PROJECT SUMMARY/ABSTRACT Dysregulation of central monoamine (dopamine, noradrenaline, and serotonin) signaling is implicated widely in neuropsychiatric and substance use disorders, but there is a fundamental knowledge gap in how monoamines signal at the level of ion channels and individual synapses. It is well-established that serotonin-releasing neurons in the dorsal raphe nucleus are driven to fire action potentials and release serotonin by noradrenergic input and activation of metabotropic alpha1-adrenergic receptors. Recently, we discovered that alpha1-adrenergic receptors depolarize serotonin neurons via coupling to the ‘orphan’ ionotropic delta 1 glutamate receptor (GluD1R) ion channels. Intriguingly, prior research showed that metabotropic glutamate receptors also couple to GluD1R in midbrain dopamine neurons and contribute to burst firing of action potentials. The signaling mechanism between these metabotropic receptors and GluD1R ion channels requires G protein signaling but what occurs downstream to activate GluD1R is not yet known. In addition to this G protein-dependent GluD1R current, we found that GluD1R channels carry a G protein-independent tonic current that depolarizes the resting membrane potential to promote action potential firing. The etiology of the tonic GluD1R current is still unresolved. Together, these findings demonstrate that ionic current carried by GluD1R plays an unexpected and critical role in potentiating the excitability of monoamine neurons. We propose to use mouse brain slice electrophysiology, fluorescence imaging, and pharmacological manipulation to record GluD1R currents in serotonin neurons to (1) delineate the postsynaptic signal transduction mechanisms between alpha1-adrenergic receptors and GluD1R; and (2) determine whether GluD1R current is sensitive to naturally occurring cysteine-modifying agents like the small transition metal zinc and the antioxidant ascorbate, in a manner similar to NMDAR current. Lastly, (3) in midbrain dopamine neurons, we will determine the role of GluD1R in metabotropic receptor-dependent AMPAR synaptic plasticity in physiological conditions and after in vivo exposure to cocaine. We will evaluate the in vivo relevance of GluD1R current using a combination of imaging and behavioral assays in wild-type mice in comparison with mice that lack expression of GluD1R and mice that express a mutated GluD1R that is ‘pore- dead’ or non-conducting. The proposed studies are expected to be significant since they will identify key molecular regulators of serotonin and dopamine neuron excitability through action on GluD1R and the mechanism through which G protein-coupled receptors act upon GluD1R to modulate their ionic current. Our preliminary data suggest these studies may reveal a metabotropic receptor-initiated signal cascade that has not yet been observed in the central nervous system. Further, we anticipate gaining a significant understanding of the contribution of GluD1R current in met...