Synaptic mechanisms underlying the rapid antidepressant properties of the (2R,6R)-hydroxynorketamine metabolite

项目总结 几十年来，一直需要改进抑郁症的药物治疗。尽管如此，新的 治疗进展缓慢，或未能达到临床实践。也许是最多的 一项有影响力的进展，是发现亚麻醉剂氯胺酮可以迅速缓解治疗- 抵抗力强的重度抑郁症。虽然已知氯胺酮的麻醉作用是通过N-甲基-D-氨基-D-天冬氨酸来实现的。 天冬氨酸受体(NMDAR)拮抗作用，目前尚不清楚这是否是其抗抑郁机制 行动。我们的实验室之前已经证明，氯胺酮可以迅速转化为各种代谢物，至少 其中一种，(2R，6R)-羟基去甲氯胺酮(HNK)，保留了快速抗抑郁药样临床前特性 氯胺酮，但由于其抑制NMDAR的亲和力较低，因此没有副作用。我的数据显示(2R，6R)- HNK促进α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic酸受体(AMPAR)的快速增强- 介导的突触传递，通过浓度依赖、NMDAR非依赖和突触- Schaffer侧支(SC)-CA1突触处谷氨酸释放概率的选择性增加(Riggs等，2019， 神经精神药理学)。我的初步数据表明，(2R，6R)-HNK的突触前效应需要 蛋白激酶活性和突触前钙通道内流。与此一致的是，研究表明 (2R，6R)-HNK导致细胞内环磷酸腺苷(CAMP)的快速蓄积， 与局部升高的钙作用汇聚，调节蛋白激酶活性。此外， (2R，6R)-HNK的行为效应与突触前谷氨酸自身下游的一种机制汇聚 抑制cAMP产生的受体，而cAMP则触发脑源性神经营养物质的释放 (2R，6R)-HNK发挥其行为效应所必需的BDNF。因此，我假设 (2R，6R)-HNK通过cAMP依赖的急剧增加而发挥其快速突触增强作用， 突触前BDNF-TrkB信号传导。我将用三个具体的目标来验证这一假设，分别使用体外和 在活体内接近。首先，我将使用急性脑片电生理学来确定突触前效应 (2R，6R)-HNK是cAMP依赖的，并与其下游靶蛋白蛋白激酶A(PKA)结合。vbl.使用 生物化学，我将验证记录后突触前cAMP的变化，以及在 PKA下游的蛋白质，已知积极参与谷氨酸的释放。第二，我要测试一下 BDNF-TrkB在(2R，6R)-HNK突触前作用中的信号转导，并评估TrkB的磷酸化变化 以及它的突触前下游靶点。最后，我将使用体内纤维光度法来确定(2R，6R)-HNK 通过增加SC-CA1区突触传递的强度来改善SC-CA1依赖任务中的学习 以脑源性神经营养因子依赖的方式突触。我的实验将测试突触前信号转导在 (2R，6R)-HNK的急性作用机制将促进我们对突触前如何 可塑性导致突触传递和行为的持续适应。

PROJECT SUMMARY There has been a need to improve depression pharmacotherapies for several decades. Despite this, new treatment advances have been slow to progress, or have failed to reach clinical practice. Perhaps the most impactful advance, has been the discovery that subanesthetic ketamine can rapidly alleviate treatment- resistant major depression. While it is known that ketamine exerts its anesthetic effects through N-methyl-D- aspartate receptor (NMDAR) antagonism, it is unclear as to whether this is its antidepressant mechanism of action. Our lab has previously shown that ketamine is rapidly converted into various metabolites, and at least one of these, (2R,6R)-hydroxynorketamine (HNK), retains the rapid antidepressant-like preclinical properties of ketamine, but lacks its adverse effects given its low affinity to inhibit the NMDAR. My data reveal that (2R,6R)- HNK promotes a rapid potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)- mediated synaptic transmission, through a concentration-dependent, NMDAR-independent, and synapse- selective increase in glutamate release probability at Schaffer collateral (SC)-CA1 synapses (Riggs et al., 2019, Neuropsychopharmacology). My preliminary data suggest that the presynaptic effects of (2R,6R)-HNK require protein kinase activity, and presynaptic calcium channel influx. Consistent with this, studies have shown that (2R,6R)-HNK leads to a rapid accumulation of intracellular cyclic adenosine monophosphate (cAMP), which converges with the actions of locally elevated calcium to regulate protein kinase activity. Additionally, the behavioral effects of (2R,6R)-HNK converge with a mechanism downstream of presynaptic glutamate auto- receptors that inhibit the production of cAMP, whereas cAMP triggers the release of brain-derived neurotrophic factor (BDNF), which is also required for (2R,6R)-HNK to exert its behavioral effects. Thus, I hypothesize that (2R,6R)-HNK exerts its rapid synaptic potentiation through an acute increase in cAMP-dependent, presynaptic BDNF-TrkB signaling. I will test this hypothesis with three specific aims, using both ex vivo and in vivo approaches. First, I will use acute slice electrophysiology to determine whether the presynaptic effects of (2R,6R)-HNK are cAMP-dependent, and engages its downstream target, protein kinase A (PKA). Using biochemistry, I will verify changes in post-recording presynaptic cAMP, as well as phosphorylation changes in proteins downstream of PKA, known to actively participate in glutamate release. Second, I will test the role of BDNF-TrkB signaling in the presynaptic actions of (2R,6R)-HNK, and assess phosphorylation changes in TrkB and its presynaptic downstream targets. Lastly, I will use in vivo fiber photometry to determine if (2R,6R)-HNK improves learning in a SC-CA1-dependent task by increasing the strength of synaptic transmission at SC-CA1 synapses in a BDNF-dependent manner. My experiments will test the role of presynaptic signal transduction in the acute mechanism of action of (2R,6R)-HNK, which will advance our understanding of how presynaptic plasticity gives rise to sustained adaptations in synaptic transmission and behavior.