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).

<ol>
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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 (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 &#34;go under&#34; 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&#39;s disease and epilepsy.

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

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 parkinsonism linked to chromosome 17, and is commonly used to study tau pathology associated with Alzheimer&#39;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 &#60; 0.004 nA/min change in slope (10 - 20 min), we first measured tonic glutamate levels (&#181;M) 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 &#181;M 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 &#8804; 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 &#34;peak matching&#34; 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.
<img alt="Figure 1" src="/files/ftp_upload/55418/55418fig1.jpg" /> 
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 &#181;M) and dopamine (DA: 2 &#181;M) did not alter the current at either glutamate oxidase or sentinel sites. Addition of glutamate (Glu: 20, 40, 60 &#181;M) produced a stepwise current increase on the glutamate oxidase site, but no change on the sentinel site. Hydrogen peroxide (H2O2: 8.8 &#181;M) produced an increase in current on both sites. Sensitivity, slope, limit of detection, and R2 values were calculated after calibration. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55418/55418fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55418/55418fig2.jpg" /> 
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. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55418/55418fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55418/55418fig3.jpg" /> 
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 (&#8593;) 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 &#181;m section of hippocampus shows location of MEA tracks in CA3 and CA1 (Mean &#177; SEM; ** p &#60; .01 Veh-Control vs. Veh-TauP301L, # p &#60; .05 Ril-Tau-P301L vs. Veh-TauP301L, ## p &#60; .01 Ril-Tau-P301L vs. Veh-TauP301L; n = 14 - 19/group). This figure reprinted with permission from John Wiley and Sons19,20. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55418/55418fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55418/55418fig4.jpg" /> 
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 &#177; SEM; * p &#60; .05 Veh-Control vs. Veh-TauP301L, ** p &#60; .01 Veh-Control vs. Veh-TauP301L, # p &#60; .05 Ril-Tau-P301L vs. Veh-TauP301L, ## p &#60; .01 Ril-Tau-P301L vs. Veh-TauP301L; n = 13 - 15/group). This figure was reprinted with permission from John Wiley and Sons20. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55418/55418fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05 M PBS</td>
			<td>2 L DI water:</td>
		</tr>
		<tr>
			<td>Store in glass beaker wrapped with tin foil. Bring pH to 7.4 with KOH if necessary.</td>
			<td>NaH2PO4&#183;H2O: 2.8 g Na2HPO4: 11.34 g Sodium Chloride: 11.68 g</td>
		</tr>
		<tr>
			<td>20 mM Ascorbic Acid</td>
			<td>50 mL DI water:</td>
		</tr>
		<tr>
			<td>Make fresh daily.</td>
			<td>Ascorbic acid: 0.18 g</td>
		</tr>
		<tr>
			<td>20 mM L-Glutamate </td>
			<td>50 mL DI water:</td>
		</tr>
		<tr>
			<td>Make fresh weekly.</td>
			<td>L-glutamate monosodium salt hydrate: 0.15 g</td>
		</tr>
		<tr>
			<td>2 mM Dopamine</td>
			<td>Dopamine hydrochloride: 0.038 g</td>
		</tr>
		<tr>
			<td>Make fresh monthly.</td>
			<td>0.1 M Perchloric acid: 10 mL Bring volume to 100 mL with DI water</td>
		</tr>
		<tr>
			<td>8.8 mM Hydrogen Peroxide</td>
			<td>50 mL DI water:</td>
		</tr>
		<tr>
			<td>Make fresh weekly.</td>
			<td>Hydrogen peroxide: 500 &#956;L</td>
		</tr>
		<tr>
			<td>70 mM potassium chloride</td>
			<td>100 mL DI water:</td>
		</tr>
		<tr>
			<td>Make fresh day of experiment.</td>
			<td>Potassium chloride: 0.08 g Sodium chloride: 0.07 g Calcium Chloride: 0.004 g</td>
		</tr>
		<tr>
			<td>200 &#956;M Glutamate</td>
			<td>20 mM glutamate: 100 &#956;L</td>
		</tr>
		<tr>
			<td>Make fresh day of experiment.</td>
			<td>Sterile saline: 10 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Calibration Solutions.

Watch this Scientific Journal Video about Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models at JoVE.com

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

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

11.2K Views.  West Virginia University. 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). 

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

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

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

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

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; 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.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Biology

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

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

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

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.

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

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

Here we describe a staged, behavioral testing 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 &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

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 &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Medicine

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

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

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

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.

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

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

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

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

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.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

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

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Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

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

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

Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis