|
Figure 2. High numbers of TG neurons respond to odorants and TRP agonists.A: Ca2+ imaging recording of fura2/AM-loaded TG neurons. Cells were challenged with 1 mM HTPA, 50 µM AITC, 300 µM men, and 3.3 µM cap. Stimulus-application is indicated by gray bars. B: Bar charts depicting the overlap of responses to the odorants vanillin, HTPA, helional, or geraniol (1 mM each) and to the TRP-agonists cap (3.3 µM), men (300 µM), and AITC (50 µM).
|
|
Figure 3. CC recording of cultured TG neurons stimulated with odorants in the presence and absence of CPZ.Exemplary CC recordings from cultured TG neurons upon stimulation with vanillin, HTPA, helional, and geraniol alone and with CPZ. Membrane potential changes and RMP of recorded neurons upon stimulation with: vanillincap-sensitive: RMP: −45 mV; vanillin: Δ10 mV, vanillin+CPZ: Δ7 mV, cap: Δ20 mV, men: Δ0 mV; vanillinmen-sensitive: RMP: −62 mV; vanillin: Δ0 mV, vanillin+CPZ: Δ0 mV, cap: Δ0 mV, men: Δ29,5 mV; HTPAcap-sensitive; RMP: −67 mV; HTPA: Δ14.6 mV, HTPA+CPZ: Δ10.3 mV, cap: Δ27.5 mV, Δ2.6 mV; HTPAmen-sensitive; RMP: −61 mV; HTPA: Δ6.3 mV, Δ3.7 mV, cap: 0 mV, men: Δ31.4 mV; helionalcap-sensitive: RMP: −73 mV; helional: Δ29.1 mV, helional+CPZ: Δ22 mV, cap Δ67 mV, men: 0 mV; helionalmen-sensitive: RMP: −50 mV; helional Δ7.2 mV, helional+CPZ: Δ7.8 mV, cap: Δ0 mV; men: Δ25 mV; geraniolcap-sensitive: RMP: −67 mV; geraniol: Δ18 mV, geraniol+CPZ: Δ14 mV, cap: Δ53 mV, men Δ0 mV; geraniolmen-sensitive: RMP: −60 mV; geraniol: Δ17.6 mV, geraniol+CPZ: Δ14.5 mV, cap: Δ2 mV; men: Δ23 mV. Capsaicin- and menthol-evoked action potentials were cut at −30 mV (vanillin), −20 mV (HTPA), 0 mV (helional), and at −10 mV (geraniol). Stimulus applications are indicated by highlighted (gray) regions.
|
|
Figure 4. CPZ and HC inhibit odorant-evoked activations of cultured TG neurons.A: Bar chart, depicting the responsiveness (%) of TG neurons to the odorants vanillin (1 mM; n = 54), HTPA (1 mM; n = 125), helional (1 mM; n = 121), and geraniol (1 mM; n = 101), as well as to cap (3.3 µM) and men (300 µM). Detailed percentages can be derived from table S1. B: Bar chart, depicting the responsiveness (%) of TG neurons to the odorants vanillin (1 mM; n = 87), HTPA (1 mM; n = 68), helional (1 mM; n = 68), and geraniol (1 mM; n = 69) as well as to cap (3.3 µM) and AITC (50 µM). Detailed percentages can be derived from table S1. (C-E) Box plot diagrams depicting the odorant-evoked membrane potential depolarization in cultured TG neurons during whole-cell CC recordings in the presence and the absence of CPZ (C), BCTC (D) or HC (E). C: Recordings from menthol- and capsaicin-sensitive neurons were subdivided. Depolarizations triggered by vanillin (n = 54), HTPA (n = 70), helional (n = 76), and geraniol (n = 59) in the presence and in the absence of CPZ, as well as percentage values of inhibitions can be derived from table 4. D: Recordings from menthol- and capsaicin-sensitive neurons were subdivided. Depolarizations triggered by HTPA (n = 55), helional (n = 45), and geraniol (n = 42) in the presence and in the absence of BCTC as well as percentage values of inhibitions can be derived from table 4. E: Recordings from AITC/cap- and cap-sensitive neurons were subdivided, respectively (original traces are depicted in fig. S2). Depolarizations triggered by vanillin (n = 87), HTPA (n = 68), helional (n = 68), and geraniol (n = 69) in the presence and in the absence of HC can be derived from table 4.
|
|
Figure 5. Odorants activate functionally expressed rTRPV1.A: Box plot diagram depicting normalized rTRPV1-mediated currents evoked by vanillin (n = 10), HTPA (n = 17), helional (n = 17), and geraniol (n = 17) during VC recordings. Outward currents recorded at +100 mV are depicted as upward facing bars, inward currents recorded at −100 mV are depicted as downward facing bars. Detailed values can be derived from table 5. B: Whole-cell VC recordings of CHO cells functionally expressing rTRPV1, challenged with vanillin, HTPA, helional, geraniol and cap. Amplitudes of currents at +100 (black) and −100 mV (gray) are plotted vs. time. Colored arrows and roman numerals assign to individual voltage ramps shown as current traces in the IV-plots depicted in C. Stimulus applications are indicated by gray bars. D: Box plot diagram depicting normalized rTRPV1-mediated currents triggered by vanillin (n = 11), HTPA (n = 9), helional (n = 10), and geraniol (n = 10) that are inhibited by CPZ (exemplary recordings are depicted in fig. S3A). Outward currents recorded at +100 mV are depicted as upward facing bars, inward currents recorded at −100 mV are depicted as downward facing bars. Detailed values can be derived from table 6.
|
|
Figure 6. Odorants activate functionally expressed rTRPM8.A: Box plot diagram depicting normalized rTRPM8-mediated currents evoked by vanillin (n = 14), HTPA (n = 17), helional (n = 17), and geraniol (n = 17) during VC recordings. Outward currents recorded at +100 mV are depicted as upward facing bars, inward currents recorded at −100 mV are depicted as downward facing bars. Detailed values can be derived from table 5. B: Whole-cell VC recordings of CHO cells functionally expressing rTRPM8, challenged with vanillin, HTPA, helional, geraniol and men. Amplitudes of currents at +100 (black) and -100 mV (gray) are plotted vs. time. Colored arrows and roman numerals assign to individual voltage ramps shown as current traces in the IV-plots depicted in C. Stimulus applications are indicated by gray bars. D: Box plot diagram depicting normalized rTRPM8-mediated currents triggered by HTPA (n = 12), helional (n = 14), and geraniol (n = 14) that are inhibited by CPZ (exemplary recordings are depicted in fig. S4A). Outward currents recorded at +100 mV are depicted as upward facing bars, inward currents recorded at −100 mV are depicted as downward facing bars. Detailed values can be derived from table 6. E: Box plot diagram depicting normalized rTRPM8-mediated currents triggered by HTPA helional, and geraniol that are inhibited by BCTC (exemplary recordings are depicted in fig. S4B). Outward currents recorded at +100 mV are depicted as upward facing bars, inward currents recorded at −100 mV are depicted as downward facing bars. Detailed values can be derived from table 6.
|
|
Figure 7. Odorants activate functionally expressed rTRPA1.A: Box plot diagram depicting normalized rTRPA1-mediated currents evoked by vanillin (n = 11), HTPA (n = 11), helional (n = 11), and geraniol (n = 8) during VC recordings. Outward currents recorded at +100 mV are depicted as upward facing bars, inward currents recorded at −100 mV are depicted as downward facing bars. Detailed values can be derived from table 5. B: Box plot diagram depicting normalized rTRPA1-mediated currents triggered by vanillin (n = 8), HTPA (n = 7), and helional (n = 9) that are inhibited by HC (exemplary recordings are depicted in fig. S3B). Outward currents recorded at +100 mV are depicted as upward facing bars, inward currents recorded at −100 mV are depicted as downward facing bars. Detailed values can be derived from table 6. C-F: Whole-cell VC recordings of CHO cells functionally expressing rTRPA1, challenged with vanillin (C), HTPA (D), helional (E), geraniol (F) and AITC. Amplitudes of currents at +100 (black) and −100 mV (gray) are plotted vs. time. Colored arrows and roman numerals assign to individual voltage ramps shown as current traces in the IV-plots depicted right sided, respectively. Stimulus applications are indicated by gray bars.
|
|
Figure 8. Odorants dose dependently inhibit TASK1 and TASK3.A: Exemplary VC recording of rTASK1-mediated voltage-driven currents upon administration of helional, vanillin, geraniol, and HTPA. B: Dose-response relations for vanillin, HTPA, helional, and geraniol, obtained from rTASK1 expressing Xenopus laevis oocytes during VC recordings. Compounds were administered in concentrations of 0.03, 0.1, 0.3, 1, 3, and 10 mM in ascending order. Data points present mean values and error bars present standard errors. C: Exemplary VC recording of TASK3-mediated voltage-driven currents during administration HTPA in arising concentrations. D: Bar diagram depicting the influence of varying concentrations of vanillin, HTPA, helional, and geraniol on voltage-driven currents via rTASK3 during VC recordings from Xenopus laevis oocytes. Bars present mean values and error bars present standard errors.
|
|
Figure 1. Different odorants activate cultured TG neurons.
A:Box plot diagram depicting membrane potential changes in TG neurons challenged with PEE (n = 31), sandalore (n = 31), sandranol (n = 31), javanol (n = 31), vanillin (n = 68), HTPA (n = 96), helional (n = 129) and geraniol (n = 108). Significant depolarization was evoked by vanillin, HTPA, helional, and geraniol. Amplitudes can be derived from table 1. B: Exemplary whole-cell CC recordings of TG neurons upon stimulation with PEE, sandalore, sandranol, javanol, vanillin, HTPA, helional, and geraniol (1 mM each). RMP: PEE: −58 mV; sandalore: −62 mV; sandranol: −62 mV; javanol: −55 mV; vanillin: −60 mV; HTPA: −61 mV; helional: −58 mV; geraniol: −55 mV. C,D: Box plot diagrams depicting densities (pA/pF) of activated (C) and inhibited (D) currents during whole-cell VC recordings from TG neurons upon stimulation with helional (n = 33) and geraniol (n = 32). Outward currents recorded at +100 mV are depicted by upward facing bars, inward currents recorded at −100 mV are depicted by downward facing bars. Detailed values can be derived from table 2. E,F: Whole-cell VC recordings from TG neurons challenged with vanillin, sandalore, helional, geraniol, cap, and ATP depicting odorant-induced activation (E) and inhibition (F) of currents. Current amplitudes at +100 (black) and −100 mV (gray) are plotted vs. time (above). Stimulus application is indicated by the gray bars. Colored arrows and roman numerals assign to individual voltage ramps depicted in the corresponding IV-plots (below).
|
