Synaptic changes in the medial prefrontal cortex in the development of compulsive alcohol drinking

摘要： 抑制饮酒的能力是戒酒的一个重大挑战，特别是在酒精存在的情况下- 关联的线索。反复酒精暴露会诱导神经适应，这种适应持续到急性戒断后，而且 增加酒精的激励突显，导致酒精摄入量的升级和抗厌恶的酒精寻求。酒精 使用还会导致与内侧前额叶皮质(MPFC)相关的认知功能障碍，这进一步加剧了 强迫饮酒和旧病复发。在啮齿类动物中，寻求酒精激活了腹侧(边缘下， MPFC的IL)和预备区(PL)，它们在控制复发行为方面扮演着截然相反的角色。而当 激活PL驱动恢复，IL中的神经元通过促进灭绝学习和抑制药物寻找 它们投射到伏隔核壳核以及基底外侧杏仁核(BLA)。然而，突触 在从受控酒精到强制酒精的转变过程中，驱动这些回路中不适应可塑性的机制- 在很大程度上，寻找仍不清楚。此应用程序中的实验将提供对特定网络的更好理解 慢性酒精暴露和戒断对mPFC执行认知功能的影响机制 减少对目标导向行为的抑制性控制。 在目标1中，我们使用膜片钳电生理和光遗传刺激来确定远距离的变化。 在长期饮酒后，谷氨酸能从BLA传入IL和PL中已识别的投射神经元， 以及在后依赖条件下。我们将在活性种群中使用靶向重组(TRAP2)与Fos2A- ICreer小鼠在戒断过程中激活的mPFC的BLA传入细胞中选择性地表达通道视紫红质， 我们将确定这些输入的变化是如何随着时间的推移而发展的(从目标导向到强迫性酒精- 正在寻找)。我们将对逆行标记的神经元进行电压钳记录，这些神经元投射回BLA，并且 我们将确定酒精诱导的突触后谷氨酸受体功能和突触前释放的变化 复职。Aim 2的实验将使用组合逆行Cre递送和Cre依赖的报告来标记 用于脊髓和谷氨酸受体高分辨形态计量学分析的相同IL和PL投射神经元 表达，以便比较导致复发行为的神经元的具体变化(通过 活性标记物磷酸化CREB的联合标记)和那些不存在的细胞(pCREB阴性细胞)。在《目标3》中，我们将再次 使用TRAP2小鼠测试mPFC中的特定集合或来自BLA的输入的光遗传操作 到mPFC(每个都在戒断过程中再次陷入陷阱)可以逆转酒精诱导的认知障碍，并减少药物- 寻找。 综上所述，这些研究将为酒精诱导的脑内突触变化提供重要的新信息。 IL的网络有助于认知灵活性和线索诱导的恢复。

SUMMARY: The ability to inhibit drinking is a significant challenge for recovering alcoholics, especially in the presence of alcohol- associated cues. Repeated alcohol exposure induces neuroadaptations that persist beyond acute withdrawal, and which increase alcohol’s incentive salience, leading to escalation of alcohol intake and aversion-resistant alcohol seeking. Alcohol use also causes deficits in cognitive functions associated with the medial prefrontal cortex (mPFC), which further fuel compulsive drinking and relapse. In rodents, alcohol seeking activates specialized networks within the ventral (infralimbic, IL) and prelimbic (PL) regions of the mPFC, which play largely opposite roles in the control of relapse behavior. While activation of the PL drives reinstatement, neurons in the IL facilitate extinction learning and inhibit drug-seeking through their projections to the Nucleus Accumbens shell, as well as the basolateral amygdala (BLA). However, the synaptic mechanisms that drive maladaptive plasticity in these circuits during the transition from controlled to compulsive alcohol- seeking remain largely unclear. The experiments in this application will provide a better understanding of network-specific mechanisms through which chronic alcohol exposure and withdrawal affect executive cognitive functions of the mPFC and diminish inhibitory control over goal-directed behavior. In Aim 1 we use patch-clamp electrophysiology and optogenetic stimulation to determine changes in long-range glutamatergic inputs from the BLA onto identified projection neurons in the IL and PL following extended access to alcohol, as well as under postdependent conditions. We will use Targeted Recombination in Active Populations (TRAP2) with Fos2A- iCreER mice to express channelrhodopsin selectively in those BLA afferents to the mPFC that are activated during withdrawal, and we will determine how alterations in these inputs develop over time (from goal-directed to compulsive alcohol- seeking). We will perform voltage-clamp recordings from retrogradely-labeled neurons that project back to the BLA, and we will determine alcohol-induced changes in postsynaptic glutamate receptor function and presynaptic release following reinstatement. Experiments in Aim 2 will use combinational retrograde Cre delivery and a Cre-dependent reporter to label the same IL and PL projection neurons for high-resolution morphometric analyses of spines and glutamate receptor expression in order to compare changes specifically in those neurons that contribute to relapse behavior (visualized via co-labeling for the activity marker phospho-CREB) and those that do not (pCREB-negative cells). In Aim 3 we will again use TRAP2 mice to test whether optogenetic manipulations of specific ensembles in the mPFC, or of inputs from the BLA to the mPFC (each again TRAPed during withdrawal) can reverse alcohol-induced cognitive deficits and reduce drug- seeking. Taken together, these studies will provide important novel information about alcohol-induced synaptic changes in networks of the IL that contribute to cognitive flexibility and cue-induced reinstatement.