Labeling of Cocaine-generated Nascent Excitatory Synapses

描述（由申请人提供）：药物成瘾可以被定义为一种极端形式的记忆，对于从未经历过药物滥用的人来说，这种记忆明显是持久的，而且是全新的。鉴于先前存在的突触/神经回路的改变已被广泛接受为形成记忆的细胞机制，我们假设药物成瘾作为一种新的、极其强大的记忆形式，不仅通过对现有突触的改变，还通过形成新的突触连接和新的神经回路来介导。这一假设虽然看起来很惊人，但与几条线索的证据是一致的，这些证据表明，在接触可卡因或其他令人上瘾的精神兴奋剂后，伏隔核（NAc）中形成了新的树突棘和过早的兴奋性突触连接，伏隔核是大脑中产生药物成瘾的关键区域。为了直接测量暴露于可卡因诱导的潜在的新突触连接/神经回路，我们建议采用并开发一种创新的成像技术，GRASP （GFP重构跨突触伙伴）技术，该技术可以标记体内新形成的突触连接。我们的策略是将GFP分成两半，一部分在潜在的突触前末端表达，另一部分在潜在的突触后末端表达。这两半不单独发出荧光，但当突触前和突触后末端相互作用形成新突触时，它们将重新构成荧光GFP。我们初步研究的概念验证数据表明，这种实验策略非常有前途。在这个应用中，我们的第一个目标是优化GRASP技术，使其可以很容易地用于新的兴奋性突触的体内标记。我们的第二个目标是利用这项技术来描述可卡因暴露后对NAc的潜在新神经投射。使用基于氟金的追踪方法，我们的初步数据显示，暴露于可卡因后，NAc收到来自外侧缰的新的、强烈的神经支配，这在盐水处理的对照动物中没有观察到。因此，拟议的工作不仅将开发一种体内GRASP技术，而且还将使用这种技术来解决一个重要的神经科学问题。因此，该提案与CEBRA应用的使命是一致的，该提案的结果将为该领域提供一种新的体内技术和潜在的新的基于电路的可卡因成瘾机制。

DESCRIPTION (provided by applicant): Drug addiction can be conceptualized as an extreme form of memory, a memory that is distinctly long-lasting and completely new for individuals who have never experienced drugs of abuse. Whereas modification of pre- existing synapses/neural circuits has been widely accepted as a cellular mechanism for forming memories, we hypothesize that drug addiction as a new and extremely robust form of memory is mediated not only by modification of the existing synapses, but also by the formation of new synaptic connections and thus new neural circuits. This hypothesis, although seemingly striking, is consistent with several lines of evidence showing that following exposure to cocaine or other addictive psychostimulants, new dendritic spines and premature excitatory synaptic connections are formed in the nucleus accumbens (NAc), a critical brain region for the development of drug addiction. To directly measure the potential new synaptic connections/neural circuits induced by exposure to cocaine, we propose to adapt and develop an innovative imaging technique, the GRASP (GFP reconstitution across synaptic partners) technique, which can label newly formed synaptic connections in vivo. Our strategy is to split GFP into two halves, one expressed in potential presynaptic terminals and the other in potential postsynaptic terminals. These two halves do not fluorescence individually but will reconstitute into a fluorescent GFP when the pre- and post-synaptic terminals interact to form new synapses. The proof-of-concept data from our preliminary studies suggest that this experimental strategy is highly promising. In this application, our first objective is to optimize the GRASP technique such that it can be readily used for in vivo labeling of new excitatory synapses. Our second objective is to use this technique to characterize a potential new neural projection to the NAc following cocaine exposure. Using a Fluoragold- based tracing approach, our preliminary data showed that following exposure to cocaine, the NAc received new, intense innervations from the lateral habenula, a projection that was not observed in saline-treated control animals. Thus, the proposed work will not only develop an in vivo GRASP technique, but also use this technique to address an important neuroscience question. As such, this proposal is consistent with the mission of CEBRA application, and the outcome of this proposal will provide the field with a novel in vivo technique and a potentially novel circuitry-based mechanism underlying cocaine addiction. PUBLIC HEALTH RELEVANCE: This application will optimize a novel investigating technique that can be used to characterize newly formed synaptic connections upon in vivo experience. With this technique, we will also determine a potential newly formed neural circuit within the nucleus accumbens upon exposure to cocaine. The proposed work will not only develop/optimize an innovative in vivo technique, but also open a new avenue toward understanding circuitry- based mechanisms for drug addiction.