Analyzing PKA-dependent synaptic plasticity in the hippocampal mossy fiber synapse using novel optogenetic tools

突触可塑性在大脑活动中起着重要作用，被认为是学习和记忆的主要细胞过程之一。虽然在许多突触中，长时程增强是由突触后机制介导的，但一些突触，尤其是海马苔藓纤维(MF)、小脑平行纤维和果蝇神经肌肉连接突触，表现为突触前可塑性，表现为突触小泡释放概率的变化。有趣的是，这种形式的突触前可塑性依赖于上述所有突触中PKA的突触前激活。这种依赖PKA的突触可塑性背后的分子机制仍然很大程度上是未知的。目前，对PKA激活水平的控制仅限于腺酰环化酶激动剂，如Forsklin(FSK)或代谢型谷氨酸受体激动剂。这些治疗既影响突触前和突触后神经元，也影响非神经细胞群体，它们的作用不仅限于突触，而且通常是不可逆的，这使得这些药物不太适合研究突触特异性的PKA激活效应。有鉴于此，在可遗传识别的细胞子集的突触中操纵PKA的能力对于研究突触前PKA依赖的机制非常有用。因此，目前的项目目标是：a)开发、优化和表征以突触特异的方式控制PKA激活水平的新型光遗传学工具；b)研究这些新型光遗传学工具对MF突触中突触传递的影响；c)检查局部突触前或突触后的PKA激活是否足以增强突触传递；d)利用超分辨显微镜阐明PKA激活后AZ机制的分子结构的变化；以及e)研究PKA下游靶点对突触增强的参与。我们将联合起来，利用Nagel教授实验室在开发新型光激活PKA工具方面的专业知识，并将其与Ashery教授实验室开发的遗传和生理方法相结合，仅针对和激活MF中的PKA，并研究其对突触可塑性和分子机制的影响。

Synaptic plasticity plays an important role in brain activity and is thought to be one of the main cellular processes underlying learning and memory. While in many synapses long-term potentiation is mediated by post-synaptic mechanisms, several synapses, most notably the hippocampal mossy fiber (MF), the cerebellar parallel fiber and Drosophila neuromuscular-junction synapses, exhibit presynaptic plasticity, which manifests as changes in the probability of synaptic vesicle release. Interestingly, this form of pre-synaptic plasticity depends on presynaptic activation of PKA in all the aforementioned synapses. The molecular mechanism underlying this PKA-dependent synaptic plasticity is still largely unknown. Currently, the control over PKA-activation levels is limited to adenylyl cyclase agonists, such as forskolin (FSK), or metabotropic glutamate receptor agonists. These treatments affect both pre- and post-synaptic neurons, as well as non-neuronal cell populations, their effect is not restricted to the synapse and is usually irreversible, facts that make these pharmacological agents less-than-ideal candidates to study synapse-specific effects of PKA activation. In light of this, the ability to manipulate PKA in the synapses of a genetically-identifiable subset of cells is highly instrumental in studying presynaptic PKA-dependent mechanisms. Accordingly, the current project aims are; a) develop, optimize and characterize novel optogenetic tools to control the level of PKA-activation in a synapse-specific manner b) investigate the effects of these novel optogenetic tools on synaptic transmission in the MF synapse, c) Examine if local pre- or post-synaptic PKA activation is sufficient to potentiate synaptic transmission, d) elucidate changes in the molecular organization of the AZ machinery following PKA activation, using super resolution microscopy and e) investigate the involvement of PKA downstream targets on synaptic potentiation. We will join forces, capitalizing on the expertise in the lab of Prof. Nagel in developing novel light activated PKA tools and combining this with the genetic and physiological approaches developed in Prof. Ashery’s lab to target and activate PKA only in MF and examine its effect on synaptic plasticity and the molecular mechanisms.