Synaptic mechanisms underlying reward seeking and compulsive drug use

项目A：多巴胺侧向抑制的通路特异性抑制需要伏隔核中的5-羟色胺受体。伏核(NAC)回路的多巴胺调制是奖赏寻求和强化学习理论的核心。尽管经过几十年的努力，NAC微电路上的急性多巴胺作用仍然令人费解。在这里，我们解剖了多巴胺在小鼠脑片中中棘神经元(MSN)之间对侧向抑制的直接作用，发现多巴胺对NAC侧向抑制的急性作用是路径特异性的。多巴胺可以有效和选择性地抑制突触前表达多巴胺D2受体的MSN(D2-MSN)的GABA能传递，而增强突触前表达多巴胺D2受体的MSN的GABA能传递。令我们惊讶的是，突触前D2受体只介导了多巴胺神经元刺激或多巴胺应用所引起的抑郁的一半。突触前5-HT1B受体是多巴胺诱导的突触抑制的重要组成部分。因此，本研究通过D2和5-HT1B受体参与D2-MSN突触的多巴胺抑制，阐明了NAc中多巴胺作用的机制。 项目B：富含亮氨酸的重复激酶2限制纹状体中的多巴胺D1受体信号，并对大量和持续饮酒产生偏见。从享乐性饮酒过渡到有问题的大量饮酒是酒精使用障碍(AUD)的一个特征。已知这种转变需要对表达多巴胺D1受体(D1R)的纹状体神经元的突触驱动和活动进行长期的改变，但这种脆弱性背后的分子机制尚不清楚。在这里，我们报道了帕金森病相关蛋白富含亮氨酸的重复蛋白激酶2(LRRK2)调节纹状体黑质通路中的D1R功能，并通过这样做，调节与酒精相关的行为，以及向重度和持续饮酒的转变。从表达D1R的纹状体神经元中特异地缺失LRRK2基因可以在细胞和突触水平上增强D1R的功能，增强酒精刺激，并促进对惩罚不敏感的重度酒精饮酒。这些发现确定了LRRK2在纹状体中的一个新的作用，并表明LRRK2限制了D1R信号，并促进了对大量和持续饮酒的弹性。 项目C：多巴胺D2受体的低表达在纹状体间接培养的棘神经元中驱动GABA和cAMP相关基因网络。背景：滥用药物劫持了多巴胺奖励系统，这是一个进化上保守的目标导向行为和学习的驱动因素。纹状体信号中释放的多巴胺主要通过激活D_1和D_2受体。这些受体的表达在纹状体MSN的两种主要类型中是分开的。D1和D2受体活性的平衡被认为是纹状体功能和控制行为的关键。D2R功能减弱与成瘾易感性有关，也与疾病的病理生理学有关。此外，反复接触刺激性药物会导致纹状体D2R的不适应下调。我们之前曾报道，在小鼠中，纹状体D2受体的低表达驱动了D1受体的超敏反应，促进了选定的可卡因诱导的行为。在这里，我们试图确定D2受体表达神经元或iMSN(间接途径中的棘神经元)的基因表达变化，这是由于基因驱动的DRD2低表达所致。 方法：对杂合子和野生型小鼠iMSN中提取的基因进行转录组学分析。翻译核糖体亲和纯化(TRAP)系统用于有条件地靶向iMSN。后续实验包括有可卡因滥用史的人死后脑组织；以及电生理学。 结果：基因驱动的Drd2低表达导致GABA和cAMP通路、神经突触形成和重塑、Na/K泵调节、脂代谢和炎症相关基因的差异表达。差异表达基因在Creb1靶基因中高度丰富，提示Creb1是上游调控因子。钠钾泵亚单位Fxyd2与Drd2的表达呈负相关。电生理实验支持IMSN-Drd2HET小鼠较高的GABA音调。 结论：与细胞间通讯有关的基因网络，更具体地说是突触的形成和调节，受到表达相同D2R的纹状体神经元中Drd2表达缺失的强烈影响。电生理实验显示了增强的纹状体抑制等功能含义，结合我们之前发表的工作，它们表明纹状体D2R的低表达足以显著改变纹状体微电路的状态。

Project A: Pathway-specific depression of lateral inhibition by dopamine requires serotonin receptors in the nucleus accumbens. Dopamine modulation of nucleus accumbens (NAc) circuitry is central to theories of reward seeking and reinforcement learning. Despite decades of effort, the acute dopamine actions on the NAc microcircuitry remain puzzling. Here, we dissect out the direct actions of dopamine on lateral inhibition between medium spiny neurons (MSNs) in mouse brain slices and find that dopamine's acute actions on NAc lateral inhibition are pathway-specific. Dopamine potently and selectively depresses GABAergic transmission from presynaptic dopamine D2 receptor-expressing MSNs (D2-MSNs), while it potentiates transmission from presynaptic dopamine D1 receptor-expressing MSNs. To our surprise, presynaptic D2 receptors mediate only half of the depression induced by either dopamine neuron stimulation or dopamine application. Presynaptic serotonin 5-HT1b receptors are responsible for a significant component of dopamine-induced synaptic depression. Thus, this study clarifies the mechanistic understanding of dopamine actions in the NAc by showing pathway-specific modulation of lateral inhibition and the involvement of both D2 and 5-HT1b receptors in dopamine depression of D2-MSN synapses. Project B: Leucine-rich repeat kinase 2 limits dopamine D1 receptor signaling in the striatum and biases against heavy and persistent alcohol drinking. Transition from hedonic alcohol drinking to problematic heavy drinking is a hallmark of alcohol use disorder (AUD). Long-lasting changes on the synaptic drive and activity of striatal neurons expressing dopamine D1 receptor (D1R) are known to be required for this transition, but the molecular mechanisms underlying this vulnerability are less understood. Here we report that the Parkinson's Disease-related protein Leucine-rich repeat kinase 2 (LRRK2) modulates D1R function within the striatonigral pathway and, by doing so, it regulates alcohol related behaviors and the transition to heavy and persistent alcohol drinking. Deletion of the Lrrk2 gene specifically from D1R-expressing striatal neurons potentiates D1R function at the cellular and synaptic level, enhancing alcohol stimulation and promoting heavy alcohol drinking that is insensitive to punishment. These findings identify a novel role for Lrrk2 function in the striatum and suggest that Lrrk2 limits D1R signaling and promotes resilience against heavy and persistent alcohol drinking. Project C: Low expression of dopamine D2 receptor drives GABA and cAMP-related gene networks in striatal indirect Medium Spiny Neurons. Background: Misused drugs hijack the dopamine reward system, an evolutionarily conserved driver of goal-directed behavior and learning. Dopamine released in the striatum signals mainly via activation of D1 and D2 receptors. The expression of these receptors is segregated in the two main types of striatal MSNs. The balance of activity of D1 and D2 receptors is thought to be critical for how the striatum functions and controls behaviors. Diminished D2R function is associated with vulnerability for addiction and also with the pathophysiology of the disorder. Further, repeated exposure to stimulant drugs leads to maladaptive downregulation of striatal D2R. We previously reported that in mice low expression of striatal D2 receptors drives D1 receptor hypersensitivity, facilitating selected cocaine-induced behaviors. Here, we sought to identify gene expression changes specific to D2 receptor expressing neurons or iMSNs (indirect pathway Medium Spiny Neurons) consequent to genetically-driven low expression of Drd2. Methods: Transcriptome analysis was performed on mRNA purified from iMSNs of heterozygous and wild type mice for the Drd2 gene. The Translating Ribosome Affinity Purification (TRAP) system was used to conditionally target iMSNs. Follow-up experiments include postmortem brain tissue from humans with cocaine misuse history; and electrophysiology. Results: Genetically-driven low expression of Drd2 resulted in differential expression of genes involved in GABA and cAMP pathways, neural synapse formation and remodeling, modulation of the Na/K pump, lipid metabolism and inflammation. Differentially expressed genes were strongly enriched in Creb1 targets, suggesting Creb1 as an upstream regulator. Fxyd2, a subunit of the sodium potassium pump, showed a negative correlation with Drd2 expression. Electrophysiology experiments supported a higher GABA tone in iMSN-Drd2HET mice. Conclusions: Genes networks implicated in cell-to-cell communication, more specifically synapse formation and regulation, are strongly impacted by deficiency in Drd2 expression within the same D2R expressing striatal neurons. Electrophysiological experiments show functional implications such as enhanced striatal inhibition, and together with our previously published work, they indicate that low striatal D2R expression is sufficient to significantly alter the state of the striatal microcircuitry.