The roles of a unique G-protein coupled dual receptor for dopamine and steroids in neuronal physiology and behavior

Steroid hormones are known to have significant effects on a wide variety of biological processes. In particular, they serve as critical modulators of neural function and behavior and play critical roles in stress responses and neurologic disorders. Until recently the biological actions of steroid hormones were believed to operate primarily through activation of cognate nuclear hormone receptors or the allosteric modulation of ion channels (Majewskaet al., 1986). However, new signaling pathways involving G-protein coupled receptors (GPCRs) for steroid hormones have been recently identified in multiple different species, implicating steroid hormones in direct fast modulation of intracellular signaling and in turn behavior (Thomas et al., 2006, Gabor et al., 2015). In mammals G protein-coupled estrogen receptor 1 (GPER), also known as G protein-coupled receptor 30 (GPR30), is expressed throughout the body including in the nervous system and has been suggested to play a variety of roles in health and behavior (Prossnitz and Barton, 2011). Despite recent progress in this area from studies using rodent models, the mechanisms underlying "non-genomic” actions of steroids remain largely elusive. This gap in our understanding presents a significant scientific and clinical challenge to a comprehensive view of the role of steroid hormones in regulating both neural function, behavior and overall health of the organism. To understand the mechanisms for this unconventional steroid signaling we sought to use a simpler system to explore the functions of GPCR’s for steroid hormones.

In 2005, Peter Evans’s group identified DopEcR, a unique GPCR in Drosophila melanogaster, which responds to ecdysone—the major steroid hormone in insects (Srivastava et al. 2005). This unconventional GPCR for steroid hormones is particularly interesting because it is a dual receptor that also responds to a structurally dissimilar compound, dopamine. DopEcR is preferentially expressed in the nervous system and has recently been implicated in modulating multiple behaviors including starvation-induced enhancement of sugar sensitivity (Inagaki et al., 2012), experience-dependent courtship suppression, habituation of the giant fiber pathway (Ishimoto et al., 2013) and ethanol-induced sedation (Petruccelli et al. 2016) in flies. DopEcR also plays a role in perception of sex pheromones in moths (Abrieux et al., 2013). More recently the mammalian GPCR for estrogen GPER has also been found to bind dopamine indicating that this unique attribute may be more prevalent among these novel GPCRs for steroids (Evans et al. 2013). Despite these previous findings, we still know little about how GPCRs for steroids modulate neurons at the cellular level and how they modulate behaviors.

Therefore we sought to forge a more comprehensive understanding of the function of steroid signaling by characterizing DopEcR function in neuronal and behavioral modulation through GPCR’s. To characterize DopEcR’s function we looked at the consequences of DopEcR signaling at three levels: behavior, neuronal morphology and finally physiology. Because changes steroid hormones levels are often associated with environmental stressors we assayed the role of DopEcR in a stress related behavior: starvation-induced sleep suppression and hyperactivity. To look at DopEcR’s role in neuronal physiology we used bioluminescent calcium imaging to measure its effect on the stimulated calcium response in a brain structure critical for behavior. Finally we used principal clock neurons in the brain (PDF+ l-LNv neurons) as a model to examine DopEcR’s role in modulating plasticity and neuronal structure.

In our present work described in Chapter 2, we found that the D1-like receptor, DopR1, modulates sleep and activity independent of starvation while DopEcR plays a role in mediating starvation-induced sleep suppression and enhanced activity. We found that knocking down EGFR in a DopEcR mutant background restored starvation induced changes in behavior, suggesting that DopEcR normally suppresses EGFR signaling to suppress sleep under starvation.

In Chapter 4, we show that the nicotine-induced Ca2+-response was selectively enhanced in the medial lobes either in DopEcR mutant or in flies with DopEcR selectively knocked down within the MBs. Using a pharmacological approach, we show that the endogenous ligands of DopEcR mediated two different responses in the MBs: the steroid ligand ecdysone enhances activity in the calyx and cell body region, whereas monoaminergic ligand dopamine reduced activity in the medial lobes. In Chapter 5, we find that reducing DopEcR in PDF neurons results in reduced basal levels of bouton numbers. The reduction in bouton number is independent of cAMP signaling but instead relies on inhibition of EGFR signaling. Signifying that DopEcR may modulate EGFR associated signaling to make changes in the in the brain.

These results demonstrate that DopEcR is able to modulate neuronal excitability, physical structure of neurons and the behavior of the organism. Interestingly it also indicates that DopEcR’s different ligands, dopamine and ecdysone, may have unique and spatially distinct effects on different brain structures or within the same structure. Overall, this study provides a solid foundation for understanding the roles and action mechanisms of GPCR-mediated steroid signaling in regulation of neural development, physiology and behavior.