Challenges of Simultaneous Measurements of Brain Extracellular GABA and Glutamate In Vivo Using Enzyme-Coated Microelectrode Arrays

Jason J. Burmeister‡, David A. Price‡†, Francois Pomerleau, Peter Huettl, Jorge E. Quintero and

Greg A. Gerhardt*

Neuroscience, Center for Microelectrode Technology, University of Kentucky, College of Medicine, Lex-ington, KY, USA.*Corresponding Author: Department of Neuroscience, MN206, 800 Rose St., Lexington, KY 40536-0298Office: 859-323-4531, Fax: 859-257-5310, gregg@uky.edu

Highlights:

GABA and glutamate were simultaneously measured using microelectrode arrays. Signals obtained with multiple enzymes were subtracted to yield GABA. Measurement challenges include calibration mismatch and recording site mismatch. In vivo measures of GABA and glutamate were demonstrated in rat brain. Endogenous GABA was modulated with vigabatrin and detected.

ABSTRACT: Background Background: Although GABA is the major inhibitory neurotransmitter in the CNS, quan-tifying 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-aminobu-tyric acid (GABA) by employing a dual-enzyme reaction scheme including GABase and glutamate oxi-dase (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 re-ports to in vivo GABA detection. The challenges of this approach are emphasized.

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Keywords(6): GABA, glutamate, sensor, microelectrode array, -aminobutyric acid, brain

Abbreviations: -aminobutyric acid (GABA); glutamate (Glu); central nervous system (CNS); micro-electrode arrays (MEAs); m-phenylenediamine (mPD), -ketoglutarate (KG), bovine serum albumin (BSA), hydrogen peroxide (H2O2); such as dopamine (DA), ascorbate (AA), dihydroxyphenylacetic acid (DOPAC); -aminobutyric glutamic transaminase (GABGT); succinic semialdehyde dehydrogenase (SSDH); glutamate oxidase (GluOx); frontal cortex (FC); local field potentials (LFP); Glutaraldehyde (Glut).

1. Introduction-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter and glutamate (Glu) is the major excitatory neurotransmitter in the central nervous system (CNS). These neurotransmitters are among the most ubiquitous neurotransmitters in the CNS and they are implicated in a number of brain disor-ders including epilepsy, depression, schizophrenia, and drug abuse. Because of their widespread avail-ability in the brain, the ability to co-monitor both neurotransmitters would be a powerful tool in both re-search and clinical settings. Although monitoring glutamate has become more routine in many laborato-ries, quantifying in vivo GABA levels has been difficult. GABA is not electroactive and current method-ologies (e.g. microdialysis) hinder the detection of sub-second changes in extracellular GABA levels alongside poor spatial and temporal resolutions of such approaches.1-3 Thus, there is a need for the development and use of technologies for rapid, reliable and spatially resolved measures of in vivo GABA, which would be further improved upon if they could be simultaneously used to measure gluta-mate.

Other labs have developed techniques to quantify in vivo GABA alone. Optical techniques include a flu-orescent sensor, which quantifies GABA on the surface of cells,4 surface-enhanced Raman spec-troscopy (SERS),5 a fiber-optic sensor for utilizing near-infrared plasmonic electromagnetic field en-hancement using raspberry-like meso-SiO2 nanospheres,6 and a fiber optic sensor based on the NADP+ functionalized quantum dots.7 Extra-synaptic GABA has also been quantified using a whole-cell sniffer.8 Moreover, an on-line sensor with enzymes, GABase (GABA-transaminase + succinic semi-aldehyde dehydrogenase) and glutamate oxidase (GluOx), has been reported whereby GABA is con-verted to glutamate then glutamate to H2O2, which is quantified using an electrode.9

Our laboratory has developed and implemented ceramic-based microelectrode arrays (MEAs) for rapid (≤ 1 Hz) measurements of neurochemicals in vivo.10 The versatility of MEAs for this purpose is a direct result of the multiple platinum (Pt) electrodes and, in turn, the ability to utilize different geometrically ar-ranged electrode configurations. Pt recording sites can be selectively coated with oxidase enzymes to transform a molecule of interest, which is not inherently electroactive, to an oxidizable reporter mole-cule (e.g. H2O2) that is oxidized upon contact with the electrode surface. In addition, self-referencing methods can be incorporated to ascertain chemical specificity for the molecule of interest while afford-ing the opportunity to measure tonic or basal neurotransmitter levels. In turn, we have successfully used enzyme-based MEAs to reliably measure brain extracellular acetylcholine,11 choline,12,13 gluta-mate,14 lactate,15 adenosine,16 and glucose.17 Similar to the detection of acetylcholine, a dual enzyme approach is utilized here to detect GABA.18 Like Hossain and colleagues,19 detection of GABA in vitro was made possible by adding GABase to our implantable glutamate (Glu) MEA (Figure 1) and using a S2 MEA design.

Here, we describe, further validate and optimize the use of this technology to measure GABA and glu-tamate in vitro and in vivo. In addition, we discuss and address complications and challenges of the multiple enzyme approach. We discuss the fundamental principles of these cutting-edge measure-ments and provide proof-of-concept studies in vivo.

2. Material and methods

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2.1 Reagents. All chemicals were used as received unless stated otherwise. GABase, GABA, m-phenylenediamine (mPD), -ketoglutarate (KG), ascorbic acid, sodium chloride, sodium L-glutamate (Glu), glutaraldehyde, bovine serum albumin (BSA), hydrogen peroxide (H2O2, 30%), dopamine, ascor-bate, vigabatrin, dibasic sodium phosphate, and monobasic sodium phosphate were obtained from Sigma. Agarose was obtained from Green Bioresearch. Glutamate oxidase (GluOx) was obtained from US Biological Life Sciences. Glutaraldehyde was stored at -20oC. Nitrile gloves and a respirator mask or fume hood were employed while working with mPD and glutaraldehyde.

Figure 1. Schematic of S2 MEA configured for GABA-glutamate detection. A Sites. GABA-recording sites are coated with GABase and glutamate oxidase (GluOx) and thus are sensitive to GABA and glu-tamate (Glu). B Sites. Sentinel sites are coated with glutamate oxidase and are only sensitive to gluta-mate. All sites were electroplated with mPD to exclude both anionic and cationic organic molecules, such as dopamine (DA), ascorbate (AA), dihydroxyphenylacetic acid (DOPAC) and others

2.2 Microelectrode Arrays. Ceramic-based MEAs (S2 configuration) were obtained from the Center for Microelectrode Technology Revenue Center (University of Kentucky, Lexington, KY). The S2 style MEAs consisted of four Pt recording sites (15 m x 333 m) geometrically arranged as two side-by-side pairs with 100 m between pairs and 30 m between sites within a pair.10 Sites A (Figure 1; GABA-recording) were coated 4 times with a ~0.1 l mixture consisting of 0.05 U/l GluOx, 0.0125 U/l GABase, 1% Bovine Serum Albumin (BSA) and 0.1% glutaraldehyde (Glut). Sites B (Figure 1; sen-tinels, Glu-recording) were coated 4 times with a ~0.1 l mixture consisting of 0.1 U/l GluOx, 1% BSA, and 0.1% glutaraldehyde. Matching the ratio of GluOx to GABase did not affect the resulting glutamate or GABA response. The indicated ratios were chosen for convenience of preparation and efficient use of enzymes based on our previous use of GluOx biosensors. Following enzyme coating, MEAs were cured at 4C for at least 24 hours to allow more consistent enzyme immobilization. All sites were elec-

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troplated with mPD to create an exclusion layer based on size to limit the detection of interferents, (e.g. such as dopamine, DOPAC or ascorbic acid).10

2.3 In Vitro Tests. All studies were performed using constant potential amperometry with a FAST-16 mkII/III system (Quanteon; Nicholasville, KY, USA). More consistent calibration results were observed when MEA tips were soaked in 25C phosphate buffered saline (PBS, 0.05 M; pH 7.4 or 8.6) containing Glu (200 M) for at least 20 min before performing the calibration. The 0.05 M PBS is a standard buffer used to calibrate glutamate MEAs in our and other labs. The low buffer capacity was chosen because it was similar to the brain and allowed local pH shifts from molecules such as NH3 and thus would best simulate local brain pH shifts. Stirred calibrations were performed in PBS at 37C by applying a con-stant potential of +0.7 V vs. Ag/AgCl reference. Changes in current were recorded following the addi-tion of stock solutions to PBS of GABA, glutamate, H2O2, ascorbate and dopamine. Calibration data were analyzed as previously reported.13,16 For this calibration, the glutamate-only current was sub-tracted, post-acquisition, from the GABA+glutamate current. Differences in glutamate sensitivity could be corrected by signal normalization when needed. A five point moving average was applied to data for clarity of presentation. Although a sentinel site without enzymes could be used to remove background current and signals from possible interfering compounds from the glutamate site, glutamate recordings are presented without a sentinel site. Thus, the accuracy of glutamate measurements relied on the se-lectivity provided by the mPD layer. Single electrode signals were low, however, they did not show signs of poor selectivity (i.e. dilution or spike/dome effects).12,24

Agarose (0.6% by weight) was used to simulate the diffusion properties of the brain.20 Agarose was mixed with PBS (0.05 mM, pH 7.4) then heated until dissolved. Aliquots of GABA, aKG, and glutamate stock solutions were mixed into the warm agarose/PBS mixture before they were allowed to cool at 4oC for 24 hours. MEAs and reference electrode were inserted into the 37oC agarose gels and amperomet-ric currents were recorded. Current response to GABA and glutamate were calculated by subtracting the current of PBS-only gel from gels containing 20 M GABA with 1 mM KG or 20 M Glu with 1 mM KG. Sensitivities were calculated by dividing the current response by concentration.

2.4 Response Time. The temporal response of the biosensor was studied by placing the MEA tip in a drop of PBS suspended from the tip of a Ag/AgCl reference electrode. While conventional flow cells have been typically used to measure response times, the geometry of the MEAs, prevented measure-ments of the response times. The described method was the most accurate we have found for this ar-ray geometry.17 The MEA and reference electrode tips were separated by approximately 1 mm. The sample medium was quickly altered by simply allowing a new drop with different chemical makeup to fall onto the reference tip thereby replacing the original drop. The response time was defined as the time from where the signal began to rise to where it surpasses 90% of the final level. Upon replacing the drop, there was an initial period (~0.5 sec) where the signal rapidly rose then fell. This interference occurred even when the drop contained only PBS. Measured response times were corrected by remov-ing this time interval.21

2.5 In Vivo Tests. Male Fischer 344 rats (3-6 months old) were anesthetized with isoflurane (1-3%) and prepared for in vivo recordings as previously described.22 Pressure ejection using a Picospritzer (Parker Hannifin, NH, USA) was used to locally apply drugs into the brain extracellular space using glass micropipettes (15-20 m i.d., positioned 50-80 m away and centered among the four recording sites). Locally applied drugs included KCl 70 mM, CaCl2 2.5 mM, NaCl 75 mM; GABA, 5 mM or 250 M in 0.9% saline; and vigabatrin 1 mM in 0.9% saline. All drugs were maintained at pH 7.4.

3. Results and Discussion.

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Figure 2. Current versus time plot for an in vitro calibration of GABA (red) and Glutamate (blue) sensi-tive MEA sites. MEAs were coated with GABase (GABA-transaminase + succinic semialdehyde dehy-drogenase) and GluOx or GluOx alone. All sites were electroplated with m-PD, a size-exclusion barrier. MEAs were calibrated in 37°C PBS (pH 8.6) containing 1 mM -ketoglutarate to determine sensitivity, selectivity, and linearity. Inset shows a GABA calibration curve of the GABA + Glutamate site. MEAs are routinely tested against a dopamine challenge to ensure adequate selectivity over cationic interferents (not shown).

3.1 Calibration. As previously reported, electroactive interferents such as dopamine were blocked by size exclusion by electropolymerization of mPD onto the recording sites.10,16,23 Small molecules like H2O2 passed to the recording sites to be oxidized while larger molecules such as dopamine and ascor-bate were blocked. Typically, there was no detectable response from cationic or anionic interferents when mPD was employed.16,24 A balance between blocking interferents and maximum analyte sensitiv-ity was desired. GABA sensitivity was relatively low compared to glutamate; therefore, mPD plating times as short as 2 minutes were employed so as to not excessively limit H2O2 from diffusing to the recording site surfaces. However, poor selectivity can yield falsely high basal levels and other arti-facts.24 Although higher selectivity ratios are preferable, we have found that selectivity ratios as low as 35:1 for ascorbate could be used for measuring glutamate or GABA (35 M change in ascorbate would be interpreted as a 1 M change in glutamate).

In vitro beaker tests revealed that the MEAs were more sensitive to glutamate than GABA. Diffusional losses as well as reduced GABase enzyme activity (in U/mg: GluOx 6.3; GABase >0.5, contribute to the lower GABA sensitivity. Increasing the ratio of GABase to GluOx did not significantly increase the sensitivity of the GABA-recording sites. However, other ratios of GABase to GluOx including 1U GABase to 1U GluOx produced usable GABA sensors. Commercially available GABase is composed of γ-aminobutyric acid aminotransferase (GABGT, Scheme 1) and succinic semialdehyde dehydroge-nase (SSDH, Scheme 2). SSDH is not utilized in the detection of GABA; however, it is included in GABase from commercial suppliers. Unlike glutamate biosensors, which cure at room temperature, the GABA sensors were cured and stored at 4oC to protect GABase enzyme activity. Although, crosslinking can make enzymes more thermally stable, GABase is inherently less stable at elevated temperatures relative to GluOx.25,26,27

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Inclusion of -ketoglutarate (KG), a GABase substrate, was essential for observing GABA signals in vitro.19 While 100 M KG produced slightly non-linear responses, increasing the concentration of KG enhanced the sensitivity and linearity of the GABA response in vitro. As others have reported, GABA signals can still be detected in the absence KG if glutamate is present in the calibration solution be-cause KG is a product of glutamate oxidation by GluOx.9,19 All sites responded to glutamate, and self-referencing revealed the “pure” GABA response in vitro (Figure 2).

KG is a co-substrate in the conversion of GABA to succinate semialdehyde and glutamate. GABA sensitivity was found to increase with KG concentration up to 1 mM. Above 1 mM KG, MEA sensitiv-ity to GABA was independent of KG. The sensitivity at 0.1 mM KG was approximately half that of 1 mM KG for 20 mM GABA. KG levels are known to vary for different brain regions from 0.6 mM for cortex, 1.9 mM for striatum to 2.7 mM for substantia nigra.28 If the GABA biosensors were to be used in a brain region where KG is known to be below 1 mM (e.g. the cortex), an appropriate calibration should be performed using an KG concentration that matches the brain concentration (e.g. 0.6 mM). Also, as others have reported by quantifying in vivo glutamate, GABA levels may be corrected for differ-ences in KG derived from endogenous glutamate.19 In vivo results support that endogenous KG are sufficient to reliably quantify GABA. Local in vivo applications of 5 mM GABA into the dorsal striatum provided reproducible responses. Importantly, these responses were not significantly altered by the lo-cal application of GABA solution containing 0.1 mM KG. Taken together with our data from in vitro studies, the endogenous levels of brain KG were sufficient to allow immobilized GABase to properly function.19,28

Initial trials calibrated at pH 7.4 yielded unrealistically high in vivo GABA levels compared to extracellu-lar values reported in the literature. This indicated that the calibration conditions did not match the mi-croenvironment of the brain (i.e. the sensor was more sensitive in brain tissue). Agarose gel (0.6% by weight) and PBS were used to simulate the diffusion properties of the brain.21 Although much less com-plex than the brain, the agarose gel allowed diffusion to be studied without biological changes such as release and uptake. Figure 3 shows comparisons of the sensitivities for GABA and glutamate in agarose gel at pH 7.4, pH 7.4 stirred PBS and pH 8.6 stirred PBS. A stirred GABA calibration at pH 7.4 yielded a sensitivity that was around 42% of the sensitivity measured in the agarose gel. However, glu-tamate sensitivity in pH 7.4 stirred PBS was within 10% of the sensitivity measured in agarose suggest-ing that diffusional losses of enzyme products were not the only cause of the sensitivity differences. The variability between multiple MEA calibrations was around 10%. Because NH3 was produced by the deamination of glutamate, the local pH in the enzyme matrix could, possibly, increase when glutamate was present. In a stirred calibration, enzyme reaction products were swept away keeping the enzyme pH to that of the buffer. However, we would expect that limited diffusion in the brain would slow the loss of reaction products. We theorize that NH3 produced from the enzymatic deamination of glutamate shifted the pH of the enzyme matrix higher, thus, increasing GABase activity and accounting for most of the difference in sensitivity between the agarose and stirred calibrations. While understanding the possible effect of NH3, produced from deamination of glutamate, on pH would be informative, we think calibration in the agarose gel provides a close approximation of the expected enzyme-matrix pH in the brain. The buffer capacity of pH 8.6 PBS is not extensive, but sufficient for the concentration ranges of glutamate and GABA employed in the present study, which consistently yielded linear Pearson’s coeffi-cients. The range of GABA and glutamate concentrations employed for MEA calibrations is well within the physiological and non-physiological ranges of the endogenous neurotransmitters.

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Figure 3. Comparison of GABA (red) and glutamate (blue) sensitivities in stirred pH 7.4 PBS and stirred pH 8.6 PBS to pH 7.4 agarose gel.

To test the effect of higher pH, MEAs were calibrated in pH 8.6 PBS. In stirred calibrations, the GABA sensitivity at pH 8.6 was nearly identical to the GABA sensitivity in agarose gel. In contrast, glutamate sensitivity was much less dependent upon pH. Glutamate sensitivity in pH 8.6 stirred calibration was around 13% lower than that of the pH 7.4 stirred calibration. The wider working pH range of GluOx made glutamate detection much less sensitive to pH changes. For convenience, GABA sensors were routinely calibrated in a stirred beaker at pH 8.6 rather than in agarose. The detection limit for GABA was less than 0.2 M and 0.05M for glutamate (see Table 1.) These limits are low enough to discrim-inate micromolar levels of GABA and glutamate. GABA showed linearity over the 0-60 M range tested (R2=0.969+0.009). As others have reported GABA response saturates above around 100 M.19 In addi-tion, selectivity over ascorbic acid was adequate for in vivo use. Differences in the enzyme matrix thick-ness and composition contributed to the glutamate sensitivity on the GABA + glutamate site being 62% that of the glutamate only site. This was corrected by normalization prior to signal subtraction when cal-culating GABA concentrations.

Possible pH shifts can affect immobilized enzyme pH thus causing changes in GABA sensitivity. How-ever, because the brain is well buffered in vivo, brain pH changes are relatively small (pH 0.1 to < 0.5).29 In addition, MEA enzyme layers, because of their thickness, tend to slow responses, and in turn, essentially insulate the MEA from the effects of rapid pH changes. Thus, for most applications, pH shifts would have a minimal effect on detected GABA levels. Although effects from localized pH shifts should be considered when interpreting results. While it is possible that local pH could be measured and used to correct MEA responses for GABA and glutamate, this process may pose technical chal-lenges.30

Table 1: In vitro performance of the GABA and glutamate MEA

Site GABA+Glu GABA+Glu GlutamateAnalyte GABA Glutamate GlutamateSlope/Sensitivity (pA/M) -2.09 ± 0.41 -7.55 ± 0.05 -12.13 ± 0.66LOD (M) 0.18 ± 0.05 0.05 ± 0.01 0.05 ± 0.01Selectivity 35 ± 7 72 ± 2 46 ± 15

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Calibration parameters for GABA+Glu and Glutamate only sites. Values are reported as mean ± SEM for n = 6 recording sites. Calibration buffer contained 1 mM KG at pH=8.6. Selectivity was determined by dividing the analyte slope by the slope of the response to ascorbic acid. Sub micromolar detection limits were achieved for GABA and glutamate.3.2 Oxygen Dependence. Glutamate oxidase (see Eq. A3) requires O2 to convert glutamate to KG, NH3 and H2O2. Although in vivo O2 concentrations are tightly regulated in the CNS, changes in O2 con-centration could affect glutamate sensitivity and therefore GABA sensitivity. The MEAs used in this study have been used to quantify O2. 13 , 23 Experiments in our lab, where glutamate and O2 were simul-taneously monitored using different sites on a single MEA, showed that N2 induced anoxia and death caused O2 concentrations to decrease by 10 µM while the measured glutamate concentration in-creased, as expected. However, there could be conditions where O2 concentrations around the record-ing sites could affect the measured glutamate and GABA response. Because physiological levels of glutamate are in the micromolar range and O2 is in the millimolar range there is typically a relative abundance of O2. Biosensors for metabolites like glucose and lactate that are in the millimolar range show greater O2 dependence.15 Although errors related to insufficient in vivo O2 levels were not ob-served in our hands, care should be taken in experiments where local O2 concentrations would be al-tered, but O2 concentrations could be monitored efficiently with the same MEA technology to deter-mine if this were an experimental problem. Calibration solutions can then be manipulated in their O2

levels once any observed changes during in vivo studies are measured and used to determine the ef-fects on the analytical response of the MEAs. From our experience, typical in vivo changes in O2 due to more physiological changes are in the low micromolar range while anoxia and seizure events produce larger changes (also unpublished data). 31-32

3.3 Response Time. The temporal response of the GABA/glutamate MEAs was studied using a falling drop method, which is capable of rapidly switching samples.33 The uncorrected 90% response time to H2O2 was similar to the initial unstable period (0.52+0.05 sec) indicating that the actual response time is less than 0.01 sec. The corrected response time estimate for glutamate was 0.19+0.05 sec. The fall-ing drop method did not accurately measure the response time for GABA due to turbulence in the drop. Approximately 5 seconds were required to achieve steady-state. However, the temporal response to GABA is estimated to be less than 1 sec based on the time required to establish a steady response in agarose samples. The rapid rise following GABA applications in vivo also supports this estimate (see Figure 4).

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Figure 4. Locally applied GABA in the rat frontal cortex. 25-100 nl GABA (25 nl increments; 250 M, pH 7.4) was locally applied in the frontal cortex (in mm, AP:+3.2; ML: +0.8; DV: -3.0). The GABA signal was resolved by subtracting current from glutamate sites from current on GABA+glutamate sites. Sig-nals were multiplied by their individual slopes to convert current into concentration. Signals following GABA application were reproducible. Glutamate sites were unresponsive to locally applied GABA. The sensitivities for GABA was 1.1 pA/M and for glutamate it was 4.8 pA/M. A 10 pA signal corresponded to about 9M for GABA and 2M for glutamate.

3.4 In Vivo Measures. Figure 4 shows the dose-response to GABA applications in the rat frontal cor-tex (FC). Applications of 25, 50, 75, and 100 nl of the 250 M GABA solution resulted in 3.8, 10, 18, and 18.8 M GABA peaks, respectively. GABA signals rapidly rose within a few seconds then returned to baseline in less than 20 sec. Robust responses were observed on the GABA+glutamate detecting sites while very small responses were observed on the glutamate-only sites (<1 M). Decreases in baseline on the glutamate-only site following GABA applications were probably because of a dilution of the endogenous glutamate surrounding the MEA.15 Lower baseline variations were observed on the subtracted GABA response due to removal of unresolved local field potentials (LFP) that are detected on both the GABA+glutamate and the glutamate sites.34 GABA detected at the recording sites in-creased with increasing GABA doses.

Figure 5 shows a representative figure of a sensor response to potassium-evoked release of GABA and glutamate in the FC, motor cortex and dorsal striatum. (AP: +1.0 mm; ML: -2.5 mm; DV: -2.7 mm (motor cortex), -6.2 mm and -6.7 mm (dorsal striatum)) Endogenous GABA release was readily mea-sured following local, potassium-induced depolarizations. Variations in detected GABA and glutamate were seen for the various brain locations. For instance, large amplitude GABA responses were ob-served in the motor cortex (80 µM) while in the dorsal striatum, the amplitudes of GABA release were smaller (6 µM or no change). Glutamate release in these regions was between 10 µM and 15 µM. As we have reported before, when signals are subtracted, transient artifacts are possible because of differ-ences in enzyme thickness, physical location of the recording sites, or poor selectivity.12, 53

Figure 5: KCl-evoked GABA/Glutamate release in the rat motor cortex and dorsal striatum. KCl (100 nl; 70 mM, pH 7.4) was locally applied in the motor cortex and two areas of the dorsal striatum. The sensi -tivities for GABA and glutamate were 1.2 and 18.4 pA/M, respectively. A 50 pA signal corresponded to about 42M for GABA and 2.7M for glutamate.

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Figure 6: Modulation of endogenous GABA with vigabatrin. Inhibition of GABA-transaminase, the degradation enzyme for GABA, with vigabatrin (arrow = 350 nl; 1 mM, pH 7.4) elevated endogenous GABA levels in the FC (in mm, AP: +3.2; ML: -1.0; DV: -5.0). The inset shows a 3 min window (shaded area) before and after local application of vigabatrin. The sensitivities for GABA and glutamate were 0.69 and 12.1 pA/M, respectively. A 25 pA signal corresponded to 36M for GABA and 2M for glu-tamate.

Vigabatrin was used to modulate endogenous GABA by inhibiting GABA transaminase.36 Figure 6. shows the sensor response to applying vigabatrin in the rat FC (AP: +3.2; ML: -1.0; DV: -5.0 mm). Fol-lowing local application of vigabatrin, endogenous GABA levels increased approximately 80 M in ~60 sec. GABA levels returned to their original baseline after approximately 60 minutes. With the exception of the first 30 seconds following vigabatrin application, glutamate was unaffected by the drug applica-tion. The inset shows a 3 min window (shaded area) spanning the local application of vigabatrin where both glutamate and GABA were briefly affected. This transient effect was likely due to local inhibition of immobilized GABase. After this initial period the immobilized enzymes did not appear to be irreversibly affected by vigabatrin. The remaining effect of vigabatrin showed an effect exclusively on endogenous GABA.

4. Conclusions

Here we present the challenges of employing the GABA/glutamate biosensor using a dual-enzyme im-mobilization approach. Increasing the pH of in vitro stirred calibrations was necessary to approximate the MEA response to GABA in vivo. This did not severely affect the glutamate response. Previously, others have only shown in vitro use of the GABA/glutamate biosensor.19 Here we show further valida-tion of this approach by demonstrating rapid dynamic changes in GABA levels in the cortex or striatum after local applications. As the primary inhibitory neurotransmitter of the CNS, GABA’s role in brain ac-tivity is critical in understanding normal and abnormal neuronal function of inhibitory neurotransmission, which has been understudied to date. Having a method to rapidly and spatially record GABA and gluta-mate signaling in the brain, might further help elucidate the mechanisms of action of these primary neu-rotransmitters. Because this approach is dependent on pH, aKG, and potential differences between in vitro calibration and in vivo measures, results must be carefully scrutinized with these pitfalls in mind. The reliance on αKG limits the technique’s rapid implementation. A possible solution for determining the absolute concentration of GABA would be to employ a correction factor based on published con-

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centration values of αKG in the area of the brain to be measured. However, if the concentration of αKG remains unknown, one could still use the biosensor to measure changes in GABA concentration over time based on a within subject experimental design. While not optimal, use of the biosensors in this manner would permit rapid detection of GABA signaling. In addition, these pitfalls must be considered with experimental design considerations to minimize their effects. However, the current approach per-mits a platform for further refinement and investigation of the dynamic changes in GABA and glutamate simultaneously in vivo in the CNS.

5. Appendices

Eq. (A.1) γ-Aminobutyric Glutamic Transaminase (GABGT)

γ-aminobutyric acid + -ketoglutarate ⇌ succinate semialdehyde + L-glutamate

Eq. (A.2) Succinic Semialdehyde Dehydrogenase (SSDH)

succinate semialdehyde + NAD+ + H2O ⇌ succinate + NADH + 2 H+

Eq. (A.3) L-Glutamate Oxidase

L-glutamate + O2 + H2O ⇌ -ketoglutarate + NH3 + H2O2

AUTHOR INFORMATIONCorresponding Author* Greg A. Gerhardt, Department of Neuroscience, 800 Rose St., Lexington, KY 40536-0298

Present Addresses† Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285 U.S.A.

ACKNOWLEDGMENT Funding: This work was supported by the National Institutes of Health [grant numbers NS39787, DA017186, T32 AG000242]; and DARPA [grant number N66001-09-C- 2080]

Author ContributionsThe manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally.

Declarations of interestPortions of this were initially presented at the Society for Neuroscience Annual meeting 2011. GAG is principal owner of Quanteon LLC, JEQ, FP, PH, and JJB serve as consultants to Quanteon LLC.

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Visual Abstract

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