Anatomy and Neuroscience - Research Publications
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ItemPurinergic mechanisms in the control of gastrointestinal motility(SPRINGER, 2008-09)For many years, ATP and adenosine have been implicated in movement regulation of the gastrointestinal tract. They act through three major receptor subtypes: adenosine or P1 receptors, P2X receptors and P2Y receptors. Each of these major receptor types can be subdivided into several different classes and is widely distributed amongst various neurons, muscle types, glia and interstitial cells that regulate intestinal functions. Several key roles for the different receptors and their endogenous ligands have been identified in physiological and pharmacological studies. For example, adenosine acting at A(1) receptors appears to inhibit intestinal motility in various pathological conditions. Similarly, ATP acting at P2Y receptors is an important component of inhibitory neuromuscular transmission, acting as a cotransmitter with nitric oxide. ATP acting at P2X and P2Y(1) receptors is important for synaptic transmission in simple descending excitatory and inhibitory reflex pathways. Some P2Y receptor subtypes prefer uridine nucleotides over purine nucleotides. Thus, roles for UTP and UDP as enteric transmitters in place of ATP cannot be excluded. ATP also appears to be important for sensory transduction, especially in chemosensitive pathways that initiate local inhibitory reflexes. Despite this evidence, data are lacking about the roles of either adenosine or ATP in more complex motility patterns such as segmentation or the interdigestive migrating motor complex. Clarification of roles for purinergic transmission in these common, but understudied, motility patterns will depend on the use of subtype-specific antagonists that in some cases have not yet been developed.
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ItemNo Preview AvailableThe lymphatic anatomy of the breast and its implications for sentinel lymph node biopsy: A Human Cadaver Study(SPRINGER, 2008-03)BACKGROUND: Current understanding of the lymphatic system of the breast is derived mainly from the work of the anatomist Sappey in the 1850s, with many observations made during the development and introduction of breast lymphatic mapping and sentinel node biopsy contributing to our knowledge. METHODS: Twenty four breasts in 14 fresh human cadavers (5 male, 9 female) were studied. Lymph vessels were identified with hydrogen peroxide and injected with a lead oxide mixture and radiographed. The specimens were cross sectioned and radiographed to provide three dimensional images. Lymph (collecting) vessels were traced from the periphery to the first-tier lymph node. RESULTS: Lymph collecting vessels were found evenly spaced at the periphery of the anterior upper torso draining radially into the axillary lymph nodes. As they reached the breast some passed over and some through the breast parenchyma, as revealed in the cross-section studies. The pathways showed no significant difference between male and female specimens. We found also perforating lymph vessels that coursed beside the branches of the internal mammary vessels, draining into the ipsilateral internal mammary lymphatics. In some studies one sentinel node in the axilla drained almost the entire breast. In most more than one sentinel node was represented. CONCLUSION: These anatomical findings are discordant with our current knowledge based on previous studies and demand closer examination by clinicians. These anatomical studies may help explain the percentage of false-negative sentinel node biopsy studies and suggest the peritumoral injection site for accurate sentinel lymph node detection.
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ItemIdentification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors(BMC, 2008-06-19)BACKGROUND: Branchiomotor neurons comprise an important class of cranial motor neurons that innervate the branchial-arch-derived muscles of the face, jaw and neck. They arise in the ventralmost progenitor domain of the rhombencephalon characterized by expression of the homeodomain transcription factors Nkx2.2 and Phox2b. Phox2b in particular plays a key role in the specification of branchiomotor neurons. In its absence, generic neuronal differentiation is defective in the progenitor domain and no branchiomotor neurons are produced. Conversely, ectopic expression of Phox2b in spinal regions of the neural tube promotes cell cycle exit and neuronal differentiation and, at the same time, induces genes and an axonal phenotype characteristic for branchiomotor neurons. How Phox2b exerts its pleiotropic functions, both as a proneural gene and a neuronal subtype determinant, has remained unknown. RESULTS: To gain further insights into the genetic program downstream of Phox2b, we searched for novel Phox2b-regulated genes by cDNA microarray analysis of facial branchiomotor neuron precursors from heterozygous and homozygous Phox2b mutant embryos. We selected for functional studies the genes encoding the axonal growth promoter Gap43, the Wnt antagonist Sfrp1 and the transcriptional regulator Sox13, which were not previously suspected to play roles downstream of Phox2b and whose expression was affected by Phox2b misexpression in the spinal cord. While Gap43 did not produce an obvious phenotype when overexpressed in the neural tube, Sfrp1 induced the interneuron marker Lhx1,5 and Sox13 inhibited neuronal differentiation. We then tested whether Sfrp1 and Sox13, which are down-regulated by Phox2b in the facial neuron precursors, would antagonize some aspects of Phox2b activity. Co-expression of Sfrp1 prevented Phox2b from repressing Lhx1,5 and alleviated the commissural axonal phenotype. When expressed together with Sox13, Phox2b was still able to promote cell cycle exit and neuronal differentiation, but the cells failed to relocate to the mantle layer and to extinguish the neural stem cell marker Sox2. CONCLUSION: Our results suggest novel roles for Sfrp1 and Sox13 in neuronal subtype specification and generic neuronal differentiation, respectively, and indicate that down-regulation of Sfrp1 and Sox13 are essential aspects of the genetic program controlled by Phox2b in cranial motoneurons.
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ItemEffects of NGF, NT-3 and GDNF family members on neurite outgrowth and migration from pelvic ganglia from embryonic and newborn mice(BMC, 2008-07-25)BACKGROUND: Pelvic ganglia are derived from the sacral neural crest and contain both sympathetic and parasympathetic neurons. Various members of the neurotrophin and GDNF families of neurotrophic factors have been shown to play important roles in the development of a variety of peripheral sympathetic and parasympathetic neurons; however, to date, the role of these factors in the development of pelvic ganglia has been limited to postnatal and older ages. We examined the effects of NGF, NT-3, GDNF, neurturin and artemin on cell migration and neurite outgrowth from explants of the pelvic ganglia from embryonic and newborn mice grown on collagen gels, and correlated the responses with the immunohistochemical localization of the relevant receptors in fixed tissue. RESULTS: Cell migration assays showed that GDNF strongly stimulated migration of tyrosine hydroxylase (TH) cells of pelvic ganglia from E11.5, E14.5 and P0 mice. Other factors also promoted TH cell migration, although to a lesser extent and only at discrete developmental stages. The cells and neurites of the pelvic ganglia were responsive to each of the GDNF family ligands--GDNF, neurturin and artemin--from E11.5 onwards. In contrast, NGF and NT-3 did not elicit a significant neurite outgrowth effect until E14.5 onwards. Artemin and NGF promoted significant outgrowth of sympathetic (TH+) neurites only, whereas neurturin affected primarily parasympathetic (TH-negative) neurite outgrowth, and GDNF and NT-3 enhanced both sympathetic and parasympathetic neurite outgrowth. In comparison, collagen gel assays using gut explants from E11.5 and E14.5 mice showed neurite outgrowth only in response to GDNF at E11.5 and to neurturin only in E14.5 mice. CONCLUSION: Our data show that there are both age-dependent and neuron type-dependent differences in the responsiveness of embryonic and neo-natal pelvic ganglion neurons to growth factors.
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ItemThe Drosophila STIM1 orthologue, dSTIM, has roles in cell fate specification and tissue patterning(BMC, 2008-10-24)BACKGROUND: Mammalian STIM1 and STIM2 and the single Drosophila homologue dSTIM have been identified as key regulators of store-operated Ca2+ entry in cells. STIM proteins function both as molecular sensors of Ca2+concentration in the endoplasmic reticulum (ER) and the molecular triggers that activate SOC channels in the plasma membrane. Ca2+ is a crucial intracellular messenger utilised in many cellular processes, and regulators of Ca2+ homeostasis in the ER and cytosol are likely to play important roles in developmental processes. STIM protein expression is altered in several tumour types but the role of these proteins in developmental signalling pathways has not been thoroughly examined. RESULTS: We have investigated the expression and developmental function of dSTIM in Drosophila and shown that dSTIM is widely expressed in embryonic and larval tissues. Using the UAS-Gal4 induction system, we have expressed full-length dSTIM protein and a dsRNAi construct in different tissues. We demonstrate an essential role for dSTIM in larval development and survival, and a tissue-specific role in specification of mechanosensory bristles in the notum and specification of wing vein thickness. CONCLUSION: Our studies show that dSTIM regulates growth and patterning of imaginal discs and indicate potential interactions with the Notch and Wingless signaling pathways. These interactions may be relevant to studies implicating STIM family proteins in tumorigenesis.
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ItemSub region-specific modulation of synchronous neuronal burst firing after a kainic acid insult in organotypic hippocampal cultures(BMC, 2008-07-02)BACKGROUND: Excitotoxicity occurs in a number of pathogenic states including stroke and epilepsy. The adaptations of neuronal circuits in response to such insults may be expected to play an underlying role in pathogenesis. Synchronous neuronal firing can be induced in isolated hippocampal slices and involves all regions of this structure, thereby providing a measure of circuit activity. The effect of an excitotoxic insult (kainic acid, KA) on Mg2+-free-induced synchronized neuronal firing was tested in organotypic hippocampal culture by measuring extracellular field activity in CA1 and CA3. RESULTS: Within 24 hrs of the insult regional specific changes in neuronal firing patterns were evident as: (i) a dramatic reduction in the ability of CA3 to generate firing; and (ii) a contrasting increase in the frequency and duration of synchronized neuronal firing events in CA1. Two distinct processes underlie the increased propensity of CA1 to generate synchronized burst firing; a lack of ability of the CA3 region to 'pace' CA1 resulting in an increased frequency of synchronized events; and a change in the 'intrinsic' properties limited to the CA1 region, which is responsible for increased event duration. Neuronal quantification using NeuN immunoflurescent staining and stereological confocal microscopy revealed no significant cell loss in hippocampal sub regions, suggesting that changes in the properties of neurons within this region were responsible for the KA-mediated excitability changes. CONCLUSION: These results provide novel insight into adaptation of hippocampal circuits following excitotoxic injury. KA-mediated disruption of the interplay between CA3 and CA1 clearly increases the propensity to synchronized firing in CA1.
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ItemThe L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo(BMC, 2008-04-08)BACKGROUND: Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth. RESULTS: We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. CONCLUSION: We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we suggest that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. Our finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion.
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ItemImmunohistochemical characterization of nodose cough receptor neurons projecting to the trachea of guinea pigs(BioMed Central, 2008-10-19)BACKGROUND: Cough in guinea pigs is mediated in part by capsaicin-insensitive low threshold mechanoreceptors (cough receptors). Functional studies suggest that cough receptors represent a homogeneous population of nodose ganglia-derived sensory neurons. In the present study we set out to characterize the neurochemical profile of cough receptor neurons in the nodose ganglia. METHODS: Nodose neurons projecting to the guinea pig trachea were retrogradely labeled with fluorogold and processed immunohistochemically for the expression of a variety of transporters (Na+/K+/2C1- co-transporter (NKCC1), alpha1 and alpha3 Na+/K+ ATPase, vesicular glutamate transporters (vGlut)1 and vGlut2), neurotransmitters (substance P, calcitonin gene-related peptide (CGRP), somatostatin, neuronal nitric oxide synthase (nNOS)) and cytosolic proteins (neurofilament, calretinin, calbindin, parvalbumin). RESULTS: Fluorogold labeled ~3 per cent of neurons in the nodose ganglia with an average somal perimeter of 137 +/- 6.2 mum (range 90-200 microm). All traced neurons (and seemingly all nodose neurons) were immunoreactive for NKCC1. Many (> 90 per cent) were also immunoreactive for vGlut2 and neurofilament and between 50 and 85 per cent expressed alpha1 ATPase, alpha3 ATPase or vGlut1. Cough receptor neurons that did not express the above markers could not be differentiated based on somal size, with the exception of neurofilament negative neurons which were significantly smaller (P < 0.05). Less than 10 per cent of fluorogold labeled neurons expressed substance P or CGRP (and these had somal perimeters less than 110 microm) and none expressed somatostatin, calretinin, calbindin or parvalbumin. Two distinct patterns of nNOS labeling was observed in the general population of nodose neurons: most neurons contained cytosolic clusters of moderately intense immunoreactivity whereas less than 10 per cent of neurons displayed uniform intensely fluorescent somal labeling. Less than 3 per cent of the retrogradely traced neurons were intensely fluorescent for nNOS (most showed clusters of nNOS immunoreactivity) and nNOS immunoreactivity was not expressed by cough receptor nerve terminals in the tracheal wall. CONCLUSION: These data provide further insights into the neurochemistry of nodose cough receptors and suggest that despite their high degree of functional homogeneity, nodose cough receptors subtypes may eventually be distinguished based on neurochemical profile.
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ItemNo Preview AvailableComparison of voice acquisition methodologies in speech research(SPRINGER, 2008-11)The use of voice acoustic techniques has the potential to extend beyond work devoted purely to speech or vocal pathology. For this to occur, however, researchers and clinicians will require acquisition technologies that provide fast, accurate, and cost-effective methods for recording data. Therefore, the present study aimed to compare industry-standard techniques for acquiring high-quality acoustic signals (e.g., hard drive and solid-state recorder) with widely available and easy-to-use, computer-based (standard laptop) data-acquisition methods. Speech samples were simultaneously acquired from 15 healthy controls using all three methods and were analyzed using identical analysis techniques. Data from all three acquisition methods were directly compared using a variety of acoustic correlates. The results suggested that selected acoustic measures (e.g., f 0, noise-to-harmonic ratio, number of pauses) were accurately obtained using all three methods; however, minimum recording standards were required for widely used measures of perturbation.
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ItemUsing a 3D virtual muscle model to link gene expression changes during myogenesis to protein spatial location in muscle(BMC, 2008-10-22)BACKGROUND: Myogenesis is an ordered process whereby mononucleated muscle precursor cells (myoblasts) fuse into multinucleated myotubes that eventually differentiate into myofibres, involving substantial changes in gene expression and the organisation of structural components of the cells. To gain further insight into the orchestration of these structural changes we have overlaid the spatial organisation of the protein components of a muscle cell with their gene expression changes during differentiation using a new 3D visualisation tool: the Virtual Muscle 3D (VMus3D). RESULTS: Sets of generic striated muscle costamere, Z-disk and filament proteins were constructed from the literature and protein-interaction databases. Expression profiles of the genes encoding these proteins were obtained from mouse C2C12 cells undergoing myogenesis in vitro, as well as a mouse tissue survey dataset. Visualisation of the expression data in VMus3D yielded novel observations with significant relationships between the spatial location and the temporal expression profiles of the structural protein products of these genes. A muscle specificity index was calculated based on muscle expression relative to the median expression in all tissues and, as expected, genes with the highest muscle specificity were also expressed most dynamically during differentiation. Interestingly, most genes encoding costamere as well as some Z-disk proteins appeared to be broadly expressed across most tissues and showed little change in expression during muscle differentiation, in line with the broader cellular role described for some of these proteins. CONCLUSION: By studying gene expression patterns from a structural perspective we have demonstrated that not all genes encoding proteins that are part of muscle specific structures are simply up-regulated during muscle cell differentiation. Indeed, a group of genes whose expression program appears to be minimally affected by the differentiation process, code for proteins participating in vital skeletal muscle structures. Expression alone is a poor metric of gene behaviour. Instead, the "connectivity model of muscle development" is proposed as a mechanism for muscle development: whereby the closer to the myofibril core of muscle cells, the greater the gene expression changes during muscle differentiation and the greater the muscle specificity.