The bone covering the dorsal OB was carefully drilled and thinned with a dental drill. In some experiments, 1.5% agar dissolved in Ringer’s solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 10 mM Glucose; pH 7.5) was applied to reduce brain pulsation. Dextran-conjugated Oregon Green Selleckchem BI-2536 BAPTA-1 (Invitrogen) was diluted in Ringer’s solution and used at a final concentration of 10%–20%. Glomeruli in OMP-spH knockin mice were visualized by two-photon fluorescence imaging
(Bozza et al., 2004), and a dye-filled glass pipette (tip I.D., 2.5 μm) was inserted into the center of the target glomerulus. Small square pulses were delivered from a current isolator (2 Hz for 2–10 min). The pulses were transferred to pseudoexponential waveforms by a capacitor (2350 pF) that bridged the outputs of the isolator (Figure S1). The waveforms were composed of sharp
currents (5–10 μA amplitude, 1–2 ms duration) that were followed by small tail currents (τ ≈25 ms). As previously reported, this electroporation method was effective within a 20–30 μm diameter and adequately labeled single glomerular-specific cells in the OB (Nagayama et al., 2007). A combination of liquid-dilution and flow-dilution methods was used for dilution of odors. Odorants were first Compound C cost diluted in mineral oil to 0.01%–10% in glass tubes. Filtered nitrogen was used as the odor vapor carrier to avoid oxidation. The saturated vapor for each odorant was then diluted fivefold through mixture with pure air. The final concentrations were adjusted to 0.002%–2% with two separate mass flow controllers for clean air and odor vapor. The total airflow was fixed at 0.5 l/min throughout STK38 the experiment. To avoid cross-contamination, multiple Teflon tubes were used for different
odorants that were delivered in parallel. One suction tube and multiple odor delivery tubes were banded and then placed in front of the nostrils of the mice. To deliver odorants, the suction was stopped with a solenoid valve, and the diluted odorants were blown toward the nostrils from a distance of 1 cm. The odorants were usually presented for 3 s and with an interstimulus interval of more than 60 s to avoid potential sensory adaptations. A constant vacuum pipe was placed over the heads of the mice for quick exhaustion of the odorants. A homologous series of aliphatic aldehydes with different carbon chain lengths were used (propylaldehyde [3CHO], butylaldehyde [4CHO], valeraldehyde [5CHO], hexylaldehyde [6CHO], heptylaldehyde [7CHO], octylaldehyde [8CHO], nonylaldehyde [9CHO]) to stimulate the olfactory epithelium. The three layers in the OB were distinguished based on anatomical features. The glomerular layer (GL) was identified by the glomerular spH image and an expected thickness of 100–150 μm from the surface of OB.