ROLE OF THE SEROTONIN TRANSPORTER AND ORGANIC CATION TRANSPORTER 3 IN SEROTONERGIC MODULATION OF EMOTION-REGULATING CIRCUITRY

Dysfunctional serotonergic neuromodulation in mood-regulating circuits underlies many psychiatric diseases, thusunderstanding regulation of serotonin (5-HT) transmission is of fundamental importance. The 5-HT transporter(SERT) clears 5-HT from extracellular fluid with high-affinity, and is considered a primary controller of the strengthand duration of 5-HT signaling. Our studies have revealed that organic cation transporter 3 (OCT3), a low-affinity,but high-capacity transporter of monoamines, plays a critical role in 5-HT clearance as well. Though circuitsmodulating arousal and emotion are highly complex, processing within the basolateral amygdala (BLA) isconsidered essential, especially for fear conditioning. The BLA receives dense input from 5-HT neurons in dorsalraphe nucleus (DRN), and BLA principal neurons have numerous fear-regulatory outputs, including denseprojections to medial entorhinal cortex (mEC), which serves as a gateway for fear memory information flow intoand out of hippocampus. Like SERT, OCT3 is highly expressed in BLA, ideally positioning these transporters topowerfully control extracellular 5-HT and its local neuro-modulatory efficacy. Proposed studies test the hypothesisthat 5-HT clearance by OCT3 and SERT in BLA facilitates acquisition and consolidation of fear memory by bufferingthe rise of 5-HT that normally restrains BLA-mEC neuronal activation by excitatory fear memory-promoting limbicinputs. We posit that fear conditioning stimuli, which lead to fear memory, co-activate limbic and DRN 5-HT inputsto BLA along with activating the hypothalamic-pituitary-adrenal stress axis. OCT3 is potently inhibited bycorticosterone, indicating that diminished OCT3 clearance allows 5-HT to rise high enough during fear conditioningto activate 5-HT receptors and effectively buffer limbic excitation of BLA-mEC neurons, decreasing their output andreducing fear memory. We will use state-of-the-art conditional gene deletion strategies to separately andcollectively deplete SERT and OCT3 from DRN neurons, combined with optogenetic activation and inhibition ofDRN 5-HT neurons projecting directly to BLA. AAV shRNA will be used to knockdown SERT and/or OCT3 on allcell types in BLA. These approaches will be used to determine the relative contributions of SERT and OCT3 to 1)5-HT clearance in BLA in vivo using high-speed chronoamperometry; 2) 5-HT modulation of BLA-mEC neuronalactivity using in vivo single neuron and whole-cell patch clamp recording in brain slices; 3) fear conditioningbehavior. Because of their important roles in fear conditioning and 5-HT signaling in BLA, we will interrogate thefunctional contributions of 5-HT2A and 5-HT1A receptors in this circuit. Serotonergic neurotransmission potentlymodulates behavior, and its dysregulation is strongly implicated in psychiatric diseases. Proposed, discoverydriven, studies will provide unprecedented mechanistic insights into the role 5-HT, and its regulation by SERT andOCT3, play specifically within the DRN-BLA-mEC fear conditioning hub.