Animals
Adult transgenic mice in which either the T1R3 or TRPM5 promoter drives expression of GFP were used. Animals were cared for in compliance with the Colorado State University Animal Care and Use Committee. Specifically, pIRES2-eGFP containing the encephalomyocarditis virus internal ribosome entry site (IRES) and enhanced green fluorescent protein (eGFP) was purchased from Clontech (Palo Alto, CA). The wheat germ agglutinin (WGA) cDNA was a gift from Dr N.V. Raikhel. The T1r3 and Trpm5 genes were subcloned from BACs obtained by screening a C57BL6 mouse BAC library. The construct T1R3-GFP contained 5' to 3': 13 kb of the mouse T1r3 gene including the 5' flanking region and the entire 5' untranslated region, the WGA cDNA, IRES and eGFP. WGA was included in the construct for the purpose of tracing studies that are unrelated to the studies described here. The construct TRPM5-GFP contained 5' to 3': 11 kb of mouse Trpm5 5' flanking sequence, Trpm5 Exon 1 (untranslated), Intron 1, and the untranslated part of Exon 2, and eGFP. The constructs were separated from the vector by restriction endonuclease digestion, purified from agarose gels using a Qiagen kit and microinjected into B6C3 mouse zygotes according to standard methods [36]. Founder transgenic mice were bred to wild-type C57BL6/J mice and their transgenic offspring were used for further experiments.
Taste cell isolation
Circumvallate and foliate taste buds from TRPM5-GFP and T1R3-GFP mice were isolated using the protocol of Béhé et al. [17]. Briefly, mice were killed with CO2 and cervical dislocation. An enzyme cocktail consisting of 1 mg/ml collagenase A or B (Roche, Indianapolis, IN), 3 mg/ml Dispase II (Roche, Indianapolis, IN), and 1 mg/ml trypsin inhibitor (Sigma, St. Louis, MO) dissolved in Tyrode's was injected beneath the epithelium of the tongue. After incubation for 40–45 min. in oxygenated nominally Ca2+ free Tyrode's, the epithelium was gently separated from the underlying connective tissue and placed in Ca2+ free Tyrode's containing 1 mM BAPTA for 10 min. Taste buds were removed by gentle suction with a fire-polished pipet and plated onto cover slips coated with Cell Tak (BD Biosciences, Bedford, MA) or poly-L-lysine (Sigma, St. Louis, MO). This procedure resulted in isolated taste cells, small cell clusters, and entire taste buds. All data for both Ca2+ imaging and whole cell recording were obtained from isolated taste cells or cell clusters so that the presence or absence of GFP expression could be verified.
Ca2+imaging
Intracellular Ca2+ measurements were obtained using ~2 μM fura-2 AM (Molecular Probes, Invitrogen Corporation). Images were acquired with the CCD Sensicam QE camera (COOKE Corporation) through a 40× oil immersion objective lens of an inverted Nikon Diaphot TMD microscope. Excitation wavelengths of 350 nm and 380 nm were used and with an emission wavelength ~510 nm. Calcium levels were reported as F350/F380 versus time. Images were captured every 1–5 seconds using Imaging Workbench 5.2 (Indec Biosystems, Inc.) and graphed using OriginPro 7.5 software. Responses were measured to m-3M3FBS (PLC activator, 10 μM, Calbiochem, San Diego, CA) and high potassium solution (55 mM). All solutions were bath applied using a gravity flow perfusion system (Automate Scientific Inc., San Francisco, CA) and laminar flow perfusion chambers (RC-25F, Warner Scientific Inc., Hamden, CT).
Whole cell recording
Voltage dependent currents were measured using the whole cell patch clamp technique [37]. Patch electrodes were pulled from LE-16 glass (Dagan Corporation, MN) with a Flaming/Brown micropipette puller (Model P-97, Sutter Instruments, Novato, CA). Pipette resistance ranged from 2 to 5 MΩ. Seals were obtained using gentle suction and entry into the cell was made further suction, or with a 1 ms depolarizing pulse to the pipette. Whole cell currents were measured using an Axopatch 200A patch clamp amplifier and recorded using pClamp 9 software (Molecular Devices, Sunnyvale, CA). Signals were filtered at 5 kHz and recorded at 100 μs. Membrane capacitance was compensated electronically and external solutions were delivered by gravity flow perfusion (Automate Scientific Inc., San Francisco, CA) at a rate of 2–3 ml/min. Voltage-gated currents were elicited by 100 ms step depolarizations from a holding potential of -80 mV.
Solutions
Ca2+imaging experiments
Tyrode's solution contained the following (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, 10 glucose, and 1 pyruvic acid, adjusted to pH 7.4 with NaOH. High K+ solution contained the following (in mM): 90 NaCl, 55 KCl, 1 MgCl, 1 CaCl2, 10 HEPES, 10 glucose, and 1 pyruvic acid. The PLC activator m-3M3FBS was diluted to 10 μM in Tyrode's solution. Ca2+/Mg2+ free Tyrode's solution contained 1 mM BAPTA.
Patch clamp experiments
Tyrode's was identical to that for Ca2+ imaging, except CaCl2 was 2 mM. For recording Ca2+ current, extracellular solution contained (in mM): 136 tetraethylammonium bromide, 10 BaCl2, 1 MgCl2, 10 HEPES, 10 glucose, 1 pyruvate, and 200 nM TTX to block voltage Na+ currents. Intracellular pipet solution contained (in mM): 140 KCl, 2 MgCl2, 1 CaCl2, 11 EGTA, 10 HEPES, and 2.5 ATP, adjusted to pH 7.2 with KOH.
Tissue preparation for immunocytochemistry
Mice were killed in a charged CO2 container followed by cervical dislocation. Tongues were removed and immediately placed into fresh 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) in 0.1 M phosphate buffer for approximately two hours. Tongues were then put into a 20% sucrose solution in 0.1 M phosphate buffer overnight for cryoprotection. Forty micron sections were cut on a Leitz 1729 digital Kryostat and collected in 0.1 M phosphate buffered saline (PBS, pH 7.2). Following sectioning, the slices were washed in PBS three times for ten minutes each at room temperature. All sections were incubated in blocking solution for 1–2 hours at room temperature. Blocking solution contained 0.3% Triton X-100, 1% normal goat serum, and 1% bovine serum albumin in 0.1 M PBS. All chemicals were purchased from Sigma Chemical Corporation (St. Louis, MO) unless otherwise noted
Antibodies
Anti-SNAP-25 polyclonal antibodies raised in rabbit against a 12 residue synthetic peptide (C)ANQRATKMLGSG based on mouse SNAP-25 residues 195–206 were purchased from Calbiochem (San Diego, CA, cat #567343). The secondary Cy-5 anti-rabbit antibody (cat# 111-175-1444) was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). PA1-740 neutralizing peptide was purchased from Affinity BioReagents (Golden, CO, cat# PEP-032)
Immunocytochemistry
Sections from mouse circumvallate papillae were incubated with the primary antibody overnight at 4°C. The primary antibody was diluted (1:1000 for anti-TRPM5; 1:500 for anti-T1R3; 1:200 for anti-SNAP-25) in blocking solution. Sections were then washed three times for ten minutes each in PBS at room temperature then incubated with the secondary antibody (1:400) at room temp for two hours. Following incubation with the secondary antibody, sections were washed three times for ten minutes in PBS and mounted on RITE-ON micro slides (Becton, Dickinson and Company, Portsmouth, NH) using Flouromount-G (Southern Biotechnology Associates, Birmingham, AL, cat# 0100-01) and cover slipped (VWR Scientific, Media, PA). Slides were stored at 4°C.
Controls for light microscopy consisted of omitting the primary antiserum and secondary antibody separately. No immunoreactivity was seen under these conditions. We also performed a peptide block for the primary anti-SNAP-25 in which we incubated PA1-740 neutralizing peptide (Affinity Bioreagents, Golden, CO) at equal weight of peptide per volume antibody overnight at 4 degrees C. We found no immunoreactivity following peptide block.
Confocal microscopy
Images were captured with an Olympus FVX-IHRT Fluoview Confocal Laser Scanning Microscope. Lasers included Argon 488 nm, HeNe 543 nm, and HeNe 622. Fluoview software was used for data acquisition. Sequential scanning techniques were used and showed no differences from simultaneous scans. Images were processed and printed using Adobe Photoshop CS2 software.