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In JoVE (1)
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Articles by Qizhi Gong in JoVE
JoVE Neuroscience
Lentivirus-gemedieerde genetische manipulatie en visualisatie van olfactorische sensorische neuronen In vivo
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
We presenteren een lentivirale techniek voor genetische manipulatie en visualisatie van een olfactorische sensorische neuron axon en de terminal arborization
Other articles by Qizhi Gong on PubMed
Conditional Ablation of Mature Olfactory Sensory Neurons Mediated by Diphtheria Toxin Receptor
The vertebrate olfactory epithelium provides an excellent model system to study the regulatory mechanisms of neurogenesis and neuronal differentiation due to its unique ability to generate new sensory neurons throughout life. The replacement of olfactory sensory neurons is stimulated when damage occurs in the olfactory epithelium. In this study, transgenic mice, with a transgene containing human diphtheria toxin receptor under the control of the olfactory marker protein promoter (OMP-DTR), were generated in which the mature olfactory sensory neurons could be specifically ablated when exposed to diphtheria toxin. Following diphtheria toxin induced neuronal ablation, we observed increased numbers of newly generated growth associated protein 43 (GAP43)-positive immature olfactory sensory neurons. OMP-positive neurons were continuously produced from the newly generated GAP43-positive cells. The expression of the signal transduction components adenylyl cyclase type III and the G-protein alpha subunit G(alpha olf) was sensitive to diphtheria toxin exposure and their levels decreased dramatically preceding the disappearance of the OMP-positive sensory neurons. These data validate the hypothesis that OMP-DTR mice can be used as a tool to ablate the mature olfactory sensory neurons in a controlled fashion and to study the regulatory mechanisms of the neuronal replacement.
Olfactory Sensory Neuron-specific and Sexually Dimorphic Expression of Protocadherin 20
Olfactory sensory axons navigate from the nasal cavity to the olfactory bulb and sort from among 1,000 different odorant receptor-expressing types to converge upon the same two or three glomeruli. To achieve this task during development, it is likely that multiple classes of regulatory molecules, including cell adhesion molecules, are involved. Cell adhesion molecules have been shown to be important in controlling axon guidance, fasciculation, and synapse formation. To gain further understanding of the involvement of adhesion molecules in olfactory circuitry development, we examined the dynamic and cell type specific expression of a novel protocadherin, PCDH20, in the olfactory system. PCDH20 is specifically expressed in newly differentiated olfactory sensory neurons and their axons during development. PCDH20 expression is down-regulated in the adult olfactory system, except in a small olfactory sensory neuron population. These small, discrete numbers of PCDH20-positive glomeruli in the adult olfactory bulb are consistently clustered in the ventral-caudal region in both male and female mice. However, adult males have higher numbers of PCDH20-positive glomeruli with a broader distribution, whereas adult females have fewer PCDH20-positive glomeruli with a more restricted distribution. The gender difference in PCDH20 expression may reflect olfactory receptor expression differences for gender-specific social discrimination.
Expressing Exogenous Functional Odorant Receptors in Cultured Olfactory Sensory Neurons
Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment.
Neurogenesis and Neurite Outgrowth in the Spinal Cord of Chicken Embryos and in Primary Cultures of Spinal Neurons Following Knockdown of Class III Beta Tubulin with Antisense Morpholinos
Microtubules are the primary cytoskeletal constituent of extending neurites. We used antisense morpholinos to knock down expression of neuron-specific Class III beta tubulin in the right half of the neural tube of chicken embryos in ovo. There was a significant (p < 0.01) reduction in the number of Class III beta tubulin immunostained interneurons 24 h following electroporation of the morpholinos when compared with the contralateral side of the neural tube. However, neural crest-derived sensory neurons labeled with the fluorescently tagged morpholinos developed distinct processes. Moreover, there was no significant difference in the number of interneurons labeled on either side of the neural tube with a second marker of developing neurons, anti-microtubule associated protein (MAP) 1b. Neural tubes were also excised and dissociated following antisense or control morpholino electroporation. The resulting neurons were cultured for 48 h and immunostained with anti-Class III beta tubulin and anti-MAP 1b. Neurons that had taken up the antisense morpholino had significantly shorter neurites (p < 0.01) than neurons from the same neural tubes that did not; they also had significantly shorter neurites (p < 0.05) than labeled neurons from neural tubes electroporated with a control morpholino. Thus, normal expression of Class III beta tubulin may not be necessary for neurogenesis in the early avian spinal cord in situ, but is required for neurite outgrowth in vitro.
Influence of Olfactory Epithelium on Mitral/tufted Cell Dendritic Outgrowth
Stereotypical connections between olfactory sensory neuron axons and mitral cell dendrites in the olfactory bulb establish the first synaptic relay for olfactory perception. While mechanisms of olfactory sensory axon targeting are reported, molecular regulation of mitral cell dendritic growth and refinement are unclear. During embryonic development, mitral cell dendritic distribution overlaps with olfactory sensory axon terminals in the olfactory bulb. In this study, we investigate whether olfactory sensory neurons in the olfactory epithelium influence mitral cell dendritic outgrowth in vitro. We report a soluble trophic activity in the olfactory epithelium conditioned medium which promotes mitral/tufted cell neurite outgrowth. While the trophic activity is present in both embryonic and postnatal olfactory epithelia, only embryonic but not postnatal mitral/tufted cells respond to this activity. We show that BMP2, 5 and 7 promote mitral/tufted cells neurite outgrowth. However, the BMP antagonist, Noggin, fails to neutralize the olfactory epithelium derived neurite growth promoting activity. We provide evidence that olfactory epithelium derived activity is a protein factor with molecular weight between 50-100 kD. We also observed that Follistatin can effectively neutralize the olfactory epithelium derived activity, suggesting that TGF-beta family proteins are involved to promote mitral/tufted dendritic elaboration.
Secreted TARSH Regulates Olfactory Mitral Cell Dendritic Complexity
Olfactory sensory neurons synapse with mitral cells to form stereotyped connections in the olfactory bulb (OB). Mitral cell apical dendrites receive input from olfactory sensory neurons expressing the same odorant receptor. During development, this restricted dendritic targeting of mitral cells is achieved through eliminating elaborated dendritic trees to a single apical dendrite. Through a genome-wide microarray screen, we identified TARSH (Target of NESH SH3) as a transiently expressed molecule in mitral cells during the dendritic refinement period. TARSH expression is restricted to pyramidal neurons along the main olfactory pathway, including the anterior olfactory nucleus and piriform cortex. The dynamic TARSH expression is not altered when odor-evoked activity is blocked by naris closure or in AC3 knockout mice. We also demonstrate that TARSH is a secreted protein. In dissociated OB cultures, secreted TARSH promotes the reduction of mitral cell dendritic complexity and restricts dendritic branching and outgrowth of interneurons. Dendritic morphological changes were also observed in mitral cells overexpressing TARSH themselves. We propose that TARSH is part of the genetic program that regulates mitral cell dendritic refinement.
Mitochondrial OPA1, Apoptosis, and Heart Failure
Mitochondrial fusion and fission are essential processes for preservation of normal mitochondrial function. We hypothesized that fusion proteins would be decreased in heart failure (HF), as the mitochondria in HF have been reported to be small and dysfunctional.
Olfactory Sensory Axon Growth and Branching is Influenced by Sonic Hedgehog
Olfactory sensory neuron (OSN) axons extend from the olfactory epithelium to the olfactory bulb without branching until they reach their target region, the glomerulus. In this report, we present evidence to support the involvement of sonic hedgehog in promoting rat olfactory sensory axons to branch and to enter into the glomerulus. Sonic hedgehog (Shh) protein is detected in the glomeruli of the olfactory bulb, whereas its transcript is expressed in the mitral and tufted cells, suggesting that Shh in the glomeruli is produced by mitral and tufted cells. In primary OSN cultures, Shh-N peptide promotes olfactory axon branching. When Shh function is neutralized in vivo by its antibody, growth of newly generated OSN axons into the glomeruli is vastly reduced.
