material, S1 showed a heterogeneous appearance, with regions exhibiting a double layer of AChE activity (layers II and IV) adjacent to poorly reactive regions. In NADPHd preparations, activity in S1 was strongest in layers I to IV although, as in AChE material, there were consistent patches of reduced NADPHd activity which corresponded to poorly reactive regions in the AChE sections. Each of the primary sensory areas of the isocortex showed a different pattern of distribution of SMI-32+ neurons. In V1, SMI-32+ neurons were distributed in two layers (III and V) throughout the tangential extent of that region. In S1, SMI-32+ neurons were concentrated in layer V, but large and discrete patches within S1 had additional SMI-32+ neurons in layer III. In primary auditory cortex there was a dense band of SMI-32+ neurons in layer V, with only occasional labeled pyramidal neurons in layer III. In the secondary sensory areas (V2 and S2) SMI-32+ neurons were either distributed in layers III and V (V2) or solely within layer V (S2). The tangential and laminar distribution of Type I reactive NADPH diaphorase neurons in the tammar wallaby cortex was more like that seen in eutheria than in polyprotodontid metatheria.

CD15 immunoreactivity in the developing brain of a marsupial, the tammar wallaby ( Macropus eugenii).

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.

The anatomy of the cerebral cortex of the echidna (Tachyglossus aculeatus).

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.

Tactile neural mechanisms in monotremes.

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 (

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

We have studied the organization of the hypothalamus in an Australian diprotodontid metatherian mammal, the wallaby ( Macropus eugenii), using cytoarchitectural, histochemical and immunohistochemical techniques. Coronal sections of adult brains were processed for Nissl staining, histochemical reactivity (cytochrome oxidase, nicotinamide adenine dinucleotide phosphate diaphorase and acetylcholinesterase) and immunohistochemistry (antibodies to tyrosine hydroxylase, calbindin, calretinin, non-phosphorylated neurofilament protein, oxytocin and vasopressin). The distribution of immunoreactive neurons for these substances was mapped with the aid of a computer-linked microscope. In general, the wallaby hypothalamus showed a similar nuclear organization to that seen in rodents. The paraventricular nucleus could be divided into several subdivisions based on the different cellular parcellation, similar to that described in rodents. The ventromedial hypothalamic nucleus had cell-sparse dorsomedial and cell-dense ventrolateral subdivisions as seen in eutheria, suggesting a similar functional compartmentalization in all theria. The positions of tyrosine hydroxylase-positive neurons in the wallaby hypothalamus were also similar to those in eutheria. Oxytocin and vasopressinergic neurons were found in all the same major nuclear groups as seen in eutheria, although a nucleus circularis could not be identified. The general similarities between wallaby and eutherian hypothalamus indicate that the basic chemo- and cytoarchitectural features of the hypothalamus are common to eutheria and metatheria and validate the use of the wallaby as a mammalian model of wide applicability in investigations of hypothalamic functional development.

Neuronal classes in the isocortex of a monotreme, the Australian echidna (Tachyglossus aculeatus).

We have used Valverde-Golgi and Golgi-Colonnier techniques to analyze cortical neuronal morphology in four regions (frontal cortex, primary motor cortex, primary somatosensory cortex, primary visual cortex) of the isocortex of the echidna (Tachyglossus aculeatus). Eight classes of neurons could be identified--pyramidal, spinous bipolar, aspinous bipolar, spinous bitufted, aspinous bitufted, spinous multipolar, aspinous multipolar and neurogliaform. All except the pyramidal neurons were morphologically similar to neuronal classes seen in eutherian and metatherian isocortex. Pyramidal neurons made up a small proportion of all cortical neurons encountered in our preparations of echidna cortex (34% in visual cortex, 35% in somatosensory cortex, 41% in frontal cortex and 49% in motor cortex) compared to both reported values in eutherian cortex and values we found in rat cortex impregnations prepared in an identical fashion to the echidna material (75% in rat motor and 78% in rat somatosensory cortex). Many pyramidal neurons in the echidna isocortex were atypical (30-42% depending on region) with inverted somata, short or branching apical dendrites and/or few basal dendrites, very different from the usual pyramidal neuron morphology in eutherian cortex. Dendritic spine density on apical and basal dendrites of echidna pyramidal neurons in somatosensory cortex and apical dendrites of motor cortex pyramidal neurons was also lower than that found in the rat. The present findings are consistent with both pyramidal neurons and the many diverse types of non-pyramidal neurons having already emerged as discrete morphological entities very early in mammalian cortical evolution, at the time of divergence of the therian and prototherian lineage.

Dynamics of expression of apoptosis-regulatory proteins Bid, Bcl-2, Bcl-X, Bax and Bak during development of murine nervous system.

We have used immunohistochemistry and immunoblotting to examine the expression of Bid and four other Bcl-2 family proteins (Bcl-2, Bcl-X, Bax and Bak) in the developing and adult murine central nervous system (CNS). Bid protein is widespread in embryonic and postnatal brain, and its expression is maintained at a high level late into the adulthood. Bid is expressed both in the germ disc, early neural tube, proliferating stem cells of ventricular zones, and in postmitotic, differentiated neurons of the developing central and peripheral nervous system. As the differentiation proceeds, the neurons express higher levels of Bid than the stem cells of the paraventricular zone. Both in embryonic and postnatal life, Bid protein is present in the most vital regions of brain, such as the limbic system, basal ganglia, mesencephalic tectum, Purkinje cells in cerebellum, and the ventral columns of spinal cord. The p15 cleaved form of Bid was detectable in the brain specimens at fetal stages of development, consistent with caspase-mediated activation of this pro-apoptotic Bcl-2 family protein. Among the Bcl-2 family proteins only Bid and Bcl-XL continue to be expressed at high levels in the adult brain.

Identification of dendritic cells in aortic atherosclerotic lesions in rats with diet-induced hypercholesterolaemia.

We have previously identified dendritic cells (DCs) in the intima of human large arteries. These vascular DCs are common in atherosclerotic lesions but their immature forms are also present in normal arterial intima. Pathophysiological studies on vascular DCs are limited because they have only been studied in human specimens obtained at operation or post-mortem. The aim of the current study was to determine whether DCs participate in the development of atherosclerotic lesions in hypercholesterolemic rats. Male Wistar rats were divided into a control (n=13) and experimental cohort (n=48). The experimental animals were fed an atherogenic diet and 1% saline, while the controls were fed standard rat cubes and water. The aortas were obtained from both groups at 10, 20, and 30 weeks following commencement of the diet. An en face immunohistochemical technique, routine section immunohistochemistry, and transmission electron microscopy were used to detect the presence of DCs in the aortas. Examination of the aortas showed that S100+ cells with dendritic cell morphology were present in the aortic intima of hypercholesterolemic rats. The S100+ DCs displayed immunopositivity for OX-62 and MHC Class II antibodies. Within various types of atherosclerotic lesions, these cells were clustered throughout the intima but were especially prominent around arterial branch-points where they co-localized with various cell types, including T-cells and macrophages. Ultrastructural analysis confirmed the presence of cells with characteristics typical of DCs. These features included the presence of a well-developed tubulovesicular system, dendritic processes, and a lack of secondary lysosomes and phagosomes. This study establishes the presence of DCs in the aortic intima of rats with diet-induced atherosclerosis. The presence of DCs in this model of experimental atherogenesis could provide a new approach to investigating the function of DCs and may help clarify the immune-inflammatory mechanisms underlying atherosclerosis.

Fluororuby as a marker for detection of acute axonal injury in rat spinal cord.

Axonal damage is a common pathological consequence of spinal cord injury. Previous studies have detected axonal injury with silver stains for degeneration or immunohistochemistry for alterations in components such as beta-amyloid precursor protein, neurofilament or ubiquitin. Fluororuby has recently been introduced as a neuronal tracer in studies of spinal cord injury and regeneration. Our study was carried out to determine whether Fluororuby can be used to identify injured axons and monitor the time course of axonal damage. Adult rats underwent needle puncture injury to the white matter in the midline and lateral spinal cord at T11. At the same time, 0.05 microl of Fluororuby was injected into the cord at the same sites. After survival times ranging from 6 h to 3 weeks, spinal cords were cut into longitudinal frozen sections and examined with confocal microscopy. Fluororuby was found to label key features of axonal injury including axonal swelling, retraction balls and disrupted axons. Damaged axons close to the injury site were consistently labeled within 6 h, with indications of swollen and disconnected axons spreading further from the site during the first week. Fewer injured axons were labeled after 1 week survival, but the marker revealed longer distances of degenerating axons both distal and rostral to the injury site. Our findings indicate that Fluororuby is a quick, sensitive, reliable and technically simple fluorescent marker for early stages of acute axonal injury and degeneration.