Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).
Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer’s disease, Parkinson’s disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer’s disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer’s disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.
Measuring neurotransmitter alterations in the brain is an essential tool for neuroscientists studying diseases of the central nervous system (CNS) that are often characterized by neurotransmitter dysregulation. Though microdialysis in combination with high pressure liquid chromatography (HPLC/EC) has been the most widely used method to measure changes in extracellular neurotransmitter levels1,2,3,4, the spatial and temporal resolution of microdialysis probes may not be ideal for neurotransmitters, such as glutamate, that are tightly regulated in the extracellular space5,6. Because of the recent advances in genetics and imaging, there are additional methods that can be used to map glutamate in vivo. Using genetically encoded glutamate fluorescent reporters (iGluSnFR) and two-photon imaging, researchers are able to visualize glutamate release by neurons and astrocytes both in vitro and in vivo7,8,9. Notably, this allows for recording from a larger field of view and does not disrupt the intrinsic connections of the brain. While these new optical techniques allow for visualization of glutamate kinetics and measurement of sensory evoked responses and neuronal activity, they lack the ability to quantify the amount of glutamate in the extracellular space in discrete brain regions.
An alternative method is the enzyme-linked microelectrode array (MEA) that can selectively measure extracellular neurotransmitter levels, such as glutamate, through the use of a self-referenced recording scheme. The MEA technique has been used to study alterations in extracellular glutamate following traumatic brain injury10,11,12, aging13,14, stress15,16, epilepsy17,18, Alzheimer's disease19,20, and injection of a viral mimic21 and represents an improvement over the spatial and temporal limitations inherent in microdialysis. Whereas microdialysis restricts the ability to measure near the synapse22,23, MEAs have a high spatial resolution that allows for selective measures of extracellular glutamate spillover near synapses24,25. Second, the low temporal resolution of microdialysis (1 – 20 min) limits the ability to investigate the fast dynamics of glutamate release and clearance occurring in the millisecond to second range26. Because differences in the release or clearance of glutamate may not be evident in measures of tonic, resting glutamate levels, it may be essential that glutamate release and clearance be directly measured. MEAs allow for such measures due to their high temporal resolution (2 Hz) and low limits of detection (< 1 µM). Third, MEAs allow for examination of subregional variations in neurotransmitters within a particular brain region, such as the rat or mouse hippocampus. For example, using MEAs we can separately target the dentate gyrus (DG), cornu ammonis 3 (CA3) and cornu ammonis 1 (CA1) of the hippocampus, which are connected via a trisynaptic circuit27, to examine subregional differences in extracellular glutamate. Because of the size of microdialysis probes (1 – 4 mm length) and the damage caused by implantation28,29, subregional differences are difficult to address. Furthermore, the optical systems only allow stimulation through external stimuli, such as a whisker stimulation or light flicker, which does not permit subregional stimulation7. A final benefit of MEAs over other methods is the ability to study these subregions in vivo without disrupting their extrinsic and intrinsic connections.
Here, we describe how a recording system (e.g., FAST16mkIII) in combination with MEAs, consisting of a ceramic-based multisite microelectrode, can be differentially coated on the recording sites to allow for interfering agents to be detected and removed from the analyte signal. We also demonstrate these arrays can be used for amperometry-based studies of in vivo glutamate regulation within the DG, CA3, and CA1 hippocampal subregions of anesthetized rTg(TauP301L)4510 mice, a commonly used mouse model of Alzheimer's disease. In addition, we provide confirmation of the sensitivity of the MEA system to the fast dynamics of glutamate release and clearance by treating the mice with riluzole, a drug shown in vitro to decrease glutamate release and increase glutamate uptake30,31,32,33, and demonstrating these respective changes in vivo in the TauP301L mouse model.
1. Coating the Microelectrode Array with Enzymes or Matrix Layer
2. Electroplating with m-Phenylenediamine for Improved Selectivity
3. Calibrate the MEA for Glutamate Detection and Selectivity (Figure 1)21
4. Assemble the Micropipette
NOTE: Micropipettes (capillary glass) should have a tip with an internal diameter of 10 – 15 µm.
5. Plate the Miniature Reference Electrode for In Vivo Use
6. General Animal Surgery for MEA Recordings
7. Cleaning Coated MEAs after Use
8. Analysis
While this technology can be used to measure alterations in glutamatergic signaling in many types of animal models, such as traumatic brain injury, aging, stress, and epilepsy, here we demonstrate how the MEA technology can be used to examine glutamatergic alterations in transgenic mouse model of human tauopathy19,20. The rTg(TauP301L)4510 mouse expresses the P301L mutation in tau associated with frontotemporal dementia and parkinsonism linked to chromosome 17, and is commonly used to study tau pathology associated with Alzheimer's disease, neurodegenerative tauopathies and frontotemporal dementia. We also demonstrate that glutamatergic signaling can be modified by pharmacological intervention. We treated TauP301L mice with riluzole, an FDA-approved disease-modifying drug for amyotrophic lateral sclerosis (ALS) that modulates glutamatergic signaling by stabilizing the inactivate state of voltage-gated sodium channels, leading to a decrease in glutamate release, and a potentiation of glutamate uptake via an increase in glutamate transporter expression, leading to increased glutamate clearance30,31,32,33.
We have chosen this particular dataset for demonstrative purposes for four reasons. First, this dataset demonstrates the temporal resolution of the system, as shown by our ability to measure the fast dynamics of transient release and uptake of glutamate. In addition, this preclinical animal model exhibits alterations in tonic glutamate, glutamate release, and glutamate clearance, allowing for an example of alterations in each measure. Second, utilizing this dataset, the high spatial resolution that allows for measurement of subregion differences in the DG, CA3, and CA1 of the hippocampus is demonstrated. Third, this data allows for demonstration of how pharmacological intervention can be used to mitigate glutamatergic alterations observed in preclinical animal models. Finally, because this preclinical model has alterations in glutamate release, the need for careful interpretation of glutamate clearance in the presence of altered glutamate release can be explained.
After reaching a stable baseline, as indicated by < 0.004 nA/min change in slope (10 – 20 min), we first measured tonic glutamate levels (µM) by averaging extracellular glutamate levels over 10 s prior to any application of solutions. We observed increased tonic glutamate levels in the CA3 and CA1 regions of TauP301L mice, and riluzole restored tonic glutamate levels to that of controls (Figure 3A). Next, KCl was locally delivered (via a micropipette) to each of the three sub-regions of one hemisphere every 2 – 3 min. After 10 reproducible signals, which is indicative of an intact glutamate neuronal system34, the results were averaged for each group and the average amplitude compared. Representative traces (Figure 3B) revealed increased glutamate release in TauP301L mice following application of KCl in all three subregions (only CA3 is shown), an effect that was rescued by riluzole treatment (Figure 3C).
The MEA was then moved to the opposite hemisphere and allowed to reach a stable baseline (10 – 20 min) before exogenous glutamate was applied to examine glutamate clearance (Figure 4). Varying volumes (50 – 250 nL) of 200 µM sterile-filtered glutamate solution were applied into the extracellular space every 2 – 3 min, and the net area under the curve (AUC) was used as a measure of glutamate uptake. This rapid application of glutamate into the extracellular space allows for mimicking of endogenous glutamate release and measurement of glutamate uptake. Because glutamate transporters exhibit Michaelis-Menten kinetics35, a range of volumes (50 – 250 nL) of exogenous glutamate is injected to expose differences in uptake. To do so, each animal receives 1 – 2 injections at 50 nL increments within the 50 – 250 nL range. Though one should always confirm that the volume injected per group per region is not statistically different at the p ≤ 0.05 level, the best way to ensure net AUC is related to uptake, and not the amount of glutamate applied or diffusion, is to ensure that both amplitude (Figure 4A) and Trise (a measure of diffusion from the point source (micropipette) to the MEA in Figure 4B) do not differ10,11,19,20. This allows for "peak matching" the amplitudes (Figure 4C) such that differences in net AUC are assumed to be due to differences in uptake and not the amount applied or diffusion. Peak matching is particularly important when animals have existing differences in glutamate release. Because a range of volumes is injected, the variance for both amplitude and Trise are often large and adding additional animals does not decrease the variance for these measures, as they are inherent to the design. Because the amplitude and Trise were similar among the groups in each subregion (Figure 4A, 4B), we assume that differences in net AUC (Figure 4C, 4D) are due to differences in glutamate uptake. Riluzole improved glutamate clearance compared to controls in all three subregions (Figure 4D). Finally, an MEA with an attached micropipette is used to locally apply a dye to confirm MEA placement after brain sectioning, as demonstrated in Figure 3D.
These data indicate that the MEA technology is capable of measuring neurotransmitter release within small subregions of the hippocampus24,25, unlike previous techniques (e.g., microdialysis), which only recorded from large sample areas and often damaged neuronal circuits28,29. In addition, the MEAs provide us with high temporal resolution to measure the fast kinetics of glutamate release and clearance25.
Figure 1: In Vitro Calibration of a Self-referencing Microelectrode Measuring the Change in Current (pA) on a Glutamate Oxidase Site (GluOx; red) vs. a Sentinel Site (Sent; blue). The interferents ascorbic acid (AA: 250 µM) and dopamine (DA: 2 µM) did not alter the current at either glutamate oxidase or sentinel sites. Addition of glutamate (Glu: 20, 40, 60 µM) produced a stepwise current increase on the glutamate oxidase site, but no change on the sentinel site. Hydrogen peroxide (H2O2: 8.8 µM) produced an increase in current on both sites. Sensitivity, slope, limit of detection, and R2 values were calculated after calibration. Please click here to view a larger version of this figure.
Figure 2. A Sample Electrochemistry Worksheet. This worksheet includes columns for time of injection or injection number (TIME/TTL), brain coordinates (AP, mL, DV), the amount of pressure applied (nitrogen), the length of pressure (time), and the volume injected by the pressure ejector. Any notes, such as odd signals or clogging of the micropipette should be recorded in the notes column. Please click here to view a larger version of this figure.
Figure 3. Extracellular Tonic and Potassium Chloride (KCl)-evoked Release of Glutamate in the DG, CA3, and CA1 Regions of the Hippocampus. (A) In the CA3 and CA1 regions of the hippocampus, tonic glutamate levels were significantly increased in vehicle-treated TauP301L mice (Veh-TauP301L), an effect attenuated by riluzole treatment. (B) Baseline-matched representative recordings of KCl-evoked glutamate release in the CA3 showed riluzole treatment (Ril-TauP301L) attenuated the significant increase in the amplitude of glutamate release observed in Veh-TauP301L mice. Local application of KCl (↑) produced a robust increase in extracellular glutamate that rapidly returned to tonic levels. (C) The significantly increased KCl-evoked glutamate release observed in Veh-TauP301L mice in the DG, CA3, and CA1 after local application of KCl was attenuated with riluzole treatment. (D) Cresyl violet-stained 20 µm section of hippocampus shows location of MEA tracks in CA3 and CA1 (Mean ± SEM; ** p < .01 Veh-Control vs. Veh-TauP301L, # p < .05 Ril-Tau-P301L vs. Veh-TauP301L, ## p < .01 Ril-Tau-P301L vs. Veh-TauP301L; n = 14 – 19/group). This figure reprinted with permission from John Wiley and Sons19,20. Please click here to view a larger version of this figure.
Figure 4. Glutamate Uptake Following Exogenous Glutamate Application in the DG, CA3, and CA1 Regions of the Hippocampus. (A) The amplitude of the locally-applied glutamate signal was similar among groups in each region. (B) Trise, an indicator of glutamate diffusion from the micropipette, was similar among the groups in each region. (C) Representative glutamate signals in the CA3 from local application of glutamate in Veh-Controls, Veh-TauP301L, and Ril-TauP301L mice. (D) Riluzole treatment reduced the significant increases in the net area under the curve (AUC) observed in Veh-TauP301L mice in all 3 regions of the hippocampus, indicating improved glutamate uptake in riluzole-treated TauP301L mice (Mean ± SEM; * p < .05 Veh-Control vs. Veh-TauP301L, ** p < .01 Veh-Control vs. Veh-TauP301L, # p < .05 Ril-Tau-P301L vs. Veh-TauP301L, ## p < .01 Ril-Tau-P301L vs. Veh-TauP301L; n = 13 – 15/group). This figure was reprinted with permission from John Wiley and Sons20. Please click here to view a larger version of this figure.
0.05 M PBS | 2 L DI water: |
Store in glass beaker wrapped with tin foil. Bring pH to 7.4 with KOH if necessary. | NaH2PO4·H2O: 2.8 g Na2HPO4: 11.34 g Sodium Chloride: 11.68 g |
20 mM Ascorbic Acid | 50 mL DI water: |
Make fresh daily. | Ascorbic acid: 0.18 g |
20 mM L-Glutamate | 50 mL DI water: |
Make fresh weekly. | L-glutamate monosodium salt hydrate: 0.15 g |
2 mM Dopamine | Dopamine hydrochloride: 0.038 g |
Make fresh monthly. | 0.1 M Perchloric acid: 10 mL Bring volume to 100 mL with DI water |
8.8 mM Hydrogen Peroxide | 50 mL DI water: |
Make fresh weekly. | Hydrogen peroxide: 500 μL |
70 mM potassium chloride | 100 mL DI water: |
Make fresh day of experiment. | Potassium chloride: 0.08 g Sodium chloride: 0.07 g Calcium Chloride: 0.004 g |
200 μM Glutamate | 20 mM glutamate: 100 μL |
Make fresh day of experiment. | Sterile saline: 10 mL |
Table 1: Calibration Solutions.
The MEA technique allows for measurement of fast kinetics of neurotransmitter release and uptake in vitro and in vivo. Hence, the technology produces a wide variety of data output including tonic neurotransmitter levels, evoked neurotransmitter release, and neurotransmitter clearance. However, because use of MEAs is a relatively complex procedure, there are numerous factors that may need to be optimized for successful use. For example, during calibration, one may note that there are no signal waveforms (oscilloscope screen) or that responses to stimulation are minimal, or absent. Likely due to a system malfunction, restarting the recording system, or computer, may resolve the issue. Additionally, the problem could be a connection error. Before calibrating, it is pertinent to double-check that the MEA and reference electrode connections are connected properly. The replacement of the DIP socket on the headstage may resolve MEA connection issues. If the MEA is not completely in solution or a poorly maintained reference electrode is used, these may also interfere with the recording during calibration. Slowed signal responses during calibration is another common issue, which may result from a thicker than needed BSA/glutaraldehyde mixture, or a slowly spinning stir bar. In the event that interferents are giving a signal during calibration, it is possible that there was an error in electroplating (e.g. did not electroplate long enough) the mPD exclusion layer. In such a case, repeating the electroplating procedure is necessary.
During surgery, one anesthetic level may not be sufficient for all mice; some mice may require higher levels of anesthesia to "go under" and some mice may require less. It is essential to start at a low level of anesthesia and slowly raise it until the mouse is under light anesthesia. Deep anesthesia may be tested once the mouse is in the stereotaxic device by a tail or toe pinch. When choosing coordinates for implanting the MEA, it is also important to note and correct for possible brain atrophy that can occur in many neurodegenerative animal models36,37,38. In addition, it is imperative during surgery that blood does not accumulate on the MEA, as this can affect the recording properties of the MEA and result in slow temporal resolution or greatly minimized signal responses. If blood clots around the electrode, simply remove the MEA and rinse with saline.
During recording, one of the most common problems that can occur is that of a noisy signal. Interference from other electronic devices, such as auxiliary lighting, additional instruments, and drills are the mostly likely culprits, so it is best to avoid plugging them into the same electrical circuit and/or outlet as the recording system. Additionally, if the recording system is not grounded properly, one will observe oscillations on the oscilloscope of the recording system as well as noise stemming from all sites, and not just the recording sites. The recommended grounding mat under the recording system must be connected to a grounded pipe to avoid these issues. If it is determined that none of these are the error, the error may lie with the reference electrode, or the MEA itself. When encountering noise issues, using a new, unused MEA to verify the operation of the system can be a useful troubleshooting approach. If disconnecting or replacing the reference electrode results in little to no change, the reference electrode or half-cell may have malfunctioned. Changing the reference electrode may be the only way to resolve the problem. Once recording is complete, a final benefit of the MEA is the ability to clean and reuse them. MEAs can be re-used approximately three times as long as the MEA continues to perform adequately during calibration.
To conclude, though troubleshooting may occasionally be required, use of the MEA technique is advantageous for examining the fast release and clearance of neurotransmitter within desired sub-regions of the brain. The current studies focused on the use of the MEA technology in anesthetized TauP301L mice to take advantage of the MEA methodology for examining glutamate release and uptake in sub-regions of the hippocampal trisynaptic pathway. Future studies will address additional tonic and phasic issues in awake mice, as this has been very beneficial for linking behavioral events to the dynamic measures of tonic and phasic glutamate in awake rats and more recently, awake nonhuman primates. While this technology was developed for basic science studies, the future holds promise for the implementation of the recording technology for clinical research and intraoperative neurochemical monitoring for treatment and evaluation of brain disorders such as Parkinson's disease and epilepsy.
The authors have nothing to disclose.
This work was supported by the National Institute of General Medical Sciences (MNR; U54GM104942), NIA (MNR; R15AG045812), Alzheimer’s Association (MNR; NIRG-12-242187), WVU Faculty Research Senate Grant (MNR), and WVU PSCOR Grant (MNR).
FAST-16mkIII-8 channel | Quanteon | 16mkIII | |
Microelectrode arrays | CenMet | W4 or 8-TRK | |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A-3059 | 10 g (expires after 1 month) |
Glutaraldehyde | Sigma-Aldrich | G-6257 | 100 mL (expires after 6 months) |
Glutamate Oxidase | US Biological or Sigma Aldrich | G4001-01 or 100646 | 50 UI (expires after 6 months) |
Hamilton Syringes | Hamilton | #701 | 2 syringes |
Methanol | BDH | UN1230 | 4 L |
m-Phenylenediamine dihydrochloride (mPD) | ACROS Organics | 1330560250 | 25 g |
Reference Electrodes (RE-5B) | BAS | MF-2079 | 3 electrodes |
Magnetic stir plate | Cole-parmer | EW-04804-01 | Can purchase from different supplier |
Glutamate | Sigma-Aldrich | G-1626 | 100 g |
Ascorbic Acid | TCI | 50-81-7 | 500 g |
Dopamine Hydrochloride | Alfa Aesar | 62-31-7 | 5 g |
Perchloric acid | VWR | UN2920 | 500 mL |
Postassium chloride | VWR | 7447-40-7 | 1 kg |
Sodium chloride | VWR | 7647-40-7 | 1 kg |
Calcium Chloride | MP | 153502 | 100 g |
Sodium Hydroxide | BDH | 1310732 | 500 g |
Glass pressure ejection pipettes | CenMet | ||
Sticky wax | Kerrlab | 625 | Can purchase from different supplier |
Microsyringe | World Precision Instruments | MF28G-5 | |
Modeling clay | WalMart | Can purchase from different supplier | |
Picospritzer III | Parker | ||
Silver wire | AM systems | 782000 | |
Hydrochloric acid | BDH | 7647010 | 2.5 L |
Platinum wire | AM Systems | 778000 | |
Solder gun | Lowes or Home Depot | Can purchase from different supplier | |
Multimeter | WalMart | Can purchase from different supplier | |
PhysioSuite | Kent Scientific | Can purchase from different supplier | |
SomnoSuite | Kent Scientific | Can purchase from different supplier | |
Stereotaxic device | Stoelting | Can purchase from different supplier | |
Digital Lab Standard | Stoelting | Can purchase from different supplier | |
Meiji EMZ microscope | Meiji | EMZ-5 | |
Drill | Dremel | Micro | |
Metricide | Metrex | 102800 | |
Scalpel | VWR | Can purchase from different supplier | |
Surgery scissors | VWR | Can purchase from different supplier | |
Sterile cotton swabs | Puritan | 25806 | Can purchase from different supplier |
Eye ointment | Puralube Vet Ointment | Obtain from the vet | |
Iodine swabs | VWR | S48050 | Can purchase from different supplier |
Alcohol swabs | Local drug store | Can purchase from different supplier | |
Sterile surgery drape | Dynarex | 4410 | Can purchase from different supplier |
Sterile saline | Teknova | S5815 | Can make own soltuion using filters |
Hydrogen Peroxide (3%) | Local drug store | Can purchase from different supplier | |
Heating Pad | WalMart | Can purchase from different supplier |