Parallel on-chip analysis of single vesicle neurotransmitter release.

Real-time investigations of neurotransmitter release provide a direct insight on the mechanisms involved in synaptic communication. Carbon fiber microelectrodes are state-of-the-art tools for electrochemical measurements of single vesicle neurotransmitter release. Yet, they lack high-throughput capabilities that are required for collecting robust statistically significant data across multiple samples. Here, we present a chip-based recording system enabling parallel in vitro measurements of individual neurotransmitter release events from cells, cultured directly on planar multielectrode arrays. The applicability of this cell-based platform to pharmacological screening is demonstrated by resolving minute concentration-dependent effects of the dopamine reuptake inhibitor nomifensine on recorded single-vesicle release events from PC12 cells. The experimental results, showing an increased half-time of the recorded events, are complemented by an analytical model for the verification of drug action.

[1]  Arto Heiskanen,et al.  Fully automated microchip system for the detection of quantal exocytosis from single and small ensembles of cells. , 2008, Lab on a chip.

[2]  Robert H. Chow,et al.  Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells , 1992, Nature.

[3]  M. Egan,et al.  Neurobiology of schizophrenia , 1997, Current Opinion in Neurobiology.

[4]  Andrew G Ewing,et al.  Only a Fraction of Quantal Content is Released During Exocytosis as Revealed by Electrochemical Cytometry of Secretory Vesicles. , 2010, ACS chemical neuroscience.

[5]  F. Gonon,et al.  Presynaptic regulation of dopaminergic neurotransmission , 2003, Journal of neurochemistry.

[6]  A. Ewing,et al.  Amperometric monitoring of stimulated catecholamine release from rat pheochromocytoma (PC12) cells at the zeptomole level. , 1994, Analytical chemistry.

[7]  D. Bruns,et al.  Real-time measurement of transmitter release from single synaptic vesicles , 1995, Nature.

[8]  Weihua Huang,et al.  Monitoring dopamine release from single living vesicles with nanoelectrodes. , 2005, Journal of the American Chemical Society.

[9]  P. Garris,et al.  Comparison of Dopamine Uptake in the Basolateral Amygdaloid Nucleus, Caudate‐Putamen, and Nucleus Accumbens of the Rat , 1995, Journal of neurochemistry.

[10]  Yong Chen,et al.  Coupling of electrochemistry and fluorescence microscopy at indium tin oxide microelectrodes for the analysis of single exocytotic events. , 2006, Angewandte Chemie.

[11]  Khajak Berberian,et al.  Transparent Electrode Materials for Simultaneous Amperometric Detection of Exocytosis and Fluorescence Microscopy. , 2012, Journal of biomaterials and nanobiotechnology.

[12]  Maruf Hossain,et al.  Automated targeting of cells to electrochemical electrodes using a surface chemistry approach for the measurement of quantal exocytosis. , 2010, ACS chemical neuroscience.

[13]  Edgar D Goluch,et al.  Stochastic sensing of single molecules in a nanofluidic electrochemical device. , 2011, Nano letters.

[14]  R. Wightman,et al.  Spatio-temporal resolution of exocytosis from individual cells. , 1998, Annual review of biophysics and biomolecular structure.

[15]  Manfred Lindau,et al.  Parallel recording of neurotransmitters release from chromaffin cells using a 10×10 CMOS IC potentiostat array with on-chip working electrodes. , 2013, Biosensors & bioelectronics.

[16]  M. Armstrong‐James,et al.  Carbon fibre microelectrodes , 1979, Journal of Neuroscience Methods.

[17]  A. Ewing,et al.  Carbon-ring microelectrode arrays for electrochemical imaging of single cell exocytosis: fabrication and characterization. , 2012, Analytical chemistry.

[18]  D. Sulzer,et al.  Dopamine neurons release transmitter via a flickering fusion pore , 2004, Nature Neuroscience.

[19]  R. S. Martin,et al.  Microchip-based electrochemical detection for monitoring cellular systems , 2013, Analytical and Bioanalytical Chemistry.

[20]  J. A. Jankowski,et al.  Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[21]  K. Weltmann,et al.  Formation of PTFE-like films in CF4 microwave plasmas , 2010 .

[22]  Gregory M Dittami,et al.  Electrically evoking and electrochemically resolving quantal release on a microchip. , 2010, Lab on a chip.

[23]  J. Castracane,et al.  Amperometric detection of quantal catecholamine secretion from individual cells on micromachined silicon chips. , 2003, Analytical chemistry.

[24]  Fwu-Shan Sheu,et al.  Microelectrode array biochip: tool for in vitro drug screening based on the detection of a drug effect on dopamine release from PC12 cells. , 2006, Analytical chemistry.

[25]  E Neher,et al.  Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Raynaud,et al.  Nomifensine: a new potent inhibitor of dopamine uptake into synaptosomes from rat brain corpus striatum , 1974, The Journal of pharmacy and pharmacology.

[27]  Zhen Yan,et al.  Parkin controls dopamine utilization in human midbrain dopaminergic neurons derived from induced pluripotent stem cells , 2012, Nature Communications.

[28]  E. Pothos,et al.  Quantitative and Statistical Analysis of the Shape of Amperometric Spikes Recorded from Two Populations of Cells , 2000, Journal of neurochemistry.

[29]  M. Lindau,et al.  Improved surface-patterned platinum microelectrodes for the study of exocytotic events. , 2009, Analytical chemistry.

[30]  H. Shiku,et al.  Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy , 2012, Proceedings of the National Academy of Sciences.

[31]  R. Westerink,et al.  Heterogeneity of catecholamine-containing vesicles in PC12 cells. , 2000, Biochemical and biophysical research communications.

[32]  E. Pothos,et al.  Presynaptic Recording of Quanta from Midbrain Dopamine Neurons and Modulation of the Quantal Size , 1998, The Journal of Neuroscience.

[33]  Fwu-Shan Sheu,et al.  In situ temporal detection of dopamine exocytosis from L-dopa-incubated MN9D cells using microelectrode array-integrated biochip , 2006 .

[34]  G. Gerhardt,et al.  Determination of diffusion coefficients by flow injection analysis , 1982 .

[35]  Tao Xu,et al.  Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity , 1998, Nature Neuroscience.

[36]  Bo Zhang,et al.  Spatially and temporally resolved single-cell exocytosis utilizing individually addressable carbon microelectrode arrays. , 2008, Analytical chemistry.

[37]  E. Neher.  Vesicle Pools and Ca2+ Microdomains: New Tools for Understanding Their Roles in Neurotransmitter Release , 1998, Neuron.

[38]  A. Ewing,et al.  Highlights of 20 years of electrochemical measurements of exocytosis at cells and artificial cells , 2011 .

[39]  D. Sulzer,et al.  Analysis of exocytotic events recorded by amperometry , 2005, Nature Methods.

[40]  J. W. Schultze,et al.  Optimization of passivation layers for corrosion protection of silicon-based microelectrode arrays , 2000 .

[41]  W. Schuhmann,et al.  Scanning electrochemical microscopy in neuroscience. , 2010, Annual review of analytical chemistry.

[42]  A. Pasquarelli,et al.  Diamond microelectrodes arrays for the detection of secretory cell activity , 2011 .

[43]  Kevin D. Gillis,et al.  Microwell device for targeting single cells to electrochemical microelectrodes for high-throughput amperometric detection of quantal exocytosis. , 2011, Analytical chemistry.

[44]  Lisa J. Mellander,et al.  Temporal resolution in electrochemical imaging on single PC12 cells using amperometry and voltammetry at microelectrode arrays. , 2011, Analytical chemistry.

[45]  R. Wightman.  Probing Cellular Chemistry in Biological Systems with Microelectrodes , 2006, Science.

[46]  Pradyumna S. Singh,et al.  Stochasticity in single-molecule nanoelectrochemistry: origins, consequences, and solutions. , 2012, ACS nano.

[47]  Christian Amatore,et al.  Indium Tin Oxide devices for amperometric detection of vesicular release by single cells. , 2012, Biophysical chemistry.

[48]  S. Amara,et al.  The Dopamine Transporter in Mesencephalic Cultures Is Refractory to Physiological Changes in Membrane Voltage , 2001, The Journal of Neuroscience.

[49]  Khajak Berberian,et al.  Electrochemical imaging of fusion pore openings by electrochemical detector arrays. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[50]  L. Greene,et al.  Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[51]  B. Wolfrum,et al.  Printed carbon microelectrodes for electrochemical detection of single vesicle release from PC12 cells. , 2012, Analytical chemistry.