A self-referencing technique utilizing two microelectrodes on a ceramic-based multisite array is employed for confirmation and elimination of interferences detected by enzyme-based microelectrodes. The measurement of L-glutamate using glutamate oxidase was the test system; however, other oxidase enzymes such as glucose oxidase can be employed. One recording site was coated with Nafion with L-glutamate oxidase and bovine serum albumin (BSA) cross-linked with glutaraldehyde while the other had Nafion with BSA cross-linked with glutaraldehyde. Differences in the chemistry of the two recording sites allowed for identification and elimination of interfering signals to be removed from the analyte response. The electrode showed low detection limits (LOD = 0.98 +/- 0.09 microM, signal-to-noise ratio of 3), fast response times (T90 approximately 1 s), and excellent linearity (R2 = 0.999 +/- 0.000) over the concentration range of 0-200 microM for calibrations of L-glutamate in vitro. The selectivity and dimensions of the multisite electrode allow in vivo glutamate measurements. This electrode has been applied to in vivo measurements of the clearance of locally applied glutamate and release of glutamate in the prefrontal cortex of anesthetized rats. In addition, a aimilar approach has been applied to the development of a microelectrode for measures of glucose.

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L-glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system. It plays major roles in normal neurophysiology and many brain disorders by binding to membrane-bound Glu receptors. To overcome the spatial and temporal limitations encountered in previous in vivo extracellular Glu studies, we employed enzyme-coated microelectrode arrays to measure both basal and potassium-evoked release of Glu in the anesthetized rat brain. We also addressed the question of signal identity, which is the predominant criticism of these recording technologies. In vivo self-referencing recordings demonstrated that our Glu signals were both enzyme-and voltage-dependent, supporting the identity of L-glutamate. In addition, basal Glu was actively regulated, tetrodotoxin (TTX)-dependent, and measured in the low micromolar range (approximately 2 lM) using multiple self-referencing subtraction approaches for identification of Glu. Moreover, potassium-evoked Glu release exhibited fast kinetics that were concentration-dependent and reproducible. These data support the hypothesis that Glu release is highly regulated, requiring detection technologies that must be very close to the synapse and measure on a second-by-second basis to best characterize the dynamics of the Glu system.

This paper describes the development and characterization of ceramic-based multisite arrays for electrochemical recordings in biological systems. These electrodes represent a parallel technology to the design of microelectrodes using silicon substrates. The ceramic substrates are stronger than silicon and are nonconducting, which makes them better suited for in vivo electrochemical measurements. The current designs are based on formation of four-site (50 x 50 microns with 200 microns spacing) electrodes on ceramic wafers using photolithography. The recording sites and connecting lines are made of Pt with a polyimide coating to insulate the connecting lines. The resulting electrodes are cut from the wafers producing a 1 cm length microelectrode that tapers to a approximately 2-5 microns tip. Electrochemical measures of dopamine and hydrogen peroxide support that the sensitivity, selectivity, and response characteristics of the electrodes exceed those of previously published silicon substrate-based microelectrodes. This is the first demonstration of microarrays formed from ceramic substrates, and the data presented support the hypothesis that these microelectrodes may be useful for a variety of neurochemical and electrophysiological applications. Preliminary in vivo electrochemical recordings are presented.

Traumatic brain injury (TBI) survivors often suffer from a wide range of post-traumatic deficits, including impairments in behavioral, cognitive, and motor function. Regulation of glutamate signaling is vital for proper neuronal excitation in the central nervous system. Without proper regulation, increases in extracellular glutamate can contribute to the pathophysiology and neurological dysfunction seen in TBI. In the present studies, enzyme-based microelectrode arrays (MEAs) that selectively measure extracellular glutamate at 2 Hz enabled the examination of tonic glutamate levels and potassium chloride (KCl)-evoked glutamate release in the prefrontal cortex, dentate gyrus, and striatum of adult male rats 2 days after mild or moderate midline fluid percussion brain injury. Moderate brain injury significantly increased tonic extracellular glutamate levels by 256% in the dentate gyrus and 178% in the dorsal striatum. In the dorsal striatum, mild brain injury significantly increased tonic glutamate levels by 200%. Tonic glutamate levels were significantly correlated with injury severity in the dentate gyrus and striatum. The amplitudes of KCl-evoked glutamate release were increased significantly only in the striatum after moderate injury, with a 249% increase seen in the dorsal striatum. Thus, with the MEAs, we measured discrete regional changes in both tonic and KCl-evoked glutamate signaling, which were dependent on injury severity. Future studies may reveal the specific mechanisms responsible for glutamate dysregulation in the post-traumatic period, and may provide novel therapeutic means to improve outcomes after TBI.