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In JoVE (2)
- Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures
- Electrophysiological Recording in the Drosophila Embryo
Other Publications (19)
- Hua Xi Kou Qiang Yi Xue Za Zhi = Huaxi Kouqiang Yixue Zazhi = West China Journal of Stomatology
- Hua Xi Kou Qiang Yi Xue Za Zhi = Huaxi Kouqiang Yixue Zazhi = West China Journal of Stomatology
- Hua Xi Kou Qiang Yi Xue Za Zhi = Huaxi Kouqiang Yixue Zazhi = West China Journal of Stomatology
- BMC Biology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- BMC Biology
- Neuron
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- The Journal of Cell Biology
- Journal of Surgical Oncology
- Surgery
- Journal of Laparoendoscopic & Advanced Surgical Techniques. Part A
- PloS One
- The Review of Scientific Instruments
- The Review of Scientific Instruments
- Journal of Laparoendoscopic & Advanced Surgical Techniques. Part A
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Molecular and Cellular Neurosciences
- Proceedings of the National Academy of Sciences of the United States of America
Articles by Kaiyun Chen in JoVE
JoVE General
Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures
1Department of Biological Sciences, University of Illinois, 2Department of Biological Sciences, Vanderbilt University
This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.
JoVE General
Electrophysiological Recording in the Drosophila Embryo
1Department of Biological Sciences, University of Illinois, 2Department of Biological Sciences, Vanderbilt University
Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.
Other articles by Kaiyun Chen on PubMed
[Soft Tissue Changes of Patients with Skeletal Class II Malocclusion After Orthodontic and Surgical Treatments]
One of the prime concerns of both the orthodontist and the oral surgeon must be the final soft tissue profile and the esthetic appearance of the patient. The purpose of this study was to evaluate the amount, direction, and predictability of the soft tissue changes associated with simultaneous skeletal changes.
[Characteristic Changes of Acetylcholine Receptor in Rat Lateral Pterygoid Muscles After Functional Mandibule Advancement]
The purpose of this study was to evaluate the maximum binding(Bmax) and affinity(Kd value) changes of acetylcholine receptor (n-AchR) in rat lateral pterygoid muscles after functional mandibule advancement.
[Study on Mechanical Compression Regulating the Proliferation of Young Growing Rat Masticatory Myocyte in Vitro]
To establish an experimental model which was the masticatory myocyte culture and force stimulation in vitro and study the proliferation changes of cultured masticatory myocytes caused by mechanical compression in young rat.
Discs-large (DLG) is Clustered by Presynaptic Innervation and Regulates Postsynaptic Glutamate Receptor Subunit Composition in Drosophila
Drosophila discs-large (DLG) is the sole representative of a large class of mammalian MAGUKs, including human DLG, SAP 97, SAP102, and PSD-95. MAGUKs are thought to be critical for postsynaptic assembly at glutamatergic synapses. However, glutamate receptor cluster formation has never been examined in Drosophila DLG mutants. The fly neuromuscular junction (NMJ) is a genetically-malleable model glutamatergic synapse widely used to address questions regarding the molecular mechanisms of synapse formation and growth. Here, we use immunohistochemistry, confocal microscopy, and electrophysiology to examine whether fly NMJ glutamate receptor clusters form normally in DLG mutants. We also address the question of how DLG itself is localized to the synapse by testing whether presynaptic innervation is required for postsynaptic DLG clustering, and whether DLG localization requires the presence of postsynaptic glutamate receptors.
The 4.1 Protein Coracle Mediates Subunit-selective Anchoring of Drosophila Glutamate Receptors to the Postsynaptic Actin Cytoskeleton
Glutamatergic Drosophila neuromuscular junctions contain two spatially, biophysically, and pharmacologically distinct subtypes of postsynaptic glutamate receptor (GluR). These receptor subtypes appear to be molecularly identical except that A receptors contain the subunit GluRIIA (but not GluRIIB), and B receptors contain the subunit GluRIIB (but not GluRIIA). A- and B-type receptors are coexpressed in the same cells, in which they form homotypic clusters. During development, A- and B-type receptors can be differentially regulated. The mechanisms that allow differential segregation and regulation of A- and B-type receptors are unknown. Presumably, A- and B-type receptors are differentially anchored to the membrane cytoskeleton, but essentially nothing is known about how Drosophila glutamate receptors are localized or anchored. We identified coracle, a homolog of mammalian brain 4.1 proteins, in yeast two-hybrid and genetic screens for proteins that interact with and localize Drosophila glutamate receptors. Coracle interacts with the C terminus of GluRIIA but not GluRIIB. To test whether coracle is required for glutamate receptor localization, we immunocytochemically and electrophysiologically examined receptors in coracle mutants. In coracle mutants, synaptic A-type receptors are lost, but there is no detectable change in B-type receptor function or localization. Pharmacological disruption of postsynaptic actin phenocopies the coracle mutants, suggesting that A-type receptors are anchored to the actin cytoskeleton via coracle, whereas B-type receptors are anchored at the synapse by another (yet unknown) mechanism.
Increased Synaptic Microtubules and Altered Synapse Development in Drosophila Sec8 Mutants
Sec8 is highly expressed in mammalian nervous systems and has been proposed to play a role in several aspects of neural development and function, including neurite outgrowth, calcium-dependent neurotransmitter secretion, trafficking of ionotropic glutamate receptors and regulation of neuronal microtubule assembly. However, these models have never been tested in vivo. Nervous system development and function have not been described after mutation of sec8 in any organism.
A Single Vesicular Glutamate Transporter is Sufficient to Fill a Synaptic Vesicle
Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.
Nonvesicular Release of Glutamate by Glial XCT Transporters Suppresses Glutamate Receptor Clustering in Vivo
We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named "genderblind" (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200-300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses.
Regulation of Glutamate Receptor Subunit Availability by MicroRNAs
The efficacy of synaptic transmission depends, to a large extent, on postsynaptic receptor abundance. The molecular mechanisms controlling receptor abundance are poorly understood. We tested whether abundance of postsynaptic glutamate receptors (GluRs) in Drosophila neuromuscular junctions is controlled by microRNAs, and provide evidence that it is. We show here that postsynaptic knockdown of dicer-1, the endoribonuclease necessary for microRNA synthesis, leads to large increases in postsynaptic GluR subunit messenger RNA and protein. Specifically, we measured increases in GluRIIA and GluRIIB but not GluRIIC. Further, knockout of MiR-284, a microRNA predicted to bind to GluRIIA and GluRIIB but not GluRIIC, increases expression of GluRIIA and GluRIIB but not GluRIIC proportional to the number of predicted binding sites in each transcript. Most of the de-repressed GluR protein, however, does not appear to be incorporated into functional receptors, and only minor changes in synaptic strength are observed, which suggests that microRNAs primarily regulate Drosophila receptor subunit composition rather than overall receptor abundance or synaptic strength.
Expression of X-linked Inhibitor of Apoptosis Protein in Human Colorectal Cancer and Its Correlation with Prognosis
X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis family of proteins and deregulation of XIAP can result in tumorigenicity. The objective of this study was to evaluate the prognostic significance of XIAP expression in colorectal cancer (CRC).
Adenovirus-mediated SiRNA Targeting Mcl-1 Gene Increases Radiosensitivity of Pancreatic Carcinoma Cells in Vitro and in Vivo
Myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic member of the B cell lymphoma/leukemia-2 (Bcl-2) family, has been shown to be involved in apoptosis and the cell cycle. Mcl-1 is overexpressed in many malignancies, including pancreatic cancer. The aim of this study was to investigate the effect of siRNA targeted against Mcl-1 on the radiosensitivity of human pancreatic carcinoma cells.
Laparoscopic Inguinal Hernia Repair: a New Approach
Over a 5-year period, patients, who underwent laparoscopic total peritoneum intraperitoneal onlay mesh (TPIPOM) hernioplasty were retrospectively examined. The investigation focused on technique feasibility and complication incidence, in particular complication related to this novel hernia therapy.
Neurexin in Embryonic Drosophila Neuromuscular Junctions
Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described.
Electron Cyclotron Emission Reconstruction Image and M/n = 3/2 Mode in HT-7 Tokamak
Electron cyclotron emission reconstruction image has been used for flux surface reconstruction. The reconstruction image is based on plasma rigid rotation which is obtained from Mirnov diagnostic. From the reconstructed two-dimensional flux surface, the classical m/n = 3/2 mode is visualized, which is of similar spatial structure as neoclassical 3/2 mode observed in some other tokamaks [B. Esposito et al., Phys. Rev. Lett. 100, 045006 (2008)].
Note: Tangential X-ray Diagnosis for Investigating Fast MHD Events in EAST Tokamak
A tangential x-ray diagnosis has been installed in the experimental advanced superconducting tokamakvacuum vessel for the study of fast magnetohydrodynamics (MHD) events. This system is based on absolute x-ray ultraviolet detectors with a collimator which is processed by laser machine. The first experimental results have proved its ability to measure the small-scale and transient MHD perturbations.
Towards a Near-zero Recurrence Rate in Laparoscopic Inguinal Hernia Repair for Pediatric Patients
The aim of this study was to assess whether the median or lateral umbilicus ligament covering the internal hernia opening region after the purse-string knot could eliminate recurrence in laparoscopic inguinal hernia repair in pediatric patients of all ages.
Membrane Penetration by Synaptotagmin is Required for Coupling Calcium Binding to Vesicle Fusion in Vivo
The vesicle protein synaptotagmin I is the Ca(2+) sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca(2+) binding by the C(2)B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca(2+) binding results in vesicle fusion remains controversial. Ca(2+)-dependent interactions between synaptotagmin and SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C(2)B Ca(2+)-binding motif, suggesting that these interactions are facilitatory rather than essential. Here we use Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca(2+)-binding pockets of synaptotagmin. Mutation of this residue in the C(2)A domain (F286) resulted in a ∼50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C(2)B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in syt(null) mutants, which is more severe than the phenotype of C(2)B Ca(2+)-binding mutants. Thus, mutation of a single, C(2)B hydrophobic residue required for Ca(2+)-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C(2)B domain of synaptotagmin, is the critical effector interaction for coupling Ca(2+) binding with vesicle fusion.
Pre and Postsynaptic Roles for Drosophila CASK
CASK ('calcium/calmodulin-dependent serine protein kinase'), also known in Drosophila as 'Caki' or 'Camguk/CMG', and in C. elegans as 'Lin-2', is thought to play an important role in cell-cell junction formation and at synapses in particular. To understand the role of CASK in synapse formation and function, we functionally and morphologically analyzed Drosophila embryonic and larval glutamatergic neuromuscular junctions (NMJs) after pan-cellular and tissue-specific manipulation of CASK expression. Our results show that Drosophila CASK is associated with both pre and postsynaptic membranes. Loss of presynaptic CASK led to less evoked synaptic transmission, fewer spontaneous synaptic events, and reduced synaptic vesicle cycling. These changes were accompanied by a reduction in the number of synapses but no change in overall NMJ size. Loss of postsynaptic CASK, on the other hand, caused reduced spontaneous synaptic current amplitudes and smaller glutamate-gated currents. These changes were accompanied by loss of postsynaptic glutamate receptors, but the receptor loss was subtype-specific: Only receptors containing GluRIIA subunits were lost in CASK mutants. Receptors containing GluRIIB were unaffected.
Tomosyn-dependent Regulation of Synaptic Transmission is Required for a Late Phase of Associative Odor Memory
Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. We tested this hypothesis in Drosophila, in which the role of the PKA pathway in associative learning has been well established. We first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. We next assayed memory performance 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that tomosyn is involved in a specific component of late associative memory.
