Changes in synaptic function and structure associated with chronic cocaine

在这里，我们概述了四个项目，并总结了进展情况。 项目1：慢性可卡因治疗如何影响突触功能和形态？ 在这第一年，我们解决了两个悬而未决的问题，关于可卡因的行动，在脑桥核：a）什么是禁欲或戒毒的作用，在可卡因诱导的可塑性，和B）可卡因的行动是否在脑桥核是细胞特异性的，或者说影响中型多刺神经元从两个途径（那些直接和间接的预测到黑质）。 a）、 虽然在单次可卡因注射后仅几个小时就足以检测腹侧被盖区中多巴胺能传递的变化（Ungless等人，Nature 2001），仅在重复可卡因注射和2周戒断后观察到丘脑核中的传递增加（Kourrich et al.，J Neurosci 2007）。类似地，在最后一次注射后一个月观察到脊髓中棘密度增加（罗宾逊和Kolb，欧洲神经科学杂志1999）。这些观察结果似乎表明，药物戒断是在发育中的塑料变化的一个关键因素。我们通过比较可卡因治疗后的突触传递与两种在戒断期程度上不同的方案来验证这一假设。我们发现，长期的可卡因戒断是不需要的可卡因诱导的可塑性的突触。此外，树突棘密度的增加在没有长期戒断可卡因的情况下发展。 B） 为了解决可卡因诱导的可塑性是否会影响中型多刺神经元的亚群，我们使用BAC转基因小鼠，表达GFP的D1-多巴胺受体启动子下，然后用荧光标记的纹状体黑质通路的中型多刺神经元的亚群。我们发现，慢性可卡因触发可塑性D1表达中型多刺神经元和形态学变化也仅限于纹状体黑质通路的神经元，而不存在于D2表达中型多刺神经元的mesamatergic突触。 项目2：只有一小部分可卡因使用者成为强迫性吸毒者。在成瘾的动物模型中似乎也是这种情况（Deroche-Gamonet等人，2004 Science 305：1014-1017）。与那些暴露但不上瘾的人相比，暴露并上瘾的受试者的大脑有什么不同？ 在慢性接触可卡因后，表现出成瘾样行为的小鼠和没有成瘾样行为的小鼠之间，突触功能和形态是否有差异？ 为了回答这些问题，我们必须首先能够在暴露于滥用药物的动物群体中识别出成瘾动物。为此，我们使用一组行为测试来对动物的成瘾性行为的发展进行评分，并继续研究和比较具有高成瘾分数和低成瘾分数的动物的突触特性。 训练小鼠通过将鼻子戳进一个孔中来自愿施用可卡因，该孔导致通过植入颈静脉中的插管递送可卡因输注。 训练后，允许小鼠每天自我给药40天，并测量3种行为特征： - 坚持寻求药物（在没有药物可用期间主动戳鼻子） - 药物的动机（在具有渐进比率的会话期间达到的断点） - 厌恶性后果（当药物与厌恶性刺激配对时，持续寻求药物） 实验正在进行中，以研究与低成瘾分数相比，高成瘾分数动物树突中的神经递质传递和钙信号传导的电生理学特性。请注意，本研究中的所有动物都主动自我施用可卡因，因此可卡因摄入的总水平预计在种群中不会有很大差异，而是药物寻求行为的强迫性质。 项目3：自愿酒精暴露对特定脑区神经元功能和形态的影响是什么？ 在与Kathleen Grant博士的实验室合作中，我们正在研究与轻度和重度乙醇饮用相关的神经元和突触形态的变化。这项研究涉及非人类灵长类动物，这些灵长类动物自愿饮用乙醇2年，其中包括反复戒断。 研究中所有受试者的平均每日乙醇消耗量为2.8 g/kg，但实际上在5.6 - 1.9 g/kg之间变化。血液乙醇水平相应地变化，显示出偏态分布，其中少数受试者消耗了绝大多数总乙醇。尸检后，从这些受试者的尾状核和壳核收集脑组织，并进行diolistic染色和形态学分析。共聚焦图像正在采集和树突和棘分析将进行调查的变化，棘密度，棘形态和树突分支之间的轻，重乙醇饮用者。

Here we outline four projects and summarize the progress. Project 1: How does chronic cocaine treatment affect synaptic function and morphology? During this first year we addressed two unresolved questions regarding the actions of cocaine in the nucleus accumbens: a) what is the role of abstinence or withdrawal in the cocaine-induced plasticity, and b) whether cocaine actions in the accumbens are cell-specific or rather affect medium spiny neurons from both pathways (those with direct and indirect projections to substantia nigra). a) While just a few hours after a single cocaine injection are enough to detect changes in the glutamatergic transmission in the ventral tegmental area (Ungless et al., Nature 2001), an increase in transmission in the nucleus accumbens is seen only after repeated cocaine injections and 2 weeks of withdrawal (Kourrich et al., J Neurosci 2007). Similarly, increases in spine density in the accumbens were observed after one month of the last injection (Robinson and Kolb, Eur J Neurosci 1999). These observations together seem to indicate that drug withdrawal is a critical factor in the developing of plastic changes in the accumbens. We tested this hypothesis by comparing synaptic transmission after cocaine treatment with two protocols that differ in the extent of the withdrawal period. We found that prolonged cocaine withdrawal is not required for the cocaine induced plasticity in glutamatergic synapses. Furthermore, the increase in dendritic spine density develops without prolonged withdrawal from cocaine. b) To address whether cocaine induced plasticity affects a subpopulation of medium spiny neurons, we use BAC transgenic mice that express GFP under the D1-dopamine receptor promoter and then label with fluorescence the subpopulation of medium spiny neurons of the striatonigral pathway. We found that chronic cocaine triggers plasticity of glutamatergic synapses onto D1 expressing medium spiny neurons and that morphological changes were also restricted to the neurons of the striatonigral pathway and not present in D2-expressing medium spiny neurons. Project 2: Only a fraction of cocaine users become compulsive drug-takers. This also seems to be the case in animal models of addiction (Deroche-Gamonet et al., 2004 Science 305:1014-1017). 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? In order to answer these questions, we must first be able to identify addicted animals within the population of animals exposed to the drug of abuse. With this purpose, we use a set of behavioral tests to score animals for the development of addictive like behaviors and proceed to study and compare the synaptic properties of animals with high and low addictive scores. Mice are trained to administer cocaine voluntarily by poking the nose into a hole that results in the delivery of a cocaine infusion through a cannula implanted into the jugular vein. After training, mice are allowed to self-administer in daily sessions for 40 days and 3 behavioral characteristics are measured: - Perseverance of drug seeking (active nose pokes during periods of no drug availability) - Motivation for the drug (break point reached during sessions with progressive ratio) - Aversive consequences (perseverance of drug seeking when drug paired to aversive stimulus) Experiments are underway to study the electrophysiological properties of glutamatergic transmission and calcium signaling in dendrites from animals with high addiction scores in comparison to those with a low addiction score. Note that all animals in this study actively self-administered cocaine and so total levels of cocaine intake are not expected to vary much across the population but rather the compulsive nature of the drug seeking behavior. Project 3: What is the effect of voluntary ethanol exposure on neuronal function and morphology in specific brain regions? In collaboration with the laboratory of Dr. Kathleen Grant, we are studying the changes in neuronal and synaptic morphology that are associated with light and heavy ethanol drinking. This study involves non-human primates that undergo 2 years of voluntarily ethanol drinking in a schedule that includes repeated withdrawal. The average daily ethanol consumption was 2.8 g/kg for all the subjects in the study but actually varied from 5.6 to 1.9 g/kg. Blood ethanol levels varied accordingly showing a skewed distribution where a few subjects consumed the vast majority of total ethanol. Upon necropsy, brain tissue was collected from the caudate and putamen of these subjects and processed for diolistic staining and morphological analysis. Confocal images are being acquired and dendrite and spine analysis will be performed to investigate changes in spine density, spine morphology and dendritic branching between light and heavy ethanol drinkers.