Role of the serotonin transporter and organic cation transporter 3 in serotonergic modulation of emotion-regulating circuitry

情绪调节回路中的多巴胺能神经调节功能障碍是许多精神疾病的基础， 了解5-羟色胺（5-HT）传递的调节是非常重要的。5-HT转运蛋白 SERT以高亲和力从细胞外液中清除5-HT，被认为是细胞内5-HT强度的主要控制者。 和5-HT信号传导的持续时间。我们的研究表明，有机阳离子转运蛋白3（OCT 3），一种低亲和力， 而且是单胺的高容量转运体，在5-HT清除中也起关键作用。虽然电路 调节唤醒和情绪是高度复杂的，在基底外侧杏仁核（BLA）内的处理是 被认为是必不可少的，特别是对于恐惧条件反射。BLA接受来自背侧5-HT神经元的密集输入 中缝核（DRN）和BLA主神经元有许多恐惧调节输出，包括密集的 内侧内嗅皮层（mEC）是恐惧记忆信息流入的通道。 和海马体。与SERT一样，OCT 3在BLA中高度表达，理想地将这些转运蛋白定位于 有力地控制细胞外5-HT及其局部神经调节功效。拟议中的研究验证了这一假设 BLA中OCT 3和SERT对5-HT的清除通过缓冲作用促进恐惧记忆的获得和巩固， 5-HT的升高通常通过兴奋性恐惧记忆促进边缘系统抑制BLA-mEC神经元活化， 输入。我们认为导致恐惧记忆的恐惧条件刺激共同激活边缘系统和DRN的5-HT输入 沿着激活下丘脑-垂体-肾上腺应激轴。OCT 3被以下物质有效抑制： 皮质酮，表明减少OCT 3清除允许5-HT在恐惧条件反射过程中升高到足够高的水平 激活5-HT受体并有效缓冲BLA-mEC神经元的边缘兴奋，降低其输出， 减少恐惧记忆。我们将使用最先进的条件性基因删除策略来单独和 从DRN神经元共同消耗SERT和OCT 3，结合光遗传学激活和抑制 DRN 5-HT神经元直接投射到BLA。AAV shRNA将用于敲低所有细胞上的SERT和/或OCT 3。 BLA中的细胞类型。这些方法将用于确定SERT和OCT 3对1） 使用高速计时电流法的体内BLA中的5-HT清除; 2）BLA-mEC神经元的5-HT调节 活动使用在体单神经元和全细胞膜片钳记录在脑切片; 3）恐惧条件反射 行为由于它们在BLA的恐惧条件反射和5-HT信号传导中的重要作用，我们将询问 5-HT 2A和5-HT 1A受体在该回路中的功能贡献。5-羟色胺能神经传递 调节行为，其失调与精神疾病密切相关。提议，发现 驱动，研究将提供前所未有的机制洞察的作用5-HT，其调控SERT和 OCT 3，在DRN-BLA-mEC恐惧调节中枢中发挥作用。

Dysfunctional serotonergic neuromodulation in mood-regulating circuits underlies many psychiatric diseases, thus understanding 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 strength and 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 circuits modulating arousal and emotion are highly complex, processing within the basolateral amygdala (BLA) is considered essential, especially for fear conditioning. The BLA receives dense input from 5-HT neurons in dorsal raphe nucleus (DRN), and BLA principal neurons have numerous fear-regulatory outputs, including dense projections to medial entorhinal cortex (mEC), which serves as a gateway for fear memory information flow into and out of hippocampus. Like SERT, OCT3 is highly expressed in BLA, ideally positioning these transporters to powerfully control extracellular 5-HT and its local neuro-modulatory efficacy. Proposed studies test the hypothesis that 5-HT clearance by OCT3 and SERT in BLA facilitates acquisition and consolidation of fear memory by buffering the rise of 5-HT that normally restrains BLA-mEC neuronal activation by excitatory fear memory-promoting limbic inputs. We posit that fear conditioning stimuli, which lead to fear memory, co-activate limbic and DRN 5-HT inputs to BLA along with activating the hypothalamic-pituitary-adrenal stress axis. OCT3 is potently inhibited by corticosterone, indicating that diminished OCT3 clearance allows 5-HT to rise high enough during fear conditioning to activate 5-HT receptors and effectively buffer limbic excitation of BLA-mEC neurons, decreasing their output and reducing fear memory. We will use state-of-the-art conditional gene deletion strategies to separately and collectively deplete SERT and OCT3 from DRN neurons, combined with optogenetic activation and inhibition of DRN 5-HT neurons projecting directly to BLA. AAV shRNA will be used to knockdown SERT and/or OCT3 on all cell 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 neuronal activity using in vivo single neuron and whole-cell patch clamp recording in brain slices; 3) fear conditioning behavior. Because of their important roles in fear conditioning and 5-HT signaling in BLA, we will interrogate the functional contributions of 5-HT2A and 5-HT1A receptors in this circuit. Serotonergic neurotransmission potently modulates behavior, and its dysregulation is strongly implicated in psychiatric diseases. Proposed, discovery driven, studies will provide unprecedented mechanistic insights into the role 5-HT, and its regulation by SERT and OCT3, play specifically within the DRN-BLA-mEC fear conditioning hub.