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。 项目1：慢性可卡因治疗如何影响突触功能和形态？ 在这第一年里，我们解决了关于可卡因在伏隔核中的作用的两个悬而未决的问题：a)戒断或戒断在可卡因诱导的可塑性中扮演了什么角色，以及b)伏隔核中的可卡因作用是细胞特异性的，还是影响了来自两条通路的中等棘神经元(那些直接和间接投射到黑质的神经元)。 A)虽然仅在注射一次可卡因几个小时后就足以检测到腹侧被盖区谷氨酸能传递的变化(Ungless等人，自然，2001)，但只有在反复注射可卡因并戒断2周后，伏隔核传递才会增加(Kourrich等人，J Neurosci 2007)。同样，在最后一次注射的一个月后观察到伏隔核内脊柱密度的增加(Robinson和Kolb，EUR J Neurosci 1999)。这些观察结果似乎共同表明，停药是伏隔核发生可塑性变化的关键因素。我们通过比较可卡因治疗后的突触传递和两种戒断时间不同的方案来验证这一假设。我们发现，可卡因诱导的谷氨酸能突触的可塑性并不需要长时间的可卡因戒断。此外，树突棘密度的增加不会导致长期戒断可卡因。 B)为了研究可卡因诱导的可塑性是否影响中等刺神经元亚群，我们使用了在D1-多巴胺受体启动子下表达GFP的BAC转基因小鼠，然后用荧光标记纹状体黑质通路的中等刺神经元亚群。我们发现，慢性可卡因可触发谷氨酸能突触向表达D2的中棘神经元可塑性，并且形态变化也仅限于纹状体黑质通路的神经元，而在表达D2的中棘神经元中不存在。 项目2：只有一小部分可卡因吸毒者成为强迫症吸毒者。在成瘾的动物模型中似乎也是如此(Deroche-Gamonet等人，2004年科学305：1014-1017)。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？ 为了回答这些问题，我们首先必须能够在暴露于滥用药物的动物种群中识别出成瘾的动物。为此，我们使用一套行为学测试来对动物的成瘾行为的发展进行评分，并继续研究和比较成瘾评分高和低的动物的突触特性。 训练小鼠通过将鼻子戳进一个洞来自愿给药，这个洞通过植入颈静脉的插管输送可卡因输液。训练结束后，小鼠在每天的训练中自我管理40天，并测量3个行为特征： -坚持不懈地寻找毒品(在没有药物可供使用的时期活跃地戳鼻子) -药物的动机(在治疗期间达到累进比率的临界点) -厌恶后果(当毒品与厌恶的刺激配对时，寻求毒品的毅力) 目前正在进行实验，以研究成瘾分数高的动物与成瘾分数低的动物树突中谷氨酸能传递和钙信号的电生理特性。请注意，这项研究中的所有动物都主动自我注射可卡因，因此可卡因的总摄入量预计不会在整个种群中有太大差异，而是强迫性寻求毒品的行为。 项目3：自愿酒精暴露对特定脑区的神经元功能和形态有何影响？ 与凯瑟琳·格兰特博士的实验室合作，我们正在研究与轻度和重度酒精饮酒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，他们自愿饮酒两年，包括反复戒酒。 所有研究对象的平均每日酒精消耗量为2.8g/kg，但实际范围为5.6至1.9g/kg。血液酒精水平相应地变化，显示出扭曲的分布，其中少数受试者消耗了全部酒精的绝大多数。在尸检时，从这些受试者的尾状核和壳核中收集脑组织，并对其进行二辉绿染色和形态分析。目前正在获取共聚焦图像，并将进行树突和脊椎分析，以调查轻度和重度饮酒者之间脊柱密度、脊柱形态和树突分支的变化。

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.