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

Podsumowanie

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.

Streszczenie

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 "de-orphanize" 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.

Wprowadzenie

The sense of smell allows terrestrial animals to interact with their volatile chemical environment to drive behaviors and emotions. Fundamentally, the odor detection process begins with the very first interaction of odorant molecules with the olfactory system, at the level of odorant receptors (ORs)¹. In mammals, ORs are individually expressed in olfactory sensory neurons (OSNs) located in the olfactory epithelium². They belong to the G-protein coupled receptor (GPCR) family and more precisely to the rhodopsin-like sub-family (also called class A). ORs couple with the stimulatory G protein G_olf whose activation leads to cAMP production followed by the opening of cyclic nucleotide gated channels and the generation of action potentials. It is accepted that an odor percept relies on a specific pattern of activated ORs^3,4 and therefore 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. And through such combinatorial coding, it is postulated that organisms can detect and discriminate between a myriad of volatile odor molecules. One of the keys to understanding how odors are perceived is to understand how and which ORs are activated by a given odor.

In an attempt to elucidate odorant-OR interactions, in vitro functional assays have played an essential role. The identification of agonist odorous ligands for orphan ORs (OR de-orphanization) has been a very active field for the past twenty years, through the use of various in vitro, ex vivo and in vivo functional assays^{5,6,7,8,9,10,11,12,13,14,15,16,17}.

In vitro assay systems are best suited for the detailed functional characterization of ORs, including identifying the functional domains and critical residues of ORs, as well as potential engineering applications. However, further development of valuable in vitro systems for ORs has been a challenge, in part due to difficulty with culturing OSNs and functional expression of ORs in heterologous cells. The first challenge had been to establish protocols that allowed for the cell surface expression of functional ORs in the mapping of odorant-OR interactions. A number of independent groups have utilized various approaches^{5,6,7,8,9,10,11,12,14,18,19,20}. One of the earliest achievements was made by Krautwurst et al. in tagged the N-terminus of ORs with a shortened sequence of rhodopsin (Rho-tag) and observed an improved surface expression in human embryonic kidney (HEK) cells¹³. Variations made to the tag attached to the OR sequence is still a path explored for improving OR expression and functionality^19,21. Saito et al. then identified receptor-transporting protein 1 (RTP1) and RTP2 which facilitate OR trafficking.²² A shorter version of RTP1, called RTP1S, has also been shown to be even more effective than the original protein²³. The development of a cell line (Hana3A) which stably expresses G_olf, REEP1, RTP1, and RTP2 ²⁴, coupled with the use of cyclic adenosine monophosphate (cAMP) reporters has enabled identification of odorant-OR interactions. The mechanism by which the RTP family of proteins promotes cell surface expression of ORs remains to be determined.

One caveat of these established methods is that they rely on odorant stimulation in liquid phase, meaning that odorants are pre-dissolved into a stimulation medium and stimulate cells by replacing the medium. This is very distinct from the physiological conditions where odorant molecules reach the olfactory epithelium in vapor phase and activate ORs by dissolution into the nasal mucosa. To more closely resemble physiologically relevant stimulus exposure, Sanz et al.²⁰ proposed an assay based on vapor stimulation by applying a drop of odorant solution to hang beneath the inner face of a plastic film placed on the top of cell wells. They recorded the calcium responses by monitoring fluorescence intensity. This method was the first to use air-phase odorant stimulation, but it did not allow a large screening of OR activation.

Here, we developed a new method that enables real-time monitoring of in vitro OR activation via vapor phase odorant stimulation by the Glosensor assay (Figure 1). This assay has been used previously in the context of liquid odorant stimulation^{18,19,25,26,27,28,29,30,31}. The monitoring chamber of the luminometer is first equilibrated with vaporized odorant prior to plate reading (Figure 1A). Odorant molecules are then solvated into the buffer, bathing Hana3A cells expressing the OR of interest, RTP1S and the Glosensor proteins (Figure 1B). If the odorant is an agonist of the OR, the OR will switch to an activated conformation and bind the G_olf, activating the adenylyl cyclase (AC), and ultimately cause cAMP levels to rise. This rising cAMP will bind to and activate the Glosensor protein to generate luminescence catalyzing luciferin. This luminescence is then recorded by the luminometer and enables OR activation monitoring. This method is of high interest in the context of OR deorphanization as it brings in vitro systems closer to the natural perception of odors.

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Protokół

1. Hana3A Cells Culture

1. Prepare M10 (Minimum Essential Medium (MEM) plus 10 % v/v fetal bovine serum (FBS)) and M10PSF (M10 plus 100 µg/mL penicillin-streptomycin and 1.25 µg/mL amphotericin B).
2. Culture the cells in 10 mL of M10PSF in a 100 mm cell culture dish in an incubator set at 37 °C and 5% carbon dioxide (CO₂).
3. Divide the cells every 2 days at a 20% ratio: when 100% confluence of cells (approximately 1.1 x 10⁷ cells) is observed under a phase-contrast microscope, aspire the media and wash the cells gently with 10 mL of phosphate-buffered saline (PBS).
4. Aspirate PBS and add 3 mL of 0.05% trypsin-ethylene diamine tetraacetic acid (EDTA, 0.48 mM). Let act for approximately 1 min, until the cells dissociate from the plate.
5. Add 5 mL of M10 to inactivate the trypsin and eventually detach the cells still attached to the plate by pipetting up and down.
6. Transfer the volume (8 mL) to a 15 mL tube and centrifuge at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells into 5 mL of M10PSF by pipetting up and down to break any cell mass. Avoid creating bubbles in the tube.
7. Transfer 1 mL of the resuspended cells solution in a new 100 mm cell culture dish and add 9 mL of fresh M10PSF. Incubate at 37 °C and 5% CO₂.

2. Preparation of the Cells for Transfection

1. Evaluate the confluence, or the number of cells, by observing them under a phase-contrast microscope. At least 10% confluence (approximately 1.1 x 10⁶ cells) is needed for one plate.
2. Aspirate the media and wash the cells gently with 10 mL of PBS. Aspirate PBS and add 3 mL of EDTA. Let act for approximately 1 min at room temperature, until the cells dissociate from the plate.
3. Add 5 mL of M10 to inactivate the trypsin and eventually detach the cells still attached to the plate by pipetting up and down. Transfer the volume (8 mL) to a 15 mL tube and centrifuge at 200 x g for 5 min.
4. Aspirate the supernatant and resuspend the cells into 5 mL of M10PSF by pipetting up and down to break any cell mass. Avoid creating bubbles in the tube.
5. Depending on the number of plates to be transfected, transfer an appropriate amount of cells in a reservoir with the proper corresponding volume of M10PSF. One 96-well plate should be plated with 1/10 of a 100% confluent 100 mm dish (approximately 1.1 x 10⁶ cells) diluted in M10PSF to reach a total volume of 6 mL. For one 96-well plate starting with a 100% confluence 100 mm dish, add 500 µL from the 5 mL of resuspended cells to 5.5 mL of fresh M10PSF. Mix the cells and M10PSF without generating air bubbles.
6. Pipette 50 µL of the suspended cells into each well of the 96-well plate using a multichannel pipette. Incubate overnight at 37 °C and 5 % CO₂.

3. Plasmid Transfection

1. Observe the 96-well plate under a phase-contrast microscope to assure a cell confluence between 30% and 50%.
2. Prepare a first transfection mix that contains the plasmids common to the entire plate (RTP1S, OR and Glosensor protein, see Table of Materials) following the volumes in Table 1. Notice that the quantity of Rho-tagged OR should be divided by the number of ORs if several ORs.
   NOTE: We strongly advice to add an empty vector negative control (here Rho-pCI) and any positive control (OR known to respond to the tested odorant) to the experiment plan.
3. Prepare a second transfection mix containing 500 µL of MEM and 20 µL of Lipofectamine 2000 reagent (valid for one 96-well plate, see Table of Materials). Add the second mix to the first one, and gently mix by pipetting up and down and incubate for 15 min at room temperature. Add 5 mL of M10 and mix gently.
4. Replace the M10PSF in the previously platted 96-well plate by 50 µL of the final transfection media. Incubate in an incubator set to 37 °C and 5% CO₂ and vacuum the chamber of the luminometer overnight following the procedure described in step 6.

4. Substrate Incubation

1. Observe the 96-well plate under a phase-contrast microscope to assure a cell confluence between 60% and 100%. Prepare a stimulation solution of Hank’s Balanced Salt Solution (HBSS) containing 10 mM of hydroxyethyl piperazineethanesulfonic acid (HEPES) and 5 mM of D-glucose.
2. Dilute 75 µL of the cAMP reagent (see Table of Materials) solution to 2.75 mL of the stimulation solution. Remove the transfection medium from the 96-well plate and wash the cells by adding 50 µL of fresh stimulation solution to each well.
3. Remove the stimulation solution and add 25 µL of cAMP reagent solution prepared in step 4.2 to each well. Incubate the 96-well plate at room temperature in a dark and odor-free environment (for example, a clean empty drawer far away from chemicals or any odorant source) for 2 h.

5. Odorant Stimulation

1. First, equilibrate the luminometer chamber with volatile odorant molecules. Dilute the odorant to the desired concentration in 10 mL of mineral oil (Figure 2A). Before the end of the cAMP reagent incubation time, add 25 µL of the odorant solution in a new 96-well plate (not the one containing the cells). Incubate this odorant plate at room temperature in the luminometer chamber for 5 min (Figure 2B) (no luminometer recording is required here).
2. Set the luminometer to record the luminescence with 0 s of delay during 20 cycles of plate measurement of 90 s with 0.7 s of interval between cycles.
3. Right before reading the plate, remove the odorant plate from the chamber. Add 25 µL of odorant between the wells of the 96-well plate containing the cells (do not add the odorant in the wells containing the cells) and quickly start the luminescence measurement of all wells for 20 cycles within 30 min (Figure 2C).

6. Removal of remaining odorant inside the Luminometer

1. Open the door of the luminometer. Insert the tube connected to the vacuum pump.
2. Vacuum odorants in the reading chamber extensively (at least 2 h, preferably overnight) between two odorants to avoid cross contamination of odor volatiles from one experiment to another. Replace with fresh air by sending compressed air during 5 min before incubating the next odorant.

7. Data Analysis

1. Export the data from the luminometer software.
2. Average the replicates of the same OR for each recording time. Calculate the normalized OR response to any eventual control (e.g., Empty vector, Figure 3A and representative results section) by dividing the control averaged value to the OR averaged value at each recording time (Figure 3B and representative results section).
3. Normalize the each OR response to their basal activity by dividing the averaged OR response at 0 s to each recording time response (See Figure 3C and representative results section).
4. Calculate the area under the curve of each OR to obtain a single OR response value. To do so, sum all the luminescence values of each recording time for each OR.

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Wyniki

We screened the response of three mouse ORs, Olfr746, Olfr124 and Olfr1093 using cinnamaldehyde vapor stimulation (Figure 3). Simultaneously, we used an empty vector control (Rho-pCI) to assure that the odorant-induced activities of the tested ORs were specific (Figure 3A). The real-time activation of the ORs upon vapor odorant stimulus was monitored over 20 measurement cycles. The data for each well were first normalized to the empty vector con...

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Dyskusje

The perception of odor is fundamentally dependent on the activation of ORs. Consequently, understanding of their functionality is required to crack the complex mechanisms that the brain use to perceive its volatile chemical environment. However, the understanding of this process has been hampered by the difficulties in establishing a robust method to screen the OR repertoire for functionality against odorants in vitro. Cell surface and heterologous expression of ORs has been partially solved by the creation of t...

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Materiały

Name	Company	Catalog Number	Comments
0.05 % trypsin-EDTA	Gibco	25300-054	0.05% Trypsin - EDTA (1x), phenol red - store at 4°C
100 mm cell culture dish	BD Falcon	353003	100 mm x 20 mm cell culture dish
15 mL tube	BD Falcon	352099	17 mm x 120 mm conical tubes
96-well plate	Corning	3843	96 well, with LE lid white with clear bottom Poly-D-lysine coated Polystyrene
Amphotericin	Gibco	15290-018	Amphotericin B 250 µg/mL - store at 4 °C
centrifuge machine	Jouan	C312	Centrifuge machine with swinging bucket rotor for 15 mL
Class II Type A/B3 fumehood	NUAIRE	NU-407-500	fumehood for cell culturing
FBS	Gibco	16000-044	Fetal Bovine Serum - store at -20 °C
GloSensor cAMP Reagent	Promega	E1290	GloSensor cAMP Reagent luminescent protein substrate - store at -20 °C
Incubator 37 °C; 5 % CO2	Fisher Scientific	11-676-604	Incubator for cell culturing
Lipofectamine 2000 reagent	Invitrogen	11668-019	Lipofectamine 2000 Reagent 1mg/ml transfection reagent - store at 4 °C
Luminometer POLARstar OPTIMA	BMG LABTECH	discontinued	96 well plate reader for luminescence
Mineral oil	Sigma	M8410	Solvent for odorants - store at room temperature
Minimum Essential Medium (MEM)	Corning cellgro	10-010-CV	Minimum Essential Medium Eagle with Earle’s salts & L-glutamine - store at 4 °C
Penicillin/Streptomycin	Sigma Aldrich	P4333	Penicillin-Streptomycin solution stabilized with 10,000 U of penicillin and 10 mg streptomycin - store at -20 °C
pGlosensor	Promega	E2301	pGloSensor-22F cAMP luminescent protein plasmid - store at 4 °C
Phase contrast microscope	Leica	090-131.001	phase contrast microscope with x4, x10, x20 objectives
RTP1S	H. Matsunami lab	-	100 ng/µL plasmid - store at 4 °C