Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that is essential for normal brain function. It is involved in multiple neuronal activities, including plasticity, information processing, and network synchronization. Abnormal GABA levels result in severe brain disorders and therefore GABA has been the target of a wide range of drug therapeutics. GABA being non-electroactive is challenging to detect in real-time. To date, GABA is detected mainly via microdialysis with a high-performance liquid chromatography (HPLC) system that employs electrochemical (EC) and spectroscopic methodology. However, these systems are bulky and unsuitable for real-time continuous monitoring. As opposed to microdialysis, biosensors are easy to miniaturize and are highly suitable for in vivo studies; they selectively oxidize GABA into a secondary electroactive product (usually hydrogen peroxide, H2O2) in the presence of enzymes, which is then detected by amperometry. Unfortunately, this method requires a rather cumbersome process with prereactors and relies on externally applied reagents. Here, we report the design and implementation of a GABA microarray probe that operates on a newly conceived principle. It consists of two microbiosensors, one for glutamate (Glu) and one for GABA detection, modified with glutamate oxidase and GABASE enzymes, respectively. By simultaneously measuring and subtracting the H2O2 oxidation currents generated from these microbiosensors, GABA and Glu can be detected continuously in real-time in vitro and ex vivo and without the addition of any externally applied reagents. The detection of GABA by this probe is based upon the in-situ generation of α-ketoglutarate from the Glu oxidation that takes place at the Glu microbiosensor. A GABA sensitivity of 36 ± 2.5 pA μM-1cm-2, which is 26-fold higher than reported in the literature, and a limit of detection of 2 ± 0.12 μM were achieved in an in vitro setting. The GABA probe was successfully tested in an adult rat brain slice preparation. These results demonstrate that the developed GABA probe constitutes a novel and powerful neuroscientific tool that could be employed in the future for in vivo longitudinal studies of the combined role of GABA and Glu (a major excitatory neurotransmitter) signaling in brain disorders, such as epilepsy and traumatic brain injury, as well as in preclinical trials of potential therapeutic agents for the treatment of these disorders.

Glutamate (GLU) and γ-aminobutyric acid (GABA) are the major excitatory (E) and inhibitory (I) neurotransmitters in the brain, respectively. Dysregulation of the E/I ratio is associated with numerous neurological disorders. enzyme-based microelectrode array biosensors present the potential for improved biocompatibility, localized sample volumes, and much faster sampling rates over existing measurement methods. However, enzymes degrade over time. To overcome the time limitation of permanently implanted microbiosensors, we created a microwire-based biosensor that can be periodically inserted into a permanently implanted cannula. Biosensor coatings were based on our previously developed GLU and reagent-free GABA shank-type biosensor. In addition, the microwire biosensors were in the same geometric plane for the improved acquisition of signals in planar tissue including rodent brain slices, cultured cells, and brain regions with laminar structure. We measured real-time dynamics of GLU and GABA in rat hippocampal slices and observed a significant, nonlinear shift in the E/I ratio from excitatory to inhibitory dominance as electrical stimulation frequency increased from 10 to 140 Hz, suggesting that GABA release is a component of a homeostatic mechanism in the hippocampus to prevent excitotoxic damage. Additionally, we recorded from a freely moving rat over fourteen weeks, inserting fresh biosensors each time, thus demonstrating that the microwire biosensor overcomes the time limitation of permanently implanted biosensors and that the biosensors detect relevant changes in GLU and GABA levels that are consistent with various behaviors. Near real-time measurement of neurotransmitter levels in the brain is expected to reveal important clues to the underlying mechanisms of neurological disorders, such as epilepsy, addiction, motor control 1-3 , and secondary damage after stroke 4 and traumatic brain injury 5,6. Two important and ubiquitous neurotransmitters in the brain are l-glutamine (GLU) and γ-aminobutyric acid (GABA), which are the major excitatory and inhibitory neurotransmitters, respectively. A balance of excitatory (E) and inhibitory (I) electrical signaling in local networks (E/I ratio) is observed in awake states and in sleep, with the exception of slow-wave sleep in humans and monkeys 7. Dysregulation in E/I is closely linked to dysregulation of GLU and/or GABA dynamics 8. Prolonged, excessive GLU release often leads to excitotoxic neuronal cell death directly or through a cascade of secondary cellular injury 9,10. Furthermore, insufficient release of GABA is associated with seizures 6,11 and autism 12. Most studies so far have only looked at GLU and GABA in isolation 13,14. However, a more meaningful interpretation of the mechanisms of disease and secondary injury can be obtained by knowing the regional E/I balance 15. Furthermore,

The effect of process parameters on the electrochemical performance of a glutamate (Glu) microbiosensor was studied. Glu is a major excitatory neurochemical that is essential for regulation of several brain functions. Glu dysregulation is associated with several neuropathological conditions such as epilepsy, Parkinson's and Alzheimer's disease. Here, an enzymatic microbiosensor in the form of a ceramic-shank substrate enabled platinum (Pt) microelectrode array (MEA) was employed to investigate the effect of key process parameters such as MEA surface cleaning methods, the effects of GluOx concentration, GluOx loading and sizeexclusion layer thicknesses on Glu sensitivity and selectivity towards common interferents. A two-step cleaning procedure demonstrated a 57.4% increase in hydrogen peroxide sensitivity, which is due to 80-fold and 2-fold reductions respectively in the charge transfer resistances of the Pt microelectrode grains and grain boundaries. The microbiosensor demonstrated an excellent combination of sensitivity (530 ± 34 nA cm −2 μM −1 ), selectivity (841 ± 54) and long-term stability (2 months). These results provide a detailed account of changes in electrochemical properties and sensitivities of Pt microelectrodes toward peroxide, the rationale for choosing a higher GluOx concentration (0.4 U/μl) instead of 0.1 U/μl, which is the most commonly used concentration in the literature and recommendations on the enzyme loading and placement of size-exclusion coatings and their deposition conditions, which significantly affects the biosensor performance.

A high-resolution, three-dimensional, optical imaging technique for the murine brain was developed to identify the effects of different therapeutic windows for preclinical brain research. This technique tracks the same cells over several weeks. We conducted a pilot study of a promising drug to treat diffuse axonal injury (DAI) caused by traumatic brain injury, using two different therapeutic windows, as a means to demonstrate the utility of this novel longitudinal imaging technique. DAI causes immediate, sporadic axon damage followed by progressive secondary axon damage. We administered minocycline for three days commencing one hour after injury in one treatment group and beginning 72 hours after injury in another group to demonstrate the method’s ability to show how and when the therapeutic drug exerts protective and/or healing effects. Fewer varicosities developed in acutely treated mice while more varicosities resolved in mice with delayed treatment. For both treatments, the drug arrested development of new axonal damage by 30 days. In addition to evaluation of therapeutics for traumatic brain injury, this hybrid microlens imaging method should be useful to study other types of brain injury and neurodegeneration and cellular responses to treatment.