The authors thank Judith Lengyel, Maria Karg, Gr&#233;goire Friz, Rachel Haab, and Marie Claire Pflimlin from Roche and Tifany Schaer and Deborah Paolucci from HiQScreen for their excellent technical assistance.

Xenopus laevis are housed and handled according to the Geneva Canton Animal Guidelines.
1. Radioligand Binding
<ol>
	<li>Preparation of the assay plate
	<ol>
		<li>Prepare 1.5 L of assay buffer with 5 mM KCl, 1.25 mM CaCl2, 1.25 mM MgCl2, 120 mM NaCl, and 15 mM Tris; adjust the pH with HCl to 7.4.</li>
		<li>Prepare the compounds to be tested at 50.76 &#181;M (e.g., a 5 &#181;L compound at 10 mM into a 980 &#181;L assay buffer in a microcentrifuge tube). Mix them well using a vortex machine at high speed for 2&#8211;5 s.</li>
		<li>Prepare a pre-dilution of the reference compound flumazenil by pipetting 1 &#181;L of the compound (10 mM) to 1,970 &#181;L of assay buffer. Mix them well using a vortex machine at high speed for 2&#8211;5 s.</li>
		<li>Dilute a [3H]flumazenil stock solution with the assay buffer at 4 &#176;C to 4 nM in a 50 mL polypropylene tube.</li>
		<li>Pipette 50 &#181;L/well of assay buffer into columns 1 and 3&#8211;11 of a 96-well microplate as shown in the plate layout represented in Figure 2.</li>
		<li>Pipette 73.2 &#181;L of the compounds diluted at 50.76 &#181;M (see step 1.1.2) into column 12 of the plate.</li>
		<li>Dilute each compound, using a geometrical progression over 9 steps (1E-10 &#8211; 1E-06 M), with a 23.12 &#181;L transfer volume and fill columns 3&#8211;11. Mix the dilution thoroughly. Change the tip after every dilution.</li>
	</ol>
	</li>
	<li>Dilute the cell membranes
	<ol>
		<li>Thaw the cell membranes previously isolated from an HEK293 overexpressing human GABAA receptor (0.025&#8211;0.15 mg/mL) to obtain 80 mL at room temperature (RT) and transfer the suspension to the assay buffer at 4 &#176;C.</li>
		<li>Resuspend the membrane solution still at 4 &#176;C with a tissue homogenizer for 30&#8211;40 s at 10,000&#8211;12,000 rpm.</li>
	</ol>
	</li>
	<li>Dilute diazepam for the non-specific binding (NSB) control
	<ol>
		<li>Dilute a 4 mM diazepam stock solution with the assay buffer to a concentration of 40 &#181;M in 5 mL.</li>
	</ol>
	</li>
	<li>Start the reaction
	<ol>
		<li>Pipette 50 &#181;L of 4 nM [3H]flumazenil into each well of the 96-well plate and keep it in an ice water container.</li>
		<li>Add 100 &#181;L of the GABAA receptor subtype membranes preparation to have a protein concentration of 0.5mg/mL.</li>
		<li>Pipette 50 &#181;L of 40 &#181;M diazepam into column 2.</li>
		<li>Incubate the plate for 1 h on ice.</li>
		<li>Add 50 &#181;L of assay buffer in each well of a 96-well filter plate for the subsequent membrane separation and radioactivity measurement and stop the reaction afterward.</li>
	</ol>
	</li>
	<li>Stop the reaction
	<ol>
		<li>Prepare a washing buffer with 50 mM Tris-HCl, pH 7.4.</li>
		<li>Filter the solution using a 96-well cell harvester. Wash the plate 3x with 300 &#181;L of ice-cold washing buffer per well.</li>
		<li>Seal the bottom of the plate with a plastic film and add 40 &#181;L of scintillation cocktail for liquid scintillation counting in each well and wipe it with ethanol 70%.</li>
		<li>Gently shake the plate for at least 1 h at RT. Let it stand for at least another hour at RT.</li>
		<li>Measure the radioactivity (in CPM) by placing the plate on a scintillation counter. Allow a measuring for 3 min per well.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Determine the mean for the 8 replicates of non-specific binding (NSB) and the total binding (TB) as well as for the duplicates or triplicates of the samples. Calculate the % specific binding (SB) for the mean of each sample according to the following equation: 
		<img alt="Equation 1" src="/files/ftp_upload/57842/57842eq1.jpg" /> 
		Here, 
		total SB = the mean TB minus the mean NSB.</li>
		<li>Plot in abscissa the % SB versus in ordinate the inhibitor concentrations. Fit the data using the single site competition analysis equation: 
		<img alt="Equation 2" src="/files/ftp_upload/57842/57842eq2.jpg" /> 
		Here, 
		y = the % of SB, 
		A = the minimum of y, 
		B = the maximum of y, 
		C = the IC50, 
		X = the log10 of the concentration of the competing compound, 
		D = the slope of the curve (a Hill coefficient).</li>
		<li>Calculate the binding affinity (Ki) using the half maximal inhibitory concentration (IC50), the dissociation constant (Kd) of [3H]flumazenil on the respective membranes9, and the concentration of [3H]flumazenil in the assay according to the following data-using equation: 
		<img alt="Equation 3" src="/files/ftp_upload/57842/57842eq3.jpg" /></li>
	</ol>
	</li>
</ol>
2. Receptor Expression and Recordings in Xenopus Oocytes
<ol>
	<li>Ovaries harvesting and oocyte preparation
	<ol>
		<li>Prepare the sterile Barth solution with 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.82 mM MgSO4.7H2O, 0.33 mM Ca(NO3)2.4H2O, and 0.41 mM CaCl2.6H2O, at pH 7.4, supplemented with 100 unit/mL of penicillin, 100 &#181;g/mL streptomycin and 0.25 &#181;g/mL of amphotericin B.</li>
		<li>Prepare the 1x OR2 medium (no CaCl2) with 88.5 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 1 mM MgCl2.6H2O, at pH 7.4.</li>
		<li>Sacrifice a Xenopus Laevis female by deep anesthesia for 20 min in cooled Tricaine methanesulfonate (at a concentration of 150 mg/L, adjusted at pH 7.4) and sodium bicarbonate (300 mg/L) followed by decapitation.</li>
		<li>Harvest the ovaries rapidly with clean scissors and forceps and place them in 2 Petri-dishes (10 cm) filled with 40 mL of 1x Barth solution and antibiotics/antimycotic.</li>
		<li>Store the non-dissociated ovaries for up to 2 weeks in Barth solution at 4 &#176;C.</li>
		<li>For the dissociation, cut the ovaries with a clean razor blade in small fractions (1&#8211;2 cm3) and incubate them in 50 mL of OR2 medium without CaCl2 (OR2-noCaCl2) containing 0.2% collagenase (Type I) at 17&#8211;19 &#176;C in a 100 mL spinner flask with a slow agitation for 4&#8211;5 h. Verify that the magnetic bar is placed approximately 3&#8211;5 cm above the bottom of the spinner flask to avoid crushing the oocytes.</li>
		<li>After 4&#8211;5 h, verify that most of the oocytes are freed from their follicle (i.e., they swim around individually). Wash them 5x with 200 mL of OR2 supplemented with 1.8 mM CaCl2.</li>
		<li>Transfer the defolliculated oocytes to Petri-dishes (10 cm) filled with Barth solution and antibiotics/antimycotics and keep them at least overnight at 17 &#176;C before the cDNA or mRNA injection.</li>
		<li>On the next day, select, under a binocular, a healthy oocyte presenting a clear and distinct animal versus a vegetal pole (dark brown for the animal pole versus light yellow for the vegetal pole). Use clean Pasteur pipettes and dispose the oocytes one by one into a sterile conical 96-injection well plate. 
		NOTE: No special care is needed in the placement of the oocytes for the mRNA injections; whereas, for the cDNA injections, it is indispensable to orient the oocytes with the animal pole facing upward.</li>
	</ol>
	</li>
	<li>mRNA or cDNA automatic injection
	<ol>
		<li>Synthesize the mRNAs using a commercially available kit following the recommended instructions. Clean the instruments and the table with RNase and wear the appropriate protecting gloves. 
		NOTE: The quality of the mRNA is determinant to yield a good expression, and it is indispensable to prevent mRNA degradation during the injection procedure.</li>
		<li>Prepare cDNAs using a commercially available kit and dissolve the desired amount in bidistilled water at a concentration of 0.2 &#181;g/&#181;L.</li>
		<li>Inject 10&#8211;50 nL of mRNA solution at a concentration of 0.2 &#181;g/&#181;L, or 10 nL of cDNA solution at a concentration ranging from 0.02&#8211;0.2 &#181;g/&#181;L, preferably in batches of 95 oocytes.</li>
		<li>For membrane proteins requiring multiple subunits (i.e., heteromeric &#945;1&#946;2&#947;2 GABAA receptors), mix the corresponding mRNAs or cDNAs in the desired ratio (i.e., 1:1:1 or 1:10:1).</li>
		<li>Keep the oocyte at 17 &#176;C to prevent the expression of heat shock proteins by the oocytes; store the microplate in a thermo-controlled storage area.</li>
		<li>Dissolve the test compounds which were tested positive in the binding assay in OR2 at 0.1&#8211;1,000 &#181;M for electrophysiological recordings and dispose them off in a 96-well flat bottom polypropylene plate.</li>
	</ol>
	</li>
	<li>Plasmids for expression
	<ol>
		<li>Follow the instruction manuals of commercially available kits for the mRNA synthesis with bacterial T7 or T6 promotors and make 20 &#181;L of a solution at 0.2 &#181;g/&#181;L in RNase-free water.</li>
		<li>Prepare at least 20 &#181;L of a solution containing a eukaryote expression vector for the cDNA expression, typically at 0.2 &#181;g/&#181;L dissolved in bidistilled water.</li>
	</ol>
	</li>
	<li>Microinjection in the Oocyte and Visualization of its Quality
	<ol>
		<li>Inject 10&#8211;50 nL of the solution containing plasmid (see step 2.3.1 or 2.3.2) using a glass micro-injection needle with a tip diameter ranging up to 100 &#181;m mounted on a micromanipulator equipped with a pressure ejection system or with an automated injection system. 
		NOTE: In the example discussed here, oocytes are injected by batches of 95 with an automated system suitable for use with cDNA or mRNA.</li>
		<li>Fill the injection needle with 1 &#181;L of a dye such as methylene blue at 1% and inject the oocytes using the standard procedure (either in the nucleus for cDNA or in the cytoplasm for mRNA).</li>
		<li>Dispose the injected oocytes for about 1 min in boiling water. Cut the oocytes in half with a razor blade under a binocular. 
		NOTE: The dye remains localized as shown in Figure 3E, allowing a precise observation of the injection.</li>
	</ol>
	</li>
	<li>Two-electrode voltage clamp recordings
	<ol>
		<li>Place the well plate containing the oocytes on the automated system, which uses the principle illustrated in Figure 4. 
		NOTE: In contrast to the patch clamp system illustrated in Figure 5, the true membrane potential of the cell is read by the voltage electrode.</li>
		<li>Program the automated recording system using the icon-based interface with, for example, the scheme illustrated in Figure 6 for the determination of the concentration activity relationship.</li>
	</ol>
	</li>
	<li>Curve fitting
	<ol>
		<li>Analyze the results using the appropriate software, using the illustrated concentration activation curve, plot the current amplitude as a function of the logarithm of the agonist concentration.</li>
		<li>Observe the sigmoidal curve that can be subsequently fitted with the empirical Hill equation in the form: 
		<img alt="Equation 4" src="/files/ftp_upload/57842/57842eq4.jpg" /> 
		Here, 
		Y= the fraction of evoked current, 
		EC50 = the concentration for a 50% activation, 
		x = the concentration of the compound, 
		nH = the Hill coefficient or apparent cooperativity.</li>
		<li>Normalize the currents to a unity by dividing the amplitude of each response versus the value recorded at the highest concentration to analyze the data obtained from a series of cells.</li>
		<li>Determine the average and standard errors and realize the curve fitting using standardly available software.</li>
	</ol>
	</li>
</ol>
3. Electrophysiological Recordings in Brain Slices
NOTE: Hippocampal rat slices are prepared in accordance with the national and institutional guidelines.
<ol>
	<li>Preparation of hippocampus slices
	<ol>
		<li>Prepare the dissection artificial cerebro-spinal fluid (dACSF) with 124 mM NaCl 124, 2.5 mM KCl, 1.25 mM KH2PO4, 2 mM MgSO4.7H2O, 2.5 mM CaCl2 2H2O, 26 mM NaHCO3, 10 mM glucose, and 4 mM saccharose gassed in the bottle with a mixture of 95% O2 and 5% CO2.</li>
		<li>Prepare the recording artificial cerebro-spinal fluid (rACSF) with 124 mM NaCl, 5 mM KCl, 1.25 mM KH2PO4, 2 mM MgSO4.7H2O, 2.5 mM CaCl2 2H2O, 25 mM NaHCO3, and 10 mM glucose gassed in the bottle with a mixture of 95% O2 and 5% CO2.</li>
		<li>Anesthetize the rats using a mixture of 2.5% isoflurane and pure oxygen and decapitate them.</li>
		<li>Cut the scalp following the midline with a fine pair of scissors. Cut the skull along the midline without damaging the underlying tissue. Remove the skull with tweezers and use a fine spatula to scoop out the brain.</li>
		<li>Place the brain in the gassed dACSF solution at RT and dissect the left hippocampal formation with a fine spatula.</li>
		<li>Cut transverse slices (400 &#181;m thick) from the medium part of the hippocampus with a tissue chopper and transfer them to the recording chamber with the use of a painting brush. Maintain the slices at RT for 45 min.</li>
		<li>Perfuse the slices with the gassed rACSF at 35 &#176;C and at a rate of 1.5 mL/min. Bubble the solution with a mixture of 95% O2 and 5% CO2. 
		NOTE: After this step, the slices are ready for the electrophysiological experiments.</li>
	</ol>
	</li>
	<li>Single population spike recording
	<ol>
		<li>Place a brain slice into the microscope-mounted chamber.</li>
		<li>Perfuse the slice at a rate of 3 mL/min with rACSF.</li>
		<li>Pull a borosilicate glass micropipette with the pipette puller having a resistance of ~2 M&#937;. 
		NOTE: This micropipette is used as a recording electrode and is electrically connected to the amplifier.</li>
		<li>Fill the micropipette with a solution containing 2 M NaCl and place it into the pipette holder of the amplifier.</li>
		<li>Position the recording micropipette in the stratum pyramidale in the CA1 region of the hippocampal slice using the right micromanipulator. 
		NOTE: The location is schematically represented in Figure 7.</li>
		<li>Place an insulated bipolar platinum/iridium electrode into the holder on the left micromanipulator. 
		NOTE: This electrode is used as the stimulation electrode and is electrically connected to the stimulus generator.</li>
		<li>Position the stimulation electrode in the Schaffer collaterals in the CA1 region of the hippocampal slice using the right micromanipulator. 
		NOTE: The location is schematically represented in Figure 7.</li>
		<li>Deliver a current pulse to the stimulation electrode using the stimulus generator every 30 s (100 &#181;s durations, starting at 10 &#181;A) and gradually increase the stimulation strength until a population spike (PS) appears.</li>
		<li>Adjust the stimulus strength to evoke a PS corresponding to 45% of the maximum amplitude that can be obtained. PSs are filtered at 2.4 KHz and digitized at 20 KHz using the signal amplifier.</li>
	</ol>
	</li>
	<li>Paired-pulse inhibition
	<ol>
		<li>Prepare the brain slices (step 3.1) and proceed as described previously (steps 3.2.1&#8211;3.2.7).</li>
		<li>Deliver two current pulses (100 &#181;s durations at a 20 ms interval) to the stimulation electrode every 30 s using the stimulus generator. Set the stimulus strength to evoke a PS corresponding to 45% of the maximum amplitude.</li>
	</ol>
	</li>
	<li>Compound testing
	<ol>
		<li>Make dilutions of the compounds to be tested in gassed ACSF so that the final concentration of DMSO not higher than 0.1%.</li>
		<li>Add DMSO to the control solution at the same concentration than that in the compound solution.</li>
		<li>Record a single (step 3.2) or a paired-pulse (step 3.3) PS evoked by Schaffer collateral stimulations every 30 s for at least 30 min. The PS shape should be stable during this baseline period.</li>
		<li>Prepare a beaker with gassed rACSF containing a fixed concentration of the compound to be tested.</li>
		<li>Perfuse the hippocampal slice with this solution while still recording the single or paired-pulse PS.</li>
		<li>Evaluate the recovery from the compound effect by perfusing the slice with gassed rACSF without the compound. 
		NOTE: For reversible effects, the PS reverts to its initial shape observed during the baseline period before the compound application.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Average the PS traces recorded during the experiment in blocks of 4 using the data analysis software.</li>
		<li>Measure the PS amplitudes of the averaged traces.</li>
		<li>Normalize the amplitudes as a percentage of the baseline values recorded 10 min before perfusing a compound.</li>
		<li>Express the data as mean &#177; SEM.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pfl&#252;gers Archiv. 391, 85-100 (1981).</li><li>Adelsberger, H., Brunswieck, S., Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Block+by+picrotoxin+of+a+GABAergic+chloride+channel+expressed+on+crayfish+muscle+after+axotomy.">Block by picrotoxin of a GABAergic chloride channel expressed on crayfish muscle after axotomy.</a> European Journal of Neuroscience. 10, 179-187 (1998).</li><li>Hapfelmeier, G., 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=Isoflurane+slows+inactivation+kinetics+of+rat+recombinant+a1b2g2L+GABAA+receptors:+enhancement+of+GABAergic+transmission+despite+an+open-channel+block.">Isoflurane slows inactivation kinetics of rat recombinant a1b2g2L GABAA receptors: enhancement of GABAergic transmission despite an open-channel block.</a> Neuroscience Letters. 307, 97-100 (2001).</li><li>Bertrand, S., Bertrand, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+electrophysiological+characterization+of+neuronal+nicotinic+acetylcholine+receptors.">Overview of electrophysiological characterization of neuronal nicotinic acetylcholine receptors.</a> Current Protocols in Pharmacology. , 17 (2004).</li></ol>

The authors Fr&#233;d&#233;ric Knoflach and Maria-Clemencia Hernandez are employees of F. Hoffmann-La Roche AG, 4070 Basel, Switzerland, a pharmaceutical company. The author Daniel Bertrand is an employee of HiQScreen S&#224;rl 6, rte de Compois, 1222 V&#233;senaz Geneva, Switzerland, providing screening facilities to pharmaceutical companies.

The development of novel compounds active at a ligand-gated ion channel such as the GABAA receptors requires the use of multiple approaches. Typically, the first step is often conducted using binding assays; however, such measurements are insufficient to characterize the physiological activity of the compound or its precise pharmacology. In particular, a compound that binds to a receptor may enhance or inhibit its function or even produce no functional effect (be silent) (i.e., bind to the receptor without modifying its function). Thus, a functional test is required for a full compound characterization.
Binding assays: 
The main advantage of the binding assay is that it can be efficiently conducted on a large number of samples ranging up to several thousands and that it provides binding affinities for the active molecules. As described herein, the first step consists of establishing a method for the assessment of the desired receptor subtypes. Namely, while binding can be conducted on native receptors from fractions or entire brains, such a method will prevent the determination of the affinities at specific receptor subtypes. It is, therefore, necessary to use recombinant systems to screen molecules at the desired target. Nowadays, a transient or stable transfection of the desired receptor subunit cDNAs in cell lines offers an efficient and reliable method to express the receptor subtypes of interest (e.g., GABAA &#945;5&#946;3&#947;2). Traditional binding assays make use of radioligands, but nowadays techniques are available that avoid the inconvenience of handling radioactivity (e.g., quantitative fluorescence-based ligand-binding).
Functional expression in a host system: 
To express genes in a host system, the coding sequence of interest must be inserted into a plasmid that contains the adequate promotors. Typically, for in vitro mRNA synthesis, the bacterial T7 or T6 promotors are used. For a cDNA expression, the transcription of the gene of interest must be regulated by a eukaryote promotor such as CMV (cytomegalovirus) or equivalent. Nowadays, several plasmids, including the two promotors (i.e., pUNIV, pCMV-6AC, psf-CMV/T7, pCI-neo, pcDNA), are available allowing either an in vitro synthesis of mRNA or a cDNA expression as shown in Figure 15. An expression of the GABAA receptors can be faithfully obtained either in cell lines or in Xenopus oocytes. The main advantages of the latter are the lack of an endogenous receptor expression, the simplicity of manipulations, and the availability of an automated system for micro-injections and recordings. The procedures described in this protocol illustrate the efficiency of the automated system which allows the injection of 96 oocytes in about 7 min and requires no micromanipulation skills. Remembering that a single ovary from a Xenopus contains between 10,000&#8211;30,000 oocytes, it is clear that a large number of cell measurements can be conducted on a single preparation. Moreover, as an expression can be conducted using cDNA injections, the same plasmids containing the genes of interest that are developed for the binding experiments can be used for a functional characterization without the need for further molecular biology manipulations.
Given its large size and high surface expression, a single oocyte yields a number of receptors that is approximately equivalent to the confluent cells in a 35 mm Petri dish. However, an oocytes preparation and injection represents a fraction of the cost of a cell line, as the experiments do not require a specific culture medium and sterile manipulations. The activation of a single GABAA channel yields a few picoamperes (10-12) and currents ranging up to tens of microampere (10-6) are easily recorded in a single oocyte, which confirms the concomitant activity of several millions of receptors during a single response.
A first step in the characterization of ligand-gated ion channels is often the determination of the apparent affinity of the receptors. The experiments that need to be conducted consist of applying a series of brief agonist test pulses and plotting the amplitude of the evoked current or, in certain cases, the area under the curve (AUC) as a function of the logarithm of the agonist concentration. As it is easily feasible to microinject different plasmid concentrations and ratio in the same batch of oocytes, this system of expression is particularly appropriate for such a characterization. Moreover, a batch-to-batch comparison revealed a high degree of stability for different EC50s. The influence of the subunit composition on the receptor&#39;s apparent affinity is readily visualized using a spider plot, as exemplified in Figure 10.
The results of the two-electrode voltage clamp obtained in Xenopus oocytes are often compared with recordings made using patch clamp electrodes. Whereas it would go beyond the scope of this work to go into a detailed comparison between these two systems, a few points can be examined. While a patch clamp in cells offers advantages for a biophysical characterization with a fast drug application, its requirements are more complex than those of the two-electrode recordings in Xenopus oocytes. A first and non-negligible difficulty is the expression of a given protein with the necessity of a cell culture, a transient or stable transfection, and the identification of the cells that are expressing the desired construct. In addition, it is indispensable to examine the possible contribution of the endogenous expression in the considered cell. Moreover, a patch clamp recording requires a skilled person to conduct the micromanipulation under a high-magnification microscope, while that person also needs to perform an adequate analysis during the experiment to assess, for example, the quality of the seal, the access resistance etc. In contrast, a two-electrode voltage clamp recording, especially one using an automated electrophysiological system, does not require any specific skills and can be conducted by laboratory technicians.
When acquiring an electrophysiological set-up, cost consideration is certainly an important factor that needs to be carefully analyzed. For example, while the cost of an automated system is relatively high, a closer comparison reveals that the installation of a complete electrophysiological rig includes buying equipment such as good micromanipulators, a binocular lens, an amplifier, a perfusion system, and an anti-vibration table, while also acquiring data and performing an analysis. A cost comparison between a two-electrode voltage clamp set-up versus a patch clamp set-up reveals even further discrepancies. Moreover, the automated system offers the advantage of fully automated equipment and runs unattended day and night.
The pharmacological properties of a compound are determined by its mode of action at the receptor. For example, competitive inhibitors are molecules that can enter the same binding pocket, or orthosteric site, as the agonist itself causes a competition. Non-competitive inhibitors are compounds that inhibit the receptors by interacting at another site and, in the case of a ligand-gated ion channel, that can enter and block the ionic pore, for example. It would go beyond the scope of this work to examine the different mechanisms of interaction, but further information can be obtained from a pharmacological textbook and/or from other work such as Bertrand and Bertrand&#39;s22.
In the case of allosteric modulators, the compound binds at a site that is distinct from the orthosteric site, but the presence of the molecule modifies the energy barrier between the active and other states. Positive allosteric modulators (PAMs) are compounds that reduce the energy barrier from the resting to the active state and, therefore, enhance the effect of the agonist. To characterize possible PAM effects, it is, therefore, necessary to determine if an exposure to the compound enhances the response to the agonist and, if so, at which concentration. The experiments presented in Figure 11 and Figure 14 illustrate protocols that can be successfully used for the characterization of PAMs at the GABAA receptors.
In some cases, exposure to the PAM alone might be sufficient to activate the receptors. Such activity can be determined by a slight modification of the experimental protocol presented here. Namely, instead of using a co-application of the modulator during the exposure to the GABA, the protocol can be modified for the application of first the compound alone and then in the presence of the GABA. A characterization of the direct agonist activity will be made by the determination of the amplitude of the response evoked by the compound itself and compared to the GABA-evoked current.
Experiments conducted in brain slices, such as illustrated in Figure 7, are used to confirm the compound activity at native receptors in a definite inhibitory circuit before going further in animal models and along the pathway towards clinical trials. The relatively simple data recording and analysis protocol allows the ranking of multiple compounds by evaluating their differential modulatory effect on PS amplitude. Non-specific effects of compounds which are not related to the GABA system may also be detected (e.g., when changes in the PS shape are observed). Direct, GABAergic neurotransmission can be assessed in these cases by measuring the effects of the compounds on inhibitory postsynaptic currents (IPSCs) using the whole-cell patch clamp technique8.

Here, we present protocols for the discovery of compounds active at GABAA receptors, from the binding to the physiology and pharmacology. In the search for novel molecules specific for GABAA receptors, it is crucial to define, as precisely as possible, the subtype of interest and the assessment of the specificity of the newly identified compounds (e.g., for a review, see Rudolph and Knoflach1or Sieghart2). A typical path in drug discovery and the steps that need to be achieved are illustrated in Figure 1.
Binding assays have been and are still largely used as the first step in drug discovery. In the case of GABAA receptors, they are optimized to identify compounds that bind to the benzodiazepine binding site of the receptor where therapeutically useful and safe drugs bind. Other techniques, using fluorometric imaging plate reader (FLIPR) membrane potential red dye-based assays3, detect compounds like barbiturates that bind to additional sites that are less desirable due to their unwanted side-effect profile. In addition, the utilized dyes can directly activate GABAA receptors, thus questioning the utility of these assays for drug discovery4. Binding assays can only provide the evidence that a given compound can bind to a specific receptor class. In vitro assays with cell membranes are used to identify selective human GABAA &#945;5&#946;3&#947;2 receptor ligands. Transiently transfected HEK293 cells expressing the human GABAA &#945;5&#946;3&#947;2, &#945;1&#946;3&#947;2, &#945;2&#946;3&#947;2, and &#945;3&#946;3&#947;2 receptors are used to prepare membranes for these assays. The effect of the ligands is detected by measuring the scintillation of [3H]flumazenil bound to the membrane receptors (an inhibition of [3H]flumazenil binding). The main advantage of this technique is that a rapid and efficient determination of the compound-binding affinity at the receptor of interest is provided at the receptor of interest.
Functional studies are essential to evaluate the functional activity of the compounds and to propose a physiological and pharmacological explanation of the mechanisms caused by the binding of the compounds to the receptors. Today, it is well-recognized that functional GABAA receptors result from the assembly of five subunits around an axis of pseudosymmetry formed by the ionic pore and result from the assembly of five identical subunits. Most of the GABAA receptors are composed of two or more different subunits. The major brain GABAA receptor, for instance, is composed of the &#945;1, &#946;2, and &#947;2 subunits in a stoichiometry of 2, 2, and 1 respectively5,6. A reconstitution in a host system such as the Xenopus oocytes or HEK293 cells offers the possibility of rapidly exploring the pharmacological properties of the receptors.
The pharmacological properties of the compounds are then explored with extracellular recordings in brain slices7. This method allows an exploration of the effect of the compounds on neurotransmission and provides an effective way to confirm the functional effects of the compounds determined in heterologous expression systems at the level of native receptors in the overall neuronal environment. GABAergic neurotransmission can also be assessed at the molecular level by measuring the effects of the compounds on inhibitory postsynaptic currents (IPSCs)8. But the protocol used here and based on whole-cell patch clamp recordings in brain slices is more elaborate and yields a lower throughput.
Finally, the strengths and weaknesses of the in vitro screening cascade used for the identification of &#945;5&#946;3&#947;2-selective ligands are discussed in the perspective of the different techniques and their intrinsic limitations. This work should provide experts and non-experts in the field of GABAA receptors a helpful review of the combination of different in vitro approaches used to tackle the discovery of new modulators of these ligand-gated ion channels.

<table border="0" cellpadding="0" cellspacing="0" style="width:1024px;" width="1023">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:465px;">General equipment</td>
			<td style="width:169px;"></td>
			<td style="width:136px;"></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">96 well cell harvester -(Filtermate 196)&#160;</td>
			<td style="width:169px;">Packard</td>
			<td style="width:136px;"></td>
			<td>To filter membranes with bound radioligand</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Microtiter plate liquid scintillation counter (Top Count NXT)</td>
			<td>Packard</td>
			<td></td>
			<td style="width:253px;">Microplate Scintillation and Luminescence counter</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Vial liquid scintillation counter (Beta-Couter 2500 TR)</td>
			<td>Packard</td>
			<td></td>
			<td style="width:253px;">Vial Scintillation and Luminescence counter</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Liquid&#160; handler (Biomek 2000)</td>
			<td style="width:169px;">Beckman coulter</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">To prepare compound dilutions</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:465px;">Vortex (Vortex Genie 2)</td>
			<td>Scientific industries Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">To mix compound stock solutions</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Tissue homogenizer (Polytron PT1200E)</td>
			<td>Kinematica AG</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">To resuspend the membrane preparations</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">XLfit5 software</td>
			<td style="width:169px;">Microsoft Excel 
			&#160;add-on by IDBS</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">Curve fitting software</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Smart Pull</td>
			<td>UniPix</td>
			<td style="width:136px;">-</td>
			<td>Electrode Puller</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">RoboInject</td>
			<td>MultiChannel Systems</td>
			<td style="width:136px;">-</td>
			<td>Automated Injection (Xenopus Oocytes)</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">HiClamp</td>
			<td>MultiChannel Systems</td>
			<td style="width:136px;">-</td>
			<td>Automated Voltage Clamp (Xenopus Ooocytes)</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">DataMining</td>
			<td>MultiChannel Systems</td>
			<td style="width:136px;">-</td>
			<td>Data Analysis Software</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">DataMerger</td>
			<td>MultiChannel Systems</td>
			<td style="width:136px;">-</td>
			<td>Data Processing Software</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Digidata 1550</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Low noise data acquisition system</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">CyberAmp 380 or equivalent</td>
			<td style="width:169px;">Axon Instruments</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">Programmable Signal Conditioner</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">pClamp program suite</td>
			<td>Molecular Devices</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">Software for data acquisition and analysis software</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Antivibraton table&#160;</td>
			<td style="width:169px;">TMC</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">To avoid microelectrode vibrations&#160;</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Patchstar Micromanipulators</td>
			<td style="width:169px;">Scientifica</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">To accurately position microelectrodes in brain slices</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Faraday Cage&#160;</td>
			<td style="width:169px;">Sutter Instruments</td>
			<td style="width:136px;"></td>
			<td style="width:253px;">To isolate recordings from noise</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">McIlwain Tissue Chopper</td>
			<td style="width:169px;">Campden Instruments LTD&#160;</td>
			<td></td>
			<td>To prepare brain slices</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Borosilicate glass micropipette&#160;</td>
			<td style="width:169px;">Warner Instruments</td>
			<td>GC150TF-10</td>
			<td style="width:253px;">To record extracellular potentials in brain slices</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Twisted pair, platinum iridium wires stimulation electrode</td>
			<td style="width:169px;">World Precision Instruments</td>
			<td></td>
			<td style="width:253px;">To stimulate the brain slices with current</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Stimulus generator for current and voltage driven stimulation</td>
			<td>MultiChannel Systems</td>
			<td>STG4000</td>
			<td style="width:253px;">Delivers the current to the stimulation electrode</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Plasticware</td>
			<td style="width:169px;"></td>
			<td style="width:136px;"></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Microtiter plate round bottom</td>
			<td style="width:169px;">Corning</td>
			<td style="width:136px;">#3365</td>
			<td>Used for the binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">GF/C glassfibre filter-bottom 96-well microplate with 1.2&#181;m poresize, for cell harvesting assays using a vacuum manifoldwell filter&#160;</td>
			<td style="width:169px;">Packard&#160;</td>
			<td>#6005174</td>
			<td style="width:253px;">used for the binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">50 mL Tubes</td>
			<td style="width:169px;">Falcon&#160;</td>
			<td>#352070</td>
			<td style="width:253px;">used for the binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Safe-lock tubes 1.5 mL</td>
			<td style="width:169px;">Eppendorf</td>
			<td>#0030 120086</td>
			<td style="width:253px;">used for the binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Safe-lock tubes 2 mL</td>
			<td style="width:169px;">Eppendorf</td>
			<td>#0030 120094</td>
			<td style="width:253px;">used for the binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Shipping tubes 180ml&#160;</td>
			<td style="width:169px;">Semadeni Europe AG&#160;</td>
			<td>#3722</td>
			<td style="width:253px;">used for the binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Pony vial Polyethylene 6ml&#160;</td>
			<td style="width:169px;">Perkin Elmer&#160;</td>
			<td>#6013329</td>
			<td style="width:253px;">used for the binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Top Seal&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td>#60051859</td>
			<td style="width:253px;">used for the binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Spinner Flask</td>
			<td>Bellco</td>
			<td>BELLCO # 1965-00100</td>
			<td>Spinner Flask</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">96&#160; Well Plate (Conical)</td>
			<td style="width:169px;">Thermo Scientific Milian</td>
			<td>56368</td>
			<td style="width:253px;">Injection plate for the oocytes</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">96&#160; Well Plate (Flat bottom)</td>
			<td style="width:169px;">Corning (Vitaris Switzerland)</td>
			<td>3364-cor</td>
			<td style="width:253px;">Compound Plate for the HiClamp</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Glass capillary&#160;</td>
			<td style="width:169px;">Hilgenberg (Germany)</td>
			<td style="width:136px;">-</td>
			<td style="width:253px;">borosilicate glass 1.2 O.D. 0.76 I.D. with filament</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">RNA preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Ambion mMessage mMachine</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>for the in vitro synthesis</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Assay buffer: KCl 5 mM; CaCl2 1.25 mM; MgCl2 1.25 mM; NaCl 120 mM; Tris 15 mM pH adjust with HCl to 7.4; store up to 3 months at 4&#176;C.</td>
			<td></td>
			<td></td>
			<td>Used for binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Washing buffer: Tris 50mM -HCl pH 7.4; store at 4&#176;C.</td>
			<td></td>
			<td></td>
			<td>Used for binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Stock solution of test compounds 10mM in DMSO</td>
			<td></td>
			<td></td>
			<td>Used for binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Flumazenil (RO0151788) 10mM in DMSO</td>
			<td></td>
			<td></td>
			<td>Used for binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Diazepam 4mM in DMSO</td>
			<td></td>
			<td></td>
			<td>Used for binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">[3H]-Flumazenil 60-85 Ci/mmol in Ethanol; store at -20&#176;C</td>
			<td></td>
			<td></td>
			<td>Used for binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Microscint 40&#160;</td>
			<td>Packard&#160;</td>
			<td>#6013641</td>
			<td>Used for binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Ultima Gold&#160;</td>
			<td style="width:169px;">Perkin Elmer&#160;</td>
			<td style="width:136px;">#6013329</td>
			<td>Used for binding assay</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">MS-222</td>
			<td style="width:169px;">Sigma</td>
			<td>Sigma # A5040</td>
			<td>MS-222</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">AB/AM</td>
			<td style="width:169px;">Sigma</td>
			<td>Sigma # A5955</td>
			<td>antibiotics / antimycotics</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Collagenase</td>
			<td style="width:169px;">Sigma</td>
			<td>Sigma # C1030</td>
			<td>Collagenase Type I</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Rnase</td>
			<td style="width:169px;">Sigma</td>
			<td>Sigma # R2020-250 mL</td>
			<td>RNaseZAP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">EndoFree Plasmid maxi Kit</td>
			<td style="width:169px;">Qiagen</td>
			<td>Qiagen # 12362</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Salts for artificial cerebrospinal fluid (NaCl, KCl, etc.)</td>
			<td style="width:169px;">Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Membranes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;">Frozen membrane preparations from transient or stably transfected HEK293 overexpressing different human GABAA receptor subtypes: &#945;1&#946;3&#947;2, &#945;2&#946;3&#947;2, &#945;3&#946;3&#947;2, &#945;5&#946;3&#947;2. See Ballard et al 2009 for detailed methods.</td>
			<td></td>
			<td></td>
			<td>Used for binding assay</td>
		</tr>
	</tbody>
</table>

methods for the discovery of novel compounds modulating a gamma-aminobutyric acid receptor type a neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Watch this Scientific Journal Video about Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission at JoVE.com

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

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13.4K 조회수.  Roche Innovation Center Basel. Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

비디오: Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

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신경과학

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

동적 클램프 기법을 사용 랫 Midbrain Dopaminergic 뉴런에서 NMDA 수용체의 전도의 응용

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

를 사용하여 화합물 Bioactivity위한 이점 Nanoinjection 및 화면에 대한 전기 생리학 분석 Drosophila melanogaster 거대 섬유 시스템

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

마우스 모델에서 정맥 주사 자기 관리를위한 방법

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

인간 Midbrain위한 고해상도 기능성 자기 공명 영상 방법

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

에서 항 정신병 약물의 작용 메커니즘을 공부하는 방법<em&gt; 예쁜 꼬마 선충</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

뉴런의 유전자 인코딩 된 부 자연스러운 아미노산을 사용하여 신경 세포 단백질의 광 제어

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

급성 Hippocampal 조각에 작은 볼륨 재활용-, 관류-침수 형 챔버 시스템 유지에 시 냅 스가 소성을 기록

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

일시적인 수용 체 잠재력 Vanilloid 타입-1의 Ultrapotent 주 작동 근의 순수한 작은 섬유 신경 병 마우스 모델 수립

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

소설 수동 전체 CNS 조직에 광학 투명도의 급속 한 생산을 위한 방법 삭제

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...