M. Leandro Heien

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• Dong, Y., Ning, G., Ewing, A. G., & Heien, M. L. (2014). Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells. PLoS ONE, 9(3).
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  Abstract: A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP) functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD). Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM). PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising therapeutic agent for the treatment of PD. © 2014 Dong et al.
• Vreeland, R. F., Laude, N. D., Lambert, S. M., & Heien, M. L. (2014). Microwave-plasma dry-etch for fabrication of conducting polymer microelectrodes. Analytical Chemistry, 86(3), 1385-1390.
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  Abstract: An inexpensive dry etch technology based on a low-pressure microwave plasma generated in a countertop microwave oven is characterized for the patterning of a conductive polymer microelectrode. The etch process is described, and the microwave-generated plasma is characterized by emission spectroscopy. The plasma is generated with an atmospheric mixture of mostly nitrogen and oxygen. A 10 μm wide band microelectrode composed of PEDOT:Tosylate, an optically transparent conductive polymer, is fabricated on a plastic substrate. Conductive polymer etch rates are approximately 280-300 nm/minute. A patterned microelectrode is characterized by atomic force microscopy. The horizontal distance of a 10-90% height of a plasma-etched 150 nm thick electrode was measured to be 360 ± 200 nm (n = 5). Electrodes are further characterized using steady-state cyclic voltammetry, and they have an electroactive area congruent with their geometric area. Finally, a complete device is assembled and used as a separation platform for biogenic amines. A microwave-etched 250 μm PEDOT:PSS electrode is employed for end-channel electrochemical detection on this microchip, where an electrophoretic separation of dopamine and catechol and a micellar electrokinetic chromatography separation of dopamine and serotonin are performed. Both mass and concentration LODs are comparable to other electrochemical detectors in an end-channel configuration. With the added advantages of easy processing, robustness, optical transparency, and low cost, we expect microwave-etched polymer films to be a viable alternative to traditional electrodes. © 2014 American Chemical Society.
• Atcherley, C. W., Laude, N. D., Parent, K. L., & Heien, M. L. (2013). Fast-scan controlled-adsorption voltammetry for the quantification of absolute concentrations and adsorption dynamics. Langmuir, 29(48), 14885-14892.
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  Abstract: Fast-scan cyclic voltammetry has depended on background subtraction to quantify small changes in neurotransmitter concentration. Because of this requirement, measurements of absolute concentrations using fast-scan cyclic voltammetry have been limited. Here we develop and characterize fast-scan controlled-adsorption voltammetry (FSCAV), which enables direct measurements of absolute concentrations in vitro without the use of flow injection to change the concentration. This enables probing the diffusion-controlled adsorption dynamics of biogenic amines and other adsorbing species. An implicit finite-difference model of mass-transport-limited adsorption was developed and is in agreement with experimental results. Optimization of FSCAV yielded a sensitivity of 81 ± 11 nA/μM for dopamine, corresponding to a limit of detection of 3.7 ± 0.5 nM. Through the combination of novel instrumentation and validated computer simulations, we show that FSCAV is an important measurement tool that can be used to determine absolute concentrations and study mass-transport-limited adsorption. © 2013 American Chemical Society.
• Atcherley, C. W., Vreeland, R. F., Monroe, E. B., Sanchez-Gomez, E., & Heien, M. L. (2013). Rethinking data collection and signal processing. 2. Preserving the temporal fidelity of electrochemical measurements. Analytical Chemistry, 85(16), 7654-7658.
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  PMID: 23919317;Abstract: Direct electrochemical measurements of biological events are often challenging because of the low signal relative to the magnitude of the background and noise. When choosing a data processing approach, the frequency and phase content of the data must be considered. Here, we employ a zero-phase (infinite impulse response (IIR)) filter to remove the noise from the analytical signal, while preserving the phase content. In fast-scan cyclic voltammetry, the frequency content of the signal is a function of the scan rate of the applied waveform. Fourier analysis was used to develop a relationship between scan rate and the filter cutoff frequency to maximize the reduction in noise, while not altering the true nature of the analytical signal. The zero-phase filter has the same effect as traditional filters with regards to increasing the signal-to-noise ratio. Because the zero-phase filter does not introduce a change to ΔEpeak, the heterogeneous electron rate transfer constant (0.10 cm/s) for ferrocene is calculated accurately. The zero-phase filter also improves electrochemical analysis of signaling molecules that have their oxidation potential close to the switching potential. Lastly, a quantitative approach to filtering amperometric traces of exocytosis based on the rise time was developed. © 2013 American Chemical Society.
• Berglund, E. C., Makos, M. A., Keighron, J. D., Phan, N., Heien, M. L., & Ewing, A. G. (2013). Oral administration of methylphenidate blocks the effect of cocaine on uptake at the drosophila dopamine transporter. ACS Chemical Neuroscience, 4(4), 566-574.
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  PMID: 23402315;PMCID: PMC3629741;Abstract: Although our understanding of the actions of cocaine in the brain has improved, an effective drug treatment for cocaine addiction has yet to be found. Methylphenidate binds the dopamine transporter and increases extracellular dopamine levels in mammalian central nervous systems similar to cocaine, but it is thought to elicit fewer addictive and reinforcing effects owing to slower pharmacokinetics for different routes of administration between the drugs. This study utilizes the fruit fly model system to quantify the effects of oral methylphenidate on dopamine uptake during direct cocaine exposure to the fly CNS. The effect of methylphenidate on the dopamine transporter has been explored by measuring the uptake of exogenously applied dopamine. The data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter and the inhibition is concentration dependent. The peak height increased to 150% of control when cocaine was used to block the dopamine transporter for untreated flies but only to 110% for methylphenidate-treated flies. Thus, the dopamine transporter is mostly inhibited for the methylphenidate-fed flies before the addition of cocaine. The same is true for the rate of the clearance of dopamine measured by amperometry. For untreated flies the rate of clearance changes 40% when the dopamine transporter is inhibited with cocaine, and for treated flies the rate changes only 10%. The results were correlated to the in vivo concentration of methylphenidate determined by CE-MS. Our data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter for cocaine uptake, and the inhibition is concentration dependent. © 2013 American Chemical Society.
• Heien, M., Monroe, E. B., & Heien, M. L. (2013). Electrochemical generation of hydroxyl radicals for examining protein structure. Analytical chemistry, 85(13).
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  The use of hydroxyl radicals to covalently label the solvent-exposed surface of proteins has been shown to be a powerful tool to examine the structure of proteins and intermolecular interfaces. Current methods to generate hydroxyl radicals for footprinting experiments rely on the laser photolysis of H2O2 or the synchrotron radiolysis of water, which adds significant costs and/or complexity to the experiments. In this work, we develop the electro-Fenton reaction as a means to generate hydroxyl radicals for structural footprinting mass spectrometry experiments to complement current laser and synchrotron-based methods, while reducing the costs and complexity of initiating such experiments. The use of an electrochemical flow cell also enables control of the timing and extent of the radical generation process, while reducing the complexity typically associated with radical footprinting experiments. Ubiquitin, a model protein, was labeled with electro-Fenton generated hydroxyl radicals and top-down proteomics was used to verify oxidation sites on the protein surface.
• Monroe, E. B., & Heien, M. L. (2013). Electrochemical generation of hydroxyl radicals for examining protein structure. Analytical Chemistry, 85(13), 6185-6189.
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  PMID: 23777226;Abstract: The use of hydroxyl radicals to covalently label the solvent-exposed surface of proteins has been shown to be a powerful tool to examine the structure of proteins and intermolecular interfaces. Current methods to generate hydroxyl radicals for footprinting experiments rely on the laser photolysis of H2O2 or the synchrotron radiolysis of water, which adds significant costs and/or complexity to the experiments. In this work, we develop the electro-Fenton reaction as a means to generate hydroxyl radicals for structural footprinting mass spectrometry experiments to complement current laser and synchrotron-based methods, while reducing the costs and complexity of initiating such experiments. The use of an electrochemical flow cell also enables control of the timing and extent of the radical generation process, while reducing the complexity typically associated with radical footprinting experiments. Ubiquitin, a model protein, was labeled with electro-Fenton generated hydroxyl radicals and top-down proteomics was used to verify oxidation sites on the protein surface. © 2013 American Chemical Society.
• Omiatek, D. M., Bressler, A. J., Cans, A., Andrews, A. M., Heien, M. L., & Ewing, A. G. (2013). The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry. Scientific Reports, 3.
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  PMID: 23486177;PMCID: PMC3596796;Abstract: Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the weak base hypothesis that amphetamine reduces synaptic vesicle transmitter and quantal size.
• Ariansen, J. L., Heien, M. L., Hermans, A., Phillips, P. E., Hernadi, I., Bermudez, M. A., Schultz, W., & Wightman, R. M. (2012). Monitoring extracellular pH, oxygen, and dopamine during reward delivery in the striatum of primates. Frontiers in Behavioral Neuroscience.
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  PMID: 22783176;PMCID: PMC3389715;Abstract: Dopamine projections that extend from the ventral tegmental area to the striatum have been implicated in the biological basis for behaviors associated with reward and addiction. Until recently, it has been difficult to evaluate the complex balance of energy utilization and neural activity in the striatum. Many techniques such as electrophysiology, functional magnetic resonance imaging (fMRI), and fast-scan cyclic voltammetry have been employed to monitor these neurochemical and neurophysiological changes. In this brain region, physiological responses to cues and rewards cause local, transient pH changes. Oxygen and pH are coupled in the brain through a complex system of blood flow and metabolism as a result of transient neural activity. Indeed, this balance is at the heart of imaging studies such as fMRI. To this end, we measured pH and O 2 changes with fast-scan cyclic voltammetry in the striatum as indices of changes in metabolism and blood flow in vivo in three Macaca mulatta monkeys during reward-based behaviors. Specifically, the animals were presented with Pavlovian conditioned cues that predicted different probabilities of liquid reward. They also received free reward without predictive cues. The primary detected change consisted of pH shifts in the striatal extracellular environment following the reward predicting cues or the free reward. We observed three types of cue responses that consisted of purely basic pH shifts, basic pH shifts followed by acidic pH shifts, and purely acidic pH shifts. These responses increased with reward probability, but were not significantly different from each other. The pH changes were accompanied by increases in extracellular O 2. The changes in pH and extracellular O 2 are consistent with current theories of metabolism and blood flow. However, they were of sufficient magnitude that they masked dopamine changes in the majority of cases. The findings suggest a role of these chemical responses in neuronal reward processing.
• Heien, M., Laude, N. D., Atcherley, C. W., & Heien, M. L. (2012). Rethinking data collection and signal processing. 1. Real-time oversampling filter for chemical measurements. Analytical chemistry, 84(19).
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  Minimizing noise in chemical measurements is critical to achieve low limits of detection and accurate measurements. We describe a real-time oversampling filter that offers a method to reduce stochastic noise in a time-dependent chemical measurement. The power of this technique is demonstrated in its application to the separation of dopamine and serotonin by micellar electrokinetic chromatography with amperometric detection. Signal-to-noise ratios were increased by almost an order of magnitude, allowing for limits of detection of 100 and 120 amol, respectively. Real-time oversampling filters can be implemented using simple software algorithms and require no change to existing experimental apparatus. The application is not limited to analytical separations, and this technique can be used to improve the signal-to-noise ratio in any experiment where the necessary sampling rate is less than the maximum sampling rate of the analog-to-digital converter. Theory, implementation, and the performance of this filter are described. We propose that this technique should be the default mode of operation for an analog-to-digital converter.
• Larsen, S. T., Heien, M. L., & Taboryski, R. (2012). Amperometric noise at thin film band electrodes. Analytical Chemistry, 84(18), 7744-7749.
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  PMID: 22928986;Abstract: Background current noise is often a significant limitation when using constant-potential amperometry for biosensor application such as amperometric recordings of transmitter release from single cells through exocytosis. In this paper, we fabricated thin-film electrodes of gold and conductive polymers and measured the current noise in physiological buffer solution for a wide range of different electrode areas. The noise measurements could be modeled by an analytical expression, representing the electrochemical cell as a resistor and capacitor in series. The studies revealed three domains; for electrodes with low capacitance, the amplifier noise dominated, for electrodes with large capacitances, the noise from the resistance of the electrochemical cell was dominant, while in the intermediate region, the current noise scaled with electrode capacitance. The experimental results and the model presented here can be used for choosing an electrode material and dimensions and when designing chip-based devices for low-noise current measurements. © 2012 American Chemical Society.
• Larsen, S. T., Vreeland, R. F., Heien, M. L., & Taboryski, R. (2012). Characterization of poly(3,4-ethylenedioxythiophene):Tosylate conductive polymer microelectrodes for transmitter detection. Analyst, 137(8), 1831-1836.
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  PMID: 22383043;Abstract: In this paper we investigate the physical and electrochemical properties of micropatterned poly(3,4-ethylenedioxythiophene):tosylate (PEDOT:tosylate) microelectrodes for neurochemical detection. PEDOT:tosylate is a promising conductive polymer electrode material for chip-based bioanalytical applications such as capillary electrophoresis, high-performance liquid chromatography, and constant potential amperometry at living cells. Band electrodes with widths down to 3 μm were fabricated on polymer substrates using UV lithographic methods. The electrodes are electrochemically stable in a range between -200 mV and 700 mV vs. Ag/AgCl and show a relatively low resistance. A wide range of transmitters is shown to oxidize readily on the electrodes. Kinetic rate constants and half wave potentials are reported. The capacitance per area was found to be high (1670 ± 130 μF cm -2) compared to other thin film microelectrode materials. Finally, we use constant potential amperometry to measure the release of transmitters from a group of PC 12 cells. The results show how the current response decreases for a series of stimulations with high K + buffer. © 2012 The Royal Society of Chemistry.
• Laude, N. D., Atcherley, C. W., & Heien, M. L. (2012). Rethinking data collection and signal processing. 1. Real-time oversampling filter for chemical measurements. Analytical Chemistry, 84(19), 8422-8426.
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  PMID: 22978644;Abstract: Minimizing noise in chemical measurements is critical to achieve low limits of detection and accurate measurements. We describe a real-time oversampling filter that offers a method to reduce stochastic noise in a time-dependent chemical measurement. The power of this technique is demonstrated in its application to the separation of dopamine and serotonin by micellar electrokinetic chromatography with amperometric detection. Signal-to-noise ratios were increased by almost an order of magnitude, allowing for limits of detection of 100 and 120 amol, respectively. Real-time oversampling filters can be implemented using simple software algorithms and require no change to existing experimental apparatus. The application is not limited to analytical separations, and this technique can be used to improve the signal-to-noise ratio in any experiment where the necessary sampling rate is less than the maximum sampling rate of the analog-to-digital converter. Theory, implementation, and the performance of this filter are described. We propose that this technique should be the default mode of operation for an analog-to-digital converter. © 2012 American Chemical Society.
• Santillo, M. F., Ewing, A. G., & Heien, M. L. (2011). Trends in computational simulations of electrochemical processes under hydrodynamic flow in microchannels. Analytical and Bioanalytical Chemistry, 399(1), 183-190.
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  PMID: 20734034;Abstract: Computational modeling and theoretical simulations have recently become important tools for the development, characterization, and validation of microfluidic devices. The recent proliferation of commercial user-friendly software has allowed researchers in the microfluidics community, who might not be familiar with computer programming or fluid mechanics, to acquire important information on microsystems used for sensors, velocimetry, detection for microchannel separations, and microfluidic fuel cells. We discuss the most popular computational technique for modeling these systems-the finite element method-and how it can be applied to model electrochemical processes coupled with hydrodynamic flow in microchannels. Furthermore, some of the limitations and challenges of these computational models are also discussed. © 2010 Springer-Verlag.
• Zhang, B., L., M., Santillo, M. F., Mellander, L., & Ewing, A. G. (2011). Temporal resolution in electrochemical imaging on single PC12 cells using amperometry and voltammetry at microelectrode Arrays. Analytical Chemistry, 83(2), 571-577.
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  PMID: 21190375;PMCID: PMC3023167;Abstract: Carbon-fiber-microelectrode arrays (MEAs) have been utilized to electrochemically image neurochemical secretion from individual pheochromocytoma (PC12) cells. Dopamine release events were electrochemically monitored from seven different locations on single PC12 cells using alternately constant-potential amperometry and fast-scan cyclic voltammetry (FSCV). Cyclic voltammetry, when compared to amperometry, can provide excellent chemical resolution; however, spatial and temporal resolution are both compromised. The spatial and temporal resolution of these two methods have been quantitatively compared and the differences explained using models of molecular diffusion at the nanogap between the electrode and the cell. A numerical simulation of the molecular flux reveals that the diffusion of dopamine molecules and electrochemical reactions both play important roles in the temporal resolution of electrochemical imaging. The simulation also reveals that the diffusion and electrode potential cause the differences in signal crosstalk between electrodes when comparing amperometry and FSCV. © 2010 American Chemical Society.
• Adams, K. L., Engelbrektsson, J., Voinova, M., Zhang, B., Eves, D. J., Karlsson, R., Heien, M. L., Cans, A., & Ewing, A. G. (2010). Steady-state electrochemical determination of lipidie nanotube diameter utilizing an artificial cell model. Analytical Chemistry, 82(3), 1020-1026.
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  PMID: 20039639;PMCID: PMC2814003;Abstract: By exploiting the capabilities of steady-state electrochemical measurements, we have measured the inner diameter of a lipid nanotube using Fick's first law of diffusion in conjunction with an imposed linear concentration gradient of electroactive molecules over the length of the nanotube. Fick's law has been used in this way to provide a direct relationship between the nanotube diameter and the measurable experimental parameters △i (change in current) and nanotube length. Catechol was used to determine the △i attributed to its flux out of the nanotube. Comparing the nanotube diameter as a function of nanotube length revealed that membrane elastic energy was playing an important role in determining the size of the nanotube and was different when the tube was connected to either end of two vesicles or to a vesicle on one end and a pipet tip on the other. We assume that repulsive interaction between neck regions can be used to explain the trends observed. This theoretical approach based on elastic energy considerations provides a qualitative description consistent with experimental data. © 2010 American Chemical Society.
• Dominak, L. M., Omiatek, D. M., Gundermann, E. L., Heien, M. L., & Keating, C. D. (2010). Polymeric crowding agents improve passive biomacromolecule encapsulation in lipid vesicles. Langmuir, 26(16), 13195-13200.
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  PMID: 20695558;PMCID: PMC2919175;Abstract: Large solutes such as high molecular weight proteins can be difficult to encapsulate in lipid vesicles. Passive trapping of these macromolecular solutes during vesicle formation typically results in concentrations inside the vesicles that are much lower than in the external solution. Here, we investigated the effect of macromolecular crowding on passive encapsulation of biological macromolecules with molecular weights ranging from 52 kDa to 660 kDa within both individual giant lipid vesicles (GVs, >3 μm diameter) and populations of 200 nm diameter large unilamellar vesicles (LUVs). Fluorescently labeled biomacromolecules were encapsulated during vesicle formation in the presence or absence of three weight percent poly(ethylene glycol) (PEG; 8 kDa) or dextran 500 kDa, which served as crowding agents. Encapsulation efficiency of the labeled biomolecules was higher for the lower molecular weight solutes, with internal concentrations essentially equal to external concentrations for labeled biomacromolecules with hydrodynamic radii (rh) less than 10 nm. In contrast, internal concentrations were reduced markedly for larger solutes with rh ≥ 10 nm. Addition of PEG or dextran during vesicle formation improved encapsulation of these larger proteins up to the same levels as observed for the smaller proteins, such that internal and external concentrations were equal. This observation is consistent with PEG and dextran acting as volume excluders, reducing the hydrodynamic radius of the biomacromolecules and increasing their encapsulation. This work demonstrates a simple and general route to improved encapsulation of otherwise poorly encapsulated macromolecular solutes in both GV and LUVs up to their concentration in the solution present during vesicle formation. © 2010 American Chemical Society.
• Dong, Y., Heien, M. L., Kurczy, M. R., & Ewing, A. G. (2010). Probing Exocytosis at Single Cells Using Electrochemistry. Chemical Cytometry: Ultrasensitive Analysis of Single Cells, 159-174.
• Heien, M. L., Piehowski, P. D., Winograd, N., & Ewing, A. G. (2010). Lipid detection, identification, and imaging single cells with SIMS. Methods in Molecular Biology, 656, 85-97.
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  PMID: 20680585;Abstract: Time-of-flight secondary ion mass spectrometry (ToF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Many of its biological applications have been in the study of membrane lipids, such as phospholipids and cholesterol. For these studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high-energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Cluster ion sources are able to increase ionization, leading to increased information collected from a surface. In this chapter, we highlight the utility of SIMS to image lipids at single cells. Particularly, we will describe sample preparation, data collection, and the analysis of lipids for two systems; rat oligodendrocytes and Tetrahymena thermophila. SIMS spectra yield information regarding lipid identity and concentration across cell surface. © 2010 Springer Science+Business Media, LLC.
• Keithley, R. B., Wightman, R. M., & Heien, M. L. (2010). Erratum to "Multivariate concentration determination using principal component regression with residual analysis" [Trends Anal. Chem. 28 (2009) 1127-1136] (DOI:10.1016/j.trac.2009.07.002). TrAC - Trends in Analytical Chemistry, 29(1), 110-.
• Kurczy, M. E., Piehowski, P. D., T., C., Heien, M. L., Winograd, N., & Ewing, A. G. (2010). Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation. Proceedings of the National Academy of Sciences of the United States of America, 107(7), 2751-2756.
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  PMID: 20133641;PMCID: PMC2840282;Abstract: Mass spectrometry imaging has been used here to suggest that changes in membrane structure drive lipid domain formation in mating single-cell organisms. Chemical studies of lipid bilayers in both living and model systems have revealed that chemical composition is coupled to localized membrane structure. However, it is not clear if the lipids that compose the membrane actively modify membrane structure or if structural changes cause heterogeneity in the surface chemistry of the lipid bilayer. We report that time-of-flight secondary ion mass spectrometry images of mating Tetrahymena thermophila acquired at various stages during mating demonstrate that lipid domain formation, identified as a decrease in the lamellar lipid phosphatidylcholine, follows rather than precedes structural changes in the membrane. Domains are formed in response to structural changes that occur during cell-to-cell conjugation. This observation has wide implications in all membrane processes.
• Kurczy, M. E., Piehowsky, P. D., Willingham, D., Molyneaux, K. A., Heien, M. L., Winograd, N., & Ewing, A. G. (2010). Nanotome Cluster Bombardment to Recover Spatial Chemistry After Preparation of Biological Samples for SIMS Imaging. Journal of the American Society for Mass Spectrometry, 21(5), 833-836.
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  PMID: 20219392;PMCID: PMC2856613;Abstract: A C60+ cluster ion projectile is employed for sputter cleaning biological surfaces to reveal spatio-chemical information obscured by contamination overlayers. This protocol is used as a supplemental sample preparation method for time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of frozen and freeze-dried biological materials. Following the removal of nanometers of material from the surface using sputter cleaning, a frozen-patterned cholesterol film and a freeze-dried tissue sample were analyzed using ToF-SIMS imaging. In both experiments, the chemical information was maintained after the sputter dose, due to the minimal chemical damage caused by C60+ bombardment. The damage to the surface produced by freeze-drying the tissue sample was found to have a greater effect on the loss of cholesterol signal than the sputter-induced damage. In addition to maintaining the chemical information, sputtering is not found to alter the spatial distribution of molecules on the surface. This approach removes artifacts that might obscure the surface chemistry of the sample and are common to many biological sample preparation schemes for ToF-SIMS imaging. © 2010 American Society for Mass Spectrometry.
• Makos, M. A., Han, K., Heien, M. L., & Ewing, A. G. (2010). Using in vivo electrochemistry to study the physiological effects of cocaine and other stimulants on the Drosophila melanogaster dopamine transporter. ACS Chemical Neuroscience, 1(1), 74-83.
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  Abstract: Dopamine neurotransmission is thought to play a critical role in addiction, reinforcing mechanisms of drugs of abuse. Electrochemical techniques have been employed extensively for monitoring in vivo dopamine changes in the brains of model organisms including rats, mice, and primates. Here, we investigated the effects of several stimulants on dopamine clearance using recently developed microanalytical tools for in vivo electrochemical measurements of dopamine in the central nervous system of Drosophila melanogaster. A cylindrical carbon-fiber microelectrode was placed in the protocerebral anterior medial region of the Drosophila brain (an area dense with dopamine neurons), while a micropipet injector was positioned to exogenously apply dopamine. Background-subtracted fast-scan cyclic voltammetry was carried out to quantify changes in dopamine concentration in the adult fly brain. Clearance of exogenously applied dopamine was significantly decreased in the protocerebral anterior medial area of the wild-type fly following treatment with cocaine, amphetamine, methamphetamine, or methylphenidate. In contrast, dopamine uptake remained unchanged when identical treatments were employed in fumin mutant flies that lack functional dopamine transporters. Our in vivo results support in vitro binding affinity studies predicting that these four stimulants effectively block normal Drosophila dopamine transporter function. Furthermore, we found 10 μM to be a sufficient physiological cocaine concentration to significantly alter dopamine transporter uptake in the Drosophila central nervous system. Taken together, these data indicate dopamine uptake in the Drosophila brain is decreased by psychostimulants as observed in mammals. This validates the use of Drosophila as a model system for future studies into the cellular and molecular mechanisms underlying drug addiction in humans. © 2009 American Chemical Society.
• Makos, M. A., Omiatek, D. M., Ewing, A. G., & Heien, M. L. (2010). Development and characterization of a voltammetric carbon-fiber microelectrode pH sensor. Langmuir, 26(12), 10386-10391.
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  PMID: 20380393;Abstract: This work describes the development and characterization of a modified carbon-fiber microelectrode sensor capable of measuring real-time physiological pH changes in biological microenvironments. The reagentless sensor was fabricated under ambient conditions from voltammetric reduction of the diazonium salt Fast Blue RR onto a carbon-fiber surface in aprotic media. Fast-scan cyclic voltammetry was used to probe redox activity of the p-quinone moiety of the surface-bound molecule as a function of pH. In vitro calibration of the sensor in solutions ranging from pH 6.5 to 8.0 resulted in a pH-dependent anodic peak potential response. Flow-injection analysis was used to characterize the modified microelectrode, revealing sensitivity to acidic and basic changes discernible to 0.005 pH units. Furthermore, the modified electrode was used to measure dynamic in vivo pH changes evoked during neurotransmitter release in the central nervous system of the microanalytical model organism Drosophila melanogaster. © 2010 American Chemical Society.
• Omiatek, D. M., Cans, A., Heien, M. L., & Ewing, A. G. (2010). Analytical approaches to investigate transmitter content and release from single secretory vesicles. Analytical and Bioanalytical Chemistry, 397(8), 3269-3279.
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  PMID: 20480152;Abstract: The vesicle serves as the primary intracellular unit for the highly efficient storage and release of chemical messengers triggered during signaling processes in the nervous system. This review highlights conventional and emerging analytical methods that have used microscopy, electrochemistry, and spectroscopy to resolve the location, time course, and quantal content characteristics of neurotransmitter release. Particular focus is on the investigation of the synaptic vesicle and its involvement in the fundamental molecular mechanisms of cell communication. © 2010 Springer-Verlag.
• Omiatek, D. M., Dong, Y., Heien, M. L., & Ewing, A. G. (2010). Only a fraction of quantal content is released during exocytosis as revealed by electrochemical cytometry of secretory vesicles. ACS Chemical Neuroscience, 1(3), 234-245.
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  Abstract: [Figure presented] The primary method for neuronal communication involves the release of chemical messengers that are packaged intracellularly in vesicles. Although experiments measuring release at single cells have classically been thought to assess the entire content of vesicles, there is evidence in the literature that suggests that the total transmitter stored in vesicles is not expelled during exocytosis. In this work, we introduce a novel technology using a microfluidic-based platform to electrochemically probe individual PC12 cell vesicles isolated from the cell environment. We measure the total vesicular content using methodology that circumvents the biophysical processes of the cell associated with exocytosis. Direct comparisons of amperometric data from release experiments at single PC12 cells versus our cell-free model reveal that on average vesicles release only 40% of their total transmitter load. The data support the intriguing hypothesis that the average vesicle does not open all the way during the normal exocytosis process, resulting in incomplete distention of the vesicular contents. In addition, we have shown that vesicular catecholamine levels can be altered with pharmacological manipulation and variances observed from these treatments can be resolved at the single vesicle level in a high-throughput manner, a process that we have termed electrochemical cytometry. Upon establishing that release in exocytotic processes proceeds in an incomplete manner, we related electrochemical data quantified from both single cell release experiments and electrochemical cytometry of vesicles to vesicular volume from electron microscopy measurements to investigate the location of intravesicular catecholamine stores retained postfusion. © 2010 American Chemical Society.
• Hanson, J. N., Motala, M. J., Heien, M. L., Gillette, M., Sweedler, J., & Nuzzo, R. G. (2009). Textural guidance cues for controlling process outgrowth of mammalian neurons. Lab on a Chip - Miniaturisation for Chemistry and Biology, 9(1), 122-131.
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  PMID: 19209344;PMCID: PMC2819304;Abstract: We explore textural cues as a mechanism for controlling neuronal process outgrowth in primary cultures of mammalian neurons. The work uses a form of decal transfer lithography to generate arrays of PDMS posts of various dimensions and spacings on glass substrates that are rendered growth-compliant by subsequent treatment with a protein activator. Hippocampal neurons plated on these substrates are used to determine how the posts direct process growth by acting as attachment points or guidance cues. Textural features varying over a large range, even as large as 100 μm in diameter, dramatically affect process growth. Indeed, two growth regimes are observed; at the smaller feature sizes considered, process branching strongly aligns (at right angles) along the post mesh, while neuronal outgrowth on the larger feature sizes elicits process wrapping. The latter behavior most strongly manifests in neurons plated initially at ∼100 cells/mm2, where the cells were able to form networks, while for isolated neurons, the cells exhibit poorer viability and development. Bag cell neurons from Aplysia californica also display regular growth patterns, but in this case are guided by contact avoidance of the posts, a behavior qualitatively different than that of the hippocampal neurons. © The Royal Society of Chemistry.
• Heien, M. L., & Ewing, A. G. (2009). Quantitative chemical analysis of single cells.. Methods in molecular biology (Clifton, N.J.), 544, 153-162.
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  PMID: 19488699;Abstract: Exocytosis, the fusion of intracellular vesicles with the membrane and subsequent release of vesicular contents, is important in intercellular communication. The release event is a rapid process (milliseconds), hence detection of released chemicals requires a detection scheme that is both sensitive and has rapid temporal dynamics. Electrochemistry at carbon-fiber microelectrodes allows time-resolved exocytosis of electroactive catecholamines to be observed at very low levels. When coupled with constant-potential amperometry, the number of molecules released and the kinetics of quantal release can be determined. The rapid response time (milliseconds) of microelectrodes makes them well suited for monitoring the dynamic process of exocytosis.
• Keithley, R. B., Wightman, R. M., & Heien, M. L. (2009). Multivariate concentration determination using principal component regression with residual analysis. TrAC - Trends in Analytical Chemistry, 28(9), 1127-1136.
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  Abstract: Data analysis is an essential tenet of analytical chemistry, extending the possible information obtained from the measurement of chemical phenomena. Chemometric methods have grown considerably in recent years, but their wide use is hindered because some still consider them too complicated. The purpose of this review is to describe a multivariate chemometric method, principal component regression, in a simple manner from the point of view of an analytical chemist, to demonstrate the need for proper quality-control (QC) measures in multivariate analysis and to advocate the use of residuals as a proper QC method. © 2009 Elsevier Ltd. All rights reserved.
• Makos, M. A., Kim, Y., Han, K., Heien, M. L., & Ewing, A. G. (2009). In vivo electrochemical measurements of exogenously applied dopamine in Drosophila melanogaster. Analytical Chemistry, 81(5), 1848-1854.
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  PMID: 19192966;PMCID: PMC2787772;Abstract: Carbon-fiber microelectrodes coupled with electrochemical detection have been used extensively for the analysis of biogenic amines. In order to determine the functional role of these amines, in vivo studies have primarily used rats and mice as model organisms. Here, we report on the development of these microanalytical techniques for in vivo electrochemical detection of dopamine in the adult Drosophila melanogastercentral nervous system (CNS). A triple-barrel micropipet injector was used to exogenously apply three different concentrations of dopamine, and a cylindrical carbon-fiber microelectrode was placed in the protocerebral anterior medial brain area where dopamine neurons are densely populated. Background- subtracted fast-scan cyclic voltammetry was used to measure dopamine concentration in the fly CNS. Distinct differences are shown for the clearance of exogenously applied dopamine in the brains of wild type flies versus fumin (fmn) mutants lacking a functional dopamine transporter. The current response due to oxidation of dopamine increased significantly from baseline for wild type flies following cocaine incubation. Interestingly, the current remained unchanged for mutant flies under the same conditions. These data confirm the accepted theory that cocaine blocks dopamine transporter function and validates the use of in vivo electrochemical methods to monitor dopamine uptake in Drosophila. Furthermore, after incubation with tetrodotoxin (TTX), a sodium channel blocker, there was a significant increase in peak oxidation current in the wild type flies; however, the current did not significantly change in the fmn mutant. These data suggest that factors that affect neuronal activity via ion channels such as TTX also influence the function of the dopamine transporter in Drosophila. © 2009 American Chemical Society.
• Makos, M. A., Kuklinski, N. J., Heien, M. L., Ewing, A. G., Kuklinski, N. J., Berglund, E. C., & Ewing, A. G. (2009). Chemical measurements in Drosophila. TrAC - Trends in Analytical Chemistry, 28(11), 1223-1234.
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  Abstract: The fruit fly, Drosophila melanogaster, has been extensively used as a model organism in genetics research and has significantly contributed to understanding molecular, cellular and evolutionary aspects of human behavior. Recently, research has focused on developing analytical methods to obtain highly sensitive chemical quantification along with spatiotemporal information from Drosophila melanogaster. We review a number of these advances in capillary electrophoresis, high-performance liquid chromatography, mass spectrometry, and technologies involving intact organisms, including in vivo electrochemistry. © 2009 Elsevier Ltd. All rights reserved.
• Omiatek, D. M., Santillo, M. F., Heien, M. L., & Ewing, A. G. (2009). Hybrid capillary-microfluidic device for the separation, lysis, and electrochemical detection of vesicles. Analytical Chemistry, 81(6), 2294-2302.
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  PMID: 19228035;PMCID: PMC2656409;Abstract: The primary method for neuronal communication involves the extracellular release of small molecules that are packaged in secretory vesicles. We have developed a platform to separate, lyse, and electrochemically measure the contents of single vesicles using a hybrid capillary-microfluidic device. This device incorporates a sheath-flow design at the outlet of the capillary for chemical lysis of vesicles and subsequent electrochemical detection. The effect of sheath-flow on analyte dispersion was characterized using confocal fluorescence microscopy and electro-chemical detection. At increased flow rates, dispersion was minimized, leading to higher separation efficiencies but lower detected amounts. Large unilamellar vesicles (diameter ̃ 200 nm), a model for secretory vesicles, were prepared by extrusion and loaded with an electroactive molecule. They were then separated and detected using the hybrid capillary-microfluidic device. Determination of size from internalized analyte concentration provides a method to characterize the liposomal suspension. These results were compared to an orthogonal size measurement using dynamic light scattering to validate the detection platform.
• Piehowski, P. D., Davey, A. M., Kurczy, M. E., Sheets, E. D., Winograd, N., Ewing, A. G., & Heien, M. L. (2009). Time-of-flight secondary ion mass spectrometry imaging of subcellular lipid heterogeneity: Poisson counting and spatial resolution. Analytical Chemistry, 81(14), 5593-5602.
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  PMID: 19530687;PMCID: PMC2758657;Abstract: Mass spectrometric imaging is a powerful tool to interrogate biological complexity. One such technique, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging, has been successfully utilized for subcellular imaging of cell membrane components. In order for this technique to provide insight into biological processes, it is critical to characterize the figures of merit. Because a SIMS instrument counts individual events, the precision of the measurement is controlled by counting statistics. As the analysis area decreases, the number of molecules available for analysis diminishes. This becomes critical when imaging subcellular features; it limits the information obtainable, resulting in images with only a few counts of interest per pixel. Many features observed in low intensity images are artifacts of counting statistics, making validation of these features crucial to arriving at accurate conclusions. With TOF-SIMS imaging, the experimentally attainable spatial resolution is a function of the molecule of interest, sample matrix, concentration, primary ion, instrument transmission, and spot size of the primary ion beam. A model, based on Poisson statistics, has been developed to validate SIMS imaging data when signal is limited. This model can be used to estimate the effective spatial resolution and limits of detection prior to analysis, making it a powerful tool for tailoring future investigations. In addition, the model allows comparison of pixel-to-pixel intensity and can be used to validate the significance of observed image features. The implications and capabilities of the model are demonstrated by imaging the cell membrane of resting RBL-2H3 mast cells. © 2009 American Chemical Society.
• Santillo, M. F., Heien, M. L., & Ewing, A. G. (2009). Temporal analysis of protozoan lysis in a microfluidic device. Lab on a Chip - Miniaturisation for Chemistry and Biology, 9(19), 2796-2802.
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  PMID: 19967116;Abstract: A microfluidic device was fabricated and characterized for studying cell lysis of Arcella vulgaris, a nonpathogenic amoeba, over time. The device contains a series of chambers which capture cells allowing them to be subsequently exposed to a constant flow of biocidal agent. With this microfluidic system, individual cells are observed as they undergo lysis. This allows high-throughput measurements of individual lysis events, which are not possible with conventional techniques. Differences in lysis and decay times for Arcella were seen at different flow rates and concentrations of benzalkonium chloride, a biocidal detergent. The efficacy of benzalkonium chloride, chlorhexidine digluconate, phenol, sodium dodecyl sulfate, and Triton X-100 were compared, revealing information on their mechanisms of action. The presented device allows cell capture, controlled exposure to chemical biocides, and observation of lysis with single-cell resolution. Observations at the single cell level give insight into the mechanistic details of the lysis of individual Arcella cells vs. the population; decay times for individual Arcella cells were much shorter when compared to a population of 15 cells. © 2009 The Royal Society of Chemistry.
• Dong, Y., Heien, M. L., Maxson, M. M., & Ewing, A. G. (2008). Amperometric measurements of catecholamine release from single vesicles in MN9D cells. Journal of Neurochemistry, 107(6), 1589-1595.
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  PMID: 19094057;PMCID: PMC2652702;Abstract: MN9D cells have been used as a successful model to investigate dopamine pharmacology and to test the specific effects of drugs for the treatment of Parkinson's disease. However, quantitative measurements of quantal release from these cells have not been carried out. In this work, we used amperometry to investigate catecholamine release from MN9D cells. Amperometric events were observed in both undifferentiated and differentiated (butyric acid-treated) cells. An increase in quantal size and half-width was observed for differentiated cells versus undifferentiated cells; however, the number of events per cell and the amplitude remained constant. In transmission electron microscopy images, no obvious cluster of small synaptic vesicles was observed, and large dense-core vesicles were present in the cell body of undifferentiated cells; however, after differentiation, vesicles were concentrated in the cell processes. In differentiated cells, l-DOPA caused an increase in quantal size and half-width, which could be blocked by the vesicular monoamine transporter inhibitor, reserpine. © 2008 The Authors.
• Piehowski, P. D., Carado, A. J., Kurczy, M. E., Ostrowski, S. G., Heien, M. L., Winograd, N., & Ewing, A. G. (2008). MS/MS methodology to improve subcellular mapping of cholesterol using TOF-SIMS. Analytical Chemistry, 80(22), 8662-8667.
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  PMID: 18925746;PMCID: PMC2597061;Abstract: Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype CeoVquadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum. © 2008 American Chemical Society.
• Piehowski, P. D., Kurczy, M. E., Willingham, D., Parry, S., Heien, M. L., Winograd, N., & Ewing, A. G. (2008). Freeze-etching and vapor matrix deposition for ToF-SIMS imaging of single cells. Langmuir, 24(15), 7906-7911.
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  PMID: 18570446;PMCID: PMC2527754;Abstract: Freeze-etching, the practice of removing excess surface water from a sample through sublimation into the vacuum of the analysis environment, has been extensively used in conjunction with electron microscopy. Here, we apply this technique to time-of-flight secondary-ion mass spectrometry (ToF-SIMS) imaging of cryogenically preserved single cells. By removing the excess water which condenses onto the sample in vacuo, a uniform surface is produced that is ideal for imaging by static SIMS. We demonstrate that the conditions employed to remove deposited water do not adversely affect cell morphology and do not redistribute molecules in the topmost surface layers. In addition, we found water can be controllably redeposited onto the sample at temperatures below -100 °C in vacuum. The redeposited water increases the ionization of characteristic fragments of biologically interesting molecules 2-fold without loss of spatial resolution. The utilization of freeze-etch methodology will increase the reliability of cryogenic sample preparations for SIMS analysis by providing greater control of the surface environment. Using these procedures, we have obtained high quality spectra with both atomic bombardment as well as C60+ cluster ion bombardment. © 2008 American Chemical Society.
• Zhang, B., Adams, K. L., Luber, S. J., Eves, D. J., Heien, M. L., & Ewing, A. G. (2008). Spatially and temporally resolved single-cell exocytosis utilizing individually addressable carbon microelectrode arrays. Analytical Chemistry, 80(5), 1394-1400.
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  PMID: 18232712;PMCID: PMC2653425;Abstract: We report the fabrication and characterization of carbon microelectrode arrays (MEAs) and their application to spatially and temporally resolve neurotransmitter release from single pheochromocytoma (PC12) cells. The carbon MEAs are composed of individually addressable 2.5-μm-radius microdisks embedded in glass. The fabrication involves pulling a multibarrel glass capillary containing a single carbon fiber in each barrel into a sharp tip, followed by beveling the electrode tip to form an array (10-20 μm) of carbon microdisks. This simple fabrication procedure eliminates the need for complicated wiring of the independent electrodes, thus allowing preparation of high-density individually addressable microelectrodes. The carbon MEAs have been characterized using scanning electron microscopy, steady-state and fast-scan voltammetry, and numerical simulations. Amperometric results show that subcellular heterogeneity in single-cell exocytosis can be electrochemically detected with MEAs. These ultrasmall electrochemical probes are suitable for detecting fast chemical events in tight spaces, as well as for developing multifunctional electrochemical microsensors. © 2008 American Chemical Society.
• Cheer, J. F., Aragona, B. J., Heien, M. L., Seipel, A. T., Carelli, R. M., & Wightman, R. M. (2007). Coordinated Accumbal Dopamine Release and Neural Activity Drive Goal-Directed Behavior. Neuron, 54(2), 237-244.
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  PMID: 17442245;Abstract: Intracranial self-stimulation (ICSS) activates the neural pathways that mediate reward, including dopaminergic terminal areas such as the nucleus accumbens (NAc). However, a direct role of dopamine in ICSS-mediated reward has been questioned. Here, simultaneous voltammetric and electrophysiological recordings from the same electrode reveal that, at certain sites, the onset of anticipatory dopamine surges and changes in neuronal firing patterns during ICSS are coincident, whereas sites lacking dopamine changes also lack patterned firing. Intrashell microinfusion of a D1, but not a D2 receptor antagonist, blocks ICSS. An iontophoresis approach was implemented to explore the effect of dopamine antagonists on firing patterns without altering behavior. Similar to the microinfusion experiments, ICSS-related firing is selectively attenuated following D1 receptor blockade. This work establishes a temporal link between anticipatory rises of dopamine and firing patterns in the NAc shell during ICSS and suggests that they may play a similar role with natural rewards and during drug self-administration. © 2007 Elsevier Inc. All rights reserved.
• Cheer, J. F., Wassum, K. M., Sombers, L. A., Heien, M. L., Ariansen, J. L., Aragona, B. J., Phillips, P. E., & Wightman, R. M. (2007). Phasic dopamine release evoked by abused substances requires cannabinoid receptor activation. Journal of Neuroscience, 27(4), 791-795.
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  PMID: 17251418;Abstract: Transient surges of dopamine in the nucleus accumbens are associated with drug seeking. Using a voltammetric sensor with high temporal and spatial resolution, we demonstrate differences in the temporal profile of dopamine concentration transients caused by acute doses of nicotine, ethanol, and cocaine in the nucleus accumbens shell of freely moving rats. Despite differential release dynamics, all drug effects are uniformly inhibited by administration of rimonabant, a cannabinoid receptor (CB1 ) antagonist, suggesting that an increase in endocannabinoid tone facilitates the effects of commonly abused drugs on subsecond dopamine release. These time-resolved chemical measurements provide unique insight into the neurobiological effectiveness of rimonabant in treating addictive disorders. Copyright © 2007 Society for Neuroscience.
• Heien, M. L., & Sweedler, J. V. (2007). Neurochemical challenge. Analytical and Bioanalytical Chemistry, 387(1), 3-4.
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  PMID: 17111104;Abstract: The effect of neurotransmitters on the firing pattern of the postsynaptic cells with which the new neuronal type forms a synapse was investigated. Two sensors capable of detecting the different neurotransmitters which is an electrochemical sensor for neurotransmitter A and a cannula interfaced with capillary electrophoresis for detection of neurotransmitter B was developed. To determine the biological function of the neurotransmitter the electrical output of the circuit as a response to an input stimulus was measured. The detection limit of neurotransmitter A was limited to 40 nmol L-1 and the detection limit of the neurotransmitter B was 80 nmol L-1. the closest position of the dual sensor was approximately 5 μm from the synapse.
• Heien, M. L., & Sweedler, J. V. (2007). Solution to the neurochemical challenge. Analytical and Bioanalytical Chemistry, 388(1), 3-.
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  PMID: 17340082;Abstract: An analysis to find a solution to address a neurochemical challenge by calculating the average time for a molecule to diffuse the required distance is addressed. The time taken by an average molecule to diffuse 5 μm is calculated by using a diffusion coefficient and a distance of 5μm. The sensors should respond to concentration changes on the time scale to detect individual release events. The average concentration for the molecules in the vesicle of the sampled volume is calculated. To detect the neurotransmitter, vesicles with a detection limit of 40 nmol L1 are released. The solution is obtained by using Poisson distribution. Three release events are required to detect neurotransmitter A with a probability of 0.013, while detection of Neurotransmitter B requires five vesicles with a probability of 0.036. The use of Poisson distribution along with 100 simulations would provide 50 events with Neurotransmitter A and 200 with Neurotransmitter B.
• Kyubong, J. o., Heien, M. L., Thompson, L. B., Zhong, M., Nuzzo, R. G., & Sweedler, J. V. (2007). Mass spectrometric imaging of peptide release from neuronal cells within microfluidic devices. Lab on a Chip - Miniaturisation for Chemistry and Biology, 7(11), 1454-1460.
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  PMID: 17960271;Abstract: Microfluidic devices are well suited for manipulating and measuring mass limited samples. Here we adapt a microfluidic device containing functionalized surfaces to chemically stimulate a small number of neurons (down to a single neuron), collect the release of neuropeptides, and characterize them using mass spectrometry. As only a small fraction of the peptides present in a neuron are released with physiologically relevant stimulations, the amount of material available for measurement is small, thereby requiring minimal sample loss and high-sensitivity detection. Although a number of detection schemes are used with microfluidic devices, mass spectrometric detection is used here because of its high information content, allowing the characterization of the released peptide complement. Rather than using an on-line approach, off-line analysis is used; after collection of the peptides onto a surface, mass spectrometric imaging interrogates that surface to determine the peptides released from the cell. The overall utility of this scheme is demonstrated using several device formats with measurement of neuropeptides released from Aplysia californica bag cell neurons. © The Royal Society of Chemistry.
• Rubakhin, S. S., Hatcher, N. G., Monroe, E. B., Heien, M. L., & Sweedler, J. V. (2007). Mass spectrometric imaging of the nervous system. Current Pharmaceutical Design, 13(32), 3325-3334.
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  PMID: 18045186;Abstract: Mass spectrometric imaging (MSI) integrates multiple fields of analytical and biomedical research with the goal of generating chemical maps that present the identity and location of the elements, molecules, and molecular complexes that comprise biological structures. Rapid advances in the development of MSI, which include a broad range of sampling and mass spectrometry strategies, allow the increasingly information-rich creation of chemical images of structurally complex tissues, individual cells, and even single chromosomes. Here we describe a variety of MSI techniques available to investigate the nervous system, with particular focus on the capability of MSI to examine both normal and diseased brain function, An important investigative tool, MSI offers tremendous potential in fundamental studies of brain chemistry, localization of pharmaceutical compounds, and the discovery of biomarkers for different neuropathologies. © 2007 Bentham Science Publishers Ltd.
• Wightman, R. M., Heien, M. L., Wassum, K. M., Sombers, L. A., Aragona, B. J., Khan, A. S., Ariansen, J. L., Cheer, J. F., E., P., & Carelli, R. M. (2007). Dopamine release is heterogeneous within microenvironments of the rat nucleus accumbens. European Journal of Neuroscience, 26(7), 2046-2054.
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  PMID: 17868375;Abstract: Many individual neurons within the intact brain fire in stochastic patterns that arise from interactions with the neuronal circuits that they comprise. However, the chemical communication that is evoked by these firing patterns has not been characterized because sensors suitable to monitor subsecond chemical events in micron dimensions have only recently become available. Here we employ a voltammetric sensor technology coupled with principal component regression to examine the dynamics of dopamine concentrations in the nucleus accumbens (NAc) of awake and unrestrained rats. The sensor has submillimeter dimensions and provides high temporal (0.1 s) resolution. At select locations spontaneous dopamine transient concentration changes were detected, achieving instantaneous concentrations of ∼50 nm. At other locations, transients were absent even though dopamine was available for release as shown by extracellular dopamine increases following electrical activation of dopaminergic neurons. At sites where dopamine concentration transients occur, uptake inhibition by cocaine enhances the frequency and magnitude of the rapid transients while also causing a more gradual increase in extracellular dopamine. These effects were largely absent from sites that did not support ongoing transient activity. These findings reveal an unanticipated spatial and temporal heterogeneity of dopamine transmission within the NAc that may depend upon the firing of specific subpopulations of dopamine neurons. © The Authors (2007).
• Heien, M. L., & Wightman, R. M. (2006). Phasic dopamine signaling during behavior, reward, and disease states. CNS and Neurological Disorders - Drug Targets, 5(1), 99-108.
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  PMID: 16613556;Abstract: The neurotransmitter dopamine is important in reward processing, however its precise modulatory role is still being investigated. Carbon-fiber microelectrodes can be used to monitor dopamine on a subsecond time scale in the striatum and nucleus accumbens of rats during behavior, and this approach is providing new insights into the mechanisms that control its extracellular concentration as well as the conditions under which it is released. Three main processes govern the amount of dopamine measured extrasynaptically: exocytotic release, neuronal uptake, and diffusion away from the release site. By monitoring local extracellular dopamine concentrations in the striatum following electrical stimulation of dopamine-containing neurons, release, uptake and diffusion can be individually examined and quantified. Dopaminergic neurons have been shown to fire in two firing modes, tonic and bursts at higher frequency. Electrical stimulation can be designed to mimic either mode to examine their effects on dopamine release. Burst firing causes a transient increase in extracellular dopamine while tonic firing causes a new steady-state level. In behaving primates, dopaminergic neurons display short-latency, phasic firing to primary reward and conditioned cues associated with reward. These bursts code differences between actual and predicted rewards. In rats, transient dopamine release in terminal regions that mimics that seen during burst firing has been demonstrated during reward-related cues. Taken together, these studies indicate that phasic dopamine release is a critical mediator of reward-related processes. © 2006 Bentham Science Publishers Ltd.
• Cheer, J. F., L., M., Garris, P. A., Carelli, R. M., & Wightman, R. M. (2005). Simultaneous dopamine and single-unit recordings reveal accumbens GABAergic responses: Implications for intracranial self-stimulation. Proceedings of the National Academy of Sciences of the United States of America, 102(52), 19150-19155.
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  PMID: 16380429;PMCID: PMC1323210;Abstract: Intracranial self-stimulation (ICS) is a motivated behavior that results from contingent activation of the brain reward system. ICS with stimulating electrodes placed in the medial forebrain bundle (MFB) is particularly robust. However, the neurons that course through this pathway use a variety of neurotransmitters including dopamine and GABA. For this reason, the neurotransmitters that are central to this behavior, and the specific roles that they subserve, remain unclear. Here, we used extracellular electrophysiology and cyclic voltammetry at the same electrode in awake rats to simultaneously examine cell firing and dopamine release in the nucleus accumbens (NAc) during ICS and noncontingent stimulation of the MFB. ICS elicited dopamine release in the NAc and produced coincident time-locked changes (predominantly inhibitions) in the activity of a subset of NAc neurons. Similar responses were elicited with noncontingent stimulations. The changes in firing rate induced by noncontingent stimulations were reversed by the GABAA receptor antagonist bicuculline. Most time-locked unit activity was unaffected by D1 or D2-like dopamine-receptor antagonists, or by inhibition of evoked dopamine release, although, for a minority of units, the D1 dopamine-receptor antagonist SCH23390 attenuated neural activity. Thus, neurons in the NAc are preferentially inhibited by GABAA receptors after MFB stimulation, a mechanism that may also be important in ICS. © 2005 by The National Academy of Sciences of the USA.
• L., M., Khan, A. S., Ariansen, J. L., Cheer, J. F., E., P., Wassum, K. M., & Wightman, R. M. (2005). Real-time measurement of dopamine fluctuations after cocaine in the brain of behaving rats. Proceedings of the National Academy of Sciences of the United States of America, 102(29), 10023-10028.
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  PMID: 16006505;PMCID: PMC1177422;Abstract: Dopamine neurotransmission has been implicated in the modulation of many cognitive processes. Both rapid (phasic) and slower (tonic) changes in its extracellular concentration contribute to its complex actions. Fast in vivo electrochemical techniques can measure extracellular dopamine on a rapid time scale but without the selectivity afforded with slower techniques that use chemical separations. Cyclic voltammetry improves chemical resolution over other electrochemical methods, and it can resolve dopamine changes in the brains of behaving rodents over short epochs (
• Cheer, J. F., Wassum, K. M., L., M., E., P., & Wightman, R. M. (2004). Cannabinoids Enhance Subsecond Dopamine Release in the Nucleus Accumbens of Awake Rats. Journal of Neuroscience, 24(18), 4393-4400.
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  PMID: 15128853;Abstract: Dopaminergic neurotransmission has been highly implicated in the reinforcing properties of many substances of abuse, including marijuana. Cannabinoids activate ventral tegmental area dopaminergic neurons, the main ascending projections of the mesocorticolimbic dopamine system, and change their spiking pattern by increasing the number of impulses in a burst and elevating the frequency of bursts. Although they also increase time-averaged striatal dopamine levels for extended periods of time, little is known about the temporal structure of this change. To elucidate this, fast-scan cyclic voltammetry was used to monitor extracellular dopamine in the nucleus accumbens of freely moving rats with subsecond timescale resolution. Intravenous administration of the central cannabinoid (CB1) receptor agonist, R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl]-(1-naphthalenyl) methanone mesylate, dose-dependently produced catalepsy, decreased locomotion, and reduced the amplitude of electrically evoked dopamine release while markedly increasing the frequency of detected (nonstimulated) dopamine concentration transients. The CB1 receptor antagonist [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl) -4-methylpyrazole-3-carboxamide] reversed and prevented all agonist-induced effects but did not show effects on dopamine release when injected alone. These data demonstrate that doses of a cannabinoid agonist known to increase burst firing produce ongoing fluctuations in extracellular dopamine on a previously unrecognized temporal scale in the nucleus accumbens.
• Forry, S. P., Murray, J. R., L., M., Locascio, L. E., & Wightman, R. M. (2004). Probing electric fields inside microfluidic channels during electroosmotic flow with fast-scan cyclic voltammetry. Analytical Chemistry, 76(17), 4945-4950.
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  PMID: 15373427;Abstract: Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used in microfluidic channels. This method offers the advantage that it can resolve electroactive species not separated in the channel. In addition, this method provides a route to investigate the distribution of applied electrophoretic fields in microfluidic channels. To probe this, microelectrodes were inserted at various distances into channels and cyclic voltammograms recorded at 300 V/s were repeated at 0.1-s intervals. The use of a battery-powered laptop computer and potentiostat provided galvanic isolation between the applied electrophoretic field and the electrochemical measurements. In the absence of an external field, the peak potential for oxidation of the test solute, Ru(bpy)32+, was virtually unaltered by insertion of the microelectrode tip into the channel. When an electrophoretic field was applied, the peak potential for Ru(bpy)32+ oxidation shifted to more positive potentials in a manner that was directly proportional to the field in the channel. The shifts in peak potential observed with FSCV enabled direct compensation of the applied electrochemical potential. This approach was used to explore the electrophoretic field at the channel terminus. It was found to persist for more than 50 μm from the channel terminus. In addition, the degree of analyte dispersion was found to depend critically on the electrode position outside the channel.
• L., M., Johnson, M. A., & Wightman, R. M. (2004). Resolving neurotransmitters detected by fast-scan cyclic voltammetry. Analytical Chemistry, 76(19), 5697-5704.
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  PMID: 15456288;Abstract: Carbon-fiber microelectrodes are frequently used as chemical sensors in biological preparations. In this work, we evaluated the ability of cyclic voltammograms recorded at fast-scan rates to resolve neurochemicals when analyzed by principal component regression. A calibration set of 30 cyclic voltammograms was constructed from 9 different substances at a variety of concentrations. The set was reduced by principal component analysis, and it was found that 99.5% of the variance in the data could be captured with five principal components. This set was used to evaluate cyclic voltammograms obtained with one or two compounds present in solution. In most cases, satisfactory predictions of the identify and concentration of analytes were obtained. Chemical dynamics were also resolved from a set of fast-scan cyclic voltammograms obtained with the electrode implanted in a region of a brain slice that contains dopaminergic terminals. Following stimulation, principal component regression of the data resolved the changes in dopamine and pH that were evoked. In a second test of the method, vesicular release was measured from adrenal medullary cells and the data were evaluated with a calibration set composed of epinephrine and norepinephrine. Cells that secreted one or the other were identified. Overall, the results show that principal component regression with appropriate calibration data allows resolution of substances that give overlapping cyclic voltammograms.
• Song, Y., Heien, M. L., Jimenez, V., Wightman, R. M., & Murray, R. W. (2004). Voltammetric detection of metal nanoparticles separated by liquid chromatography. Analytical Chemistry, 76(17), 4911-4919.
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  PMID: 15373423;Abstract: Fast-scan cyclic voltammetric (FSCV) detection of the separation of small (
• E., P., Stuber, G. D., L., M., Wightman, R. M., & Carelli, R. M. (2003). Erratum: Subsecond dopamine release promotes cocaine seeking (Nature (2003) 422 (614-618)). Nature, 423(6938), 461-.
• L., M., E., P., Stuber, G. D., Seipel, A. T., & Wightman, R. M. (2003). Overoxidation of carbon-fiber microelectrodes enhances dopamine adsorption and increases sensitivity. Analyst, 128(12), 1413-1419.
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  PMID: 14737224;Abstract: The voltammetric responses of carbon-fiber microelectrodes with a 1.0 V and a 1.4 V anodic limit were compared in this study. Fast-scan cyclic voltammetry was used to characterize the response to dopamine and several other neurochemicals. An increase in the adsorption properties of the carbon fiber leads to an increase in sensitivity of 9 fold in vivo. However the temporal response of the sensor is slower with the more positive anodic limit. Increased electron transfer kinetics also causes a decrease in the relative sensitivity for dopamine vs. other neurochemicals, and a change in their cyclic voltammograms. Stimulated release in the caudate-putamen was pharmacologically characterized in vivo using Ro-04-1284 and pargyline, and was consistent with that expected for dopamine.
• Robinson, D. L., Venton, B. J., Heien, M. L., & Wightman, R. M. (2003). Detecting subsecond dopamine release with fast-scan cyclic voltammetry in vivo. Clinical Chemistry, 49(10), 1763-1773.
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  PMID: 14500617;Abstract: Background: Dopamine is a potent neuromodulator in the brain, influencing a variety of motivated behaviors and involved in several neurologic diseases. Measurements of extracellular dopamine in the brains of experimental animals have traditionally focused on a tonic timescale (minutes to hours). However, dopamine concentrations are now known to fluctuate on a phasic timescale (subseconds to seconds). Approach: Fast-scan cyclic voltammetry provides analytical chemical measurements of phasic dopamine signals in the rat brain. Content: Procedural aspects of the technique are discussed, with regard to appropriate use and in comparison with other methods. Finally, examples of data collected using fast-scan cyclic voltammetry are summarized, including naturally occurring dopamine transients and signals arising from electrical stimulation of dopamine neurons. Summary: Fast-scan cyclic voltammetry offers real-time measurements of changes in extracellular dopamine concentrations in vivo. With its subsecond time resolution, micrometer-dimension spatial resolution, and chemical selectivity, it is the most suitable technique currently available to measure transient concentration changes of dopamine. © 2003 American Association for Clinical Chemistry.
• Robinson, D. L., L., M., & Wightman, R. M. (2002). Frequency of dopamine concentration transients increases in dorsal and ventral striatum of male rats during introduction of conspecifics. Journal of Neuroscience, 22(23), 10477-10486.
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  PMID: 12451147;Abstract: Transient, elevated concentrations of extracellular dopamine were characterized in the dorsal and ventral striatum of male rats during solitude, brief interaction with a conspecific, and copulation. Conspecific rats were systematically presented to male rats and allowed to interact for 30 sec; the males were kept in solitude between each presentation. During these episodes, 125 dopamine concentration transients from 17 rats were detected with fast-scan cyclic voltammetry at carbon-fiber microelectrodes (peak amplitude, 210 ± 10 nM; duration, 530 ± 20 msec). The frequency of dopamine transients increased sixfold during conspecific episodes compared with solitude. However, the phasic dopamine activity habituated on the second presentation of the conspecifics. When males were allowed to copulate with receptive females, additional dopamine transients were observed at frequencies ∼20% of those during the previous interaction episodes. A subset of these transients immediately preceded intromission. Overall, phasic dopamine activity appeared to be associated with input from multiple sensory modalities and was followed by a variety of approach and appetitive behaviors, consistent with electrophysiological observations of dopaminergic neuron burst-firing. In summary, (1) dopamine concentration transients occur in awake rats during solitude, in the absence of overt external cues; (2) dopamine transients are significantly more frequent in the presence of a conspecific, although this effect habituates; and (3) dopamine transients are less frequent during copulation than during brief conspecific episodes. These results establish for the first time that transient dopamine fluctuations occur throughout the dorsal and ventral striatum and demonstrate that they are more frequent with salient stimuli that elicit a response behavior.
• Troyer, K. P., Heien, M. L., Venton, B., & Wightman, R. (2002). Neurochemistry and electroanalytical probes. Current Opinion in Chemical Biology, 6(5), 696-703.
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  PMID: 12413556;Abstract: Electroanalytical techniques have been applied to monitoring chemical events including neurotransmitter release during rodent behaviour and the release of zeptomoles of molecules from single cells. Transgenic mice models have been developed and studied to identify specific cell types in vitro. Characterization and surface modification of electroanalytical probes has enhanced the selectivity and sensitivity of measurements.