In this study, we isolated the sexually dimorphic behavioral effects of T1 neuron activation on male and female Drosophila courtship behavior. Using a dominant-active lush allele, lushD118A, we were able to increase the basal activity of the T1 neurons two-fold in the absence of cVA. This increased firing rate was sufficient to reproduce sexually dimorphic behaviors that normally occur in the presence of cVA. Furthermore, the alterations in both male and female behavior due to lushD118A and cVA were both abrogated by loss of the downstream receptor Or67d. These findings indicate that activity in the T1 circuit alone is necessary and sufficient to mediate the effects of cVA on courtship behaviors.
Introduction of recombinant LUSHD118A
into the sensillum lymph of T1 sensilla can stimulate T1 neurons from 1 spike/s to 15 spikes/s (Laughlin, 2008
). Transgenic expression of LUSHD118A
increased the basal firing rate of the T1 neurons approximately two-fold in the transgenic animals, while there was no increase in the average T2 neuron firing rates. Thus, LUSHD118A
specifically activates T1 neurons when expressed as a transgene regulated by the lush
promoter. We suspect this modest increase in firing compared to direct infusion of recombinant protein may reflect desensitization of the T1 neurons due to chronic LUSHD118A
stimulation or perhaps a structural difference when LUSHD118A
is expressed through the support cell secretory pathway. Indeed, it appears that LUSHD118A
expressed in the fly is cVA-sensitive (Supplemental Figure S1
) but not when recombinant bacterially-expressed protein is infused via the recording pipette (Laughlin, 2008
). Based on our observation of courtship behavior, this further increase in T1 firing rate by exogenous cVA has no additional effect on courtship behaviors of lushD118A
transgenic flies in which this circuit is already activated (). It is known from the work of Schlief and Wilson (2007)
that even small increases in T1 firing rate can induce large increases in the frequency of downstream projection neuron firing (Schlief and Wilson, 2007
). Indeed, the robust effects on courtship behavior in the LUSHD118A
lines support the idea that the T1 circuit is activated in these animals. This is consistent with cVA acting as an inhibitor of mating in males, to avoid unproductive courtship with other males or possibly recently mated females, but as an aphrodisiac in females when they are being courted by a cVA-producing male.
There has been recent controversy over whether cVA-induced mating behaviors are mediated by Or67d, Or65a, or both (Kurtovic, 2007
; Ejima 2007
). Our data supports the notion that cVA acts exclusively through T1 neurons expressing Or67d, thus affecting courtship behavior. This is based on several observations. First, transgenic lushD118A
specifically increases T1 activity and induces cVA-related behaviors. No effect was observed in T2 neurons. Secondly, the effects of lushD118A
and cVA are both blocked by loss of Or67d, indicating these behaviors are elicited through this pathway. Finally, in the presence of LUSHD118A
, cVA has no additional effects on behavior in these engineered flies. Together, these findings present a compelling argument for Or67d providing the major, if not exclusive, sensory pathway mediating cVA behaviors. Our findings do not support those of Ejima et al. (2007)
, where tetanus toxin expressed under the Or65a promoter blocked cVA-induced suppression of naive male courtship, while expression under the Or67d promoter had no effect (Ejima, 2007
). It is possible that the ectopic tetanus toxin expression they noted in these lines produced additional effects on courtship behavior (Ejima, 2007
). Furthermore, activation of Or65a by cVA requires ligand concentrations far above the physiologically relevant range (van der Goes van Naters and Carlson, 2007
; Laughlin, 2008
), suggesting that cVA may not be a physiologic ligand for Or65a. Indeed, cVA was recently shown to mediate male-male aggression, and these behaviors also appear to require Or67d but not Or65a (Wang, 2009
). However, given our findings, we have no explanation for why tetanus toxin expressed by the Or67d promoter-Gal4 driver failed to inhibit male courtship (Ejima, 2007
). Analysis of Or65a mutants in the future may shed light into these issues.
Our results confirm and extend the findings of Kurtovic et al. (2007)
, that demonstrated increased male-male courtship in males lacking Or67d, and increased latency to mate in females lacking this receptor (Kurtovic, 2007
). These studies revealed that Or67d is important for sexually dimorphic, cVA-related courtship behaviors, but did not address whether activity in Or67d neurons alone accounts for all of these behaviors. Here we show that activating the T1 pathway with LUSHD118A
has the opposite effects on behavior compared to the loss of function mutation in Or67d
. Furthermore, we show that activation of T1 neurons enhances female receptivity to courting males. We conclude that the T1/Or67d circuit is not only necessary but is sufficient for these behaviors.
Finally, it should be possible to identify sexual dimorphism in the downstream neurons of the T1 circuit. Indeed, the exclusive target of Or67d sensory neurons is the DA1 glomerulus (Couto, 2005
). Datta et al. recently discovered sexually dimorphic branches in the projection neurons that transmit activity in this glomerulus to higher brain centers (Datta, 2008
). It remains to be seen if these differences are truly responsible for the differences in behavior observed in male and female fruit flies. However, with the powerful genetic tools available in Drosophila (Pfeiffer, 2008
), it should be possible to elucidate the complete neuronal map of this cVA-activated circuit.