Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
J Neurosci Methods ; 329: 108435, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31600528

ABSTRACT

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, KCl-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.


Subject(s)
Brain Chemistry/physiology , Electrochemistry/standards , Electrodes, Implanted , Glutamic Acid/metabolism , Microelectrodes , gamma-Aminobutyric Acid/metabolism , 4-Aminobutyrate Transaminase , Aldehyde Oxidoreductases , Amino Acid Oxidoreductases , Animals , Ceramics , Electrochemistry/instrumentation , Electrochemistry/methods , Feasibility Studies , Male , Rats , Rats, Inbred F344
2.
J Vis Exp ; (135)2018 05 12.
Article in English | MEDLINE | ID: mdl-29806825

ABSTRACT

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.


Subject(s)
Anesthetics/adverse effects , Brain/pathology , Enzyme Assays/methods , Microelectrodes , Neurotoxicity Syndromes/etiology , Anesthetics/pharmacology , Animals , Enzyme Assays/instrumentation , Humans , Neurotoxicity Syndromes/pathology , Swine
3.
Biosens Bioelectron ; 74: 512-7, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26183072

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.


Subject(s)
Adenosine/metabolism , Conductometry/instrumentation , Enzyme-Linked Immunosorbent Assay/instrumentation , Microelectrodes , Neurons/metabolism , Tissue Array Analysis/instrumentation , Animals , Biosensing Techniques/instrumentation , Computer Systems , Equipment Design , Equipment Failure Analysis , Equipment Reuse , Extracellular Fluid/metabolism , Male , Neurotransmitter Agents/metabolism , Rats , Rats, Sprague-Dawley
4.
ACS Chem Neurosci ; 4(5): 721-8, 2013 May 15.
Article in English | MEDLINE | ID: mdl-23650904

ABSTRACT

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.


Subject(s)
Amino Acid Oxidoreductases/drug effects , Cross-Linking Reagents/pharmacology , Enzymes, Immobilized/drug effects , Glutamic Acid/analysis , Glutaral/pharmacology , Amino Acid Oxidoreductases/physiology , Biosensing Techniques , Enzymes, Immobilized/physiology , Microelectrodes
5.
J Neurosci Methods ; 202(2): 199-208, 2011 Nov 15.
Article in English | MEDLINE | ID: mdl-21896284

ABSTRACT

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.


Subject(s)
Acetylcholine/metabolism , Electrochemical Techniques/methods , Glutamic Acid/metabolism , Microelectrodes/standards , Prefrontal Cortex/metabolism , Acetylcholine/analysis , Animals , Electrochemical Techniques/instrumentation , Glutamic Acid/analysis , Male , Prefrontal Cortex/drug effects , Rats , Rats, Inbred F344 , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Time Factors
6.
J Neurosci Methods ; 198(2): 222-9, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21513736

ABSTRACT

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°). 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.


Subject(s)
Ceramics , Electrochemistry/instrumentation , Microelectrodes , Microscopy, Atomic Force , Platinum/chemistry , Surface Properties
7.
J Neurotrauma ; 27(5): 889-99, 2010 May.
Article in English | MEDLINE | ID: mdl-20233041

ABSTRACT

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.


Subject(s)
Brain Injuries/enzymology , Diffuse Axonal Injury/enzymology , Glutamic Acid/metabolism , Potassium/toxicity , Up-Regulation/physiology , Animals , Brain Injuries/pathology , Brain Injuries/physiopathology , Diffuse Axonal Injury/pathology , Diffuse Axonal Injury/physiopathology , Disease Models, Animal , Enzyme Assays/instrumentation , Enzyme Assays/methods , Glutamic Acid/analysis , Male , Microdialysis/instrumentation , Microdialysis/methods , Microelectrodes , Potassium Chloride/toxicity , Rats , Rats, Sprague-Dawley
8.
Biosens Bioelectron ; 23(9): 1382-9, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18243683

ABSTRACT

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.


Subject(s)
Acetylcholine/analysis , Brain Chemistry , Ceramics/chemistry , Choline/analysis , Microelectrodes , Animals , Male , Rats , Rats, Inbred F344
9.
Eur J Neurosci ; 24(10): 2749-57, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17156201

ABSTRACT

Microdialysis has been widely used to measure acetylcholine (ACh) release in vivo and has provided important insights into the regulation of cholinergic transmission. However, microdialysis can be constrained by limited spatial and temporal resolution. The present experiments utilize a microelectrode array (MEA) to rapidly measure ACh release and clearance in anaesthetized rats. The array electrochemically detects, on a second-by-second basis, changes in current selectively produced by the hydrolysis of ACh to choline (Ch) and the subsequent oxidation of choline and hydrogen peroxidase (H(2)O(2)) at the electrode surface. In vitro calibration of the microelectrode revealed linear responses to ACh (R(2) = 0.9998), limit of detection of 0.08 microm, and signal-to-noise ratio of 3.0. The electrode was unresponsive to ascorbic acid (AA), dopamine (DA), or norepinephrine (NE) interferents. In vivo experiments were conducted in prefrontal cortex (PFC) of anaesthetized rats. Pressure ejections of ACh (10 mm; 40 nL) through an adjoining micropipette produced a rapid rise in current, reaching maximum amplitude in approximately 1.0 s and cleared by 80% within 4-11 s. Endogenously released ACh, following local depolarization with KCl (70 mm; 40, 160 nL), was detected at values as low as 0.05 microm. These signals were volume-dependent and cleared within 4-12 s. Finally, nicotine (1.0 mm, 80 nL) stimulated ACh signals. Nicotine-induced signals reflected the hydrolysis of ACh by endogenous acetylcholinesterase (AChE) as inhibition of the enzyme following perfusion with neostigmine (10 microm) attenuated the signal (40-94%). Collectively, these data validate a novel method for rapidly measuring cholinergic transmission in vivo with a spatial and temporal resolution that far exceeds conventional microdialysis.


Subject(s)
Acetylcholine/metabolism , Prefrontal Cortex/metabolism , Analysis of Variance , Animals , Chromatography, High Pressure Liquid/methods , Electrochemistry/methods , Extracellular Space/metabolism , Male , Microdialysis/methods , Microelectrodes , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Prefrontal Cortex/drug effects , Rats , Rats, Wistar
10.
Biosens Bioelectron ; 20(9): 1772-9, 2005 Mar 15.
Article in English | MEDLINE | ID: mdl-15681193

ABSTRACT

A newly developed multisite array microelectrode for in vivo measurements of L-lactate is presented. The resulting microelectrode is composed of three functional layers. First, Nafion is used to repel interfering electroactive anions, such as ascorbate. Second, L-lactate oxidase immobilized onto the recording sites is used to convert L-lactate to hydrogen peroxide. The H2O2 produced is proportional to L-lactate concentrations and is quantified at the platinum recording sites. Third, a layer of polyurethane is coated over the L-lactate oxidase to adjust the linear range of the microelectrode to one that is compatible with in vivo measurements. This layer reduces the amount of L-lactate that diffuses to the enzyme while not significantly limiting oxygen diffusion. The resulting L-lactate microelectrodes were linear to 20 mM (R2 = 0.997 +/- 0.001) and beyond in some cases with detection limits of 0.078 +/- 0.013 mM (n = 12). The selectivity and response time of these electrodes make them suitable for in vivo measurements in brain tissue. Self-referencing recordings may be utilized to further improve the selectivity of the recordings. However this is not necessary for most applications in the brain, because the resting and stimulated levels of dopamine (DA), norepinephrine (NE), and other potentially interfering cations are two to three orders of magnitude lower than that of in vivo L-lactate, which is in the millimolar range. Preliminary in vivo measures of L-lactate in the brain of anesthetized rats support that the microelectrodes are capable of measuring rapid endogenous changes in vivo.


Subject(s)
Brain/metabolism , Ceramics , Electrochemistry/instrumentation , Electrodes, Implanted , Lactic Acid/metabolism , Microelectrodes , Animals , Biocompatible Materials , Electrochemistry/methods , Equipment Design , Equipment Failure Analysis , Lactic Acid/analysis , Materials Testing , Men , Rats , Rats, Inbred F344
11.
J Neurosci Methods ; 121(1): 41-52, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12393160

ABSTRACT

Previously, we developed technology that coupled high-speed chronoamperometry with microejections of dopamine (DA) to measure DA clearance in the brains of freely-behaving rats. Here, by varying the ejection volumes of DA across a 200-fold difference, the kinetics of striatal clearance were analyzed as a function of time and DA volume from 289 chronoamperometric signals (n=20 rats). Each DA clearance trace was fitted to a first-order exponential decay function to determine the rate constant for DA clearance (k). Additionally, the apparent Michaelis-Menten V(max) and K(m) kinetic constants were determined in freely-moving rats, enabling quantitative comparison of our values with other models of reuptake. The first-order rate constant for DA clearance, which reflects the V(max)/K(m) ratio or clearance efficiency, did not vary significantly when small volumes of DA were ejected resulting in peak DA signal amplitudes (A(max)) of <5 microM. However, following nomifensine-induced DAT inhibition, A(max) was increased and k was attenuated simultaneously with behavioral activation; and A(max) and behavior remained elevated beyond the initial period. Our results indicate that the analysis of kinetic parameters from chronoamperometric DA signals may be useful for investigating drug-induced regulation of DAT kinetics in relation to the behavior of freely-moving rats.


Subject(s)
Corpus Striatum/metabolism , Dopamine/pharmacokinetics , Electrochemistry/methods , Animals , Corpus Striatum/cytology , Corpus Striatum/drug effects , Dopamine/analysis , Dopamine Uptake Inhibitors/pharmacology , Dose-Response Relationship, Drug , Kinetics , Male , Metabolic Clearance Rate , Microinjections/methods , Motor Activity/drug effects , Nomifensine/pharmacology , Rats , Rats, Sprague-Dawley , Time Factors
12.
J Neurosci Methods ; 119(2): 163-71, 2002 Sep 30.
Article in English | MEDLINE | ID: mdl-12323420

ABSTRACT

This paper describes improvements and further characterization of a ceramic-based multisite microelectrode for in vivo measurements of L-glutamate. Improvements include increased recording area, insulation deposition using photolithography for more uniform recording sites and forming the microelectrodes using a diamond saw providing smoother microelectrode edges. The new microelectrodes are triangular in shape, 1 cm in length and taper from 1 mm to a 2-5 microm tip. Details on performing in vivo measurements are given, including microelectrode preparation, pitfalls of the recording method and approaches to enhance reproducibility of the technique. The detection limit for L-glutamate was lowered to approximately 0.5 microM and a self-referencing recording technique was utilized to remove interferents as well as decrease noise. Applications of the microelectrodes to study L-glutamate uptake and release in rat prefrontal cortex, cortex, cerebellum and striatum are included.


Subject(s)
Central Nervous System/metabolism , Ceramics , Glutamic Acid/analysis , Microdialysis/instrumentation , Animals , Ceramics/chemistry , Glutamic Acid/metabolism , Male , Microdialysis/methods , Microelectrodes/standards , Rats , Rats, Inbred F344
SELECTION OF CITATIONS
SEARCH DETAIL
...