Mammalian Ringer’s solution (Ringer’s) contained (in mM): 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 1 sodium pyruvate, 20 N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES, pH 7.2). For Ca2+-free Ringer’s, CaCl2 was removed and replaced with 1 mM ethylene glycol tetraacetic acid (EGTA). Isolation solution contains (in mM): 145 NaCl, 5 KCl, 2 EDTA, 1 sodium pyruvate, 20 HEPES, 100 μg/mL gentamycin. Stop solution contains (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 d-glucose, 1 sodium pyruvate, 20 HEPES. Cell culture medium consisted of Iscove’s Dulbecco’s modified Eagles medium (IDMEM high glucose) supplemented with 10% fetal bovine serum (heat inactivated) and 1% penicillin/streptomycin (100 IU and 100 μg/mL, respectively).
Other compounds used in this study were as follows: S(−)-nicotine (Philip Morris, Richmond, VA, >99% by GC), R(+)-nicotine (Toronto Research Chemical Inc., North York, Canada, 97% by TLC, NMR and optical rotation), noncompetitive nicotinic acetylcholine receptor antagonist mecamylamine hydrochloride (20 μM), competitive nicotinic acetylcholine receptor antagonist hexamethonium chloride (20 μM), nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium (DMPP, 10 μM), adenylate cyclase inhibitor MDL-12,330A hydrochloride (20 μM), phospholipase C antagonists U-73122 (20 μM) and neomycin (1 mM), sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (200 nM, Calbiochem, San Diego, CA), adenylate cyclase activator forskolin, phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), leupeptin, l-cysteine, papain, and DNase I (Roche Diagnostics, Indianapolis, IN). Unless otherwise specified, all compounds were from Sigma-Aldrich, Inc. (St Louis, MO).
Human Subjects and Biopsy of Olfactory Tissue
Work with human tissue described in this manuscript was conducted in accordance with the Declaration of Helsinki using procedures approved by the Institutional Review Board of Thomas Jefferson University, Philadelphia, PA. Subjects provided informed consent by signing a document describing the nature and possible consequences of participation. The subjects were five men and seven women, ages 23−61 (28.3 ± 11.8, mean ± SD), two of whom smoked and 10 who had never smoked. All subjects performed within normal limits on unilateral detection threshold testing for phenylethylalcohol using standard procedures (14
), did not report any current sinus problems or taste or smell disorders, and were not taking any medications that would exclude them from participation (anticoagulants or sinus or allergy medications). An ~1 mm3
tissue sample was excised from the middle turbinate and opposing septum under local anesthesia using established olfactory biopsy techniques (15
). Biopsies were placed immediately into IDMEM and transported to the laboratory for further processing.
The use and handling of animals was performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Institutes of Health guide (NIH Publications 80-23, revised 1978). The experimental protocols were approved by the Institutional Animal Care and Use Committee of Monell Chemical Senses Center, and all efforts were made to reduce the number of animals used and minimize animal suffering. Animals were euthanized with CO2, and the turbinates and septum (from 150 to 250 g, 50−75 day old Sprague−Dawley rats) were immediately excised and transferred into isolation solution.
Preparation of Dissociated Olfactory Sensory Neurons and Primary Cell Culture
For the acute preparation of both human and rat OSNs, the tissue was finely minced and incubated in isolation solution with 5 μL/mL papain and 5 mM l-cysteine for 15 min. Following cell dissociation, the tissue was gently triturated with a fire polished pipet, transferred into a stop solution containing 10 μg/mL leupeptin and 20 U of DNase I and filtered with a 70 μm nylon cell strainer (BD Falcon, Bedford, MA). The dissociated cells were layered on top of a 20%/40% Percoll gradient and centrifuged for 10 min at 700g. The olfactory cells were harvested from the 20%−40% interface.
Primary cultures of human olfactory epithelial cells for both calcium imaging and electrophysiology experiments were prepared as described previously (3
). Briefly, biopsies were minced coarsely and incubated in the calcium- and magnesium-free HBSS for ~20 min, triturated, and centrifuged at 600g
for 5 min. The pellet was resuspended in culture medium and transferred to a 25 cm2
culture flask and allowed to grow for 1−2 weeks until cell growth was sufficient for transfer to continuous culture. Cells were maintained in 75 cm2
culture flasks in a humidified incubator (37 °C, 5% CO2
). Cultured cells established in this fashion grew to confluence in 1 week. Based on previous experiments an optimum number of mature neurons (based on assays of odorant responsiveness or immunocytochemical detection of molecular markers such as olfactory marker protein (3
) were present by day 4−5 after plating. Cultures could be frozen at passage 2−3, thawed, and used to generate new cultures with similar growth rates and proportions of mature cells, and typically became senescent after 6−10 passages. The current protocol is identical to earlier studies (3
) in which approximately 65% of subject biopsies generated successful cultures based on proliferation and generation of cells showing calcium responses to odorants.
Measurement of Cellular Responses to Stimulation
Cellular responses were measured using ratiometric calcium imaging techniques as previously described (18
). Rat and freshly biopsied human OSNs were loaded with 5 μM acetoxymethylester of Fura-2 (Fura-2/AM) and 80 μg/mL pluronic F127 (Molecular Probes, Eugene, OR) for at least 1 h at room temperature and transferred onto coverslips (22 × 60 mm2
, No. 0, Thomas Scientific Co.) coated with 1 mg/mL concanavalin A. Cultured human olfactory cells (HOCs) were similarly loaded with Fura-2/AM after having been plated onto glass coverslips and cultured for 4−5 days. Coverslips with OSNs or HOCs were set in a recording chamber and superfused with Ringer’s solution or Ringer's containing different chemical compounds via a valve controller (VC-8, Warner Instruments, Hamden, Connecticut) and perfusion pump (Perimax 12, SPETEC, Erding, Germany). Stimulation duration was 30 s, and perfusion rate was 0.8 mL/min. There was a 20 s delay between solenoid activation and arrival of stimulus compounds at the neurons. The Photon Technology International (PTI) calcium imaging system was as follows: excitation from a DeltaRAM X monochromator was at 340 nm and 380 nm with a 510 nm emission filter. Cellular fluorescence was imaged (20×, Olympus IX70) with a Cooke CCD camera. Images were digitized and analyzed using Imagemaster (PTI) software. Since there were multiple types of cells in the dissociated tissue preparation, rat and human olfactory neurons were identified morphologically, and only cells with rounded cell bodies and a dendrite with knob (Figure A, inset) were considered to be olfactory sensory neurons. The fluorescence ratio (F340
) was calculated for regions-of-interest drawn around these cells. Cells were considered responsive if they displayed a 10% increase in intracellular calcium.
Perforated patch electrophysiology was carried out with freshly dissociated rat olfactory cells according to previously published protocols (19
). Olfactory neurons were identified morphologically, and only cells with rounded cell bodies, a dendrite with knob, and cilia were selected for recording. An Axopatch 200A amplifier (Axon Instruments) and a PC running PClamp 8 were used to apply pulse protocols and record data. Cells were recorded in perforated-patch configuration using gramicidin (120 μg/mL) as ionophore. Patch pipettes were fabricated from borosilicate glass (Kimex 51, Kimble Glass, Inc.) on a vertical pipet puller (PP-83, Narishige) and fire polished to a final bath resistance of 3−5 MΩ. After formation of a giga seal, cell membranes generally began to perforate within 5 min, and steady-state access was reached after ~15 min, at which point large voltage-activated sodium and potassium currents could be elicited by depolarizing voltage steps. Stimuli were applied via a multibarrel pipet connected to a Picospritzer unit. Cells were continuously rinsed with Ringer’s after mounting on the microscope. Prior to stimulation, the bath solution (initially 1 mM Ca2+
Ringer’s) was changed to 0 Ca2+
Ringer’s (nominally calcium-free). The pipet solution contained (in mM): 110 potassium aspartate, 36 KCl, 1 MgCl2
, 1 EGTA, 10 HEPES, 1 ATP, 0.1 GTP. The pH was adjusted to 7.2 with KOH, and the osmolarity was 305 mOsmol.