Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Summary

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).

Abstract

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.

Introduction

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 levels^1,2,3,4, the spatial and temporal resolution of microdialysis probes may not be ideal for neurotransmitters, such as glutamate, that are tightly regulated in the extracellular space^5,6. 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 vivo^7,8,9. 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 injury^10,11,12, aging^13,14, stress^15,16, epilepsy^17,18, Alzheimer's disease^19,20, 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 synapse^22,23, MEAs have a high spatial resolution that allows for selective measures of extracellular glutamate spillover near synapses^24,25. 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 implantation^28,29, 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 uptake^30,31,32,33, and demonstrating these respective changes in vivo in the TauP301L mouse model.

Protocol

1. Coating the Microelectrode Array with Enzymes or Matrix Layer

1. Preparing the protein matrix solution
   1. Weigh out 10 mg of bovine serum albumin (BSA) and transfer to the 1.5 mL microcentrifuge tube.
   2. Add 985 µL of DI water to the microcentrifuge tube containing BSA. Mix the solution by manual agitation (re-pipetting using 1,000 µL pipette ~ 3 times, until the BSA is dissolved).
      NOTE: Do not use vortex to mix the solution as doing so may introduce air into the solution. Also, set the pipette to a volume <1,000 µL (e.g. 700 µL) to avoid introducing air bubbles. The solution may be placed in a microcentrifuge if necessary.
   3. Once the BSA solution is dissolved, add 5 µL of 25% glutaraldehyde solution. Mix the solution by inverting the closed tube 3 - 5 times. The resulting solution is 1% BSA with 0.125% glutaraldehyde.
   4. Set aside the protein matrix solution for 5 min. The solution will appear light yellow in color.
2. Preparing the desired enzyme solution (e.g., glutamate oxidase)
   1. Prepare the glutamate oxidase solution by adding filtered DI water to the lyophilized, purified enzyme (e.g., 50 µL DI water added to 25 units of glutamate oxidase to yield 0.5 U/µL).
      NOTE: Aliquot stock solution (e.g., 1 µL) and store the aliquots in appropriately sized containers (e.g., 500 µL microcentrifuge tubes) at -20 °C. Aliquots may be stored for up to 6 months under these conditions.
   2. Add 4 µL of the BSA/glutaraldehyde solution to the microcentrifuge aliquoted tube containing glutamate oxidase. Mix the solution by manual agitation (re-pipetting with a 10 µL pipette. The pipette should be set to volume < 5 µL). The resulting solution is 1% BSA, 0.125% glutaraldehyde and 0.1 U/µL glutamate oxidase. Use the solution for coating immediately.
      Note: Glutamate oxidase coating solution may only be used within 15 min. Although several MEAs may be coated with one prepared solution, it is recommended not to exceed the useable time window for glutamate oxidase. The number of electrodes that can be coated within 15 min vary. Generally, 4 to 6 electrodes can be coated at a time.
3. Coating the MEA
   1. Typically, coat a pair of recording site(s) on a MEA with the desired enzyme (glutamate oxidase) and a different pair of recording site(s) ('sentinel' site(s)) is coated with the inactive protein-matrix.
   2. Use blunt-tip Hamilton microsyringes (e.g. 10 µL) to coat MEAs with L-glutamate oxidase or inactive protein matrix solution (BSA+glutaraldehyde). The procedures used for enzyme or inactive protein-matrix coatings are the same.
   3. Clean the syringes before and after use (3 rinses with DI water, 3 rinses with methanol, 5 rinses with DI water).
      NOTE: Dedicated syringes are used for applying coats of enzyme (glutamate oxidase) or protein-matrix. Do not use the same syringe for a different solution. It is recommended to perform protein-matrix coatings before enzyme coatings.
   4. Draw up glutamate oxidase or protein matrix using a 10 µL Hamilton syringe. Gently press the plunger to dispense a small bead of solution at the syringe tip (visualized using a dissecting microscope).
   5. Using the dissecting microscope to target the MEA recording sites, apply the solution to the appropriate recording sites by briefly contacting the recording sites with the solution droplet/bead, which represents one layer. Three layers are sufficient for both the protein-matrix and the enzyme coat. Thick layers can reduce the sensitivity of the MEA.
   6. Raise the solution droplet straight up and off the recordings sites (i.e. the syringe tip should not scrape across the MEA surface).
   7. Set a timer for 1 min. This is the minimum time requirement between coating applications to the recording site(s). Allow enzyme-coated MEAs to cure at 4 °C for 5-7 days.
   8. Record the number of coats applied, how long it took to apply the coatings, the electrode number, and a name and date in a laboratory notebook.

2. Electroplating with m-Phenylenediamine for Improved Selectivity

1. Preparing m-Phenylenediamine dihydrochloride (mPD) solution
   NOTE: Plate the exclusion layer, mPD, to prevent the oxidation of dopamine and other large, interferent molecules (e.g., ascorbic acid), thereby improving the selectivity of the sensor for glutamate/H₂O₂ over other potentially-interferent molecules.
   1. Measure 50 mL of 0.05 M phosphate buffered saline (PBS) in a volumetric flask and transfer 50 mL of 0.05 M PBS to a larger (150 mL) Erlenmeyer flask.
   2. Using the tubing connected to a nitrogen tank (e.g., using the tubing connected a pressure ejector), attach a pipette tip (e.g. P200 tip) to the open end of the tubing. Place the tubing with the attached pipette tip in PBS solution and cover the solution loosely with parafilm. Turn on nitrogen and gently bubble solution for 20 min.
   3. Weigh 0.045 g of mPD.
      Caution: mPD is a potentially carcinogenic substance. Weighing of mPD should be performed using a scale contained within a fume hood. Wear gloves and mask when using mPD. Ensure the hood is functional and that the sash is pulled to the appropriate working level. Immediately clean surfaces that may have been contaminated with mPD to avoid permanent staining.
   4. After bubbling, transfer all of the mPD to the de-gassed PBS solution. Cover the flask opening with parafilm and gently swirl solution to dissolve mPD in solution. Avoid aggressive mixing or use of a vortex as doing so may introduce gasses into the solution.
   5. Transfer the solution to a 50-mL beaker. A stir bar is not necessary.
2. Electroplating the electrode
   1. Obtain a reference electrode. Use reference electrodes dedicated to mPD plating only. Use a different reference electrode for calibration procedures.
   2. Position the plastic arm of the reference electrode holder over the beaker. Check for air bubbles in the glass reference electrode (may not be able to see). Gently flick the distal end of the reference electrode to remove any air bubbles at the tip.
   3. Place the reference electrode through the opening in the plastic arm and lower into the beaker containing PBS. Ensure that the reference electrode does not contact the bottom of beaker.
   4. Connect the reference electrode to the headstage (e.g. 2 pA/mV) and connect (e.g. DIP connector) the MEA to be mPD plated to the headstage.
   5. Lower the MEA into the beaker solution such that only the MEA tip is submerged in solution. Do not submerge the MEA tips in liquid beyond the black 'bubble' (the black insulation located above the MEA tip). Doing so may damage the MEA.
   6. Run a 'test' calibration to verify the functionality of the electrode channels and connection to the headstage.
      NOTE: Although hardware settings should reflect the equipment being used, details related to analyte/interferent type and the concentration do not have to be entered for the 'test' calibration. The recoding mode should be 'Amperometry' and the 'V-applied' should be -0.7 V. Verify typical recording for all channels.
   7. Cancel by selecting 'Cancel' when connectivity of all the recording sites is verified. It is not necessary to save the calibration data.
   8. Select the "Electroplating" icon on the desktop. The Electroplating Tool Menu will appear. Verify the correct settings are entered:
      P-P amplitude: 0.25
      Offset: -0.5
      Frequency: 0.05
      Duration (min): 20
   9. Select the 'pause' button to begin plating. The elapsed time field should begin counting down.
   10. Upon completion (all time elapsed), select 'another MEA' in the popped up menu to begin mPD plating an additional MEA. Replace the plated MEA with an MEA to be mPD plated and repeat the electroplating procedure using the electroplating tool.
       NOTE: It is recommended to use the prepared mPD solution no more than 2 times or within a 1 h time period. Prepare a fresh solution to prepare more than 2 MEAs.
   11. Select 'exit software' to finish electroplating. Rinse mPD-plated electrode tips with DI water (to avoid a salt residue) and store (24 h) before calibrating.
   12. Remove the reference electrode from solution and rinse with DI water before placing into the appropriate storage solution (e.g. 3 M NaCl). Ensure the reference electrode tip is submerged in solution. Slowly lower glass reference electrode into the solution to avoid damaging the glass.
   13. Dispose of the mPD solution in an appropriately labeled hazardous waste container.

3. Calibrate the MEA for Glutamate Detection and Selectivity (Figure 1)²¹

1. Preparing the solutions needed for calibration.
   NOTE: Refer to Table 1.
2. Perform calibration under constant stirring (magnetic battery operated stirrer) at 37 °C. Turn on the heating pad or circulating water bath and obtain a small stir bar. Stir slowly but fast enough that when an addition is made (below), the new plateau is reached quickly.
3. Connect the headstage (2 pA/mV) to the recording control system and insert the MEA tip into 40 mL of stirred 0.05 M PBS (use a 50-mL beaker).
4. Connect the reference electrode. Flick the end to ensure there are no air bubbles.
5. Open the recording program and click "Calibration". Make sure the settings are correct (check that sentinel sites are selected as this may change with different electrodes), choose MEA number and press "Start".
6. Allow 5 - 10 min for equilibration; begin once the baseline has stabilized (< 0.004 nA/min change).
7. Select "Baseline" and add 500 µL of 20 mM ascorbic acid (interferent; final [250 µM]) and select "Interferent" (AA is thought of as brain-wide interferent) once the current has reached a new, steady plateau, if any. Allow 0.5 - 1 min between additions. If the MEA is properly electroplated, almost no change should be observed.
8. Add 40 µL of 20 mM L-glutamate (final [20 µM]) and once the current has reached a new, steady plateau, mark 1^st addition "Analyte". Repeat three times for a total of 3 glutamate additions.
9. Add 40 µL DA. Do not select "Analyte"; select "Test Substance" - DA may only be an interferent in areas containing DA.
10. Add 40 µL Peroxide. Do not select "Analyte"; select "Test Substance".
11. Click the "Stop" button once the calibration is finished.
12. To determine the functionality of the MEA, click the calculation tab and record the following parameters in a lab notebook.
    1. For the MEA design (3,000 µm²) used in these calibrations, use a MEA with a slope of ≥ 0.004 nA/µM but lower slopes can be used for certain experiments. If there is less than a 10% difference in slope between enzyme coated and uncoated sites, then use these MEAs for evoked release only or clean/discard the MEA.
    2. Use a limit of detection (LOD) ≤ 1.5 µM. If the LOD is greater than 1.5 µM, then use these MEAs for evoked release and uptake only. Do not use these electrodes for recording tonic levels.
    3. Use a selectivity for the desired analyte (i.e., glutamate) over the interferent of > 20 arbitrary units. If selectivity is less than 20, then clean/discard the MEA.
    4. Use a linearity (R²) of the calibration curve of ≥ 0.90. If linearity is less than 0.90, then clean or discard MEA.
13. Save the file with the # of the MEA, date, and initials of the person calibrating.

4. Assemble the Micropipette

NOTE: Micropipettes (capillary glass) should have a tip with an internal diameter of 10 - 15 µm.

1. Place the micropipette centrally among all four platinum recording sites and mount 50-100 µm above the MEA using sticky wax and modeling clay.
2. To load the micropipette, use a 1 mL syringe filled with glutamate or KCl solution, a 0.22-µm sterile syringe filter, and a 97 mm long, 28-gauge pipette filler, backfill the micropipette. That is, insert the needle at the top of the micropipette and fill at the bottom of the micropipette (the end toward the MEA), making sure there are no bubbles.
3. Attach the micropipette to the pressure ejecting system at 2 - 20 psi for about 1 s.

5. Plate the Miniature Reference Electrode for In Vivo Use

1. Prepare the plating solution (50 g NaCl/90 mL 1 M HCl in a 100 mL beaker (plating bath)) and cut the bath electrode (~ 10 cm length of platinum (Pt) wire (~ 0.02" diameter)).
2. Cut a 10 - 15 cm long piece of Teflon coated silver wire (0.008" bare) and use a razor blade to scrape off ~ 1 cm of Teflon coating from each end of end of silver wire.
3. Solder a gold pin on one end and place one end of exposed silver wire into the plating bath.
4. Using a DC adapter (use only a DC adapter having an output of ~ 9 - 15 VDC max.), clamp the "red" (+) wire to the prepared silver wire (reference electrode) on the gold pin side. Clamp the "black" (-) wire to Pt bath cathode.
5. Plug in the DC adapter. Look for the correct plating process — bubbles should appear at the bath electrode; reference electrode turns a silver/gray color.
   CAUTION: Do not switch the leads [Black (-) vs Red (+)] — plating bath will be ruined if this occurs and fresh plating solution will need to be made. A bad solution will turn yellow in color: DO NOT USE!
   NOTE: The plating reaction takes 2-5 min generally: Ag⁰ + Cl^- → AgCl + e^-
6. Test the reference electrode.
   1. Set a multimeter to the 100 - 200 mV DC setting.
   2. Place a benchmark (that is, a previously tested electrode known to be working) reference electrode in 3 M NaCl and test against the newly plated reference electrode.
      NOTE: If using a new reference electrode, soak it in 3 M NaCl before use (12 h recommended).
   3. Place the "red" (+) lead on the gold pin of the reference electrode to be tested. Place the "black" (-) lead on benchmark electrode. An acceptable readout range is ± 10 mV. 0 mV is ideal across the 2 reference electrodes.

6. General Animal Surgery for MEA Recordings

1. Preparation for surgery
   1. Place surgery tools in disinfectant the night before.
   2. Remove the tools from the disinfectant and rinse thoroughly and place the tools on a sterile surgery pad. Obtain a heating pad.
   3. Calibrate two electrodes and attach the micropipette as described in procedures 3 and 4.
   4. Make the reference electrode as described in Section 5.
   5. Make 200 µM glutamate and 70 mM KCl. Refer to Table 1.
   6. Obtain the animal and record its weight on surgery sheets.
   7. Prepare the anesthesia and turn on the heating pad.
2. Perform MEA surgery
   1. Using isoflurane (4% flow in oxygen), anesthetize the animal in an induction chamber until the respiratory rate has significantly declined and the animal does not respond to toe or tail pinch. Record respiratory rate and body temperature every 15 min.
   2. Place the animal in the stereotaxic device using the ear bars to stabilize the head. Make sure the animal is secure and the head does not move. Lower the isoflurane flow to 1.5 - 3% in oxygen. Ensure that the heating pad is properly positioned under the animal and set to 37 °C to provide supplemental heat. Failure to provide supplemental heat may result in profound hypothermia and premature death of the animal.
   3. Apply eye ointment using a sterile cotton applicator and record the respiratory rate and body temperature. Using a battery-powered trimmer, shave the top of the head. Use the small surgical scissors to remove the fur near the ears.
   4. At this point, put on the sterile surgery gloves. Apply iodine and then alcohol (three times) to the scalp.
   5. Using a scalpel, make an incision straight down the middle of the scalp and spread the skin using bull dog clamps. Take a sterile cotton tip to soak up any blood. Use hydrogen peroxide to facilitate the appearance of bregma and lambda.
   6. Check that the head is properly positioned by measuring the D/V and M/L coordinates of bregma and lambda. Coordinate changes should be zero if the head is on a flat plane. Adjust the head and ear bars as necessary to ensure a flat plane.
   7. Find bregma, and zero the coordinates. Using the desired coordinates, move to the left and right and make a mark using a permanent marker. Using a cauterizer/marker, make a large square around the mark with enough room to reach the desired region.
   8. Using a sterile rotary tool, drill around the square mark. Soak up any blood using a sterile cotton applicator. Thoroughly disinfect the drill bit prior to each use.
   9. Attach the MEA to the headstage and backfill the pipette with the first desired solution. Be sure leave a gap in the pipette without solution so that the solution being expelled can be examined. Attach the tubing to the glass micropipette.
   10. Turn on the nitrogen tank and the pressure ejector (psi=5, time=0.6 s). Test (press manual red button) to ensure the pipette is not clogged before beginning.
   11. Calibrate the micropipette. Start at 0 on the reticle and place a piece of blue tape on the headstage to indicate the start point. Turn on the digital reader and reset it to zero. Go down 1 mm (DV) and count the number of ticks the solution has moved on the reticle. 10 ticks should equal 1 mm (1 mm = 250 nL), so 1 tick = 25 nL.
   12. Place the reference electrode in a remote location from the MEA such that it is still in liquid contact with the brain to complete the circuit.
   13. Find bregma with the MEA attached and zero the digital reader. Move the MEA to the desired coordinates. Slowly lower the MEA until the tip touches the brain. Zero the DV coordinate and SLOWLY lower the MEA into the brain.
   14. On the recording program, click on the desired calibrated electrode and then click "Perform Experiment." The system will then start recording tonic levels for the analyte of interest while the animal is anesthetized.
   15. Zoom to an axis that will help in observing baseline and allow the electrode to baseline for 20 - 45 min.
   16. After the baseline is stable, set the pressure ejector to .6 s and 5 psi and press the red button on the pressure ejector to eject. Record the time and pressure as well as volume/ticks moved on the injection sheet (Figure 2). Make any notes that are necessary (i.e., larger peaks, dilution effects, clogged pipette, noisy baseline/o-scope).
   17. For glutamate injections, wait 3 - 5 min between injections. For KCl injections wait 1 - 2 min between injections. Inject using the same pressure and time at least 3 times. Change time and repeat. Try not to change pressure unless the micropipette is clogged.
   18. If the micropipette is clogged, increase the pressure up to 30 psi.
   19. Record from the desired brain regions, repeating on both sides of the brain using different solutions. Record a baseline in each new region for 20 min.
   20. Take the MEA with micropipette attached, rinse with DI water and soak in PBS overnight until all the blood is gone.
   21. Euthanize the animal and save the brain in a pre-labeled tube and store in the -80 °C freezer or keep one side for fixation. To euthanize the animal, overdose with isoflurane (inhalation). Perform decapitation using surgical scissors and dissect out the desired regions.

7. Cleaning Coated MEAs after Use

1. Do not clean MEAs if unused. If there is lint or dust on the surface, clean with methanol and let dry for 24 h before coating.
2. Turn on the water bath (80 °C) and soak the tip of the MEA for 30 min. Carefully wipe the tip of the electrode with a cotton tipped applicator to remove any debris that may be left on the electrode tip. Do not get the black "insulation portion" of the MEA wet.
3. Using three different sonicators, soak the tip of the MEA in (1) Citrisolv, a commercial solvent and clearing agent (2) isopropyl, and (3) DI water for 5 min. Carefully wipe the tip of the electrode with cotton tipped applicator to remove any debris that may be left on the electrode tip.
4. Examine the tips under a dissecting microscope to ensure the layers have been successfully removed.

8. Analysis

1. To analyze the data, first export the desired experiment by double-clicking the finished experiment and selecting "export data" from the file drop-down menu.
2. Save this data to the desired file location and open up the analysis program. Click "open" in the analysis program and choose the recently saved file.
3. Give the program a few moments to upload the file and then check experiment under the event markers tab. Under output, check the boxes for the desired parameters. For example, baseline, amplitude, peak area (area under the curve), T_rise, and T₈₀.
4. Click refresh and then analyze to export the data to a spreadsheet (this may take a few minutes). The program will give a prompt to save the file to the desired location. Once saved, the file can be edited in a spreadsheet.

Results

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 tauopathy^19,20. The rTg(TauP301L)4510 mouse expresses the P301L mutation in tau associated with frontotemporal dementia and parkin...

Discussion

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	#80383	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
Battery-powered 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	#786500
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