title: Real-time monitoring of extracellular adenosine using enzyme-linked microelectrode arrays.
abstract: Throughout the central nervous system extracellular adenosine serves important neuroprotective and neuromodulatory functions. However, current understanding of the in vivo regulation and effects of adenosine is limited by the spatial and temporal resolution of available measurement techniques. Here, we describe an enzyme-linked microelectrode array (MEA) with high spatial (7500 µm(2)) and temporal (4 Hz) resolution that can selectively measure extracellular adenosine through the use of self-referenced coating scheme that accounts for interfering substances and the enzymatic breakdown products of adenosine. In vitro, the MEAs selectively measured adenosine in a linear fashion (r(2)=0.98±0.01, concentration range=0-15 µM, limit of detection =0.96±0.5 µM). In vivo the limit of detection was 0.04±0.02 µM, which permitted real-time monitoring of the basal extracellular concentration in rat cerebral cortex (4.3±1.5 µM). Local cortical injection of adenosine through a micropipette produced dose-dependent transient increases in the measured extracellular concentration (200 nL: 6.8±1.8 µM; 400 nL: 19.4±5.3 µM) [P<0.001]. Lastly, local injection of dipyridamole, which inhibits transport of adenosine through equilibrative nucleoside transporter, raised the measured extracellular concentration of adenosine by 120% (5.6→12.3 µM) [P<0.001]. These studies demonstrate that MEAs can selectively measure adenosine on temporal and spatial scales relevant to adenosine signaling and regulation in normal and pathologic states.
authors.affiliation: Department of Neurosurgery, University of Cincinnati (UC) College of Medicine, Neurotrauma Center at UC Neuroscience Institute and Mayfield Clinic, Cincinnati, OH, USA. Electronic address: jmhinz2@gmail.com.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Hinzman
authors.affiliation: University of Cincinnati (UC) College of Medicine, Cincinnati, OH, USA.
authors.forename: Justin L
authors.initials: JL
authors.lastname: Gibson
authors.affiliation: Department of Neurosurgery, University of Cincinnati (UC) College of Medicine, Neurotrauma Center at UC Neuroscience Institute and Mayfield Clinic, Cincinnati, OH, USA; Mayfield Clinic, Cincinnati, OH, USA.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Tackla
authors.affiliation: University of Cincinnati (UC) College of Medicine, Cincinnati, OH, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Costello
authors.affiliation: Department of Anatomy and Neurobiology, University of Kentucky (UK), Center for Microelectrode Technology, Lexington, KY, USA.
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Burmeister
authors.affiliation: Department of Anatomy and Neurobiology, University of Kentucky (UK), Center for Microelectrode Technology, Lexington, KY, USA.
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Quintero
authors.affiliation: Department of Anatomy and Neurobiology, University of Kentucky (UK), Center for Microelectrode Technology, Lexington, KY, USA.
authors.forename: Greg A
authors.initials: GA
authors.lastname: Gerhardt
authors.affiliation: Department of Neurosurgery, University of Cincinnati (UC) College of Medicine, Neurotrauma Center at UC Neuroscience Institute and Mayfield Clinic, Cincinnati, OH, USA; Mayfield Clinic, Cincinnati, OH, USA.
authors.forename: Jed A
authors.initials: JA
authors.lastname: Hartings
mesh_terms: D000241:Adenosine / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation; D003199:Computer Systems; D003217:Conductometry / Q000295:instrumentation*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017743:Equipment Reuse; D045604:Extracellular Fluid / Q000378:metabolism; D008297:Male; D008839:Microelectrodes; D009474:Neurons / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D046888:Tissue Array Analysis / Q000295:instrumentation*
chemical_list: D018377:Neurotransmitter Agents; D000241:Adenosine
keywords: Amperometry; Microdialysis; Voltammetry