Anal. Chem. 2006, 78, 6756-6764
In Vitro Continuous Amperometry with a Diamond Microelectrode Coupled with Video Microscopy for Simultaneously Monitoring Endogenous Norepinephrine and Its Effect on the Contractile Response of a Rat Mesenteric Artery Jinwoo Park,† James J. Galligan,*,‡,§ Gregory D. Fink,‡,§ and Greg M. Swain*,†,§
Department of Chemistry, Department of Pharmacology and Toxicology, and the Neuroscience Program, Michigan State University, East Lansing, Michigan 48824
Continuous amperometry with a diamond microelectrode and video microscopy were used to record (in vitro) endogenous norepinephrine release simultaneously with the evoked contractile response of a mesenteric artery from a healthy Sprague Dawley rat. Norepinephrine (NE) is a vasoconstricting neurotransmitter released from sympathetic nerves that innervate the smooth muscle cell layers surrounding arteries and veins. Using these two techniques along with several drugs, the NE released at sympathetic neuroeffector junctions nearby the microelectrode was measured as an oxidation current. Key to the amperometric measurement was the use of a diamond microelectrode because of the response sensitivity, reproducibility, and stability it provided. NE release was elicited by electrical stimulation at frequencies between 1 and 60 Hz, with a maximum response seen at 20 Hz. Confirmation that the oxidation current was, in fact, associated with endogenous NE came from the results of several drugs. Tetrodotoxin (TTX, 0.3 µM), a voltagedependent sodium channel antagonist that blocks nerve conduction, abolished both the oxidation current and the arterial constriction. The r2-adrenergic autoreceptor antagonist, yohimbine (1.0 µM), caused an increase in the oxidation current and the corresponding constriction. The addition of cocaine (10 µM), an antagonist that inhibits neuronal NE reuptake, caused both the oxidation current and the contractile response to increase. These results, combined with the fact that the hydrodynamic voltammetric E1/2 for endogenous NE was identical to that for a standard solution, confirmed that the oxidation current was due to NE and that this compound caused, at least in part, the contractile response. The results demonstrate that continuous amperometric monitoring of NE with a diamond microelectrode and video imaging of vascular tone allow real time local measurement of the temporal relationship between nerve-stimulated NE release and arterial constriction. Electroanalytical methods have proven useful for the in vitro and in vivo monitoring of electroactive catecholamine neurotrans6756 Analytical Chemistry, Vol. 78, No. 19, October 1, 2006
mitters, such as dopamine and norepinephrine (NE), in the central and peripheral nervous systems. These compounds can be detected with a carbon fiber microelectrode via a 2H+/2eoxidation reaction. The microelectrode is usually 5-10 µm in diameter and must be prepared appropriately for use in biological environments to maximize the response sensitivity and stability.1-3 For example, carbon electrode preparation can involve electrochemical pretreatment for activation. However, although useful for improving sensitivity, such pretreatment can introduce surface roughening, surface oxide formation, and microstructural damage.4,5 The magnitude of these physical and chemical changes and the extent to which they affect the carbon electrode response for the neurotransmitter depend on the pretreatment conditions and the initial electrode microstructure. Pretreatment that causes such physiochemical change has the benefit of increasing the carbon electrode response sensitivity for the catecholamine but the drawbacks of increasing the background current and the electrode response time. In addition, protective polymer coatings, such as Nafion, can be applied to improve the response sensitivity and selectivity for the catecholamine and to minimize fouling in the biological environment by protecting the electrode from biomolecule adsorption.2,6 Application of the polymer coating has the drawback of increasing the electrode response time. Electroanalytical methods with carbon fiber microelectrodes have been used extensively for catecholamine monitoring in the brain and the central nervous system since the pioneering work of Gonon and Adams.7-12 By comparison, there have been fewer †
Department of Chemistry. Department of Pharmacology and Toxicology. § Neuroscience Program. (1) Travis, E. R.; Wightman, R. M. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 77-103. (2) Kawagoe, K. T.; Zimmerman, J. B. Wightman, R. M. J. Neurosci. Methods 1993, 48, 225-240. (3) Wipf, D. O.; Michael, A. C.; Wightman, R. M. J. Electroanal. Chem. 1989, 269, 15-25. (4) Kawagoe, K. T.; Janikowski, J. A.; Wightman, R. M. Anal. Chem. 1991, 63, 1589-1594. (5) Adams, R. N. Anal. Chem. 1976, 48, 1128A-1138A. (6) Ponchon, J.-L.; Cespuglio, R.; Gonon, F.; Jouvet, M.; Pujol, J. F. Anal. Chem. 1979, 51, 1483-1486. (7) Gonon, F. G.; Fombarlet, C. M.; Bud, M. J.; Pujol, J. F. Anal. Chem. 1981, 53, 1386-1389. ‡
10.1021/ac060440u CCC: $33.50
© 2006 American Chemical Society Published on Web 08/29/2006
reports of in vitro or in vivo electrochemical monitoring of neurogenic processes in the peripheral nervous system, such as the release of NE from the sympathetic nerve terminals of isolated organs.13-22 The sympathetic nervous system regulates blood pressure by controlling the tone of muscular resistance arteries and capacitance veins. At sympathetic neuroeffector junctions with smooth muscle cells, the electrical response is mediated by neurotransmitter release. A triad of neurotransmitters can be released, including NE, ATP (adenosine 5′-triphosphate), and neuropeptide Y. Once released, the neurotransmitters diffuse across the synaptic cleft, bind briefly to receptor proteins in the effector cell membrane, and if present in an adequate amount, elicit a specific physiological response (e.g., contraction). However, in many respects, the pathophysiological mechanisms of neural control in arteries and veins are incompletely understood17,18-24. Long-term, we seek a better understanding of the functional differentiation of noradrenergic transmission in arteries and veins and how these control mechanisms are altered in cardiovascular disease states (e.g., hypertension). Of the neurotransmitters released from sympathetic nerves, NE is the only compound that is easily electrooxidizable; thus, it can be monitored electrochemically. To date, published reports have demonstrated that electrically or chemically elicited NE release can be electrochemically monitored in vitro in vasculature preparations.13-22 For example, continuous amperometry with a carbon fiber microelectrode has been employed to record NE release from sympathetic nerve endings at rat tail and mesenteric arteries.13-22 In the majority of these studies, rat tail artery was used because of its dense sympathetic innervation that forms a two-dimensional plexus at the external surface13-18,22. The carbon fiber microelectrode in these studies was prepared for use by either anodic polarization13-17 or coating with Nafion.18-22 Using the oxidation current or charge recorded for endogenous NE along with the effect of various vasoactive agents, researchers have learned about factors controlling release, receptor binding, and clearance at sympathetic neuroeffector junctions. (8) Nagy, G.; Gerhardt, G. A.; Oke, A. F.; Rice, M. E.; Adams, R. N.; Moore, R. B.; Szentirmay, M. N.; Martin, C. R. J. Electroanal. Chem. 1985, 188, 8594. (9) Capella, P.; Ghasemzadeh, B.; Mitchell, K.; Adams, R. N. Electroanalysis 1990, 2, 175-182. (10) Capella, P.; Ghasemzadeh, M. B.; Adams, R. N.; et al. J. Neurochem. 1993, 60, 449-453. (11) Mermet, C.; Gonon, F. G.; Stja¨rne, L. Acta Physiol. Scand. 1990, 140, 323329. (12) Gonon, F.; Bao, J. X.; Msghina, M.; Saud-Chagny, M. F.; Stja¨rne, L. J. Neurochem. 1993, 60, 1251-1257. (13) Gonon, F.; Msghina, M.; Stja¨rne, L. Neuroscience 1993, 56, 535-538. (14) Gonon, F. Bioelectrochem. Bioenerg. 1995, 38, 247-249. (15) Msghina, M.; Gonon, F.; Stja¨rne, L. J. Physiol. 1999, 515, 523-531. (16) Brock, J. A.; Bridgewater, M.; Cunnane, T. C. Br. J. Pharmacol. 1997, 120, 769-776. (17) Dunn, W. R.; Brock, J. A.; Hardy, T. A. Br. J. Pharmacol. 1999, 128, 174180. (18) Brock, J. A.; Dunn, W. R.; Boyd, N. S. F.; Wong, D. K. Y. Br. J. Pharmacol. 2000, 131, 1507-1511. (19) Dunn, W. R.; Hardy, T. A.; Brock, J. A. Br. J. Pharmacol. 2003, 140, 231238. (20) Brock, J. A.; Tan, J. H. C. Br. J. Pharmacol. 2004, 142, 267-274. (21) Stja¨rne, L. J. Auton. Nerv. Syst. 2000, 81, 236-243. (22) Kennedy, C. J. Auton. Pharmacol. 1996, 6, 337-340. (23) Stja¨rne, L. Auton. Neurosci. 2001, 87, 16-36. (24) Babalov, J. Mutafova-Yambolieva, V. N. Clin. Exp. Pharmacol. Physiol. 2001, 28, 397-401.
As stated above, we seek to better understand the functional differentiation of noradrenergic transmission in arteries and veins and how these control mechanisms are altered in hypertension. Toward this end, we have recently reported on the fabrication and electrochemical characterization of a diamond microelectrode25-27 and presented initial results from its application in electroanalytical measurements in biological tissue.26,27 This new carbon electrode provides a greater level of oxidation response sensitivity, reproducibility, and stability than does a bare carbon fiber for monitoring exogenous and endogenous NE in tissue.26,27 The superior response performance was obtained without any conventional electrode preparation, either electrochemical pretreatment or polymer coating. Impressively, the response stability of the diamond microelectrode for exogenous NE in tissue was better than 90% over an 8-h period of continuous use.26 This new electrode has three characteristics that are beneficial for in vitro electrochemical measurements: (i) no preparation (e.g., electrochemical pretreatment or polymer coating) is normally needed to ready the electrode for use, (ii) the electrode surface is relatively oxygen-free so that the background voltammetric current is low and independent of the solution pH, and there are no features present associated with redox-active carbon-oxygen functional groups; and (iii) a high level of response stability is achieved during exposure to biological environments (i.e., minimal fouling), even without the use of a protective polymer film, such as Nafion. In this manuscript, we report on the combined use of in vitro continuous amperometry with a diamond microelectrode and video microscopy to simultaneously measure endogenous NE released from sympathetic nerves innervating a rat mesenteric artery and the resulting contractile response. These two techniques, along with various vasoactive agents, were employed to study the functional relationship between the oxidation current associated with endogenous NE released at neuroeffector junctions nearby the electrode and the extent of arterial constriction. EXPERIMENTAL SECTION Diamond Film Growth and Microelectrode Preparation. The boron-doped diamond thin film was deposited on a sharpened 76-µm-diameter Pt wire (99.99%, Aldrich Chemical) by microwaveassisted chemical vapor deposition (CVD) (1.5 kW, 2.54 GHz, ASTeX, Woburn, MA), as detailed previously.25-27 The diamondcoated Pt wire was affixed to a longer copper wire using conductive Ag epoxy, and the entire assembly was insulated with polypropylene from a pipet tip. This is shown in Figure 1A. The insulation was applied by inserting the microelectrode into a polypropylene pipet and carefully heating the tapered end using the coil of a micropipet puller. This softened the polypropylene and caused it to conformally flow over the polycrystalline diamond surface.28,29 SEM images of the insulated diamond microelectrode are presented in Figure 1B and C. The conical shape of the exposed microelectrode is seen with an arrow pointing to the edge of the polypropylene insulation. The polymer layer appears thin (25) Cvacka, J.; Quaiserova´, V.; Park, J.; Show, Y.; Muck, A., Jr.; Swain, G. M. Anal. Chem. 2003, 75, 2678-2687. (26) Park, J.; Show, Y.; Quaiserova´-Mocko, V.; Galligan, J. J.; Fink, G. D.; Swain, G. M. J. Electroanal. Chem. 2005, 583, 56-68. (27) Park, J.; Quaiserova´-Mocko, V.; Peckova´, K.; Galligan, J. J.; Fink, G. D.; Swain, G. M. Diamond Relat. Mater. 2006, 15, 761-772. (28) Zhou, Z.; Misler, S. J. Biol. Chem. 1996, 271, 270-277. (29) Chow, R. H.; von Ruden, L.; Neher, E. Nature 1992, 356, 60-63.
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Figure 1. (A) Diagram of the conically shaped diamond microelectrode insulated with polypropylene. SEM images of the polypropyleneinsulated diamond microelectrode at (B) lower and (C) higher magnification. Top-view SEM images of the diamond film morphology without (D) and with (E) the polypropylene insulation layer.
and uniform over the wire. Figure 1D and E show top view images of the diamond film with and without the polypropylene coating. The microelectrode diameter at the narrowest point was 10 µm and at the widest point was 80 µm. The length of the exposed electrode was 100-200 µm. This insulation method is quite reproducible in terms of coating diamond with a thin and continuous polymer film, but precise control of the exposed electrode length is difficult to achieve. Mesenteric Artery Preparation and In Vitro Electrochemical Measurement. The Committee on Animal Use and Care at Michigan State University approved all animal use procedures. Male Sprague Dawley rats (380-430 g, Charles River Inc, Portage, MI) were euthanized with a lethal pentobarbital injection (50 mg, i.p.). The abdomen was surgically opened, and the small intestine was carefully removed and placed in oxygenated (95% O2, 5% CO2) Krebs’ buffer solution of the following composition: 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 1.2 mM NaH2PO4, and 11 mM glucose. The ileal segment was placed in a Petri dish, and the mesentery was gently stretched and pinned flat. A section of the mesentery close to the ileal wall was carefully cut free from the intestine and transferred to a small silicone elastomer-lined Teflon flow bath (4.8 mL volume). The bath was 3 cm wide, 4 cm long and 0.4 cm deep. Secondary or tertiary arteries (200-300 µm diameter) were isolated for in vitro study by carefully clearing away the surrounding adipose and connective tissues under a dissection microscope. These preparations were used in the measurement of NE release from perivascular nerves during electrical stimulation (vide infra). The bath was mounted on the stage of an inverted microscope (Olympus CKX41) and superfused continuously with warm (37 °C) buffer solution at a flow rate of 1.6 mL/min. The solution flow was controlled with a peristaltic pump. The diamond microelectrode was affixed to a micromanipulator (MP-1, Narishige Instruments, Japan) for reproducible placement in the tissue. The microcylinder electrode was positioned against the side of the blood vessel with light pressure so that the electrode moved as the vessel tone changed. Such positioning 6758
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led to amperometric currents that were unaffected by changes in the vessel diameter. This will be discussed further below. A Pt wire counter and a commercial “no leak” Ag/AgCl (3 M KCl, model EE009, Cypress Systems Inc., U.S.A.) reference electrode were also mounted in the bath to complete the electrochemical cell. All electrochemical measurements were made with an Omni 90 analogue potentiostat (Cypress Systems Inc.), an analog-todigital converter (Labmaster 125), and a computer running Axotape software (version 2.0, Axon Instruments, Foster City, CA). Continuous amperometric i-t curves were recorded at 800 mV. The analogue output from the potentiostat was low-pass-filtered at a time constant of 200 ms (5 Hz). The filtered analogue current was then digitized using the A/D converter at a sampling rate of 100 Hz, and the data were stored on a computer for further processing. The diamond microelectrode was soaked in distilled isopropyl alcohol (IPA) for at least 15 min prior to the start of a series of measurements.30 The Krebs’ buffer flowed over the electrode and the tissue sample for 30 min prior to the start of a series of measurements. In Vitro Recording of Vasoconstriction. The output of a black-and-white video camera (Hitachi, KP-111; Yokohama, Japan) attached to the microscope (Olympus CKX41) was fed to a PC Vision Plus frame-grabber board (Imaging Technology, Woburn, MA). Changes in blood vessel diameter of 0.1 µm could be resolved. The video images were analyzed using Diamtrak software (http://www.diamtrak.com, Adelaide, Australia). The digitized signal was converted to an analogue output (DAC-02 board, Keithley Metrabyte, Tauton, MA) and sent to an analogto-digital converter (Labmaster 125) in a second computer running Axotape software (version 2.0, Axon Instruments, Foster City, USA) for a permanent recording of the changes in artery diameter as a function of time. Analogue signals were sampled at 100 Hz, and the data were stored on the computer’s hard drive for subsequent analysis and display. Transmural Stimulation of Perivascular Nerves. Electrical stimulation of the sympathetic nerves was accomplished using voltage pulses delivered from an electrically isolated stimulator. Perivascular nerves were stimulated using a bipolar electrode positioned near the surface of a blood vessel but upstream from the detection electrode (see Figure 2). The distance between the stimulation and detection electrodes was maintained at ∼200 µm to minimize noise in the current response of the latter. The focal stimulator consisted of two AgCl-coated Ag wires inserted into a double-barrel glass capillary (diameter ) 180 µm). The wires were connected to an isolation unit and a Grass Instruments stimulator (S88, Quincy, MA). The tissue was stimulated with 60 pulses of a 0.3-ms pulse width at a frequency between 1 and 60 Hz and a voltage of 30-50 V. The voltage selected within this range yielded the maximum contractile response for a particular vessel. Typical firing rates for perivascular sympathetic nerves are
