Activation of the T1 neuronal circuit is necessary and sufficient to induce sexually dimorphic mating behavior in Drosophila melanogaster

Abstract

The molecular and cellular events mediating complex behaviors in animals are largely unknown. Elucidating the circuits underlying behaviors in simple model systems may shed light on how these circuits function. In drosophila, courtship behavior provides a tractable model for studying the underlying basis of innate behavior. The male-specific pheromone 11-cis-vaccenyl acetate (cVA) modulates courtship behavior and is detected by T1 neurons, located on the antenna of male and female flies. The T1 neurons express the odorant receptor Or67d and are exquisitely tuned to cVA pheromone. However, cVA-induced changes in mating behavior have also been reported upon manipulation of olfactory neurons expressing odorant receptor Or65a. These findings raise the issue of whether multiple olfactory-driven circuits underlie cVA-induced behavioral responses and what role these circuits play in behavior. Here, we engineered flies in which the Or67d circuit is specifically activated in the absence of cVA to determine the role of this circuit in behavior. We created transgenic flies that express a dominant-active, pheromone-independent variant of the extracellular pheromone receptor, LUSH. We found that, similar to the behaviors elicited by cVA, engineered male flies have dramatically reduced courtship, whereas engineered females showed enhanced courtship. cVA exposure did not enhance the dominant LUSH-triggered effects on behavior in the engineered flies. Finally, we show the effects of both cVA and dominant LUSH on courtship are reversed by genetically removing Or67d. These findings demonstrate that the T1/Or67d circuit is necessary and sufficient to mediate sexually dimorphic courtship behaviors.

Figure 1.

lush^D118A is expressed at wild-type levels and in correct spatial pattern. Top, Western blot of LUSH and LUSH^D118A protein indicates that LUSH^D118A is produced at levels similar to wild-type LUSH. α-Tubulin (α-tub) serves as a loading control. Bottom, Frozen tissue sections from wild-type (w¹¹¹⁸), lush¹ mutants, and lush¹ mutants expressing the lush^D118A transgene indicate LUSH^D118A is expressed and secreted properly.

Figure 2.

T1 neural activation is necessary and sufficient to induce sexually dimorphic courtship behavior. A, Representative traces obtained from single sensillum electrophysiological recordings of T1 neurons of wild-type (w¹¹¹⁸), lush^D118A transgenic flies, or flies expressing lush^D118A in an Or67d mutant background (lush^D118A;Or67d^GAL4). B, Quantitation of spontaneous activity in T1 neurons from flies of genotypes described above. C, Male courtship index of wild-type, lush^D118A, or lush^D118A;Or67d^GAL4 flies. (1) and (2) indicate two transgenic lines generated by independent p-element insertions. Males of the indicated genotypes were crossed to control (w¹¹¹⁸) females. D, Female courtship index of wild-type, lush^D118A, or lush^D118A;Or67d^GAL4 flies. Females of the indicated genotypes were crossed to control (w¹¹¹⁸) males. Error bars represent SEM. Significant differences from the wild-type controls calculated using Student's t test. For all bars, n ≥ 7. *p < 0.05, **p < 0.01.

Figure 3.

Sexually dimorphic cVA-triggered mating behaviors are mediated specifically by T1 neurons. cVA exposure suppresses male courtship behavior in wild-type (wt) males to a level indistinguishable from lush^D118A in the absence of cVA (p > 0.1). cVA exposure has no further suppression of courtship by lush^D118A males (p > 0.1). cVA fails to suppress courtship of Or67d^GAL4 mutant males and, when crossed to wild-type females, courtship index is enhanced due to the aphrodisiac effect of cVA on the female. This enhancement by cVA is lost when both males and females lack Or67d. Error bars represent SEM. Significant differences from the wild-type controls calculated using Student's t test. For all bars, n ≥ 6. ns, Not significant. *p < 0.05, **p < 0.01.