Name: Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimers Mouse Models (Video) | JoVE Journal
Uploaded: 2017-05-03T13:00:00.000+00:00
Duration: 10 min 46 s
Description: Learn to measure tonic and phasic glutamate in vivo using enzyme-linked microelectrode arrays in Alzheimer's mouse models. This minimally invasive method enables precise neurotransmitter detection in hippocampal subregions.

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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).

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</ol>

GG is the sole proprietor of Quanteon, LLC that makes the FAST-16 recording system used for glutamate measurements in this study. JEQ is a paid consultant for Quanteon.

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 ...

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&#39;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 (&#60; 1 &#181;M). 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&#39;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.

<table><tbody><tr><td>FAST-16mkIII-8 channel</td><td>Quanteon</td><td>16mkIII</td><td></td></tr><tr><td>Microelectrode arrays</td><td>CenMet</td><td>W4 or 8-TRK</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A-3059</td><td>10 g (expires after 1 month)</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G-6257</td><td>100 mL (expires after 6 months)</td></tr><tr><td>Glutamate Oxidase</td><td>US Biological or Sigma Aldrich</td><td>G4001-01 or 100646</td><td>50 UI (expires after 6 months)</td></tr><tr><td>Hamilton Syringes</td><td>Hamilton</td><td>#80383</td><td>2 syringes</td></tr><tr><td>Methanol</td><td>BDH</td><td>UN1230</td><td>4 L</td></tr><tr><td>m-Phenylenediamine dihydrochloride (mPD)</td><td>ACROS Organics</td><td>1330560250</td><td>25 g</td></tr><tr><td>Reference Electrodes (RE-5B)</td><td>BAS</td><td>MF-2079</td><td>3 electrodes</td></tr><tr><td>Battery-powered Magnetic stir plate</td><td>Cole-parmer</td><td>EW-04804-01</td><td>Can purchase from different supplier</td></tr><tr><td>Glutamate</td><td>Sigma-Aldrich</td><td>G-1626</td><td>100 g</td></tr><tr><td>Ascorbic Acid</td><td>TCI</td><td>50-81-7</td><td>500 g</td></tr><tr><td>Dopamine Hydrochloride</td><td>Alfa Aesar</td><td>62-31-7</td><td>5 g</td></tr><tr><td>Perchloric acid</td><td>VWR</td><td>UN2920</td><td>500 mL</td></tr><tr><td>Postassium chloride</td><td>VWR</td><td>7447-40-7</td><td>1 kg</td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>7647-40-7</td><td>1 kg</td></tr><tr><td>Calcium Chloride</td><td>MP</td><td>153502</td><td>100 g</td></tr><tr><td>Sodium Hydroxide</td><td>BDH</td><td>1310732</td><td>500 g</td></tr><tr><td>Glass pressure ejection pipettes</td><td>CenMet</td><td></td><td></td></tr><tr><td>Sticky wax</td><td>Kerrlab</td><td>625</td><td>Can purchase from different supplier</td></tr><tr><td>Microsyringe</td><td>World Precision Instruments</td><td>MF28G-5</td><td></td></tr><tr><td>Modeling clay</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Picospritzer III</td><td>Parker</td><td></td><td></td></tr><tr><td>Silver wire</td><td>AM systems</td><td>#786500</td><td></td></tr><tr><td>Hydrochloric acid</td><td>BDH</td><td>7647010</td><td>2.5 L</td></tr><tr><td>Platinum wire</td><td>AM Systems</td><td>778000</td><td></td></tr><tr><td>Solder gun</td><td>Lowes or Home Depot</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Multimeter</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>PhysioSuite</td><td>Kent Scientific</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>SomnoSuite</td><td>Kent Scientific</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Stereotaxic device</td><td>Stoelting</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Digital Lab Standard</td><td>Stoelting</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Meiji EMZ microscope</td><td>Meiji</td><td>EMZ-5</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>Micro</td><td></td></tr><tr><td>Metricide</td><td>Metrex</td><td>102800</td><td></td></tr><tr><td>Scalpel</td><td>VWR</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Surgery scissors</td><td>VWR</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Sterile cotton swabs</td><td>Puritan</td><td>25806</td><td>Can purchase from different supplier</td></tr><tr><td>Eye ointment</td><td>Puralube Vet Ointment</td><td></td><td>Obtain from the vet</td></tr><tr><td>Iodine swabs</td><td>VWR</td><td>S48050</td><td>Can purchase from different supplier</td></tr><tr><td>Alcohol swabs</td><td>Local drug store</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Sterile surgery drape</td><td>Dynarex</td><td>4410</td><td>Can purchase from different supplier</td></tr><tr><td>Sterile saline</td><td>Teknova</td><td>S5815</td><td>Can make own soltuion using filters</td></tr><tr><td>Hydrogen Peroxide (3%)</td><td>Local drug store</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Heating Pad</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr></tbody></table>

using enzyme-based biosensors to measure tonic and phasic glutamate in alzheimer's mouse models

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

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.

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 parkin...

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 ...

The overall goal of this procedure is to measure tonic and phasic extracellular glutamate changes in vivo using enzyme linked MicroElectrode Arrays. This method can help answer key questions in the neuroscience field, such as whether extracellular neurotransmitter levels and the fast dynamics of neurotransmitter release and clearance are altered in disease states. The main advantage of this technique is that the MicroElectrode Arrays can measure specific neurotransmitter changes within discrete brain regions, making it a spatially and temporally precise approach while being minimally invasive.Demonstrating the procedure will be Holly Hunsberger and Ryan Heslin from my laboratory. To begin this procedure, coat a pair of recording sites on an MEA with the desired enzyme and a different pair of recording sites with the inactive protein matrix. Then, draw up the glutamate oxidase or protein matrix using a 10 microliter Hamilton syringe.Gently press the plunger to dispense a small bead of solution at the syringe tip. To target the MEA recording sites, under a dissecting microscope, apply the solution to the appropriate recording sites by briefly contacting the recording sites with the solution droplet, which represents one layer. Set a timer for one minute between coating applications to the recording sites.Now, place the reference electrode through the opening in the plastic arm. Lower the reference electrode into a beaker containing PBS and ensure that it does not contact the bottom of the beaker. Next, connect the reference electrode to the head stage and connect the MEA to the mPD plated to the head stage.Lower the MEA into the beaker of solution such that only the MEA tip is submerged in solution. Do not submerge the MEA tip in liquid beyond the black bubble. Doing so may damage the MEA.Then, select the electroplating icon on the desktop. In the electroplating tool menu, verify the correct settings are entered. Afterward, select exit software to finish electroplating.Rinse the mPD plated electrode tips with DI water and store for 24 hours before calibrating. In this procedure, turn on a heating pad at 37 degrees Celsius and connect the head stage to the recording control system. Then, insert the MEA tip into 40 milliliters of stirred 0.5 molar PBS.Next, connect the reference electrode to the head stage. Flick the reference electrode tip to ensure there are no air bubbles. After that, open the recording program, make sure the settings are correct, choose the MEA number, and click Calibration, and press start.Allow five to 10 minutes for equilibration and begin calibration once the baseline has stabilized. Next, select Baseline and add 500 microliters of ascorbic acid. Then, select Interferent once the current has reached a new steady plateau.If the MEA is properly electroplated, no change should be observed. Next, add 40 microliters of 20 millimolar L-glutamate. Once the current has reached a new steady plateau, mark first addition as analyte.Repeat three times for a total of three glutamate additions. Then, add 40 microliters of dopamine. And select Test Substance.Subsequently, add 40 microliters of peroxide. Select Test Substance and click the stop button once the calibration is finished. In this step, place the micropipette centrally among all four platinum recording sites and mount it 50 to 100 micrometers above the MEA using sticky wax and modeling clay.Using a DC adapter, clamp the red positive wire to the prepared silver wire on the gold pin side, and clamp the black negative wire to the platinum bath cathode. Next, plug in the DC adapter and look for the correct plating process. Bubbles should appear at the bath electrode, and the reference electrode should turn a silver gray color.In this procedure, place the animal in the stereotaxic device and use the ear bars to stabilize its head. Make sure the animal is secured and the head does not move. Then, apply eye ointment using a sterile cotton applicator.After that, shave the head with a trimmer. And remove the fur near the ears using small surgical scissors. Subsequently, apply iodine and then alcohol to the scalp three times alternatively.Following this, make an incision in the middle of the scalp and spread the skin. Soak up any blood with a sterile cotton tip and apply hydrogen peroxide to facilitate the appearance of bregma and lambda. Make sure the head is properly positioned by measuring the dorsal-ventral and medial-lateral of bregma and lambda.Set the coordinates at bregma to zero and mark the target coordinates. Next, drill around the mark. Afterward, attach the MEA to the head stage.Back fill the pipette with the first desired solution. Be sure to leave a gap in the pipette without solution so that the solution being expelled can be examined. Then, attach the tubing to the glass micropipette.Find bregma with the MEA attached and set the coordinates to zero. Move the MEA to the desired coordinates and slowly lower the MEA until the tip touches the brain. Next, zero the dorsal-ventral coordinate and then slowly lower the MEA into the brain.Place the reference electrode in a remote location from the MEA such that it's still in liquid contact with the brain to complete the circuit. On the recording program, click on the desired calibrated electrode and then click perform experiment to start recording. After obtaining a stable baseline, set the pressure ejector to 0.6 seconds and 0.5 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 and make any notes if necessary. After that, remove the MEA with the micropipette attached, rinse it with DI water, and soak it in PBS overnight until all the blood is gone. In the CA3 and CA1 regions of the hippocampus, tonic glutamate levels were significantly increased in the vehicle treated TauP301L mice, an effect attenuated by riluzole treatment.Here, the baseline matched representative recordings of potassium chloride evoked glutamate release in the CA3 showed riluzole treatment attenuated the significant increase in the amplitude of glutamate release observed in Vehicle-TauP301L mice. Local application of potassium chloride produced a robust increase in extracellular glutamate that rapidly returned to tonic levels. The significantly increased potassium chloride evoked glutamate release in the DG, CA3, and CA1 observed in the Vehicle treated TauP301L mice after local application of potassium chloride was attenuated with riluzole treatment.This cresyl violet stained section of hippocampus confirms the location of MEA tracks in CA3 and CA1. While attempting this procedure, it's important to remember the troubleshooting steps listed in the manuscript. For instance, to reduce noisy signals, remember to ground the system and follow the steps in a timely manner.

using-enzyme-based-biosensors-to-measure-tonic-phasic-glutamate

Research

JoVE Journal

Neuroscience

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses in human patients.  Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

One of the pathological characteristics of AD is the formation of Amyloid β protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

In this video, a Multielectrode Array (MEA) coated with glutamate Oxidase is implanted into the brain tissue of an anesthetized mouse. Glutamate solution is injected into the extracellular space where the MEA detects the glutamate levels, enabling real-time monitoring of glutamate uptake by the astrocytes.

In Vivo Real-Time Glutamate Monitoring Using an Enzyme-Coated Microelectrode Array

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of β-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

One feature of Alzheimer's Disease is the elevation of Aβ1-42 peptide. Here we provide a protocol for preparing synthetic Aβ1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of Aβ-induced pathology and drug screening.

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

Mouse Models of Periventricular Leukomalacia

Learn to measure tonic and phasic glutamate in vivo using enzyme-linked microelectrode arrays in Alzheimer's mouse models. This minimally invasive method enables precise neurotransmitter detection in hippocampal subregions.

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

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