Here we report a novel microelectrode array recording approach to measure tonic (resting) and phasic release of dopamine (DA) in DA-rich areas such as the rat striatum and nucleus accumbens. The resulting method is tested in intact central nervous system (CNS) and in animals with extensive loss of the DA pathway using the neurotoxin, 6-hydroxyDA (6-OHDA). The self-referencing amperometric recording method employs Nafion-coated with and without m-phenylenediamine recording sites that through real-time subtraction allow for simultaneous measures of tonic DA levels and transient changes due to depolarization and amphetamine-induced release. The recording method achieves low-level measures of both tonic and phasic DA with decreased recording drift allowing for enhanced sensitivity normally not achieved with electrochemical sensors in vivo.

Background: Although GABA is the major inhibitory neurotransmitter in the CNS, quantifying in vivo GABA levels has been challenging. The ability to co-monitor both GABA and the major excitatory neurotransmitter, glutamate, would be a powerful tool in both research and clinical settings. New method: Ceramic-based microelectrode arrays (MEAs) were used to quantify gamma-aminobutyric acid (GABA) by employing a dual-enzyme reaction scheme including GABase and glutamate oxidase (GluOx). Glutamate was simultaneously quantified on adjacent recording sites coated with GluOx alone. Endogenous glutamate was subtracted from the combined GABA and glutamate signal to yield a pure GABA concentration. Results: Electrode sensitivity to GABA in conventional, stirred in vitro calibrations at pH 7.4 did not match the in vivo sensitivity due to diffusional losses. Non-stirred calibrations in agarose or stirred calibrations at pH 8.6 were used to match the in vivo GABA sensitivity. In vivo data collected in the rat brain demonstrated feasibility of the GABA/glutamate MEA including uptake of locally applied GABA, KCI-evoked GABA release and modulation of endogenous GABA with vigabatrin. Comparison with existing methods: Implantable enzyme-coated microelectrode arrays have better temporal and spatial resolution than existing off-line methods. However, interpretation of results can be complicated due to the multiple recording site and dual enzyme approach. Conclusions: The initial in vitro and in vivo studies supported that the new MEA configuration may be a viable platform for combined GABA and glutamate measures in the CNS extending the previous reports to in vivo GABA detection. The challenges of this approach are emphasized.

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain. This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

Direct electrochemical measurements of glutamate release in vivo were combined with optogenetics in order to examine light-induced control of glutamate neurotransmission in the rodent brain. Self-referenced recordings of glutamate using ceramic-based microelectrode arrays (MEAs) in hippocampus and frontal cortex demonstrated precise optical control of light-induced glutamate release through channelrhodopsin (ChR2) expression in both rat hippocampus and frontal cortex. Although the virus was only injected unilaterally, bilateral and rostro-caudal expression was observed in slice imaging, indicating diffusion and active transport of the viral particles. Methodology for the optogenetic control of glutamate signaling in the rat brain is thoroughly explained with special attention paid to MEA enzyme coating and cleaning for the benefit of other investigators. These data support that optogenetic control of glutamate signaling is robust with certain advantages as compared to other methods to modulate the in vivo control of glutamate signaling.

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.

A major goal of our research is to develop an implantable device for routine amperometric recordings of l-glutamate and other neurotransmitters in the mammalian central nervous system. Specifically, we wanted to develop a microelectrode that is (1) mass produced such that other laboratories can easily utilize the same recording technology, (2) designed to study multiple brain regions and neurotransmitters in various in vitro and in vivo systems, and (3) configured for “self-referencing” recordings, which allows for measurements of resting or tonic levels of neurotransmitters, cross-checking of the selectivity of the microelectrode measures, and improved signal-to-noise ratio by noise subtraction. The present chapter documents our current capabilities of measuring l-glutamate and several other neurotransmitters with rapid temporal resolution using mass-fabricated microelectrode arrays formed on ceramic. We have routinely demonstrated that these electrodes have fast temporal resolution (<1 s), excellent spatial resolution (microns), and low detection limits (≤200 nM) and cause minimal damage (50–100 μm) to surrounding brain tissue. While not a comprehensive assessment of the technology, this chapter contains a large amount of information regarding the fabrication, use, and potential pitfalls of this technology.

Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observe comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific "wet" coating procedures.

The medial prefrontal cortex (mPFC) is an area of the brain critical for higher cognitive processes and implicated in disorders of the CNS such as drug addiction, depression and schizophrenia. Glutamate and acetylcholine are neurotransmitters that are essential for cortical functioning, yet little is known about the dynamic function of these neurotransmitters in subregions of the mPFC. In these studies we used a novel microelectrode array technology to measure resting levels (tonic release) of glutamate and acetylcholine as well as KCl-evoked release (stimulated phasic release) in the mPFC of the anesthetized rat to further our understanding of both tonic and phasic neurotransmission in the cingulate cortex, prelimbic cortex, and infralimbic cortex of the mPFC. Studies revealed homogeneity of tonic and phasic signaling among brain subregions for each neurotransmitter. However, resting levels of glutamate were significantly higher as compared to acetylcholine levels in all subregions. Additionally, KCl-evoked acetylcholine release in the cingulate cortex (7.1 μM) was significantly greater than KCl-evoked glutamate release in any of the three subregions (Cg1, 2.9 μM; PrL, 2.0 μM; IL, 1.8 μM). Interestingly, the time for signal decay following KCl-evoked acetylcholine release was significantly longer by an average of 240% as compared to KCL-evoked glutamate release for all three brain subregions. Finally, we observed a negative relationship between acetylcholine resting levels and KCl-evoked release in the Cg1. These data suggest a homogenous distribution of both glutamatergic and acetylcholinergic innervation in the mPFC, with alterations in tonic and phasic release regulation accounting for differences between these neurotransmitters.

Amperometric measurements using microelectrode arrays (MEAs) provide spatially and temporally resolved measures of neuromolecules in the central nervous system of rats, mice and non-human primates. Multi-site MEAs can be mass fabricated on ceramic (Al2O3) substrate using photolithographic methods, imparting a high level of precision and reproducibility in a rigid but durable recording device. Although the functional capabilities of MEAs have been previously documented for both anesthetized and freely moving paradigms, the performance enabling intrinsic physical properties of the MEA device have not heretofore been presented. In these studies, spectral analysis confirmed that the MEA recording sites were primarily composed of elemental platinum (Pt degrees). In keeping with the precision of the photolithographic process, scanning electron microscopy revealed that the Pt recording sites have unique microwell geometries post-fabrication. Atomic force microscopy demonstrated that the recording surfaces have nanoscale irregularities in the form of elevations and depressions, which contribute to increased current per unit area that exceeds previously reported microelectrode designs. The ceramic substrate on the back face of the MEA was characterized by low nanoscale texture and the ceramic sides consisted of an extended network of ridges and cavities. Thus, individual recording sites have a unique Pt composition and surface profile that has not been previously observed for Pt-based microelectrodes. These features likely impact the physical chemistry of the device, which may influence adhesion of biological molecules and tissue as well as electrochemical recording performance post-implantation. This study is a necessary step towards understanding and extending the performance abilities of MEAs in vivo. (C) 2011 Elsevier B.V. All rights reserved.

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.

A major challenge in the elucidation of brain neurotransmitter events in vivo in the laboratory and in the operating room has been the development of methods to record neurotransmitters in the CNS on a second-by-second time scale. Microdialysis methods have been widely employed but are limited due to their inherent slow sampling rates (1–20 min) and poor exchange (∼10–20%) of neurotransmitters. We have developed an enzyme-based ceramic microelectrode array (MEA) for 2Hz measures of tonic (resting) and phasic (spontaneous bursts or stimulated) release of neurotransmitters. We have used the most recent designs for reproducible, sensitive, selective and rapid measures of L-glutamate and other neurotransmitters in rats, mice and monkeys. In conjunction with Ad-Tech Medical Instruments Inc., we have recently developed a new MEA design for nonhuman primate and human recordings that lays the foundation for potential use of this technology during neurosurgery for intra-operative chemical diagnostics.

Researchers and clinicians have become intrigued with coupling seizure prediction and local electrical stimulation or pharmacotherapy for treatment of the approximately one million individuals with persistent uncontrolled epilepsy. Critical for interventions aimed at halting seizure onset are—the ability to detect and characterize aberrant neuronal activity indicative of imminent seizure and the ability to maintain cognitive function normally supported by the brain tissue that must be treated to prevent seizures. This chapter reviews the research on the development and refinement of an enzyme-based, mass-fabricated microelectrode array technology that can be implanted into the mammalian central nervous system and utilized for second-by-second assessment of localized glutamate neurotransmission. Based upon the experience, extracellular fluctuations in glutamate do not exhibit the spontaneity and complexity observed in the electrical characteristics of aberrantly firing neurons. Predictive modeling based upon chemical neuronal communication may present a very accurate seizure detection system with minimal false-positives and false-negatives currently complicating electroencephalography (EEG)-based seizure prediction. Though a prediction and local intervention therapeutic paradigm could offer a dramatic improvement in treatment of refractory seizures, it is proposed that achieving the ultimate goal of enhancing the daily functioning of these patients necessitates consideration of consequences experienced by the patient when hippocampal circuitry is “shut-down” to thwart seizure onset. The prosthetic technology utilizes non-linear modeling to generate outputs characteristic of the normal functioning of the epileptic hippocampal circuit during the time when that circuit must be “shut down” to suppress seizure. The outputs are computed from afferent neuronal input recorded from an indwelling microelectrode array “upstream” from the impaired hippocampal tissue so as to bypass the malfunctioning portion of the hippocampus.

Researchers and clinicians have become intrigued with coupling seizure prediction and local electrical stimulation or pharmacotherapy for treatment of the approximately one million individuals with persistent uncontrolled epilepsy in the USA. Critical for interventions aimed at halting seizure onset are (i) the ability to detect and characterize aberrant neuronal activity indicative of imminent seizure and (ii) the ability to maintain cognitive function normally supported by the brain tissue that must be treated to prevent seizures. A major goal of our research has been the development and refinement of an enzyme-based, mass-fabricated microelectrode array technology that can be implanted into the mammalian central nervous system and utilized for second-by-second assessment of localized glutamate neurotransmission. Based upon our experience, extracellular fluctuations in glutamate do not exhibit the spontaneity and complexity observed in the electrical characteristics of aberrantly firing neurons. We believe predictive modeling based upon chemical neuronal communication may present a very accurate seizure detection system with minimal false-positives and false-negatives currently complicating electroencephalography (EEG)-based seizure prediction. Though a prediction and local intervention therapeutic paradigm could offer a dramatic improvement in treatment of refractory seizures, we propose that achieving the ultimate goal of enhancing the daily functioning of these patients necessitates consideration of consequences experienced by the patient when hippocampal circuitry is 'shut-down' to thwart seizure onset. Therapeutic termination of seizure activity may produce memory-loss and other gross impairments of information processing in the brain; therefore, the second goal of our research stated above is the development of a hippocampal prosthesis to 'patch' therapeutically impaired hippocampal circuitry. Our prosthetic technology utilizes non-linear modeling to generate outputs characteristic of the normal functioning of the epileptic hippocampal circuit (i.e. when that circuit is not epileptiform) during the time when that circuit must be 'shut down' to suppress seizure. The outputs are computed from afferent neuronal input recorded from an indwelling microelectrode array 'upstream' from the impaired hippocampal tissue so as to bypass the malfunctioning portion of the hippocampus. The present chapter documents our current capabilities of measuring glutamate in rodents, primates and humans with in vivo amperometry and our progress with a hippocampal prosthesis, as we work toward development of a 'closed-loop' system for seizure prediction and termination with maintenance of normal hippocampal function.

A major challenge in the elucidation of neurotransmitter events in vivo has been the development of microelectrode technologies, which can selectively record second-by-second changes in neurotransmitters such as glutamate, acetylcholine and GABA. Historically, microelectrodes have not had the capabilities to allow for subtraction of noise and interferents analogous to other analytical methods. Microelectrode array (MEA) technology can now be used for measures of basal or resting levels of neurotransmitters as well as evoked overflow due to stimulation or behavior-induced events [1,2}. Our group has been working on a new generation of MEAs, which are micro-fabricated using photolithographic techniques. Our new designs employ Al2O3 substrates patterned with Pt or Ir surfaces and are coated with polyimide layers for insulation. The resulting 4-16 recording site arrays are modified with enzymes and organic molecules to make them sensitive and selective for the detection of a variety of molecules, including but not limited to glutamate, glucose, aspartate, choline, acetylcholine, lactate, GABA and others such as peroxide and alcohol. We have extensively focused on the development of microelectrodes for the reproducible, sensitive, selective and rapid measures of L-glutamate, which is the major excitatory neurotransmitter in the CNS. In this report, we present the latest data from our group on the assembly, characterization, and testing of self-referencing MEAs for in vivo brain measurements of L-glutamate carried out in rats, mice and non-human primates.

A ceramic-based microelectrode array (MEA) with enzyme coatings for the accurate measurement of acetylcholine (ACh) in brain tissues is presented. Novel design features allow for self-referencing recordings for improved limits of detection and highly selective measurements of ACh and choline (Ch), simultaneously. Design and fabrication features also result in minimal tissue damage during implantation and improved enzyme coatings due to isolated recording sites. In these studies we have used a recombinant human acetylcholinesterase enzyme coating, which has better reproducibility than other commercially available enzymes. The precisely patterned recording site dimensions, low limit of detection (0.2 micro M) and fast response time ( approximately 1s) allow for second-by-second measurements of ACh and Ch in brain tissues. An electropolymerized meta-phenylenediamine (mPD) layer was used to exclude interfering substances from being recorded at the platinum recording sites. Our studies support that the mPD layer was stable for over 24h under in vitro and in vivo recording conditions. In addition, our work supports that the current configuration of the MEAs produces a robust design, which is suited for measures of ACh and Ch in rat brain.

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and is implicated in a number of brain disorders including Parkinson’s disease (PD), cognitive disturbances, epilepsy, schizophrenia, attention deficit hyperactivity disorder (ADHD) and drug abuse. While microdialysis methods have been used extensively over the last decade to investigate minute-by-minute measures of L-glutamate, the rapid time dynamics of L-glutamate signaling in the CNS has warranted a technique to measure L-glutamate release on a second-by- second basis. A major goal of the research is to develop a recording technology for recording second-by-second measurements of L-glutamate and other neurotransmitters—specifically a mass-fabricated microelectrode technology that could be (1) mass produced such that other laboratories could utilize the same recording technology and (2) configured for “self-referencing” recordings, which allows for second-by-second cross-checking of the selectivity of the micro-electrode measures and improved signal-to-noise of the recording methods. The present chapter documents current capabilities of measuring L-glutamate and several other neurotransmitters on a second-by-second basis using mass-fabricated microelectrode arrays formed on ceramic. While not a comprehensive assessment of the technology, this chapter contains a large amount of information regarding the fabrication, use, and potential pitfalls of this technology. The reader should refer to numerous articles [1–6] for additional details regarding measuring neurotransmitters in the CNS.