Hawkmoth Pheromone Transduction Involves G-Protein–Dependent Phospholipase Cβ Signaling

Illustration of the linear fit analysis of pheromone responses. A, BAL stimuli were applied every 5 min in the 120-min-long tip-recordings of hawkmoth trichoid sensilla. For each animal and experimental condition [here, one control (black) and one GDP-β-S (red) animal], we generated a linear regression model for time and respective BAL response parameters (here, latency to the first AP after the onset of the BAL response) and used a t test to determine whether the slope of the fit significantly differed from zero (solid line) or not (dashed line). B, To quantify the response kinetics to BAL stimulation, we binned the data of the first second after the BAL stimulus in 10 ms bins across all BAL stimulations for each animal. Each resulting cumulative histogram (dotted line) was fitted with a sigmoidal function (solid line) for quantification that yielded three fit parameters: maximum spike number (AP_max), time of the midpoint of the sigmoid (t_1/2; indicated by arrows at the x-axis), and slope of the midpoint (k). Depicted are examples of two animals.

Inhibition of G-protein signaling of pheromone-sensitive ORNs by GDP-β-S decreased the phasic BAL response and increased response latency during the hawkmoth's activity phase. A, Examples of high-pass filtered tip-recordings (AC) of ORN responses in control (ctrl, recording solution + DMSO, black) and with the G-protein antagonist GDP-β-S (dissolved in SLR + DMSO, red) at ZT 1–3, 100 min after the start of the recording. Arrow indicates the onset of BAL response. Response parameters (Fig. 1) during the moth's late activity phase (ZT 1–3; B) and at rest (ZT 9–11; C) in control and GDP-β-S. Data are shown as mean (line) ± standard deviation (shaded area). D, One-way ANOVA results with appropriate post hoc test for multiple comparisons (α = 0.05) for the slopes of BAL response parameters (see Materials and Methods and Fig. 2A). F_6AP slopes decreased significantly, and latency slopes increased significantly in GDP-β-S compared with control at the activity phase (ZT 1–3). No other parameters showed significant differences compared with control at either ZT. Dots show data for individual experiments; red lines indicate the mean. Raw data are provided in Extended Data Figure 3-1.

Inhibition of G-protein signaling changed kinetics of the ORN response to BAL stimulation during the activity phase. A, Peristimulus time histograms (PTSH; 10 ms bins) of the first second of the ORN response to BAL stimulation in control (black) and in GDP-β-S (red). Data are shown as mean (line) ± standard deviation (shaded area). Arrow: onset of BAL response. B, Fit parameters [total number of spikes in 1 s after onset of the BAL response (AP_max), time of the sigmoid midpoint (t_1/2), and slope (k) of the midpoint] of sigmoidal fits to the cumulative spike histograms (see Eq. 1, Materials and Methods, and Fig. 2B). One-way ANOVA with appropriate post hoc test (α = 0.05) revealed a significantly steeper slope (k closer to zero) in control compared with GDP-β-S during the activity phase (ZT 1–3). Steeper slopes indicate faster rise and fall times of the spike count in the PTSHs. The total number of spikes and sigmoid midpoint were not significantly different. Dots show fit parameter values for individual experiments; red lines indicate the mean. Raw data are provided in Extended Data Figure 4-1.

Inhibition of PLC of pheromone-sensitive ORNs with U73122 decreased the phasic BAL responses at both ZTs while decreasing SPA and increasing response latency only at ZT 1–3. A, Example high-pass filtered (AC) tip-recordings of ORN responses in control (U73343, dark purple) and in PLC inhibitor (U73122, light purple) at ZT 1–3, 100 min. The arrow indicates the onset of the BAL response. Response parameters (Fig. 1) during the activity phase (ZT 1–3; B) and at rest (ZT 9–11; C) in control and U73122. Data are shown as mean (lines) ± standard deviation (shaded areas). D, One-way ANOVA results with appropriate post hoc test for multiple comparisons (α = 0.05) for the slopes of BAL response parameters (see Materials and Methods and Fig. 2A). Compared with controls, F_6AP slopes decreased at both ZTs in U73122, but SPA and latency slopes increased only at the activity phase (ZT 1–3). Other BAL response parameters showed no significant differences between control and U73122 at either ZT. Dots show data of individual experiments; red lines indicate the mean. Raw data are provided in Extended Data Figure 5-1.

Responses to BAL stimulation at ZT 1–3 were affected by toxins that target G_αs or G_αo subunits. A, Examples of tip-recordings from ORNs with BAL stimulation in control (black), with cTox (blue), pTox (yellow), and pmTox (green); repeated measures of one animal shown for each toxin. AC: high-pass filtered recording to highlight AP response; DC: unfiltered SPA with APs; Arrow: onset of the BAL response. B, Quantification with one-way RM ANOVA with appropriate post hoc test for multiple comparisons (α = 0.05) of the same parameters as in Figure 3 and Figure 5. During the activity phase (ZT 1–3), the frequency of the phasic BAL responses (F_6AP) and the number of APs during the first 100 ms of the response [#APs (early)] decreased in cTox (blue; sustained activation of G_αs) and pTox (yellow; inhibition of G_αo), while response latency increased significantly. The effect of pmTox (green; constitutive activation of G_α12/13, G_αi, and G_αq) was not different from control. Raw data are provided in Extended Data Figure 6-1.