Name: a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate
Uploaded: 2018-07-10T15:00:00
Description: Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

The authors would like to thank the Post Baccalaureate Scholars Program Office and Novartis Institutes for BioMedical Research as a whole for funding this study.

1. Preparation of Cells
NOTE: This protocol, including data generation, uses HEK293 cells transduced with a baculovirus encoding NR1 and NR2A cells.
<ol>
	<li>Seed the appropriate number of HEK293 cells and add the NR1 and/or NR2A virus at the appropriate final concentrations (1.00 &#181;L each). For a 384-well plate, use 10,000 cells/well in a final volume of 30 &#181;L.</li>
	<li>Alternatively, seed HEK293-NR1-NR2A cells at the appropriate cell number and volume (for a 384-well plate, use ...

<ol>
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L., Westbrook, G. L., Guthrie, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+block+by+Mg2++of+NMDA+responses+in+spinal+cord+neurones.">Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones.</a> Nature. 309 (5965), 261-263 (1984).</li><li>Zhu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+NMDA+Receptor+Inhibition+and+Activation.">Mechanism of NMDA Receptor Inhibition and Activation.</a> Cell. 165 (3), 704-714 (2016).</li><li>Yildiz-Unal, A., Korulu, S., Karabay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+strategies+against+calpain-mediated+neurodegeneration.">Neuroprotective strategies against calpain-mediated neurodegeneration.</a> Neuropsychiatric Disease and Treatment. 11, 297-310 (2015).</li><li>Gascon, S., Sobrado, M., Roda, J. M., Rodriguez-Pena, A., Diaz-Guerra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxicity+and+focal+cerebral+ischemia+induce+truncation+of+the+NR2A+and+NR2B+subunits+of+the+NMDA+receptor+and+cleavage+of+the+scaffolding+protein+PSD-95.">Excitotoxicity and focal cerebral ischemia induce truncation of the NR2A and NR2B subunits of the NMDA receptor and cleavage of the scaffolding protein PSD-95.</a> Molecular Psychiatry. 13 (1), 99-114 (2008).</li><li>Uttara, B., Singh, A. V., Zamboni, P., Mahajan, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+neurodegenerative+diseases:+a+review+of+upstream+and+downstream+antioxidant+therapeutic+options.">Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options.</a> Current Neuropharmacology. 7 (1), 65-74 (2009).</li><li>Hansen, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+a+fluorescence-based+screening+assay+identifies+histamine+H3+receptor+antagonists+clobenpropit+and+iodophenpropit+as+subunit-selective+N-methyl-D-aspartate+receptor+antagonists.">Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.</a> Journal of Pharmacology and Experimental Therapeutics. 333 (3), 650-662 (2010).</li><li>Bettini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+novel+NMDA+receptor+antagonists+selective+for+NR2A-+over+NR2B-containing+receptors.">Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors.</a> Journal of Pharmacology and Experimental Therapeutics. 335 (3), 636-644 (2010).</li><li>Feuerbach, D., Loetscher, E., Neurdin, S., Koller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pharmacology+of+the+human+NMDA-receptor+subtypes+R1-2A,+R1-2B,+R1-2C+and+R1-2D+using+an+inducible+expression+system.">Comparative pharmacology of the human NMDA-receptor subtypes R1-2A, R1-2B, R1-2C and R1-2D using an inducible expression system.</a> European Journal of Pharmacology. 637 (1-3), 46-54 (2010).</li><li>Hansen, K. B., Brauner-Osborne, H., Egebjerg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+ligands+at+recombinant+NMDA+receptor+subtypes+by+electrophysiological+recordings+and+intracellular+calcium+measurements.">Pharmacological characterization of ligands at recombinant NMDA receptor subtypes by electrophysiological recordings and intracellular calcium measurements.</a> Combinatorial Chemistry and High Throughput Screening. 11 (4), 304-315 (2008).</li><li>Guo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+NMDA-receptor+calcium+influx+assay+sensitive+to+stimulation+by+glutamate+and+glycine/D-serine.">A NMDA-receptor calcium influx assay sensitive to stimulation by glutamate and glycine/D-serine.</a> Scientific Reports. 7 (1), 11608 (2017).</li><li>Hackos, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+Allosteric+Modulators+of+GluN2A-Containing+NMDARs+with+Distinct+Modes+of+Action+and+Impacts+on+Circuit+Function.">Positive Allosteric Modulators of GluN2A-Containing NMDARs with Distinct Modes of Action and Impacts on Circuit Function.</a> Neuron. 89 (5), 983-999 (2016).</li><li>Romero-Hernandez, A., Furukawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Mode+of+Antagonist+Binding+in+NMDA+Receptors+Revealed+by+the+Crystal+Structure+of+the+GluN1-GluN2A+Ligand-Binding+Domain+Complexed+to+NVP-AAM077.">Novel Mode of Antagonist Binding in NMDA Receptors Revealed by the Crystal Structure of the GluN1-GluN2A Ligand-Binding Domain Complexed to NVP-AAM077.</a> Molecular Pharmacology. 92 (1), 22-29 (2017).</li><li>Auberson, Y. 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The success of this assay depends largely on the health of the HEK cells used. Cells undergoing exponential growth and with low passage number should be used. This assay involves many transfers and additions of solutions, so using caution will ensure higher accuracy in its results. Concentrations of compounds and all other reagents should be also cross-checked to minimize errors. When replacing cell media with the assay buffer for the calcium flux assay, the plate should be tilted gently so that the cells do not detach. ...

With current advances in medicine, life expectancy has increased significantly; however, so has the prevalence of age-related diseases. Diseases of the central nervous system (CNS) such as schizophrenia, amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease, and Parkinson&#39;s disease, among others, are no exception and have been projected to increase over the next decade1,2,3. The malfunction of ionotropic glutamate receptors known as N-methyl-D-aspartate receptors (NMDAR) has been linked to Alzheimer&#39;s disease, schizophrenia, traumatic brain injury, stroke, diabetes, and glaucoma among others, which warrants the need to study their biology for the development of effective, disease-modifying therapies4,5,6,7.
NMDARs are composed of four monomers or subunits4,8,9. The structural composition of the NMDAR shows developmental and regional variability within the brain7,10. NMDARs are involved in synaptic plasticity, cognition, and the generation of rhythms for breathing and locomotion11,12,13. As a voltage-gated channel, it is largely non-conducting at resting membrane potential (-70 mV) and is blocked by magnesium to prevent further permeation of ions. The channel is activated by the binding of two ligands, glutamate and glycine/D-serine, and a simultaneous depolarization at the synaptic membrane mediated by AMPA receptors, another subclass of ionotropic glutamate receptors. The depolarization removes the magnesium blockage of the NMDAR, enabling the influx of cations, particularly calcium14,15,16. Even though the activation of NMDAR is essential for cell survival, excessive activation can lead to cell death17,18,19 through excitotoxicity. This, in addition to the complex nature of the receptor, makes it challenging to perform studies necessary for developing effective therapies.
Different methods have been developed to study the NMDAR. However, each one has accompanying caveats. For example, one widely-used technique is a fluorescence-based assay that measures NMDAR-mediated changes in intracellular calcium in a stable cell line under the control of a tetracycline-inducible promoter (Tet-On)20. However, in this system, the supramaximal concentrations of ligands that are needed and the requirement that NMDAR inhibitors are present during the measurement makes it nearly impossible to detect activity of the competitive antagonist. In other similar systems, the expression of the functional receptor causes toxicity, requiring channel blockers such as ketamine21,22 to preserve the cell cultures. These channel blockers sit at the core of the receptor and are difficult to wash out, especially in a plate-based format, so they interfere with functional studies of the receptor. Finally, in electrophysiological measurements such as patch clamping, there is limited throughput, and large-scale studies are very expensive23. Notwithstanding, the above systems are insensitive to glycine stimulation; hence, studying glycine-dependent activity of the NMDAR becomes a challenge.
Here, we describe a novel approach for studying NMDAR that overcomes the discussed limitations. Our technique capitalizes on the baculovirus expression system to express the receptor at functional levels with an optimal ratio of the subunits in as little as 16 hours. Furthermore, the use of baculovirus allows for a simple and combinatorial approach, which provides a broad characterization of the distinct recombinant NMDAR subtypes. Unlike other assays, this protocol does not require channel blockers because of the use of weak antagonists. The method&#39;s strongest advantage is that after washout of the weak antagonist, the receptor is sensitive to modulation of the individual glycine- and glutamate-binding sites in addition to dual modulation of both glycine/D-serine and glutamate ligand-binding sites. The assay recapitulates known pharmacology of the NMDAR receptor and the effects of its known positive and negative modulators. Finally, generation of this in vitro cellular assay overcomes the cellular toxicity caused by excessive calcium influx and allows for functional studies of the receptor in a high-throughput fashion, which can accelerate discoveries of NMDAR modulators in disease states.

<table>
	<tbody>
		<tr>
			<td>HEK-293</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Human NMDA (NR1/NR2A) Receptor Cell Line</td>
			<td>ChanTest Corporation</td>
			<td>CT6120</td>
			<td></td>
		</tr>
		<tr>
			<td>pFastBac Dual Expression Vector</td>
			<td>ThermoFisher Scientific</td>
			<td>10712-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well Clear Flat Bottom Microplate</td>
			<td>Corning Life Sciences</td>
			<td>3844</td>
			<td></td>
		</tr>
		<tr>
			<td>FLIPR Calcium 6-QF Assay Kit</td>
			<td>Molecular Devices</td>
			<td>R8192</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>49621</td>
			<td></td>
		</tr>
		<tr>
			<td>D-serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4250</td>
			<td></td>
		</tr>
		<tr>
			<td>L701,324</td>
			<td>Tocris</td>
			<td>907</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Boston Bio Product</td>
			<td>BB-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>E506</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>ThermoFisher Scientific</td>
			<td>P36400</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX media</td>
			<td>ThermoFisher Scientific</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>MDL105,519</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>NVP-AAM077</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>CGP070667</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Before testing the effects of small molecules, one must determine the optimal expression levels of NMDARs as well as the optimal ligand concentrations. As described, HEK293 cells were seeded at 10,000 cells per well in a 384-well plate, in the presence of 5 &#181;M CGP060667, then transduced with varying amounts of NR1 and NR2A viruses. After incubation overnight, ligand-induced calcium flux was measured (Figure 1). The results of these experiments reveal the...

Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

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

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 ...

This method can help the discovery of NMDA modulators to treat neurological diseases. The main advantage of this technique is that it allows the fissum study of NMDA-receptor activity, in response to defense concentrations or combinations of ligands in compounds. So this method can provide insight into the general capability of NMDA receptor.It can also be applied to study subtypes specific in biology and a sensitivity of NMDA receptor, in response to ligands or compounds. To measure the activity of the NMDA-receptor, prepare a 384 well plate, with HEK 293 cells, transduced with NR1, NR2, or HEK 293, NR1, NR2A cells in the presence of protective compound. Then, incubate the plate at 37 degrees Celsius, and five percent carbon dioxide for 16 hours.The next day, remove the plate from the incubator, and gently discard the cell media, by slowly flipping the plate over a compatible biowaste container. Gently tap the plate on a paper towel, to clean the media off the edges of the plate, and drain any remaining media. Subsequently, prepare the calcium 6 dye, by solubilizing one vial of calcium 6 dye, in 10 milliliters of incubation buffer.Add 30 microliters of the dye to the cells in the 384 well plate. Incubate the plate for two hours at 37 degrees Celsius, and five percent carbon dioxide. After two hours, remove the plate from the incubator, and let the cells adjust to room temperature for 10 minutes.Next, gently pour out the calcium 6 dye, and add 30 microliters of assay buffer. Repeat the wash for a total of three times, and leave 30 microliters of assay buffer on the cells, after the last wash. Then, let the cells rest for five minutes at room temperature.To prepare the ligand plate, make a four-fold stock of 400 micromolar glycine, and 400 micromolar glutamate. Transfer a minimum of 25 microliters of ligand stock into a V Bottom 384 well plate. Then transfer the ligand plate and cell plate into the FDSS.Afterward, measure the baseline fluorescence of the cell plate for 30 seconds. Transfer 10 microliters of the ligand stock into the cell plate, using the FDSS dispensing function, and measure the calcium-flux by recording calcium 6 dye fluorescence for five minutes. Afterward, calculate the maximal fluorescence ratio, by dividing the maximal fluorescence obtained by the baseline fluorescence.To test the compounds, prepare the cells, the ligand, and the compound plates. Load the ligand, compound, and cell plate into the FDSS. Next, measure the baseline fluorescence for 30 seconds.Add 10 microliters of compound stock, and measure the fluorescence for five minutes. Then, add 10 microliters of ligand stock, and measure the fluorescence for five minutes. Afterward, determine the integral of the fluorescence ratio by calculating the area under the curve of the fluorescent ratios during the last five minutes of the measurement.Using the predetermined optimal ligand and subunit expressions, the effects of a glutamate binding site antagonist, NVP-AAM077, and the glycine binding site antagonist, L701, 324, were characterized by the calcium-flux assay, for NR1, NR2A at the indicated concentrations. A concentration equal to the eZ80 of glycine or glutamate, in the presence of the saturating concentration of the other ligand, was used after addition of NVP-AAM077, and L701, 324, respectively. While attempting this procedure, it's important to remember to optimize the respective various amounts for this specific cell lines, using the experiment.And to use healthy nonconfluent cells. Special occasion should be spent on the dilution of compounds and ligands. Following this procedure, other methods like electrophysiology can be performed, in order to assign additional questions about a specific mechanism of actions, or their compounds.

Research

JoVE Journal

Neuroscience

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...