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 biological safety cabinet to ensure sterile environment.</li>
	<li>Subculture at a ratio of 10-20% in 100-mm dishes every 2-3 days.</li>
</ol>
2. Plating Cells for Transfection
<ol>
	<li>Observe the Hana3A cells under a phase-contrast microscope to ensure cell viability and to estimate confluence.</li>
	<li>Aspirate all medium from the cell culture dish.</li>
	<li>Wash cells by adding 10 mL of phosphate-buffered saline (PBS) onto the plate, swirling the dish, and aspirating the PBS.</li>
	<li>Add 3 mL of 0.05% trypsin-ethylene diamine tetraacetic acid (EDTA) onto the plate. Mix for 1 min or until all cells are afloat. 
	NOTE: Observe the progress of cells detaching from the bottom of the plate under the microscope as necessary.</li>
	<li>Inactivate trypsin by adding 5 mL of MEM with 10% FBS and pipette up and down to break up chunks of cell mass. 
	NOTE: For plating prior to transfection, the medium used should not contain antibiotics. Addition of antibiotics may decrease transfection efficiency.</li>
	<li>Transfer an appropriate amount of cells to a 15-mLl tube depending on the number of plates to be transfected. For each 96-well plate, plate 2 x 106 cells, or approximately 1/5 of a 100% confluent 100 mm dish, giving a cell count of 2 x 104 cells per well or a density of approximately 15-30% confluence per well. Centrifuge tubes at 200 x g for 5 min and aspirate the supernatant without disturbing the cell pellet. 
	NOTE: Calculate the correct amount of cells to be plated onto 96-well plates to avoid overgrowing or undergrowing cells prior to stimulation.</li>
	<li>Add an appropriate amount of MEM with 10% FBS into the 15-mL tube and pipette up and down to break up chunks of the cell mass. For each 96-well plate, resuspend the cells with 6 mL of MEM with 10% FBS. 
	NOTE: Be careful not to generate air bubbles in the tube.</li>
	<li>Add the suspended cells into a reservoir. Using a multichannel pipette, pipette 50 &#181;L of cells onto each well of a 96-well plate. Incubate overnight at 37 &#176;C with 5% CO2.</li>
</ol>
3. Transfection of Plasmids
<ol>
	<li>Prior to transfection, observe the plated cells to ensure a proper confluence of approximately 30-50% per well under a phase-contrast microscope and return to the incubator.</li>
	<li>Prepare in advance the Rho-tagged OR construct11,21,22,28, the accessory factor constructs (RTP1S22,36 and M3R23), and the biosensor variant construct29,30 by miniprep. Quantify the DNA concentration by a spectrophotometer and adjust plasmid DNA concentrations (e.g., to 100 ng/&#181;L) with distilled water as necessary.</li>
	<li>Preparation of the Transfection Mixtures
	<ol>
		<li>Prepare a plasmid transfection mixture in 500 &#181;L of MEM for each 96-well plate according to Table 1. 
		NOTE: When different ORs are tested on the same 96-well plate, the volume of the transfection mixture and the amount of plasmid DNA added must be adapted in function of the number of transfected wells with a given OR.</li>
		<li>Prepare a transfection mixture in a tube with 18 &#181;L of lipid-mediated transfection reagent in 500 &#181;L of MEM.</li>
	</ol>
	</li>
	<li>Mix the plasmid mixture with the transfection mixture by pipetting up and down. Incubate at room temperature for 15 min.</li>
	<li>Stop the reaction by adding 5 mL of MEM with 10% FBS.</li>
	<li>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 towel so that the medium is completely absorbed by the paper towel. 
	NOTE: Do not forcefully or abruptly tap the plate as one could lose cells.</li>
	<li>Transfer 50 &#181;L of the combined transfection mixture to each well of the 96-well plate and incubate overnight for 18-24 h at 37 &#176;C with 5% CO2. 
	NOTE: A time-managed transfection and stimulation schedule should precede the measurement when more than one 96-well plate are tested in one experiment in order to have all plates measured after the same transfection time and stimulus exposure time.</li>
</ol>
4. Stimulation and Measuring OR Activity Using the Real-Time cAMP Assay
<ol>
	<li>Observe the transfected cells under a phase-contrast microscope to ensure a proper confluence of 50-80% per well and return to the incubator.</li>
	<li>Prepare the stimulation medium by adding 10 mM hydroxyethyl piperazineethanesulfonic acid (HEPES) and 5 mM glucose to Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Thaw the real-time cAMP assay substrate reagent aliquots on ice. Store substrate reagent at -80 &#176;C at 55 &#181;L per tube in PCR tubes. Use 1 tube per plate.</li>
	<li>Prepare 2% equilibration solution by mixing 55 &#181;L of the substrate reagent and 2750 &#181;L HBSS/HEPES/glucose solution.</li>
	<li>Spread out a thick layer of paper towels on the bench. Gently and repeatedly tap the plate upside-down on the paper towel so that the transfection medium is completely absorbed by the paper towel.</li>
	<li>Wash the cells by pipetting 50 &#181;L of HBSS/HEPES/glucose solution to each well.</li>
	<li>Gently and repeatedly tap out the HBSS/HEPES/glucose solution from the 96-well plate.</li>
	<li>Pipette 25 &#181;L of 2% equilibration solution to each well and incubate at room temperature in dark for 2 h.</li>
	<li>Prepare in advance 1 M odorant stock solutions in DMSO and store at -20 &#176;C until used.</li>
	<li>Prior to the end of the incubation time, dilute the odorant stock solutions to working concentrations in HBSS/HEPES/glucose stimulation medium. 
	NOTE: The concentrations of the odorant dilutions prepared in this step should be doubled to yield the correct final concentrations in each well.</li>
	<li>Using a chemiluminescence plate reader and before odorant addition, measure the basal luminescence level of the plate for 2 consecutive times at a rate of 1000 ms per well34.</li>
	<li>Quickly remove the plate from the plate reader and add 25 &#181;L odorant dilutions to each well and immediately start continuous luminescence measurement of all wells for 20 cycles within 30 min. 
	NOTE: Pipette carefully to avoid contaminating neighboring wells when using different odorants and/or different concentrations of the same odorants in the same plate.</li>
</ol>
5. Data Analysis
<ol>
	<li>Export the data from the chemiluminescence plate reader software.</li>
	<li>Calculate normalized OR response for each time point using the formula 
	(luminescenceN &#8211; luminescencebasal)/(luminescencehighest &#8211; luminescencebasal) 
	where N = luminescence value of a certain well; basal = average luminescence value of the two basal luminescence values; highest = highest luminescence value of a plate or a set of plates. 
	NOTE: Depending on the purpose of the experiment, alternative graphical representations can be adopted. For example, for screening assays (see Figure 1) and for generating concentration-response curves, the luminescence value of a certain well at a desired time point during the kinetic measurement, such as the maximum value or the final value, can be used.</li>
</ol>

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</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Amphotericin B</td><td>Sigma</td><td>A2942</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10099-141</td><td></td></tr><tr><td>GloSensor cAMP reagent</td><td>Promega</td><td>E1290</td><td></td></tr><tr><td>pGloSensor-20F cAMP plasmid</td><td>Promega</td><td>E1171</td><td></td></tr><tr><td>Hana3A cells</td><td></td><td></td><td>available from authors upon request</td></tr><tr><td>HBSS, without calcium or magnesium</td><td>GIBCO</td><td>14175095</td><td></td></tr><tr><td>HEPES</td><td>Hyclone</td><td>SH30237</td><td></td></tr><tr><td>Lipofectamine2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>M3R plasmid</td><td></td><td></td><td>cloned into a mammalian expression vector such as pCI</td></tr><tr><td>MEM, with EBSS and L-glutamine</td><td>Hyclone</td><td>SH30024</td><td></td></tr><tr><td>Muscone</td><td>Santa Cruz</td><td>sc-200528</td><td></td></tr><tr><td>Musk tibetene</td><td>Sigma-Aldrich</td><td>S359165</td><td></td></tr><tr><td>OR plasmids</td><td></td><td></td><td>cloned with a Rho-tag into a mammalian expression vector such as pCI</td></tr><tr><td>PBS, without calcium or magnesium</td><td>Cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Plasmid miniprep kit</td><td>Tiangen</td><td>DP103-03</td><td></td></tr><tr><td>Puromycin</td><td>Sigma</td><td>P8833</td><td></td></tr><tr><td>RTP1S plasmid</td><td></td><td></td><td>cloned into a mammalian expression vector such as pCI</td></tr><tr><td>Trypsin-EDTA</td><td>Hyclone</td><td>SH30236</td><td></td></tr><tr><td>0.2-mL PCR tube</td><td>Axygen</td><td>PCR-02-C</td><td></td></tr><tr><td>1.5-mL Eppendorf tube</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>15-mL 17 mm x 120 mm conical tube</td><td>BD Falcon</td><td>352096</td><td></td></tr><tr><td>8-well and/or 12-well multichannel pipetman</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>96-well flat-bottomed white cell culture plate</td><td>Greiner</td><td>655098</td><td></td></tr><tr><td>100 mm x 20 mm cell culture dish</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>Class II biological safety cabinet with laminar flow</td><td></td><td></td><td></td></tr><tr><td>Cell culture incubator, with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge, with swinging bucket rotor for 15-ml conical tubes</td><td></td><td></td><td></td></tr><tr><td>Infinite F200 plate reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Phase-contrast microscope with x10 and x20 objectives</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td></td></tr><tr><td>Sterile reagent reservoirs for multichannel distribution</td><td></td><td></td><td></td></tr><tr><td>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. ...

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

Research

JoVE Journal

Neuroscience

8.9K Views.  Shanghai Jiao Tong University School of Medicine. Karakterisering van de functie van odorant receptoren serveert een onmisbaar onderdeel in het proces van deorphanization. Beschrijven we een methode voor het meten van de activering van odorant receptoren in real time via een kamp assay.

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

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein G&alpha;olf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

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 ...

cAMP Analysis with cADDis: Single-Cell to High-Throughput Screening

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer&#160;compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 &#181;M forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

Author Spotlight: Advancing Real-Time cAMP Detection in Cells Using cADDis Biosensor

The protocol presented in this study illustrates the effectiveness of the cAMP Difference Detector In Situ in measuring cAMP through two methods. One method utilizes a 96-well plate reading spectrophotometer with HEK-293 cells. The other method demonstrates individual HASM cells under a fluorescent microscope.

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is ...

Biochemistry

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Measuring Odorant Receptor Activation using a Real-Time Cyclic Adenosine Monophosphate Assay

Source: Zhang, Y. et al., <a href="https://app.jove.com/v/55831">Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay.</a> J. Vis. Exp.(2017)
This video demonstrates the protocol for measuring odorant receptor activation in transfected cells using a cyclic adenosine monophosphate (cAMP) biosensor and luminescence detection. The method is applied to study receptor-ligand interactions and signaling pathways in olfactory neurons.

1. Stimulation and Measuring Odorant Receptor (OR) Activity Using the Real-Time cAMP Assay

	Observe the transfected cells under a phase-contrast ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Real-Time Monitoring of Odorant Receptor Activation in Recombinant Cells

<a href='https://app.jove.com/v/59446/real-time-vitro-monitoring-odorant-receptor-activation-an-odorant'>Source: de March, C. A. et. al., Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase. J. Vis. Exp. (2019)</a>This video demonstrates the method to monitor the real-time activation of odorant receptors in recombinant mammalian cells using luminescence measurement. Odorant binding triggers G-protein activation and cAMP production, activating glosensor proteins that emit luminescence upon substrate binding, providing a quantitative measure of receptor response.

Source: de March, C. A. et. al., Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase. J. Vis. Exp. (2019)This ...

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

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