The work was supported by the Chinese National Science Foundation (31070972), Science and Technology Commission of Shanghai Municipality (16ZR1418300), the Program for Innovative Research Team of Shanghai Municipal Education Commission, the Shanghai Eastern Scholar Program (J50201), and the National Basic Research Program of China (2012CB910401).

1. Culturing and Maintenance of Hana3A Cells
<ol>
	<li>Maintain cells in 10 mL of minimum essential medium (MEM) with 10% fetal bovine serum (FBS), 100 &#181;g/mL penicillin-streptomycin, and 1.25 &#181;g/mL amphotericin B in a 100-mm cell culture dish in a 37 &#176;C cell culture incubator with 5% CO2. With every other passage, add 1 &#181;g/mL puromycin to maintain stable transfection of plasmids (See Introduction). 
	NOTE: Perform all steps involving cell culture in a class II biolog...

<ol>
	<li>Malakoff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+the+scent+of+avian+olfaction.">Following the scent of avian olfaction.</a> Science. 286 (5440), 704-705 (1999).</li><li>Zippel, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+of+vertebrate+olfaction:+D.M.+Stoddart.+Chapman+and+Hall,+Andover,+Great+Britain,+1980.+£15.00,+234+pp.+ISBN+0-412-21820-8.">The ecology of vertebrate olfaction: D.M. Stoddart. Chapman and Hall, Andover, Great Britain, 1980. £15.00, 234 pp. ISBN 0-412-21820-8.</a> Behav Processes. 7 (2), 198-199 (1982).</li><li>Dryer, L., Berghard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptors:+a+plethora+of+G-protein-coupled+receptors.">Odorant receptors: a plethora of G-protein-coupled receptors.</a> Trends Pharmacol Sci. 20 (10), 413-417 (1999).</li><li>Zhang, X., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+olfactory+receptor+gene+superfamily+of+the+mouse.">The olfactory receptor gene superfamily of the mouse.</a> Nat Neurosci. 5 (2), 124-133 (2002).</li><li>Glusman, G., Yanai, I., Rubin, I., Lancet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+human+olfactory+subgenome.">The complete human olfactory subgenome.</a> Genome Res. 11 (5), 685-702 (2001).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+ligands+for+odorant,+vomeronasal+and+taste+receptors.">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat Rev Neurosci. 5 (4), 263-278 (2004).</li><li>Reed, R. 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W., Chi, Q., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTP+family+members+induce+functional+expression+of+mammalian+odorant+receptors.">RTP family members induce functional expression of mammalian odorant receptors.</a> Cell. 119 (5), 679-691 (2004).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+of+accessory+factors+in+functional+expression+of+mammalian+odorant+receptors.">Synergism of accessory factors in functional expression of mammalian odorant receptors.</a> J Biol Chem. 282 (20), 15284-15293 (2007).</li><li>Li, Y. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-Cell+Luminescent+Assays+for+GPCR+Studies:+Combination+of+Sensitive+Detection+and+Real-Time+Analysis+Expands+Applications.">Live-Cell Luminescent Assays for GPCR Studies: Combination of Sensitive Detection and Real-Time Analysis Expands Applications.</a> Genetic Engineering &amp; Biotechnology News. 29, 30-31 (2009).</li><li>Geithe, C., Andersen, G., Malki, A., Krautwurst, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Butter+Aroma+Recombinate+Activates+Human+Class-I+Odorant+Receptors.">A Butter Aroma Recombinate Activates Human Class-I Odorant Receptors.</a> J Agric Food Chem. 63 (43), 9410-9420 (2015).</li><li>Noe, F., 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=OR2M3:+A+Highly+Specific+and+Narrowly+Tuned+Human+Odorant+Receptor+for+the+Sensitive+Detection+of+Onion+Key+Food+Odorant+3-Mercapto-2-methylpentan-1-ol.">OR2M3: A Highly Specific and Narrowly Tuned Human Odorant Receptor for the Sensitive Detection of Onion Key Food Odorant 3-Mercapto-2-methylpentan-1-ol.</a> Chem Senses. 42 (3), 195-210 (2016).</li><li>Geithe, C., Noe, F., Kreissl, J., Krautwurst, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+broadly+tuned+odorant+receptor+OR1A1+is+highly+selective+for+3-methyl-2,4-nonanedione,+a+key+food+odorant+in+aged+wines,+tea,+and+other+foods.">The broadly tuned odorant receptor OR1A1 is highly selective for 3-methyl-2,4-nonanedione, a key food odorant in aged wines, tea, and other foods.</a> Chem Senses. 42 (3), 181-193 (2016).</li><li>Duan, X., 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=Crucial+role+of+copper+in+detection+of+metal-coordinating+odorants.">Crucial role of copper in detection of metal-coordinating odorants.</a> Proc Natl Acad Sci U S A. 109 (9), 3492-3497 (2012).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smelling+Sulfur:+Copper+and+Silver+Regulate+the+Response+of+Human+Odorant+Receptor+OR2T11+to+Low-Molecular-Weight+Thiols.">Smelling Sulfur: Copper and Silver Regulate the Response of Human Odorant Receptor OR2T11 to Low-Molecular-Weight Thiols.</a> J Am Chem Soc. , (2016).</li><li>Wu, L., Pan, Y., Chen, G. Q., Matsunami, H., Zhuang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-transporting+protein+1+short+(RTP1S)+mediates+translocation+and+activation+of+odorant+receptors+by+acting+through+multiple+steps.">Receptor-transporting protein 1 short (RTP1S) mediates translocation and activation of odorant receptors by acting through multiple steps.</a> J Biol Chem. 287 (26), 22287-22294 (2012).</li><li>Shirasu, M., 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=Olfactory+receptor+and+neural+pathway+responsible+for+highly+selective+sensing+of+musk+odors.">Olfactory receptor and neural pathway responsible for highly selective sensing of musk odors.</a> Neuron. 81 (1), 165-178 (2014).</li><li>Block, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implausibility+of+the+vibrational+theory+of+olfaction.">Implausibility of the vibrational theory of olfaction.</a> Proc Natl Acad Sci U S A. 112 (21), E2766-E2774 (2015).</li><li>Sato-Akuhara, N., 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=Ligand+Specificity+and+Evolution+of+Mammalian+Musk+Odor+Receptors:+Effect+of+Single+Receptor+Deletion+on+Odor+Detection.">Ligand Specificity and Evolution of Mammalian Musk Odor Receptors: Effect of Single Receptor Deletion on Odor Detection.</a> J Neurosci. 36 (16), 4482-4491 (2016).</li><li>Gane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+vibration-sensing+component+in+human+olfaction.">Molecular vibration-sensing component in human olfaction.</a> PLoS One. 8 (1), e55780 (2013).</li><li>Young, J. 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Accurately measuring an OR&#39;s activation upon exposure to a certain odorant is the first step in deciphering the coding of olfactory information. The experiments shown in this study represent an example of how one can identify, using an in vitro OR expression system, responsive ORs among the human OR repertoire for the odorous chemical of interest and subsequently characterizing the receptor pharmacology using various concentrations of the chemical. Our results confirm OR5AN1 as a bona fide receptor ...

The sense of smell plays an important role in animals&#39; survival as they rely on their olfactory abilities to obtain food, avoid predators and danger, distinguish species, and select mate1,2. The realization of these functions depends on the odorant receptors (ORs), which are individually expressed at the ciliary surface of olfactory sensory neurons (OSNs) located in the olfactory epithelium (OE). ORs constitute the largest family of the G-protein coupled receptor (GPCR) superfamily with approximately 400 and 1200 diverse OR genes in human and mouse, respectively3,4,5. ORs activated by odorants lead to increased intracellular cAMP levels via the sequential activation of olfactory G-protein (Golf) and type III adenylyl cyclase (ACIII). The resulting increased level of intracellular cAMP could function as a second messenger, which opens the nucleotide-gated channel on the cell surface, triggering influx of cations including Ca2+ and action potentials, and ultimately initiating neuro-potential transmission and olfactory perception. The process of detecting and discriminating a large number of odorants by ORs is regarded as the first step of olfactory perception6,7.
Since Buck and Axel8 first successfully cloned odorant receptors and elucidated the mechanism of olfactory perception initiated by ORs, deorphanization of the OR family became one of the hotspots in this field. Various in vivo, ex vivo and in vitro methods to measure OR activation have been reported9,10,11,12. A traditional method that used Ca2+ imaging followed by single-cell RT-PCR on OSNs enabled the identification of different ORs to aliphatic odorants13,14,15. More recently, the advent of large-scale transcriptome analyses promoted the development of more high-throughput in vivo methods. The Kentucky assay identified eugenol- and muscone-responsive mouse ORs with the use of the S100a5-tauGFP reporter mouse strain and microarray analysis9. Based on the decrease in OR mRNA levels after odorant exposure, the DREAM technology employed a transcriptomic approach to determine OR activation profiles in both vertebrate and non-vertebrate species16. Similarly, given the phosphorylation of S6 in neuronal activations,the Matsunami group sequenced mRNAs from phosphorylated ribosome immunoprecipitations to identify responsive ORs12. Finally, the Feinstein group reported super sniffer mice that could serve as a platform to study odor coding in vivo, known as the MouSensor technology17.
In the in vitro realm, the challenge of culturing OSNs makes a heterologous expression system that mimics OR functional expression in vivo an ideal solution to conduct large-scale screening of odorous chemicals for ORs. Nevertheless, since cultured cell lines of non-olfactory origins differ from native OSNs, OR proteins are retained in the endoplasmic reticulum and unable to traffic to the plasma membrane, resulting in OR degradation and loss of receptor function18,19. To solve this problem, extensive works have been made to replicate OR functional expression on the cell membrane in heterologous cell lines. Krautwurst et al. first attached the first 20 amino acids of rhodopsin (Rho-tag) to the N-terminal of OR protein and this promoted the cell-surface expression of some ORs in human embryonic kidney (HEK) cells20. By conducting a serial analysis of gene expression (SAGE) library analysis from single OSNs, Saito et al. first cloned the receptor-transporting protein (RTP) family members, RTP1 and RTP2, and the receptor expression enhancer protein 1 (REEP1) that facilitated OR trafficking to the cell membrane and enhanced odorant-mediated responses of ORs in HEK293T cells21. Based on these findings, the Matsunami group successfully established the Hana3A cell line, stably transfected with RTP1, RTP2, REEP1, and G&#945;olf in HEK293T, and transiently transfected with Rho-tagged ORs, for efficient OR functional expression. Subsequent studies revealed 1) a shorter form of RTP1, RTP1S, that could more robustly promote OR function than the original RTP1 protein and 2) the type 3 muscarinic acetylcholine receptor (M3R) that could enhance OR activity via inhibition of &#946;-arrestin-2 recruitment, both of which were introduced to the heterologous expression system to optimize experimental output22,23.
Several detection methods have been used to quantify receptor activation in heterologous systems. The secreted placental alkaline phosphatase (SEAP) assay works with a reporter enzyme transcriptionally regulated by cAMP response elements (CREs), making it an attractive option for assessing OR activation. The fluorescence is readily detected in a sample of the culture medium after the incubation with SEAP detection reagent24. Using this method, the functions of ORs as well as a secondary class of chemosensory receptors expressed in the OE-the trace amine-associated receptors (TAARs) have been characterized25,26,27. Another common method, the luciferase assay, uses a firefly luciferase reporter gene under the control of the cAMP response element (CRE). Measuring luminescence generated by luciferase production provides an efficient and robust means of quantifying OR activation10,11,28.
Real-time cAMP assays have also been widely used in dynamically monitoring the function of heterologous or endogenous GPCRs. One example of such advanced assays takes advantage of a genetically encoded biosensor variant, which possesses a cAMP-binding domain fused to a mutant form of luciferase. When cAMP binds, the conformational change leads to the activation of luciferase, luminescence from which can then be measured with a chemiluminescence reader29,30. The real-time cAMP technology has been reported suitable for the de-orphaning of human odorant receptors in HEK293 and NxG 108CC15 cells31,32,33, as well as in the HEK293T-derived Hana3A cells34,35. The Krautwurst group also described in detail the real-time cAMP technology to be suitable for bi-directional large-scale OR screening approaches32,33.
Here we describe a protocol for measuring OR activation using a real-time cAMP assay in Hana3A cells. In this protocol, the luminescence of pre-equilibrated live cells is kinetically measured for 30 min following treatment with specific volatile compounds, representing a more efficient and accurate analysis of OR activation that are less susceptible to artifacts that occur in the cellular environment with prolonged time and odor-induced cell toxicities. This real-time measurement allows for a large-scale screening of both ORs and ligands, as well as characterization of specific OR-ligand pairs of interest. Using this method, we successfully identified OR5AN1 as the receptor for the musk compound muscone by performing a screening against 379 human ORs and subsequently confirming the positive screening result.

<table width="1240">
	<tbody>
		<tr height="25">
			<td height="25" style="width:405px;height:25px;">Amphotericin B</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">A2942</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">FBS</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">GloSensor cAMP reagent</td>
			<td>Promega</td>
			<td>E1290</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">pGloSensor-20F cAMP plasmid</td>
			<td>Promega</td>
			<td>E1171</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Hana3A cells</td>
			<td></td>
			<td></td>
			<td>available from authors upon request</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HBSS, w/o calcium chloride and magnesium chloride</td>
			<td>GIBCO</td>
			<td>14175095</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HEPES</td>
			<td>Hyclone</td>
			<td>SH30237</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Lipofectamine2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">M3R plasmid</td>
			<td></td>
			<td></td>
			<td>cloned into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MEM, w/EBSS and w/L-glutamine</td>
			<td>Hyclone</td>
			<td>SH30024</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Muscone</td>
			<td>Santa Cruz</td>
			<td>sc-200528</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Musk tibetene</td>
			<td>Sigma-Aldrich</td>
			<td>S359165</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">OR plasmids</td>
			<td></td>
			<td></td>
			<td>cloned with a Rho-tag into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PBS, w/o calcium or magnesium</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Penicillin-streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Plasmid miniprep kit</td>
			<td>Tiangen</td>
			<td>DP103-03</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Puromycin</td>
			<td>Sigma</td>
			<td>P8833</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RTP1S plasmid</td>
			<td></td>
			<td></td>
			<td>cloned into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Trypsin-EDTA</td>
			<td>Hyclone</td>
			<td>SH30236</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">0.2-mL PCR tube</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1.5-mL Eppendorf tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">15-mL 17 mm x 120 mm conical tube</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">8-well and/or 12-well multichannel pipetman</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">96-well flat-bottomed white cell culture plate</td>
			<td>Greiner</td>
			<td>655098</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">100 mm x 20 mm cell culture dish</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Class II biological safety cabinet with laminar flow</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell culture incubator, w/ 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Centrifuge, with swinging bucket rotor for 15-ml conical tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Infinite F200 plate reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Phase-contrast microscope with x10 and x20 objectives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Spectrophotometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sterile reagent reservoirs for multichannel distribution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sterile paper towel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live-cell measurement of odorant receptor activation using a real-time camp assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Muscone is the main aromatic component from natural musk. Recent studies identified OR5AN1 as a human receptor for muscone and other macrocyclic musk compounds based on homology to the mouse OR, MOR215-1, cloned from muscone-responsive glomeruli in behaving mice37,38. By screening the human OR repertoire, our group and the Touhara group also identified OR5AN1 as a major receptor for two macrocyclic musk compounds, cyclopentadecano...

Watch this Scientific Journal Video about Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay at JoVE.com

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

The overall goal of this real-time high-throughput cyclic AMP assay is to screen odorants against odorant receptors and vice versa. Since mice are useful for investigating odorant receptor functioning in that it allows for larger scale screenings of both odorant receptors and the ligands as well as pharmacological cartelizations of a receptor-ligand pairs of interest. The main advantage of this technique is that we can assess odorant-to-receptor activation efficiently and reliably.Heterologous odorant-to-receptor expression system using live cells. Begin by observing Hana3A cells under a phase-contrast microscope to ensure cell viability and to estimate confluence. Then aspirate all medium from the cell culture dish.And wash the cells by adding 10 milliliters of phosphate buffered saline onto the plate, swirling the dish, and aspirating the PBS. Next, add the three milliliters of 0.5%trypsin-EDTA onto the plate. Leave the trypsin on the cells for one minute or until all cells are afloat.Inactivate the trypsin by adding 5 milliliters of MEM with 10%FBS and pipetting up and down to break up chunks of cell mass. After performing a cell count, transfer 2x10 to the 6th cells per 96-well plate to be transfected to a 15 milliliter tube. It is important that you calculate the correct number of cells to be prepared onto 96-well plate to avoid over or undergrowth of cells prior to stimulation.Centrifuge the tubes at 200 times G for five minutes. And then aspirate the supernatant without disturbing the cell pallet. Add six milliliters of medium without antibiotics for each 96-well plate and pipette up and down without creating air bubbles to break up chunks of the cell mass.Transfer the suspended cells into a reservoir. Using a multi-channel pipette, pipette 50 microliters of cells into each well of the 96-well plates. Incubate overnight at 37 degrees Celsius with 5%carbon dioxide.Prior to transfection observe the plated cells under a phase-contrast microscope to ensure a proper confluence of approximately 30 to 50%per well and then return to the incubator. Follow the row tagged odorant-to-receptor construct, the accessory factor constructs, and the cyclic AMP biosensor variant construct. Then prepare a plasma transfection mixture in 500 microliters of MEM for each 96-well plate.Next, prepare a transfection mixture containing 18 microliters of lipid-mediated transfection reagent in 500 microliters of MEM. Mix the transfection mixture with the plasma mixture by pipetting up and down. Then incubate at room temperature for 15 minutes.After the incubation, stop the reaction by adding 5 milliliters of MEM with 10%FBS to the transfection mix. Next, spread out a thick layer of sterile paper towels in the cell culture hood. Take a 96-well plate with cells from the incubator.Gently and repeatedly, tap the plate upside down on the pile of paper towels so that the medium is completely absorbed by the paper towel. Transfer 50 microliters of the combined transfection mixture to each well of the 96-well plate and incubate overnight for 18 to 24 hours at 37 degrees Celsius with 5%carbon dioxide. You can manage the transfection and the stimulation schedule should persist on instrument for more than one 96-well plate is tested in one experiment so that all plates are mirrored after same transfection and distillers exposure interval.Before stimulation, observe the transfected cells under a phase-contrast microscope to ensure a proper confluence of 50 to 80%per well before returning to the incubator. Then prepare stimulation medium by adding 10 millimolar heaps and five millimolar glucose to Hanks'balanced salt solution. Thaw 55 microliters alloquats of real-time cyclic AMP assay substrate reagent alloquats on ice.Prepare 2%equal abration solution by mixing 55 microliters of the substrate reagent and 2, 750 microliters of stimulation medium. After spreading out a thick layer of paper towels on the bench, gently and repeatedly tap the plate upside down on the paper towels so that the transfection medium is absorbed by the paper towels. Wash the cells by pipetting 50 microliters of stimulation medium to each well.Gently and repeatedly tap out the stimulation medium from the 96-well plate. Pipette 25 microliters of 2%equillibration solution into each well and incubate at room temperature in the dark for two hours. Prior to the end of the incubation time, dilute thawed odorant stock solutions to working concentration and stimulation medium.Before odorant addition, use a chemiluminescence plate reader to measure the basal luminescence level of the plate. Quickly remove the plate from the plate reader and add 25 microliters of the odorant dilutions to each well. Then immediately start continuous luminescence measurement of all wells for 20 cycles within 30 minutes.379 unique human odorant receptors were screened against 30 micromolar muscone using the real-time cyclic AMP assay. Colored blocks along the X axis indicate different human odorant-receptor families. The Y axis represents normalized luminescence 30 minutes after odorant addition when the response reached the maximum for the positive control.The response of OR5AN1 is indicated. This figure shows real-time measurements of OR5AN1 activation by different concentrations of muscone and the result from a no-odor negative control. The arrow indicates the time point of oderant addition.Here, a concentration response curve of OR5AN1 against muscone 30 minutes after oderant addition is shown. The technique described here takes a total of three days starting with a stock plate of culture tiers on the first day. Once mastered, the process on the last day can be completed in three hours for each 96-well plate if it is performed properly.After watching this video, you should have a good understanding of how to to manage the activation of oderant receptors using a cyclic AMP assay to harness the results.

live-cell-measurement-odorant-receptor-activation-using-real-time

Research

JoVE Journal

Neuroscience

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis during feeding. The ability to rapidly obtain high-resolution measurements of natural antenna and proboscis movements and assess how they change in response to chemical, developmental, and genetic manipulations can aid the understanding of insect behavior. By extending our previous work on assessing aggregate insect swarm or animal group movements from natural and laboratory videos using the video analysis software SwarmSight, we developed a novel, free, and open-source software module, SwarmSight Appendage Tracking (SwarmSight.org) for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The software processes frames about 120 times faster than humans, performs at better than human accuracy, and, using 30 frames per second (fps) videos, can capture antennal dynamics up to 15 Hz. The software was used to track the antennal response of honey bees to two odors and found significant mean antennal retractions away from the odor source about 1 s after odor presentation. We observed antenna position density heat map cluster formation and cluster and mean angle dependence on odor concentration.

This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy.

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

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 ...