Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both "classical" and "modulatory" signals that could produce diverse and/or complementary effects in associated circuits. Here, we establish that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. Our findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network.

Within the extracellular matrix, matricellular proteins (MCPs) are dynamically expressed non-structural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The CCN (Cellular Communication Network Factors) family of MCPs serve regulatory roles to regulate cell function and are defined by their conserved multi-modular organization. Here we characterize the expression and neuronal requirement for the Drosophila CCN family member. Drosophila CCN (dCCN) is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. dCCN-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of dCCN results in reduced female fertility. In addition, dCCN accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila CCN family member will enhance our potential to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity.