Jason J. Burmeister

Improved ceramic-based multisite microelectrode for rapid measurements of L-glutamate in the CNS.

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.

Microelectrode array studies of basal and potassium-evoked release of l-glutamate in the anesthetized rat brain

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 µm) 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.

Ceramic-based multisite microelectrode arrays for simultaneous measures of choline and acetylcholine in CNS

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.

Diffuse Brain Injury Elevates Tonic Glutamate Levels and Potassium-Evoked Glutamate Release in Discrete Brain Regions at Two Days Post-Injury: An Enzyme-Based Microelectrode Array Study

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.

Noninvasive Blood Glucose Measurements by Near-Infrared Transmission Spectroscopy Across Human Tongues

Noninvasive blood glucose measurements are characterized in human subjects. A series of first overtone transmission spectra are collected across the tongues of five human subjects with type 1 diabetes. The noninvasive human spectra are collected by an experimental protocol that is designed to minimize chance correlations with blood glucose levels. In one treatment of the data, every fifth sample is used as a blind prediction point to validate model performance. In another rearrangement of the data, the spectra collected over the first 29 days are used to build calibration models that are then used to predict in vivo glycemia from spectra collected over the next 10 days. Of the five data sets (one for each subject), one demonstrates a complete inability to predict blood glucose levels and is deemed void of glucose-specific information. Glucose-specific information is evident in the remaining four data sets, albeit to varying degrees. For all data sets, the ability to measure glucose from spectra collected noninvasively from human subjects depends on spectral quality and reproducibility of the tongue-to-spectrometer interface. The standard error of prediction is 3.4 mM for the best calibration model. The significance of this magnitude of prediction error is discussed relative to the situations where: (1) the model is completely void of glucose-specific information and (2) glucose predictions are limited by spectral signal-to-noise and sample thickness. Overall, glucose-specific information is available from noninvasive first-overtone spectra collected across human tongues. Significant improvements are necessary, however, before clinically useful measurements are possible.

Kinetic analysis of striatal clearance of exogenous dopamine recorded by chronoamperometry in freely-moving rats

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.

Second-by-second measurement of acetylcholine release in prefrontal cortex.

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 (H2O2) at the electrode surface. In vitro calibration of the microelectrode revealed linear responses to ACh (R2 = 0.9998), limit of detection of 0.08 µm, 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 ∼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 µm. 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 µm) 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.

Ceramic-based multisite microelectrode arrays for in vivo electrochemical recordings of glutamate and other neurochemicals

Ceramic-based microelectrode arrays for electrochemical measures of neurochemicals in brain and spinal cord tissue are reviewed. Current fabrication procedures are explained, including discussions of various recording site layouts, insulating materials and recording strategies. Various coatings are employed to alter the sensitivity and the selectivity of the microelectrodes for the detection of certain neurochemicals. Several applications of these microelectrode arrays are discussed.

Phantoms for noninvasive blood glucose sensing with near infrared transmission spectroscopy

In vivo spectra from human subjects can be simulated with a phantom composed of different layers of water, fat and muscle tissue. All three components are necessary to simulate in vivo spectra collected over the combination spectral region (5000–4000 cm−1). Muscle tissue is not required, however, to accurately simulate overtone spectra (6600–5400 cm−1). The near‐IR spectral characteristics of fat and muscle tissue from several animal sources are essentially identical to those found for human tissue, hence, the animal source for these phantom components is not critical. Thickness of each tissue layer can be determined by a regression analysis where the in vivo spectrum of interest is regressed against standard absorbance spectra of the necessary model components (water, fat and muscle). In general, in vivo overtone spectra collected across human webbing tissue with a thickness of 6.7 mm can be simulated with water layer thicknesses ranging from 5.0 to 6.4 mm combined with fat layer thicknesses from 1.4 to 4.2 mm.

Tonic and Phasic Release of Glutamate and Acetylcholine Neurotransmission in Sub-regions of the Rat Prefrontal Cortex Using Enzyme-based Microelectrode Arrays

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.