pC1 neuron subset comprising pC1b and c subtypes regulates EHP in response to 2MC and 7-T, both of which upregulate cAMP activity and excitability of pC1 neurons
A-D, Optogenetic silencing of a pC1 neuron subset comprising pC1b and c subtypes increases EHP. Females of the indicated genotypes were cultured on food with or without all-trans-retinal (ATR). ΔEHP is calculated by subtracting the mean of reference EHP of females cultured in control ATR-food from the EHP of individual test females. Female genotypes are: (A) pC1a,b,c>GtACR1 (pC1-S-Gal4/UAS-GtACR1), (B) pC1d,e>GtACR1 (pC1-A-Gal4/UAS-GtACR1), (C) pC1a>GtACR1 (pC1a-split-Gal4 /UAS-GtACR1), and (D) pC1b,c>GtACR1 (Dh44-pC1-Gal4/UAS-GtACR1). Gray circles indicate the ΔEHP of individual females, and the mean ± SEM of data is presented. Gray circles with dashed borders indicate ΔEHP values beyond the axis limits (>120 min). Mann-Whitney Test (n.s. p > 0.05; *p <0.05; ****p < 0.0001). Numbers below the horizontal bar represent the mean of EHP differences between treatments.
E, Relative CRE-Luciferase reporter activity of pC1 neurons in mated females of the indicated genotypes, incubated with a piece of filter paper perfumed with solvent vehicle control or the indicated odorants. To calculate the relative luciferase activity, we set the average luminescence unit values of female incubated with the vehicle to 100%. One-way ANOVA test (n.s. p > 0.05; ***p < 0.001; ****p < 0.0001). Gray circles indicate the relative luciferase activity (%) of individual females, and the mean ± SEM of data is presented.
F, Optogenetic production of cAMP in the pC1 b and c neurons shortens EHP, whereas the same treatment in pC1a or pC1d and e neurons does not. ΔEHP is calculated by subtracting the mean of reference EHP of females incubated in the control illumination (Dim light), which does not activate a photoactivatable adenylate cyclase (PhotoAC), from the EHP of individual test females. Mann-Whitney Test (n.s. p > 0.05, ****p < 0.0001).
G, Optogenetic production of cAMP increases excitability of pC1 neurons transiently. Left, schematic of the experimental procedure. Right, peak ΔF/F in the LPC projections of pC1 neurons from freshly mated females in response to the pheromone cVA, before and after photoactivation of PhotoAC expressed in pC1 neurons. Calcium response was measured at specific time points: after 1 minute (Blue dots and box, 1 TAA = 1 Time After Activation) or 10 minutes (Purple dots and box, 10 TAA) after activation. Repeated measures (RM) one-way ANOVA test with the Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test (*p < 0.05; ***p < 0.001; ****p < 0.0001).
H, Left, schematic of the experimental procedure. Right, re-mating rate of females during optogenetic cAMP production in pC1b and c, scored as the percentage of females that copulate with naive male within 6 h after end of first mating. Female genotypes are control (+/UAS-PhotoAC), pC1b,c>UAS-PhotoAC (Dh44-pC1-Gal4/UAS-PhotoAC). Chi-square test (*p <0.05).