Pharmacology of Mammalian Olfactory Receptors

Abstract

Mammalian species have evolved a large and diverse number of odorant receptors (ORs). These proteins comprise the largest family of G-protein-coupled receptors (GPCRs) known, amounting to ~1,000 different receptors in the rodent. From the perspective of olfactory coding, the availability of such a vast number of chemosensory receptors poses several fascinating questions; in addition, such a large repertoire provides an attractive biological model to study ligand–receptor interactions. The limited functional expression of these receptors in heterologous systems, however, has greatly hampered attempts to deorphanize them. We have employed a successful approach that combines electrophysiological and imaging techniques to analyze the response profiles of single sensory neurons. Our approach has enabled us to characterize the “odor space” of a population of native aldehyde receptors and the molecular range of a genetically engineered receptor, OR-I7.

1. Introduction

In the vertebrate olfactory system, odorants are recognized by a combinatorial mechanism (1). A single receptor can recognize multiple odorants, and multiple receptors can detect the same odorant; indeed, odorants are recognized by different combinations of odorant receptors (ORs). The mammalian olfactory system employs a large number of receptors that comprise the largest family of G-protein-coupled receptors (GPCRs) known (2). The diversity of these receptors and the number of molecules they can potentially bind impose a great challenge to our understanding of peripheral olfactory processing (3). The difficulty of this challenge is exacerbated by our lack of knowledge of the mechanisms of OR expression, which limits the use of heterologous systems (4, 5). Thus, the initial excitement of the discovery of ORs in the early 1990 has slowly yielded to the realization that deorphanizing, the pairing of each of these receptors with a cognate ligand, will pose many challenges, key among which is the paltry expression of these proteins in systems suitable for deorphanization. More than two-thirds of known ORs remain orphaned. To combat this, rather than relying on artificial expression systems we have used a different approach to determine the “odor space” of ORs, which is to analyze the response profiles of single sensory neurons (1, 6-8). The success of this approach relies on the peculiarity of OR expression—an olfactory sensory neuron expresses only one receptor from the available repertoire (2). Thus, we expose individual cells to a selected pool of odorants, with specific structural and chemical properties. The cell’s responses are then analyzed using electrophysiological and imaging techniques. The odor response profile of a single cell is then, the odorant profile of a single OR. This approach allows the functional analyses of a large number of receptors in their native environment. By using a slightly different approach, sensory neurons have been previously used to express selected receptors using viral constructs containing an OR of interest (4, 9). The nasal epithelium is utilized as a proficient expression system and a single OR can be then challenged with a set of various odor molecules to determine its molecular range. We summarize below the nuances of the technique used to prepare isolated sensory neurons for calcium imaging recordings.

2. Materials

2.1. Isolation of Sensory Neurons

1. Ringer’s solution: 138 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1.5 mM MgCl₂, 10 mM HEPES, 10 mM d-glucose, pH 7.4 (see Note 1).

2. Divalent-free Ringer’s used for dissociation: 145 mM NaCl, 5.6 mM KCl, 10 mM HEPES, 10 mM d-glucose, 4 mM ethyleneglycol tetraacetic acid (EGTA), pH 7.4. This solution can be aliquoted and stored at 4 °C.

3. Enzyme mixture for sensory neurons dissociation: Divalent-free Ringer’s containing 5 mg/mL bovine serum albumin, 1 mg/mL collagenase, 2.4 U/mL dispase II, and 100 μL deoxyribonuclease II (DNase II).

4. Concanavalin A (10 mg/mL in water)-coated glass coverslips (22 × 22 mm, see Note 2).

5. Culture medium; Dulbecco’s Modified Eagle’s Medium/F-12 (DMEM/F12) supplemented with 10 % fetal bovine serum (FBS), 1× insulin–transferrin–selenium-X, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. This solution is filtered through a Nalgene filter flask and aliquoted into 50 mL portions.

2.2. Calcium Imaging Recordings

1. Loading solution: Fura-2AM plus pluronic acid (F-127, 80 μg/mL) in Ringer’s (see Note 3).

2. Inverted fluorescence microscope (IMT-2 Olympus, Tokyo, Japan) equipped with a SIT camera (C2400-08, Hamamatsu Photonics, Hamamatsu, Japan) connected to a frame grabber (LG-3, Scion, Frederick, MD).

3. Modified recording chamber (Warner, Hamdem, CT) connected to a multichannel perfusion system (custom made, see Note 4).

4. The NIH Image software for data acquisition and analysis (NIH, Bethesda, MD) with customized macros written for the shutter control (Uniblitz, Vincent associates, Rochester, NY) and time-lapse imaging.

5. Odor solutions are prepared from pure odors in DMSO at a concentration of 1 mM and stored at 4 °C.

3. Methods

Olfactory sensory neurons are not well-suited for long-term cell cultures and quickly undergo apoptosis once their axon is severed. Therefore, great care should be exercised during the dissection and dissociation of the sensory epithelium. This protocol works well for both mice and rats.

3.1. Dissection of the Sensory Epithelium

1. Once the animal has been euthanized and the head isolated, the fur is removed and the head bisected from the midline, starting from the most rostral point, cutting caudally using the nasal bones as guideline.

2. The head halves are placed on a plastic 35 mm Petri dish and covered with ice-cold divalent-free Ringer. Use small curved scissors to remove the turbinates and septal bone from the skull and transfer them to a Petri dish.

3. The olfactory sensory tissue is then carefully dissected out from the turbinates and from the septal bone gently but quickly using a pair of fine-tip forceps. The whole procedure should be done in ice-cold Ringer’s and over ice.

3.2. Dissociation of the Sensory Neurons

1. Place the tissue on a Petri dish containing divalent-free Ringer’s and carefully mince using a microsurgery scissors.

2. Transfer the tissue to a Falcon plastic conical 15 mL tube and remove the excess of divalent-free Ringer’s and add the enzyme mixture supplemented the DNase (100 μL).

3. Incubate the tissue in the enzyme mixture at 37 °C for 30 min (mouse) or 45 min (rat) on a shaker, at approximately 35 rpm.

4. After the incubation, place the tube inside a tissue culture hood and let a small piece of tissue settle at the bottom of the conical tube. Use a transfer pipette to suck off as much enzyme solution as possible and add 5 mL of pre-warmed culture media (37 °C). Gently mix with the tissue to stop the enzyme reaction and let the tissue settle at the bottom of the tube. Carefully transfer the tissue to another conical tube containing 5 mL of culture media, let the tissue settle and complete two additional washes.

5. Remove as much medium as possible and add 500 μL of fresh culture medium. The volume needed at this step can be varied depending on the amount of tissue; in general, rats require more volume.

6. For the dissociation, it works best to disperse the cells by holding the tube vertically and striking it downward on a firm surface. At this point, the solution becomes clouded as the cells have dissociated.

3.3. Fura-2AM Loading

1. Let the larger pieces of tissue settle and take 50 μL of the cell suspension from the supernatant; spot the cell suspension onto a Concanavalin A (Con A)-coated coverslip. Let the cells attach to the Con A for about 20 min.

2. Add 1.5 mL of media being careful not to disturb the spot of Con A containing the cells, and then using a suction pipette carefully remove the media from the dish, repeat this and wash about three times, avoiding to let the cells dry in between the washes.

3. After the last wash, add 1.5 mL of media containing ascorbic acid (100 μM) and incubate the cells for 45 min to 1 h.

4. Wash the dish twice with pre-warmed (37 °C) Ringer’s and then add 1 mL of Ringer containing the Fura-2AM and incubate for 45 min at room temperature. To prepare the loading solution, thaw a tube containing a 6.25 μL aliquot and then add 1 mL Ringer and 1 μL of pluronic acid and vortex for at least 1 min.

5. After the 45 min incubation period remove, the Ringer’s containing the Fura and wash twice with regular Ringer’s. Leave in regular Ringer’s for at least 10 min, before imaging the cells.

3.4. Calcium Imaging

1. The coverslip is mounted at the bottom of the perfusion chamber over a small spot of Vaseline used to prevent movements during the perfusion.

2. Recordings are made at 380 nm excitation and 510 nm emission (10). Images are acquired at a rate of 15 frames per minute. After a baseline of 40 s, odors are perfused into the chamber for 8 s in enough volume to completely replace the solution in the chamber (~200 μL). An example of Fura-2AM loaded cells is shown in Fig. 1a.

3. Odor responses are quantified as the fractional change in fluorescent light intensity: ΔF/F₀ or (F–F₀)/F₀, where F is the fluorescent light intensity at each point and F₀ is the value of emitted fluorescent light before the odor application (baseline). An example of a cell’s responses to aliphatic aldehydes of different carbon lengths is shown in Fig. 1b (see Note 5).

4. We use odorant at concentrations 0.3–300 μM, applied at intervals of at least 3 min. Odorant solutions are freshly prepared in Ringer’s by dilutions from odorant stocks made in dimethyl sulfoxide (DMSO) and applied randomly. We routinely limit the number of odor presentations to less than 15 per cell.

Fig. 1.

Calcium imaging of olfactory sensory neurons. (a) Cells were loaded with Fura-2AM for 45 min. Data are analyzed offline by drawing a region of interest around responding cells (white arrow) and determining the change in fluorescent intensity. We find that, on average, 20 % of the cells in the recording field are viable olfactory sensory neurons, as measured by the response to a high KCl stimulus (50 mM) and IBMX (2 mM, not shown). (b) Responses to aliphatic aldehydes of different carbon lengths in the cell shown in (a). An odorant is considered to elicit a response when the change in ΔF/F₀ is higher than two times the value of the standard deviation of the baseline and the decrease in ΔF/F₀ lasts more than 20 s. The profile of this cell corresponds to that of OR-I7 (9)

3.5. Discussion

Since the discovery of ORs, more than 20 years ago (11), the deorphanization of ORs has been dishearteningly slow. A critical factor contributing to the slow progress of deorphanization is the poor expression of ORs in heterologous systems that are commonly used to study receptor pharmacology, such as transfected cell lines and expression in Xenopus oocytes. Early observations indicated that intracellular trafficking of ORs was impaired and several molecular approaches were undertaken to overcome this issue (12).

The identification of a group of chaperone proteins in the laboratory of Dr. Hiroaki Matsunami has offered a promising starting point (13). The researchers co-expressed these proteins with ORs in transfected cells and coupled their expression with a luciferase reporting system, which greatly contributed to the deorphanization of several ORs. This approach has proven limited however, as most ORs still have expression issues and a large number of ORs remains orphan. The prevailing view is that various cofactor proteins (or chaperones), yet to be discovered, are necessary for the appropriate intracellular trafficking and targeting of ORs to the cell’s surface (4).

The advantage of recording responses directly from intact sensory neurons, as our methodologies described here indicate, is obvious; this is because the native transduction machinery is already present. In addition, compared with the kinetics of the responses in the luciferase assay (hours), the kinetics of the responses in the isolated cell preparation is closer to that of an in vivo environment (seconds). A small drawback of our approach, however, is that in these experiments isolated sensory neurons are surrounded by an artificial solution, instead of the natural mucus layer that naturally bathes their cilia. Therefore, the odorant–OR interaction is not under the ideal physiological conditions. Nevertheless, odor application is fast and several odors can be applied to the same cell allowing for the rapid screening of odorants and thorough characterization of an OR pharmacology (14). Lastly, a critical step is to follow the characterization of the OR pharmacological profile by its genetic identification, using single cell PCR of the recorded neuron. Only when the receptor sequence is known and reconstituted in vivo can the receptor’s pharmacological profile be confirmed.

4. Notes

1. All solutions are prepared in ultrapure water (less than 18 MΩ cm resistivity). Unless indicated all solutions and materials are prepared under sterile conditions under a tissue culture hood.

2. The Concanavalin A solution is filtered and aliquoted into 100 μL stored at −20 °C.

3. The Fura-2AM vial is aliquoted into 6.25 μL in 1.5 mL Eppendorf tubes and stored at −20 °C.

4. The perfusion chamber is modified so that the perfusion solution only fills the diamond area. Odors are applied using syringes connected to the perfusion system via a manifold.

5. When studying the responses to different molecules we use one odor as a control to test the viability throughout the recordings, for example, octanal (6). Alternatively, responses can be normalized to the cell’s response to the phosphodiesterase inhibitor 3-iso-butyl-1-xanthine (IBMX, 2 mM) or the activator of the adenylyl cyclase, Forskolin (10 μM) (14).