Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells.

[1]  H. Wu,et al.  Endothelin receptor binding and cellular signal transduction in neurohybrid NG108-15 cells , 1991, Neuroscience.

[2]  Y. Yamane,et al.  Surface Structures of Cultured Type 2 Astrocytes Revealed by Atomic Force Microscopy , 1999 .

[3]  G. Vauquelin,et al.  Desensitization of alpha 2-adrenergic receptors in NG 108 15 cells by (-)-adrenaline and phorbol 12-myristate 13-acetate. , 1989, The Biochemical journal.

[4]  E. F. Stanley,et al.  Localization of individual calcium channels at the release face of a presynaptic nerve terminal , 1994, Neuron.

[5]  T. Reese,et al.  Structural changes after transmitter release at the frog neuromuscular junction , 1981, The Journal of cell biology.

[6]  H. Kasai Voltage‐ and time‐dependent inhibition of neuronal calcium channels by a GTP‐binding protein in a mammalian cell line. , 1992, The Journal of physiology.

[7]  A. Ogura,et al.  Bradykinin-evoked acetylcholine release via inositol trisphosphate-dependent elevation in free calcium in neuroblastoma x glioma hybrid NG108-15 cells. , 1990, The Journal of biological chemistry.

[8]  E. M. Adler,et al.  Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses , 1990, Neuron.

[9]  G. Augustine,et al.  A Neuronal Sec1 Homolog Regulates Neurotransmitter Release at the Squid Giant Synapse , 1998, The Journal of Neuroscience.

[10]  Thomas C. Südhof,et al.  The synaptic vesicle cycle: a cascade of protein–protein interactions , 1995, Nature.

[11]  Mark K. Bennett,et al.  A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion , 1993, Cell.

[12]  A. Bonfiglio,et al.  Subcellular details of early events of differentiation induced by retinoic acid in human neuroblastoma cells detected by atomic force microscope. , 1995, Experimental cell research.

[13]  Paul Greengard,et al.  Induction of formation of presynaptic terminals in neuroblastoma cells by synapsin IIb , 1991, Nature.

[14]  J. Heuser,et al.  The role of coated vesicles in recycling of synaptic vesicle membrane. , 1989, Cell biology international reports.

[15]  Y. Yamane,et al.  Dynamics of astrocyte adhesion as analyzed by a combination of atomic force microscopy and immuno-cytochemistry: the involvement of actin filaments and connexin 43 in the early stage of adhesion. , 1999, Archives of histology and cytology.

[16]  Reinhard Jahn,et al.  Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution , 1998, Nature.

[17]  Benedikt Westermann,et al.  SNAREpins: Minimal Machinery for Membrane Fusion , 1998, Cell.

[18]  H. Higashida Acetylcholine release by bradykinin, inositol 1,4,5‐trisphosphate and phorbol dibutyrate in rodent neuroblastoma cells. , 1988, The Journal of physiology.

[19]  Y. Yamane,et al.  Quantitative analyses of topography and elasticity of living and fixed astrocytes. , 2000, Journal of electron microscopy.

[20]  Gero Miesenböck,et al.  Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins , 1998, Nature.

[21]  Kazuhiro Abe,et al.  Practical Scan Speed in Atomic Force Microscopy for Live Neurons in a Physiological Solution , 1997 .

[22]  D. Braunstein,et al.  Large secretory structures at the cell surface imaged with scanning force microscopy. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[23]  K. Furuya,et al.  Developmental time courses of Na and Ca spikes in neuroblastoma X glioma hybrid cells. , 1983, Brain research.

[24]  M. Mishina,et al.  Selective coupling with K+ currents of muscarinic acetylcholine receptor subtypes in NG108-15 cells , 1988, Nature.

[25]  S. Terakawa,et al.  Neurotransmitter-induced exocytosis in goblet and acinar cells of rat nasal mucosa studied by video microscopy. , 1993, The American journal of physiology.

[26]  P. De Camilli,et al.  Pathways to regulated exocytosis in neurons. , 1990, Annual review of physiology.

[27]  M. Dennis,et al.  Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release , 1979, The Journal of cell biology.

[28]  W. Betz,et al.  Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. , 1992, Science.

[29]  H. Horstmann,et al.  Transport, docking and exocytosis of single secretory granules in live chromaffin cells , 1997, Nature.

[30]  W. P. Hurlbut,et al.  Vesicle hypothesis of the release of quanta of acetylcholine. , 1980, Physiological reviews.

[31]  Y. Yamane,et al.  Comparative Atomic Force and Scanning Electron Microscopy for Fine Structural Images of Nerve Cells , 1998 .

[32]  S. W. Kuffler,et al.  From Neuron to Brain: A Cellular and Molecular Approach to the Function of the Nervous System , 1992 .

[33]  S. Hsu,et al.  Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. , 1981, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[34]  P. Haydon,et al.  Actin filament dynamics in living glial cells imaged by atomic force microscopy. , 1992, Science.

[35]  Kazuhiro Abe,et al.  Fine Surface Images That Reflect Cytoskeletal Structures in Cultured Glial Cells by Atomic Force Microscopy , 1998 .

[36]  J. Rothman,et al.  Mechanisms of intracellular protein transport , 1994, Nature.

[37]  Kazuhiro Abe,et al.  Reexamination of Fine Surface Topography of Nerve Cells Revealed by Atomic Force Microscopy , 1999 .

[38]  Z. Shao,et al.  Biological atomic force microscopy: from microns to nanometers and beyond. , 1995, Annual review of cell and developmental biology.

[39]  Kazuhiro Abe,et al.  AFM Observation of Three-Dimensional Fine Structural Changes in Living Neurons , 1996 .

[40]  Eric Henderson,et al.  Imaging of living cells by atomic force microscopy , 1994 .

[41]  T. Murphy,et al.  Synaptic Regulation of Immediate Early Gene Expression in Primary Cultures of Cortical Neurons , 1991, Journal of neurochemistry.

[42]  M. Ciotti,et al.  Native and modified uncoated neurons observed by atomic force microscopy , 1996 .

[43]  M. Nirenberg,et al.  A Neuroblastoma × Glioma Hybrid Cell Line with Morphine Receptors , 1974 .

[44]  R Llinás,et al.  Microdomains of high calcium concentration in a presynaptic terminal. , 1992, Science.

[45]  M. Grattarola,et al.  Scanning force microscopy on live cultured cells: Imaging and force‐versus‐distance investigations , 1994, Journal of microscopy.

[46]  P K Hansma,et al.  Atomic force microscopy for high-resolution imaging in cell biology. , 1992, Trends in cell biology.

[47]  L. Donnelly,et al.  Opiate‐Dependent Changes in the Sensitivity of Adenylate Cyclase to Stimulatory Agonists and 5′‐Guanylylimidodiphosphate Are Independent of G Protein Abundance and Eukaryotic ADP‐Ribosyltransferase Activity in NG108‐15 Cells , 1992, Journal of neurochemistry.

[48]  E. Ito,et al.  Bimodal effects of acetylcholine on synchronized calcium oscillation in rat cultured cortical neurons , 2000, Neuroscience Letters.

[49]  H. Lux,et al.  Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[50]  S. Schmid,et al.  Synaptic Vesicle Recycling: The Ferrari of endocytosis? , 1995, Current Biology.

[51]  E. Townes‐Anderson,et al.  Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[52]  Y. Yamane,et al.  Atomic Force Microscopic Observation of Three-Dimensional Morphological Changes of Neurons When Stimulated by a Neurotransmitter , 1999 .

[53]  R. Llinás,et al.  Calcium in synaptic transmission. , 1982, Scientific American.

[54]  Richard H Schaller Membrane trafficking in the presynaptic nerve terminal , 1995, Neuron.

[55]  Y. Yamane,et al.  Acquisition of neuronal proteins during differentiation of NG108-15 cells , 2000, Neuroscience Research.

[56]  Thorsten Lang,et al.  Ca2+-Triggered Peptide Secretion in Single Cells Imaged with Green Fluorescent Protein and Evanescent-Wave Microscopy , 1997, Neuron.

[57]  B. Jena,et al.  Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.