Monoamine neurotransmitters such as noradrenalin are released from both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), the latter mediating extrasynaptic signaling. The contribution of synaptic versus extrasynaptic signaling to circuit function and behavior remains poorly understood. To address this question, we have previously used transgenes encoding a mutation in the Drosophila Vesicular Monoamine Transporter (dVMAT) that shifts amine release from SVs to LDCVs. To circumvent the use of transgenes with non-endogenous patterns of expression, we have now used CRISPR-Cas9 to generate a trafficking mutant in the endogenous dVMAT gene. To minimize disruption of the dVMAT coding sequence and a nearby RNA splice site, we precisely introduced a point mutation using single-stranded oligonucleotide repair. A predicted decrease in fertility was used as a phenotypic screen to identify founders in lieu of a visible marker. Phenotypic analysis revealed a defect in the ovulation of mature follicles and egg retention in the ovaries. We did not detect defects in the contraction of lateral oviducts following optogenetic stimulation of octopaminergic neurons. Our findings suggest that release of mature eggs from the ovary is disrupted by changing the balance of VMAT trafficking between SVs and LDCVs. Further experiments using this model will help determine the mechanisms that sensitize specific circuits to changes in synaptic versus extrasynaptic signaling.

Octopamine is essential for egg-laying in Drosophila melanogaster, but the neuronal pathways and receptors by which it regulates visceral muscles in the reproductive tract are not known. We find that the two octopamine receptors that have been previously implicated in egg-laying-OAMB and Octβ2R-are expressed in octopaminergic and glutamatergic neurons that project to the reproductive tract, peripheral ppk(+) neurons within the reproductive tract and epithelial cells that line the lumen of the oviducts. Further optogenetic and mutational analyses indicate that octopamine regulates both oviduct contraction and relaxation via Octβ2 and OAMB respectively. Interactions with glutamatergic pathways modify the effects of octopamine. Octopaminergic activation of Octβ2R on glutamatergic processes provides a possible mechanism by which octopamine initiates lateral oviduct contractions. We speculate that aminergic pathways in the oviposition circuit may be comparable to some of the mechanisms that regulate visceral muscle contractility in mammals.