Changes in synaptic function and structure associated with chronic cocaine

为了解决上述具体问题，我们列出了五个项目和一个简短的实验设计总结。 项目1：慢性可卡因治疗如何影响突触功能和形态？ 野生型和转基因小鼠将在5-30天内每天接受被动可卡因/生理盐水注射(IP)。这之后将进行行为测试：每天用注射测试运动活动和位置偏好。然后，我们将准备急性脑片/脑片培养，并进行体外程序 不同的技术和体外程序将被应用于确定上瘾动物大脑中发生的变化。我们将调查： -形态变化：如脊椎数量、大小、运动性和树突分支的变化。 -突触强度、突触可塑性和调制的变化。 -突触蛋白表达的生化变化，如谷氨酸AMPA受体、NMDA受体、多巴胺受体和信号分子。 -棘突和树突细胞内钙信号的变化 项目2：表现出高冲动行为的动物在长期接触可卡因时表现出更易成为强迫性毒品寻求者的脆弱性(Belin等人，2008年科学320：1352-1355)。我们将比较表现出高和低冲动的小鼠大脑不同区域的突触功能和神经元结构。 野生型和转基因小鼠每天接受行为测试(5选择系列反应时间任务)，持续30-40天，然后进行体外实验。不同的技术将被用来识别高度冲动的神经元标记动物。我们将调查： -形态：棘突数目、大小、活动度和树突分支。 -生物化学：突触蛋白和信号分子的表达水平。 -棘突和树突中的钙信号 -突触强度和可塑性 项目3：只有一小部分可卡因吸毒者成为强迫症吸毒者。与那些暴露但没有上瘾的人相比，暴露并上瘾的受试者的大脑有什么不同？ 在长期接触可卡因后，表现出类似成瘾行为的小鼠和没有表现出类似上瘾行为的小鼠在突触功能和形态上是否存在差异？ 野生型小鼠和转基因小鼠将接受为期30天的可卡因/盐水自我注射，然后在最后一次暴露1、10或30天后进行行为测试，以确定成瘾评分。然后，我们将在这些小鼠身上进行体外程序。将按照项目1和2所述进行电生理、形态和生化分析。 项目4：自愿酒精暴露对特定脑区的神经元功能和形态有何影响？积极寻求乙醇的动物和那些在慢性治疗后避免使用乙醇的动物的大脑有什么不同？ 野生型和转基因小鼠将接受15-30天的自愿酒精摄入，然后进行行为测试，以确定依赖程度。然后，将对这些小鼠的脑组织进行体外程序。将如上所述进行电生理、形态和生化分析。

To address the specific questions mentioned above, we have outline five projects and a brief experimental design summary. Project 1: How does chronic cocaine treatment affect synaptic function and morphology? Wild-type and transgenic mice will receive passive cocaine/saline administration (IP) daily for 5-30 days. This will be followed by behaivoral testing: locomotor activity and place preference tested every day with injection. We will then prepare acute brain slices / brain slice cultures and perform in vitro procedures Different techniques and in vitro procedures will be applied to determine the changes that take place in the brain of addicted animals. We will investigate: - Morphological changes: such as alterations in spine numbers, size, motility and dendritic branching. - Changes in synaptic strength, synaptic plasticity and modulation. - Biochemical changes in expression of synaptic proteins such as glutamate AMPA receptors, NMDA receptors, dopamine receptors and signaling molecules. - Changes in intracellular calcium signaling in spines and dendrites Project 2: Animals that display high impulsivity behavior show increased vulnerability to become compulsive drug seekers when chronically exposed to cocaine (Belin et al., 2008 Science 320:1352-1355). We will compare the synaptic function and the structure of neurons in different brain regions of mice that display high and low impulsivity. Wild-type and transgenic mice will undergo behavioral testing (5 choice serial reaction time task) daily for 30-40 days followed by in vitro procedures. Different techniques will be used to identify neuronal markers highly impulsive animals. We will investigate: - Morphology: spine numbers, size, motility and dendritic branching. - Biochemistry: expression levels of synaptic proteins and signaling molecules. - Calcium signaling in spines and dendrites - Synaptic strength and plasticity Project 3: Only a fraction of cocaine users become compulsive drug-takers. What is different in the brain of subjects exposed and addicted in comparison to those exposed but non-addicted? Is there a difference in synaptic function and morphology between mice that display addictive-like behaviors and mice that do not after they all have been chronically exposed to cocaine? Wild-type mice and transgenic mice will receive cocaine/saline self-administration for 30 days followed by behavioral testing to determine addictive score after 1, 10 or 30 days of last exposure. We will then perform in vitro procedures on these mice. Electrophysiological, morphological and biochemical analysis will be performed as described in project 1 and 2. Project 4: What is the effect of voluntary ethanol exposure on neuronal function and morphology in specific brain regions? What is different in the brain of animals that actively seek ethanol and those that avoid it after the chronic treatment? Wild-type and transgenic mice will undergo voluntary ethanol intake for 15-30 days followed by behavioral testing to determine degree of dependence. In vitro procedures will then be performed on brain tissue from these mice. Electrophysiological, morphological and biochemical analysis will be performed as described above.