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 10,000 cells/well in a final volume of 30 &#181;L). Add tetracycline at a concentration of 2 &#181;g/mL to induce NR2A expression and add the protection compound (100 &#181;M MDL105,519 or 5 &#181;M CGP07066724).</li>
	<li>NOTE: The baculovirus encoding NR1 and NR2 should have an approximate titer between 5 x 109 - 10 x 109 infectious unit per milliliter24.</li>
</ol>
2. Measurement of NMDA-receptor Activity
<ol>
	<li>Prepare a 384-well plate with HEK293 cells transduced with NR1/NR2 or HEK293-NR1-NR2A cells in the presence of either protective compound. Use a clear bottom, black frame, poly-D-lysine coated plate. 
	NOTE: Protection with 100 &#181;M MDL105,519 is used to confer sensitivity to glycine, while 5 &#181;M CGP070667 is used to confer sensitivity to glutamate.</li>
	<li>Incubate the plate at 37 &#176;C and 5% CO2 for 16 hours (overnight).</li>
	<li>Remove the plate from the incubator and gently discard the cell media by slowing flipping the plate over a compatible biowaste container. Gently tap the plate on a paper towel to clean the media off edges of the plate and drain any remaining media.</li>
	<li>Prepare the calcium6-dye by solubilizing one vial of calcium6 dye (1 plate size) in 10 mL of incubation buffer (HBSS pH 7.5, 20 mM HEPES, 1 mM MgCl2, 1 mM probenecid). Incubate the plate for 2 hours at 37 &#176;C and 5% CO2.</li>
	<li>Remove the plate from the incubator and let the cells adjust to room temperature for 10 minutes.</li>
	<li>Gently pour out the calcium6-dye as described in step 2.3 and add 30 &#181;L of assay buffer (HBSS pH 7.5, 20 mM HEPES, 1.8 mM CaCl2, 1 mM probenecid).</li>
	<li>Repeat step 2.6 twice for a total of 3 washes. Leave 30 &#181;L of assay buffer on the cells after the last wash.</li>
	<li>Let the cells rest for 5 minutes at room temperature.</li>
	<li>Prepare the ligand plate by making a 4-fold stock of 400 &#181;M glycine and 400 &#181;M glutamate (final concentration of 100 &#181;M; optimal concentration of ligands to be determined as described below). Transfer a minimum of 25 &#181;L of ligand stock into a V-bottom 384-well plate.</li>
	<li>Load the ligand plate and cell plate into the FDSS (Functional Drug Screening System).</li>
	<li>Measure the baseline fluorescence (Fo) of the cell plate for 30 seconds. Transfer 10 &#181;L of the ligand stock into the cell plate using the FDSS dispensing function and measure the calcium flux by recording calcium6-dye fluorescence for 5 minutes.</li>
	<li>Calculate the maximal fluorescence ratio by dividing the maximal fluorescence obtained by the baseline fluorescence (Fmax/Fo).</li>
</ol>
3. Optimization of NMDAR Expression Levels
<ol>
	<li>To determine the optimal amount of baculovirus to use, perform a titration of both NR1 and NR2A viruses. Prepare a 384-well plate with HEK293 cells (recommendation of 10,000 cells/well, 30 &#181;L of media) and include the protection compounds as described in step 2.1.</li>
	<li>Add varying amounts of the NR1 virus in different rows of the plate (for example: add 0 &#181;L, 2 &#181;L, 1 &#181;L, 0.5 &#181;L, 0.25 &#181;L in rows A-E respectively). Add duplicates for data accuracy.</li>
	<li>Add varying amounts of the NR2A virus in different columns of the plate (for example: add 0 &#181;L, 2 &#181;L, 1 &#181;L, 0.5 &#181;L, 0.25 &#181;L in rows A-E respectively). Add duplicates for data accuracy.</li>
	<li>Incubate the plate at 37 &#176;C and 5% CO2 for 16 hours (overnight).</li>
	<li>Determine the optimal ratio and amount of the NR1 and NR2A viruses by performing a calcium flux measurement on the FDSS (see above).</li>
</ol>
4. Ligand Titration
<ol>
	<li>Prepare the cells as described in step 2.3.</li>
	<li>Prepare dilutions of each ligand in the presence of saturating concentrations (100 &#181;M) of the other ligand as a 4-fold stock solution. For example: for a 10 &#181;M final test concentration of glycine, prepare a 40 &#181;M stock solution of glycine including 400 &#181;M glutamate.</li>
	<li>Proceed with the FDSS measurement as described in step 2.11.</li>
</ol>
5. Compound Testing
<ol>
	<li>Prepare the cells as described in step 1.</li>
	<li>Prepare the ligand plate with a 5-fold stock of final ligand concentration in the assay buffer.</li>
	<li>Prepare the compounds plate with the 4-fold stock of desired final concentration of compounds in the assay buffer. For a final compound concentration of 10 &#181;M in the assay buffer, prepare a 40 &#181;M stock solution.
	<ol>
		<li>Keep the concentration of the dimethyl sulfoxide (DMSO) constant across all samples by preparing a pre-dilution of the compound in DMSO before further dilution into the assay buffer.</li>
		<li>For a dose response of the compound in the final assay ranging between 3 and 30 &#181;M, prepare a pre-dilution of the compound in DMSO ranging from 1 to 10 mM. Dilute those in the assay buffer to the 4-fold stock concentration. The final concentration of the DMSO should not exceed 0.5%. 
		NOTE: It is recommended to test each sample in triplicates.</li>
	</ol>
	</li>
	<li>Load the ligand, compound, and cell plate into FDSS.</li>
	<li>Measure the baseline fluorescence for 30 seconds.</li>
	<li>Add 10 &#181;L of compound stock and measure the fluorescence for 5 minutes.</li>
	<li>Add 10 &#181;L of ligand stock and measure the fluorescence for 5 minutes.</li>
	<li>Determine the integral of the fluorescence ratio by calculating the area under the curve of the fluorescent ratios during the last 5 minutes of the measurement. 
	NOTE: Alternatively, the maximal ratio of fluorescence after ligand addition can be used for analysis.</li>
</ol>

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The authors have no conflicts of interest and nothing to disclose.

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. Finally, the use of poly-D-lysine coated plates in this protocol increases attachment of the cells to the plate.
There may be differences in NMDAR expression and the amount of virus needed for subunit expression depending on the passage number and fitness of the HEK293 cells at the time of transduction. Also, the baculovirus has a short shelf life ranging from six months to a year. Therefore, the quality of the virus is an important parameter for this assay. A standard immunofluorescence assay can be used to detect and confirm the expression levels and localization of the NMDAR units before further experiments24. Other cell lines can be used to study the NMDA receptor using this protocol. However, the tolerance and expression of NMDARs using the baculovirus must be confirmed before proceeding with the protocol; otherwise, unexpected results may occur. Additionally, compounds such as topoisomerase II inhibitors have been reported to increase the efficacy of baculovirus expression29. These alternatives can be tested and optimized according to the user&#39;s preferences.
The study of biological processes in a cell type different than the original cell type raises questions within the scientific community. A limitation of this assay is the use of kidney cell lines (HEK 293) as a model system to study the ionotropic glutamate receptor, NMDAR. HEK cells were used because of their depolarized membrane potential similar to that of depolarized neurons. In addition, the similarity between NMDARs in the brain and in our model is confirmed by our supplementary studies. The activity of positive allosteric modulators such as GNE-8324, negative modulators such as ifenprodil, and channel blockers such as ketamine, as well as the voltage dependence of the NMDAR, were all confirmed in our model22,23,24. Also, our ability to adjust the amount of virus for the NR1 and NR2A subunits ensures optimal functional expression levels.
Because of the low efficiency, high cost, and minimal success outcome, the characterization of individual receptors and their unique pharmacology are currently conducted in low-throughput manual patch-clamp assays22,23. On the other hand, excitotoxicity remains a major obstacle when using stably transfected cells, because overexpression of functional NMDARs are harmful to cells due to the ligands (glutamate and glycine/D-serine) that are released into the media18. As a stronger alternative, our assay uses baculovirus-mediated expression of functional NMDARs, which overcomes excitotoxicity and yields rapid, titratable expression of the receptors. Rather than being limited by the sticky nature of channel blockers, this assay also takes advantage of antagonists of weak ligand-binding sites. These antagonists compete with endogenous ligands in the media and protect the ligand-binding sites. Hence, after washout of the antagonists, sensitivity to exogenously-added ligands of the glutamate- and glycine/D-serine- binding sites is restored.
This assay, combined with recent advances in molecular biology such as CRISPR, will help target and characterize novel regulators of NMDAR expression and function. Concurrently, this assay will help identify small molecules that may directly or indirectly modulate NMDAR activity without the need to generate stable cell lines, thereby providing compatibility with genetic- and small molecule- based screens.

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 best quantity and ratio of virus to use for each subunit (Figure 1, yellow box).
Once the optimal quantity and ratio for transduction with the NR1 and NR2A were determined, a ligand titration was performed to determine the optimal ligand concentration for activation of NMDARs. To accomplish this, one of the co-ligands was added in varying concentrations at fixed, saturating concentrations of the other co-ligand (Figure 2A-2B). Similar results were obtained for other subunit combinations of the NMDAR such as NR1/NR2B and NR1/NR2D (Figure 2C), demonstrating the assay&#39;s applicability to other NMDAR family members24. After determining optimal expression levels and ligand concentrations, one can proceed with testing the effects of NMDAR-modulators in the assay.
Consistent with published reports, this first plate-based assay to study NMDARs with sensitivity to both glycine and glutamate on a large scale reproduces data on the known properties of the NMDAR14,25,26. Here, we use the activity of two compounds, [(R)-[(S)-1-(4-bromophenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077), a glutamate site antagonist, and [7-chloro-4- hydroxy-3-(3-phenoxy)phenyl-2(H)quinolone] (L701,324), a glycine site antagonist, to exemplify the predicted results of the assay27,28 (Figure 3). Finally, each compound was tested at the EC80 of the respective natural ligand of the binding site. Reduction of the ligand concentrations is expected to increase the potency of antagonists, while it is expected to have no effect on the potency of non-competitive inhibitors or pore-blockers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58160/58160fig1.jpg" /> 
Figure 1: Titration of NR1 and NR2A subunits. Different volumes of virus that encode NMDAR subunits NR1 and NR2A were titrated to determine the amount needed for optimal functional activity of the receptor. A 1:1 (v/v) ratio NR1:NR2A was observed as the optimal ratio with 1 &#181;L of each (yellow box). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58160/58160fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58160/58160fig2.jpg" /> 
Figure 2: Ligand titration. A. In a fixed concentration of glycine (100 &#181;M), varying glutamate concentrations were titrated and calcium flux was recorded. Error bars represent standard deviation. B. Glutamate remained at a concentration of 100 &#181;M and was titrated against different concentrations of D-serine and glycine, and calcium flux was measured. There was no significant difference found between the calcium flux in glycine and in D-serine. Error bars represent standard deviation. C. Cells transduced with NR1 and NR2D baculovirus were stimulated with either 100 &#181;M glutamate and varying concentrations of D-serine or 100 &#181;M D-serine and varying concentrations of glutamate and calcium flux was measured. Error bars represent standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58160/58160fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58160/58160fig3.jpg" /> 
Figure 3: Characterizing activity of ligand binding site antagonist. Using the predetermined optimal ligand and subunit expressions, the effects of a glutamate-binding site antagonist, NVP-AAM077, and a 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 EC80 of glycine or glutamate in the presence of the saturating concentration (100 &#181;M) of the other ligand was used after addition of NVP-AAM077 and L701,324, respectively. Error bars represent standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58160/58160fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

a-high-throughput-calcium-flux-assay-to-study-nmda-receptors-with

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.2K Views.  Novartis Institutes for BioMedical Research. 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.

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

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

Bioengineering

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Biochemistry

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.

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 ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

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 ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Biology

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 ...