Adhesion GPCR Cirl as a novel regulator of dopamine neurotransmission

PROJECT ABSTRACT/SUMARY Adhesion G-protein coupled receptors (GPCRs) are the second largest class of GPCRs yet their functions and ligands remain predominantly unidentified. Polymorphisms in the gene encoding the adhesion GPCR ADGRL3 have been associated with an increased risk for substance use disorder and attention deficit hyperactivity disorder in various linkage and association studies. Disrupting the function of the ADGRL3 homologs leads to hyperactivity in four different model systems ? zebrafish, fruit flies, mice, and rats. In addition to hyperactivity, ADGRL3 knockout mice have higher dopamine levels in forebrain motor regions as well as increased sensitivity to the stimulant cocaine, which acts on the dopamine transporter. Together with dopamine?s established role in mediating locomotion, these findings suggest that ADGRL3 contributes to behavior by modulating dopamine signaling; however, a mechanistic link has yet to be established. The goal of this proposed research is to investigate the synaptic mechanisms that underlie ADGRL3 function in dopaminergic circuits using Drosophila as a model system. I have replicated the hyperactive phenotype in fruit flies that carry a null mutation for the ADGRL3 homolog, Cirl. To directly assay the role of Cirl in neurotransmission, I activated dopamine neurons acutely and found that Cirl null flies were much more sensitive to dopamine neurotransmission. Intriguingly, activating dopamine neurons in Cirl null flies throughout development rescued Cirl null hyperactivity to control levels, indicating that tonic dopamine neurotransmission during development may compensate for lack of Cirl function. To test whether Cirl functions in dopamine neurons to modulate activity, I reintroduced Cirl expression in dopamine neurons in the Cirl null background and instead found that this exacerbated the hyperactive phenotype. Thus, the hyperactivity seen in Cirl null mutants is likely not mediated by its absence from dopamine neurons, but rather in a separate population of neurons in which Cirl normally acts to reduce activity. My imaging studies have revealed that Cirl is expressed post-synaptically throughout the brain, and additionally localizes to the central complex which is involved in locomotion and motor planning and receives dense dopaminergic input. This proposal aims to first identify the dopamine neuron or group of neurons that induces hyperactivity when acutely activated, and identify their synaptic contacts in the central complex. Once I have identified this circuit, I will use optogenetics, in combination with in vivo 2-photon imaging of fluorescent biosensors to delineate the pre and postsynaptic changes in this circuit responsible for the hyperactive response to dopamine neuron activation during adulthood, as well as the reversal of this phenotype in Cirl null flies subjected to continuous dopamine neuron activation throughout development. These results will delineate how an adhesion GPCR modulates dopamine neurotransmission in vivo, and identify a novel therapeutic target for disorders caused by dysregulation of dopaminergic activity such as substance use disorder.