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

项目摘要 几十年来，一直需要改善抑郁症的药物治疗。尽管如此，新 治疗进展缓慢，或未能达到临床实践。也许最 有影响力的进展是发现亚麻醉氯胺酮可以迅速减轻治疗- 顽固性抑郁症虽然已知氯胺酮通过N-甲基-D-甲基-N- 天冬氨酸受体（NMDAR）拮抗作用，尚不清楚这是否是其抗抑郁机制。 行动上我们的实验室先前已经表明，氯胺酮迅速转化为各种代谢产物，至少 其中之一，（2 R，6 R）-羟基去甲氯胺酮（HNK），保留了 氯胺酮，但由于其抑制NMDAR的低亲和力而没有其副作用。我的数据显示，（2 R，6 R）- HNK促进α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPAR）的快速增强。 介导的突触传递，通过浓度依赖性，NMDAR非依赖性，和突触- 在Schaffer侧支（SC）-CA 1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，（2 R，6 R）-HNK的突触前效应需要 蛋白激酶活性和突触前钙通道内流。与此相一致的是，研究表明， (2R，6 R）-HNK导致细胞内环磷酸腺苷（cAMP）的快速积累， 与局部升高的钙调节蛋白激酶活性的作用一致。另夕h （2 R，6 R）-HNK的行为效应与突触前谷氨酸自分泌的下游机制一致， 抑制cAMP产生的受体，而cAMP触发脑源性神经营养素的释放， 因子（BDNF），其也是（2 R，6 R）-HNK发挥其行为效应所必需的。因此，我假设， (2R，6 R）-HNK通过cAMP依赖性， 突触前BDNF-TrkB信号传导。我将用三个具体的目标来检验这个假设， in vivo体内approaches方法.首先，我将使用急性切片电生理来确定是否突触前效应 （2 R，6 R）-HNK的活性依赖于cAMP，并与其下游靶蛋白激酶A（PKA）结合。使用 生物化学，我将验证后记录突触前cAMP的变化，以及磷酸化的变化， PKA下游的蛋白质，已知其积极参与谷氨酸释放。第二，我将测试的作用， （2 R，6 R）-HNK的突触前作用中的BDNF-TrkB信号传导，并评估TrkB中的磷酸化变化 以及突触前下游靶点。最后，我将使用体内纤维光度法来确定（2 R，6 R）-HNK是否 通过增加SC-CA 1处突触传递的强度，改善SC-CA 1依赖性任务中的学习 以BDNF依赖的方式形成突触。我的实验将测试突触前信号转导在 （2 R，6 R）-HNK急性作用机制，这将促进我们对突触前 可塑性引起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.