慢性饮酒调节中脑基因转录与多巴胺终端调节之间的同步性-动脉网

Synchrony between midbrain gene transcription and dopamine terminal regulation is modulated by chronic alcohol drinking

Nature 等信源发布 2025-02-25 11:23

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Abstract

摘要

Alcohol use disorder is marked by disrupted behavioral and emotional states which persist into abstinence. The enduring synaptic alterations that remain despite the absence of alcohol are of interest for interventions to prevent relapse. Here, 28 male rhesus macaques underwent over 20 months of alcohol drinking interspersed with three 30-day forced abstinence periods.

酒精使用障碍的特点是行为和情绪状态的紊乱，这种状态会持续到戒酒后。尽管没有酒精存在，但持久的突触改变仍然是预防复发干预措施的研究重点。在此研究中，28只雄性恒河猴经历了超过20个月的饮酒过程，期间穿插了三次为期30天的强制戒酒期。

After the last abstinence period, we paired direct sub-second dopamine monitoring via ex vivo voltammetry in nucleus accumbens core with RNA-sequencing of the ventral tegmental area. We found persistent augmentation of dopamine transporter function, kappa opioid receptor sensitivity, and putative dynorphin release – all inhibitory regulators which act to decrease extracellular dopamine.

在最后一个戒断期后，我们通过伏隔核核心的离体伏安法直接监测亚秒级多巴胺水平，并结合腹侧被盖区的RNA测序。我们发现多巴胺转运体功能、κ阿片受体敏感性以及推测的强啡肽释放持续增强——这些抑制性调节因子均作用于降低细胞外多巴胺水平。

Surprisingly, though transcript expression was not altered, the relationship between gene expression and functional readouts of these encoded proteins was highly dynamic and altered by drinking history. These results outline the long-lasting synaptic impact of alcohol use and suggest that assessment of transcript-function relationships is critical for the rational design of precision therapeutics..

令人惊讶的是，尽管转录表达没有改变，但这些编码蛋白质的基因表达与功能读数之间的关系却是高度动态的，并受到饮酒历史的影响。这些结果概述了酒精使用的持久突触影响，并表明评估转录-功能关系对于合理设计精准治疗至关重要。

Introduction

简介

Alcohol Use Disorder (AUD) is defined by numerous symptoms involving over-prioritization and -dedication to alcohol use, including craving, excessive consumption, and continued use in the face of negative consequences

酒精使用障碍（AUD）的定义涉及多个症状，包括对酒精使用的过度优先考虑和投入，如渴望、过量饮酒以及在面临负面后果时仍然继续饮酒。

. Unfortunately, AUD is a chronic relapsing disorder, and even with treatment, relapse rates after achieving abstinence remain as high as 40–60%

不幸的是，AUD是一种慢性复发性疾病，即使经过治疗，达到戒酒后的复发率仍然高达40%-60%。

，

. Decades of clinical and preclinical research has shown that protracted abstinence from chronic drinking is characterized by depression-like behaviors, increased response to alcohol-related cues, and greater stress-induced craving, making periods of abstinence a critical intervention point to treat individuals susceptible to relapse.

数十年的临床和临床前研究表明，长期戒酒的特点是抑郁样行为、对酒精相关线索的反应增强以及压力诱发的渴求增加，这使得戒酒期成为治疗易复发个体的关键干预点。

，

. Leading models of AUD neurobiology posit that complex, alcohol-induced changes in gene expression and translation result in time-dependent, neural circuit-specific functional plasticity, which in turn drive the cardinal behavioral repertories of AUD

领先的AUD神经生物学模型认为，酒精引起的基因表达和翻译的复杂变化会导致时间依赖性、神经回路特异性的功能可塑性，进而驱动AUD的核心行为特征。

，

. Central to these models, the mesolimbic dopamine circuit, consisting of dopamine neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens core (NAc), undergoes dramatic functional plasticity in response to chronic alcohol exposure and there is wide consensus that this circuit plays an essential role in many facets of AUD.

这些模型的核心是中脑边缘多巴胺回路，该回路由从腹侧被盖区（VTA）投射到伏隔核核心（NAc）的多巴胺神经元组成，在慢性酒精暴露下会发生显著的功能可塑性变化，并且广泛共识认为该回路在酒精使用障碍（AUD）的许多方面发挥着关键作用。

，

. Leveraging current detailed knowledge of alcohol-induced transcriptional plasticity and circuit function has enormous potential for developing more effective, targeted therapeutic interventions, and this possibility has captured the attention and efforts of the preclinical alcohol field at large.

利用当前对酒精诱导的转录可塑性和回路功能的详细了解，对于开发更有效、更有针对性的治疗干预措施具有巨大的潜力，这一可能性已经引起了临床前酒精研究领域的广泛关注和努力。

Although alcohol-induced plasticity in the mesolimbic dopamine system is robust, the directionality and magnitude of functional alterations to the receptors and transporters governing dopamine dynamics are highly dependent on the exact parameters of alcohol exposure and the model species used, despite consistent behavioral sequalae.

尽管酒精在中脑边缘多巴胺系统中引发的可塑性变化显著，但对控制多巴胺动态的受体和转运蛋白的功能改变的方向性和程度高度依赖于酒精暴露的确切参数和所使用的模型物种，尽管行为后果是一致的。

，

. Cross-species comparisons have allowed identification of a subset of dopaminergic regulators which display consistent alcohol-induced adaptations. These studies have demonstrated that the efficiency of the dopamine transporter and inhibitory drive of kappa-opioid receptors on presynaptic dopamine release are ubiquitously upregulated by alcohol exposure across species, strain, sex, alcohol exposure length, and alcohol exposure protocol.

跨物种比较已经允许识别出一部分多巴胺调节器，它们表现出一致的酒精诱导适应。这些研究已经证明，多巴胺转运蛋白的效率和κ阿片受体对突触前多巴胺释放的抑制驱动在不同物种、品系、性别、酒精暴露时长和酒精暴露协议中普遍因酒精暴露而上调。

，

. The consistency of these alterations makes them attractive treatment targets, due to the likelihood of these changes being conserved in human drinkers and given that most therapeutics are administered without the benefit of detailed information as to the patient’s exact alcohol use history. However, it is unknown whether these alterations persist into abstinence..

这些改变的一致性使它们成为有吸引力的治疗靶点，因为这些变化很可能在人类饮酒者中得以保留，并且鉴于大多数治疗药物都是在没有详细了解患者确切饮酒史的情况下施用的。然而，目前尚不清楚这些改变是否会持续到戒酒期间。

Additionally, to date, our knowledge of alcohol-induced transcriptional and functional plasticity signatures is derived primarily from parallel investigations, and both literatures have largely utilized non-primate model species with limited gene homology to humans. Determining the degree to which transcriptional milieus can predict circuit plasticity profiles and advancing these hypotheses to primate species are critical next steps for the field.

此外，迄今为止，我们对酒精诱导的转录和功能可塑性特征的了解主要来自于平行研究，而且两者的研究大多使用与人类基因同源性有限的非灵长类模式物种。确定转录环境在多大程度上可以预测神经回路可塑性特征，并将这些假设推广到灵长类物种，是该领域的关键下一步。

Here, we sought to determine whether alcohol-induced plasticity in dopamine reuptake and opioidergic control of axonal dopamine release persist into abstinence, and the degree to which lasting plasticity in dopamine synapses can be explained by upstream changes in somatic transcriptional profiles..

在这里，我们试图确定酒精诱导的多巴胺再摄取和阿片肽能对轴突多巴胺释放的控制是否持续到戒断期，以及多巴胺突触中持久的可塑性在多大程度上可以由细胞体转录谱的上游变化来解释。

To answer these questions, we paired a model of chronic voluntary alcohol (ethanol) drinking and protracted abstinence with within-subject measures of dopamine terminal function and assessment of gene transcription. In an effort to maximize translational relevance and implications of gene expression quantification, rhesus macaque (.

为了回答这些问题，我们将慢性自愿饮酒（乙醇）和长期戒断的模型与多巴胺终端功能的主体内测量以及基因转录评估相结合。为了最大化基因表达量化的转化相关性和影响，我们使用了恒河猴（。

Macaca mulatta)

猕猴属 mulatta种

subjects were used, given that they display behavioral diversity which mirrors individual differences in human drinkers

鉴于它们表现出的行为多样性反映了人类饮酒者个体之间的差异，这些实验对象被使用。

, as well as homology to humans in genetic sequence

以及与人类在基因序列上的同源性

and neural circuit architecture

神经回路结构

，

. Following an induction protocol, subjects were allowed to self-administer alcohol under continuous access conditions for 12 months, followed by three one-month abstinence periods interspersed with three-month periods of re-access to alcohol. At the end of the third and final abstinence period, real-time dopamine release kinetics and inhibitory regulation by axonal kappa opioid receptors were measured directly via ex vivo fast-scan cyclic voltammetry in the NAc core (17 alcohol drinkers, 11 calorically yoked or housing controls).

按照诱导方案，实验对象被允许在持续获取的条件下自我管理酒精长达12个月，随后是三个为期一个月的戒酒期，其间穿插三个月的重新获取酒精期。在第三个也是最后一个戒酒期结束时，通过NAc核心区的离体快速扫描循环伏安法直接测量了实时多巴胺释放动力学以及轴突κ阿片受体的抑制调节（17名饮酒者，11名热量匹配或环境对照）。

The findings of the voltammetry experiments included 1) that dopamine transporter and kappa opioid receptor plasticity persisted into protracted abstinence, 2) that changes in evoked dopamine release magnitude did not persist, except at the extremes of the input/output curve, 3) that G protein signaling was both necessary and sufficient for kappa opioid receptor inhibitory control of axonal dopamine release but did not account for alcohol-induced supersensitivity, 4) that kappa opioid receptor co-activation was necessary for corticotropin-releasing factor (CRF) receptor-mediated inhibition of dopamine release but was not altered by drinking history, and 5) that dynorphin release probability, measured using a time-resolved readout, was increased in protracted abstinence..

伏安法实验的结果包括：1）多巴胺转运体和κ阿片受体可塑性持续到长期戒断期；2）诱发的多巴胺释放量的变化没有持续，除了在输入/输出曲线的极端情况下；3）G蛋白信号传导对于κ阿片受体抑制轴突多巴胺释放既是必要的也是充分的，但不能解释酒精诱导的超敏反应；4）κ阿片受体的协同激活是促肾上腺皮质激素释放因子（CRF）受体介导的多巴胺释放抑制所必需的，但不受饮酒史的影响；5）使用时间分辨读数测量的强啡肽释放概率在长期戒断期增加。

To probe mechanisms that may underly functional differences observed by fast-scan cyclic voltammetry, bulk RNA-sequencing of the VTA was conducted to measure gene expression upstream of dopamine terminals. Given the involvement of the mesolimbic system in addiction-related behaviors, we hypothesized that chronic alcohol consumption and abstinence would result in robust changes in VTA gene expression, and that plasticity in dopamine terminal function would be related to changes in the expression of dopamine-specific genes.

为了探究可能导致快速扫描循环伏安法观察到的功能差异的机制，进行了VTA的批量RNA测序以测量多巴胺终端上游的基因表达。鉴于中脑边缘系统在成瘾相关行为中的参与，我们假设慢性饮酒和戒酒会导致VTA基因表达的显著变化，并且多巴胺终端功能的可塑性将与多巴胺特异性基因表达的变化有关。

Contrary to our hypothesis, we found zero differentially expressed genes in the VTA between drinkers and controls and minimal evidence for any network-level dysregulation. Unexpectedly, within-subject correlations between fast-scan cyclic voltammetry measures, such as release magnitude and rate of reuptake, and the expression of genes encoding for proteins thought to be functionally involved, such as D2-type autoreceptors and dopamine transporter, showed that VTA transcript expression and NAc function are often decorrelated in controls.

与我们的假设相反，我们发现在饮酒者和对照组之间的VTA中没有任何差异表达的基因，并且几乎没有证据表明存在任何网络水平的失调。出乎意料的是，快速扫描循环伏安法测量值（如释放幅度和再摄取速率）与被认为功能相关的蛋白质编码基因（如D2型自受体和多巴胺转运体）的表达之间的个体内相关性显示，在对照组中，VTA转录表达和NAc功能常常是不相关的。

Further, relationships between expression and function are experience-dependent, as drinking resulted in the emergence or change in directionality of many transcript × function relationships. These findings directly call into question the common assumption that transcriptional expression is positively correlated with functional outputs of the encoded proteins – instead, we show that the existence and directionality of these relationships are highly plastic and cannot be assumed.

此外，表达与功能之间的关系依赖于经验，因为饮酒导致了许多转录本×功能关系的出现或方向性改变。这些发现直接质疑了一个常见假设，即转录表达与编码蛋白质的功能输出呈正相关——相反，我们证明了这些关系的存在和方向性具有高度可塑性，不能被简单假设。

Together, this work probes the complex and dynamic relationships between function and gene expression and demonstrates multiple plasticity mechanisms by which chronic alcohol consumption and abstinence modulate the mesolimbic dopamine circuit..

这项工作共同探讨了功能与基因表达之间复杂而动态的关系，并展示了慢性饮酒和戒酒通过多种可塑性机制调节中脑边缘多巴胺回路的方式。

Results

结果

Subjects displayed wide individual difference in alcohol consumption

受试者在酒精消费方面表现出广泛的个体差异

With the goal of translationally modeling human alcohol consumption and relapse, a drinking protocol was devised such that intake was voluntary, alcohol was available for prolonged periods, and macaques had access to alcohol restored after periods of forced abstinence

为了在转化研究中模拟人类的饮酒行为和复发，设计了一种饮酒方案，其中饮酒是自愿的，酒精供应持续较长时间，并且猕猴在被迫戒断一段时间后又能重新获得酒精。

，

. Briefly, 17 alcohol drinking rhesus macaques underwent a schedule-induced polydipsia procedure to initiate voluntary alcohol consumption, followed by 12 months of 22 hours/day open access drinking, a month of forced abstinence, 3 months of open access alcohol reintroduction, a month of forced abstinence, 3 months of open access alcohol reintroduction, and one month of forced abstinence at the end of which subjects were necropsied (Fig. .

简要地说，17只饮酒的恒河猴经历了诱发性多饮程序以启动自愿酒精摄入，随后进行了12个月每天22小时的开放饮酒，一个月的强制戒断，3个月的开放饮酒重新引入，一个月的强制戒断，3个月的开放饮酒重新引入，以及最后一个月的强制戒断，在此之后对实验对象进行了尸检（图。

). Through this protocol, individual differences emerged in average alcohol intakes (g/kg) during the first open access period (Fig.

). 通过这一协议，在第一个开放获取期间，平均酒精摄入量（克/千克）出现了个体差异（图。

), and both reintroduction periods (Fig.

)，以及两个重新引入期（图。

), as well as the sum total of lifetime alcohol consumption (Fig.

），以及一生中酒精消费总量（图。

1天

). Blood was collected longitudinally over the course of alcohol access periods and assayed for blood alcohol concentration, which strongly correlated with the subject’s alcohol consumption during the collection day (Fig.

在酒精获取期间纵向收集血液，并测定血醇浓度，这与受试者在采集当天的酒精摄入量密切相关（图。

). Experiments were performed in two cohorts of subjects which each contained controls and drinkers, and did not differ in alcohol consumption levels (see Methods and Fig.

). 实验在两组受试者中进行，每组均包含对照组和饮酒者，且酒精摄入量水平无差异（见方法和图）。

S1A–D

S1A-D

), body weight, or age (Fig.

）、体重或年龄（图。

S1E, F

S1E，F

). Immediately following necropsy, the brain was blocked into sections including the NAc and the VTA

）。尸检后立即对大脑进行分区，包括NAc和VTA。

. The NAc was kept under physiological conditions for live ex vivo slice recordings whereby fast-scan cyclic voltammetry was used to measure sub-second dopamine release dynamics and probe multiple aspects of dopamine terminal function. The VTA, taken from the same subjects, was flash frozen and subsequently bulk RNA-sequencing was used to probe upstream alcohol-induced changes in transcription.

在生理条件下对NAc进行活体外切片记录，使用快速扫描循环伏安法测量亚秒级多巴胺释放动态，并探究多巴胺终端功能的多个方面。取自相同个体的VTA被快速冷冻，随后通过批量RNA测序来探究上游酒精诱导的转录变化。

Altogether, this design allowed for the investigation of how chronic drinking and protracted abstinence alter presynaptic dopamine terminal function in the NAc core as well as transcriptional changes in the VTA. A unique feature of this within-subject design is the ability to assess relationships between endogenous gene expression in the midbrain soma and functional measurement of axonal dopamine release, reuptake, and regulation (Fig. .

总体而言，这种设计允许研究慢性饮酒和长期戒酒如何改变NAc核心中的突触前多巴胺末端功能，以及VTA中的转录变化。这种主体内设计的一个独特之处在于能够评估中脑体细胞内源基因表达与轴突多巴胺释放、再摄取和调节功能测量之间的关系（图）。

1楼

）。

Fig. 1: Diagram of experimental design and rationale.

图1：实验设计和原理图。

Rhesus macaques (17 alcohol and 11 calorically yoked or housing controls) underwent a drinking (or housing control) protocol designed to uncover individual differences in drinking phenotypes between subjects. Briefly, a schedule induced polydipsia procedure was used to induce voluntary alcohol consumption, followed by 12 months of 22 hour/day open access drinking, a month of forced abstinence, 3 months of open access alcohol reintroduction, a month of forced abstinence, 3 months of open access alcohol reintroduction, and one month of forced abstinence at the end of which subjects were necropsied.

恒河猴（17只为酒精组，11只为热量匹配或饲养对照组）接受了一项饮酒（或饲养对照）方案，旨在揭示个体之间饮酒表型的差异。简而言之，通过诱导性多饮程序促使自愿酒精摄入，随后进行12个月每天22小时的开放饮酒，一个月的强制戒断，3个月的重新开放饮酒，一个月的强制戒断，3个月的重新开放饮酒，最后一个月强制戒断，之后对实验对象进行了尸检。

。

–

Drinking data from each epoch of the self-administration paradigm is shown, with color indicating the same subject throughout.

自我给药范式中每个时期的饮水数据如下所示，颜色表示始终为同一受试者。

Cumulative alcohol intake was calculated over the first period of open access.

累积酒精摄入量是在开放访问的第一阶段计算的。

Average daily alcohol intake during each of the two alcohol reintroduction periods.

两个酒精重新引入期间，平均每日酒精摄入量。

Lifetime intake in g/kg was calculated for each of the 17 alcohol-exposed subjects.

计算了17名酒精暴露受试者每人的终生摄入量（以克/千克计）。

Blood alcohol concentration (in milligram percent [i.e. mg/dL]) was collected weekly, 7 hours after session start, for each subject and was strongly positively associated with the alcohol intake on the same day (two-tailed Pearson’s correlation).

每周收集每个受试者的血液酒精浓度（以毫克百分比计 [即 mg/dL]），在每次实验开始后7小时进行采集，并且与当天的酒精摄入量呈显著正相关（双尾皮尔逊相关）。

After necropsy, the brain was blocked in coronal sections including the nucleus accumbens (NAc) core and the ventral tegmental area (VTA); dopamine dynamics were recorded from the NAc with fast-scan cyclic voltammetry, and gene expression from the upstream VTA region was assessed via bulk RNA-seq. Stimulation parameters and pharmacological manipulations were used to assess different features of dopamine terminal release in the NAc.

尸检后，大脑被切成包含伏隔核（NAc）核心区和腹侧被盖区（VTA）的冠状切片；使用快速扫描循环伏安法记录NAc中的多巴胺动态，并通过批量RNA测序评估上游VTA区域的基因表达。刺激参数和药理学操作用于评估NAc中多巴胺末梢释放的不同特征。

These effects were then correlated with gene expression measures from the VTA to assess the relationship between terminal function and upstream transcription. Unless otherwise indicated, values indicate mean ± SEM. (drinkers: .

这些效应随后与来自VTA的基因表达测量值相关联，以评估终端功能与上游转录之间的关系。除非另有说明，数值表示平均值±SEM。（饮酒者：。

= 17) Created in BioRender. Siciliano, C. (2025)

= 17) 在BioRender中创建。Siciliano, C. (2025)

https://BioRender.com/l92m987

。

Full size image

全尺寸图像

Protracted abstinence from chronic drinking results in minimal transcriptional perturbation in the VTA

长期戒断慢性饮酒在VTA中导致最小的转录扰动。

To search for potential mechanisms mediating long-term neuronal dysregulation induced by cycles of chronic alcohol consumption and abstinence, full transcriptomic RNA-sequencing was conducted on VTA tissue, which houses the somatic compartment of the mesolimbic dopamine circuit, following the alcohol self-administration procedure described above.

为了寻找介导由慢性酒精消费和戒断循环引起的长期神经元失调的潜在机制，按照上述酒精自我给药程序，对包含中脑边缘多巴胺回路体细胞区室的VTA组织进行了全转录组RNA测序。

Given the persistence of aberrant behaviors associated with heavy drinking, and the large body of work implicating the mesolimbic dopamine system in alcohol-related behaviors, we hypothesized that transcriptional networks, and dopamine neuron-specific genes in particular, would be markedly dysregulated following abstinence from chronic drinking.

鉴于与大量饮酒相关的异常行为的持续存在，以及大量研究表明中脑边缘多巴胺系统与酒精相关行为有关，我们假设在戒除慢性饮酒后，转录网络特别是多巴胺神经元特异性基因会显著失调。

In stark contrast to our hypothesis, differential expression analysis showed that, following Benjamini-Hochberg false discovery rate adjustment, zero genes were differentially expressed between drinkers and controls (Fig. .

与我们的假设形成鲜明对比的是，差异表达分析显示，在使用Benjamini-Hochberg错误发现率调整后，饮酒者和对照组之间没有任何基因存在差异表达（图。

). Also contrary to our hypothesis, but corroborating the lack of differential gene expression, dimensionality reduction via principal component analysis did not produce appreciable segregation between drinkers and controls across pairwise visualizations of the top five principal components (Figs.

). 与我们的假设相反，但证实了缺乏差异基因表达，通过主成分分析进行的降维并没有在前五个主成分的成对可视化中显示出饮酒者和对照组之间明显的分离（图。

，

). This suggests that long periods of abstinence were not characterized by gross changes in the expression of individual genes in the VTA.

). 这表明长时间的戒断并未导致VTA中个别基因表达的显著变化。

Fig. 2: Minimal changes to the expression of individual genes or coexpression networks in midbrain following chronic alcohol intake and protracted abstinence.

图2：慢性饮酒和长期戒断后中脑个别基因表达或共表达网络的最小变化。

Deep sequencing was performed on the ventral tegmental area (VTA) from 28 macaques (paired-end 150 bp, roughly 150 million read pairs per sample). Reads were aligned to the

对来自28只猕猴的腹侧被盖区（VTA）进行了深度测序（双端150 bp，每个样本大约1.5亿个读对）。读段被比对到

Macaca mulatta

猕猴

genome (Mmul_10) and read counts were calculated and normalized for each subject.

基因组（Mmul_10）并对每个受试者的读数进行计算和标准化。

A volcano plot of gene expression showing log

显示基因表达对数的火山图

(fold change) between drinkers and controls and the raw p-value. After Benjamini Hochberg FDR <0.05 correction of p-values, zero genes were significantly different between drinkers and controls. While no genes passed false-discovery rate correction, the transcripts with the five lowest

（折叠变化）在饮酒者和对照组之间以及原始p值。在对p值进行Benjamini Hochberg FDR <0.05校正后，饮酒者和对照组之间没有任何基因显著差异。尽管没有基因通过错误发现率校正，但具有五个最低值的转录本

values are labeled and highlighted in green.

值被标记并以绿色突出显示。

Dimensionality reduction via principal component analysis did not produce appreciable segregation between drinkers and controls across pairwise visualizations of the top 2 principal components.

通过主成分分析进行的降维并未在前两个主成分的成对可视化中显示出饮酒者和对照组之间的明显分离。

Adjacency plot of weighted co-expression between the top 500 genes defining modules after WGNA analysis.

WGNA分析后定义模块的前500个基因之间的加权共表达邻接图。

Tree dendrogram of weighted gene co-expression. Module eigengenes that reached a threshold of 0.99 or above were included for network analysis.

加权基因共表达的树状图。达到0.99或以上阈值的模块特征基因被纳入网络分析。

Heatmap showing module-trait relationships by group from WGCNA analysis. Color axis indicates Pearson’s

热图显示了来自WGCNA分析的组间模块与性状的关系。颜色轴表示皮尔逊相关系数。

value.

价值。

Genes and connectivity, and significant GO terms of interest of the

基因和连通性，以及感兴趣的显著GO术语

dark grey

深灰色

module most correlated with controls.

与控制最相关的模块。

Genes and connectivity, and significant GO terms of interest of the

基因和连通性，以及感兴趣的显著GO术语

sienna 3

赤褐色 3

module most correlated with alcohol drinkers. All statistical tests were two-tailed. (*

与饮酒者最相关的模块。所有统计检验均为双尾检验。(*

≤ 0.05, **

≤ 0.01, ***

≤ 0.001, ****

≤ 0.0001) (controls:

≤ 0.0001)（对照组：

= 11; drinkers:

= 11；饮酒者：

= 17).

Full size image

全尺寸图像

To evaluate whether transcriptional differences were maintained within networks of co-expressed genes, a weighted gene correlation network analysis was conducted

为了评估转录差异是否在共表达基因网络中得以维持，进行了加权基因相关网络分析。

(Fig.

（图。

2C, D

2C，D

). This analysis allows for the clustering of co-expressed genes into network modules, without a priori information of gene function

）。这种分析允许将共表达的基因聚类到网络模块中，而无需预先了解基因功能信息。

(Supplemental Dataset

（补充数据集

). Through this analysis, we found 4 modules that were correlated with the control condition, and one module correlated with having a history of alcohol self-administration (Fig.

通过此分析，我们发现了4个与对照条件相关的模块，以及一个与有酒精自我给药历史相关的模块（图。

). Using gene ontology analysis, the module most correlated with controls,

). 通过基因本体分析，与对照组最相关的模块，

dark grey

深灰色

, was enriched for pathways including vesicle-mediated transport, such as the kinesin-like protein, KIF1C, and ATPase activity, such as probable ATP-dependent RNA helicase 10, DDX10 (Fig.

，包括囊泡介导的运输途径（如驱动蛋白样蛋白 KIF1C）和 ATP 酶活性（如可能的 ATP 依赖性 RNA 解旋酶 10，DDX10）在内的通路得到了富集（图。

2楼

). The module

）。该模块

sienna

棕红色

, most correlated with alcohol drinkers, was enriched for genes involved in the postsynapse, such as semaphorin 6 A (sema6a) and misfolded protein binding, among others (Fig.

，与饮酒者最密切相关，富集了参与突触后相关基因，例如semaphorin 6 A（sema6a）和错误折叠蛋白结合等（图。

). From this data, we concluded that there were minimal global changes in gene expression within the VTA that were induced by repeated cycles of chronic alcohol drinking and long-term abstinence and thus, behavioral effects of abstinence are likely maintained through mechanisms beyond up- or downregulation of specific genes or networks..

). 从这些数据中，我们得出结论，在反复的慢性饮酒和长期戒酒过程中，VTA内基因表达的全局变化很小，因此，戒酒的行为效应可能通过特定基因或网络的上调或下调之外的机制来维持。

Alcohol consumption constrains dynamic range of accumbal dopamine release and augments reuptake rate

酒精摄入限制了伏隔核多巴胺释放的动态范围并增加了再摄取速率。

In parallel, we sought to assess if functional alterations in accumbal dopamine terminals were present in these subjects. Voltammetric detection of extracellular dopamine concentration was used to monitor evoked release and reuptake kinetics in real-time in ex vivo brain slices

同时，我们试图评估这些受试者中是否存在伏隔核多巴胺终端的功能改变。使用伏安法检测细胞外多巴胺浓度，以实时监测离体脑片中诱发释放和再摄取动力学。

. We first assessed the excitability of dopamine terminals by comparing release magnitude across a range of stimulation intensities (50-900 µA, single pulse, 4 ms, monophasic stimulations) (Fig.

我们首先通过比较一系列刺激强度（50-900 µA，单脉冲，4 ms，单相刺激）下的释放幅度来评估多巴胺终端的兴奋性（图。

). We found that, while groups displayed similar EA

）。我们发现，尽管各组表现出相似的EA

values (Excitatory Amplitude 50, or the stimulation amperage required to produce a half-maximal excitatory dopamine response), the relationship between stimulation intensity and dopamine release was altered by alcohol history such that drinkers showed decreased dopamine release evoked by high intensity stimulations.

值（兴奋幅度50，或产生半最大兴奋性多巴胺反应所需的刺激电流），酒精历史改变了刺激强度与多巴胺释放之间的关系，使得饮酒者在高强度刺激下表现出多巴胺释放减少。

Further, chronic drinking and abstinence resulted in decreased dynamic range of dopamine release, as measured by the differential between the upper and lower plateaus of the input-output curve (Fig. .

此外，慢性饮酒和戒酒导致多巴胺释放的动态范围缩小，这是通过输入-输出曲线的上下平台之间的差异来测量的（图。

3B–D

). For the remainder of experiments, release was evoked by 350 µA stimulations, near the shared EA

). 在其余的实验中，释放是由接近共享EA的350 µA刺激引发的。

for drinkers and controls (Fig.

对于饮酒者和对照组（图。

）。

Fig. 3: Chronic voluntary alcohol consumption retricts dynamic range of accumbal dopamine release.

图3：慢性自愿性酒精摄入限制了伏隔核多巴胺释放的动态范围。

An input-output curve showing the peak dopamine release (nA) evoked by single pulse stimulations across ascending intensities (50–900 µA). Curves were fit with a 4-parameter sigmoidal regression, and best-fit values are shown with 95% confidence band and half-maximal excitatory amperage (EA

输入-输出曲线显示了由不同强度（50-900 µA）单脉冲刺激引发的峰值多巴胺释放（nA）。曲线通过四参数S型回归拟合，最佳拟合值附带95%置信区间和半最大兴奋电流（EA）。

) indicated (controls: EA

`) 表示（控制：EA`

= 316.4 µA; drinkers: EA

= 316.4 微安；饮酒者：EA

= 315.9 µA).

= 315.9 微安）。

–

Comparison of best-fit values between drinkers and controls.

饮酒者与对照组的最佳拟合值比较。

Subjects with ethanol history have an attenuated upper asymptote compared to controls, indicative of decreased maximal dopamine release magnitude (unpaired

有乙醇史的受试者与对照组相比，其上限渐近线有所减弱，这表明最大多巴胺释放量减少（不成对）。

-test;

-测试；

= 2.710,

= 0.0118).

= 0.0118)。

There is no difference between groups at the bottom of the curve plateau (unpaired

曲线平台底部的组之间没有差异（未配对

-test;

-测试；

= 1.033,

= 0.3110).

= 0.3110)。

Drinkers show a decreased span of the input-output curve [upper plateau minus lower plateau] compared to control subjects suggesting a lower dynamic range of dopamine release (unpaired

饮酒者显示出输入-输出曲线的跨度减少（上平台减去下平台），与对照组相比，表明多巴胺释放的动态范围较低（未配对）。

-test;

-测试；

= 2.365,

= 0.0258).

= 0.0258)。

There was no difference in the EA

EA没有差异

between drinkers and controls (unpaired

饮酒者和对照组之间（未配对

-test;

-测试；

= 0.01,

= 0.99). (controls:

= 0.99)。（对照组：

= 3; drinkers:

= 3；饮酒者：

= 7) All statistical tests were two-tailed and values indicate mean ± SEM. (*

= 7) 所有统计检验均为双尾检验，数值表示为平均值±标准误。(*

≤ 0.05, **

≤ 0.01, ***

≤ 0.001, ****

≤ 0.0001).

≤ 0.0001)。

Full size image

全尺寸图像

Evidence suggests that ongoing alcohol intake produces a hypodopaminergic state through increased activity of inhibitory presynaptic regulators, including increased rate of dopamine reuptake from the extracellular space through augmented DAT efficiency

证据表明，持续的酒精摄入通过增加抑制性突触前调节因子的活性，包括通过增强的DAT效率加快从细胞外空间重摄取多巴胺的速度，从而产生一种低多巴胺能状态。

，

, but whether this effect is maintained throughout protracted abstinence from voluntary alcohol drinking remains untested. One pulse stimulations (350 µA, 4 ms, monophasic) were used to evoke dopamine release (Fig.

，但这种效应是否在长期戒断自愿酒精饮用的过程中持续存在仍未得到验证。使用单脉冲刺激（350 µA，4 ms，单相）来诱发多巴胺释放（图。

). Observed peak dopamine concentrations are a product of the amount of dopamine released and the rate of ongoing dopamine reuptake, parameters which can be dissociated using Michaelis-Menten modeling. Using this approach, we found no group difference in dopamine release evoked by a 350 µA stimulation (Fig. .

）。观察到的多巴胺峰值浓度是多巴胺释放量和持续的多巴胺再摄取速率的产物，这些参数可以使用米氏方程建模来分离。使用这种方法，我们发现由350 µA刺激引发的多巴胺释放没有组间差异（图. 。

). However, maximal rate of dopamine reuptake (V

). 然而，多巴胺再摄取的最大速率 (V

max

最大值

) was increased in subjects with a history of chronic alcohol self-administration (Fig.

）在有慢性酒精自我给药史的受试者中增加（图。

). These results are consistent with a hypodopaminergic state

这些结果与低多巴胺能状态一致

，

, which is consistently observed in the mesolimbic system of humans with AUD and thought to be critical to symptomatology

，这在人类AUD的中脑边缘系统中被一致观察到，并被认为对症状学至关重要。

，

, and provide evidence that this state persists in prolonged abstinence.

，并提供证据证明这种状态在长期戒断中持续存在。

Fig. 4: Chronic drinking induced long-lasting changes in dopamine reuptake concomitant with synchrony between upstream transcription and downstream dopamine release dynamics.

图4：长期饮酒诱导了多巴胺再摄取的持久变化，同时伴随着上游转录和下游多巴胺释放动力学之间的同步性。

Representative normalized concentration versus time traces, pseudo-color plots, and current by voltage traces for a drinker and a control subject.

饮酒者和对照受试者的代表性归一化浓度与时间轨迹、伪彩色图以及电流与电压轨迹。

There was no difference in dopamine release between drinkers and controls (unpaired

饮酒者和对照组之间的多巴胺释放没有差异（未配对

-test;

-测试；

= 1.13,

= 1.13，

= 0.2647).

= 0.2647)。

Alcohol history increased the rate of dopamine reuptake (V

酒精史增加了多巴胺再摄取的速率 (V

max

最大值

) (unpaired

) (未配对

-test;

-测试；

= 2.239,

= 0.0302).

= 0.0302)。

–

The best-fit linear regression is plotted and Pearson’s correlation coefficient

最佳拟合线性回归图和皮尔逊相关系数

and

和

values are reported as an inset. Covariance between expression and function emerged following drinking:

值被报告为嵌入图。饮酒后表达与功能之间的协方差显现：

values of 0.6 or greater were observed for

观察到的值为0.6或更大

(D)

the dopamine receptor 2 (

多巴胺受体2 (

DRD2

(E)

（E）

the kappa opioid receptor (

κ阿片受体 (

OPRK1

(F)

（F）

the dopamine transporter (

多巴胺转运体 (

DAT

数据

(G)

（G）

and the vesicular monoamine transporter 2 (

和囊泡单胺转运体2 (

VMAT2

）。

For

为了

DRD2

, there was a trend towards a positive correlation between gene expression and V

，基因表达与V之间存在正相关的趋势

max

最大值

in drinkers, not controls.

在饮酒者中，而非对照组中。

我

Expression of

表达

OPRK1

was not correlated with V

与V不相关

max

最大值

in either group. V

在任一组中。V

max

最大值

and transporter expression did not correlate in controls, but in drinkers, V

在对照组中，转运蛋白表达没有相关性，但在饮酒者中，V

max

最大值

showed a positive correlation with the expression of

与表达呈正相关关系

DAT

数据

(K)

（K）

and

和

VMAT2

(

）。

The mean slope of gene expression over terminal function was greater in drinkers (unpaired

饮酒者在终端功能上的基因表达平均斜率更大（未配对

-test;

-测试；

= 4.14,

= 0.001). The mean slope differed from zero in drinkers (one sample

= 0.001）。饮酒者的平均斜率与零不同（单样本

-test; t

-测试；t

= 4.18.

= 4.18。

= 0.004), but not in controls (one sample

= 0.004)，但在对照组中不显著（单样本

-test; t

-测试；t

= 1.01,

= 0.35).

= 0.35)。

，

北

The best-fit linear regression is shown with a 95% confidence band. Inset: Spearman’s

最佳拟合线性回归显示为95%置信带。插图：Spearman’s

- and

- 和

-values.

-值。

There was no correlation between expression of homosynaptic regulators and terminal release in control subjects.

在对照受试者中，同突触调节因子的表达与末梢释放之间没有相关性。

北

Alcohol history induced a positive correlation between gene expression and function. All statistical tests were two-tailed and unless otherwise indicated, values indicate mean ± SEM. (*

酒精历史诱导了基因表达与功能之间的正相关。所有统计检验均为双尾检验，除非另有说明，数值表示平均值±标准误。(*

≤ 0.05, **

≤ 0.01, ***

≤ 0.01，***

≤ 0.001, ****

≤ 0.0001,

††

≤ 0.01 vs 0) (controls:

≤ 0.01 vs 0) （对照组：

= 8 [23 slices]; drinkers:

= 8 [23片]；饮用者：

= 8 [23 slices]).

= 8 [23片]）。

Full size image

全尺寸图像

Chronic drinking induces intercompartmental synchrony between somatic transcription and axonal dopamine dynamics

慢性饮酒诱导躯体转录和轴突多巴胺动力学之间的区室同步性

We next asked how these persistent changes in dopamine terminal function were maintained through abstinence, considering that the abstinence period is 5-10 fold longer than the biological half-life/turnover rate of the dopamine transporter protein (3-6 days)

接下来，我们探讨了这些多巴胺末端功能的持续变化是如何在戒断期间维持的，考虑到戒断期比多巴胺转运蛋白的生物半衰期/周转率（3-6天）长5-10倍。

，

, and it can therefore be reasonably assumed that none of the dopamine transporters present in the brain at this point have ever interacted directly with alcohol or its metabolites. To address this question, we assessed the within-subject relationship between dopamine terminal function in the NAc and the upstream expression of genes known to encode for regulators of dopamine release and reuptake localized on accumbal dopamine terminals arising from the VTA.

，因此可以合理假设，此时大脑中存在的多巴胺转运蛋白都没有与酒精或其代谢物直接相互作用。为了解决这个问题，我们评估了NAc中多巴胺终端功能与已知编码调节多巴胺释放和再摄取的基因上游表达之间的关系，这些基因位于来自VTA的伏隔核多巴胺终端上。

Thus, dopamine release (Fig. .

因此，多巴胺释放（图。

4D–G

) and V

）和 V

max

最大值

(Fig.

（图。

4H–K

) were correlated with the VTA expression of a priori selected genes encoding for: the D2-type dopamine autoreceptor (

）与预先选择的基因在VTA中的表达相关，这些基因编码：D2型多巴胺自受体（

DRD2;

ENSMMUG00000014334), the kappa opioid receptor (

ENSMMUG00000014334)，κ阿片受体 (

OPRK1

; ENSMMUG00000065247), the dopamine transporter (

；ENSMMUG00000065247），多巴胺转运蛋白（

SLC6A3;

ENSMMUG00000005198; referred to throughout by the gene alias

ENSMMUG00000005198；全文中使用基因别名指代

DAT

数据

), and the vesicular monoamine transporter-2 (

），以及囊泡单胺转运体-2（

SLC18A2;

ENSMMUG00000014817; referred to throughout by the gene alias

ENSMMUG00000014817；全文中以其基因别名指代

VMAT2

). The receptors encoded by

）。受体由编码

DRD2

and

和

OPRK1

are G

是 G

i/o

输入/输出

coupled GPCRs which are highly expressed on presynaptic dopamine terminals in the NAc core, and activation of either receptor results in decreased dopamine release and dopamine terminal excitability

在NAc核心的突触前多巴胺末端高度表达的耦合GPCR，任一受体的激活都会导致多巴胺释放和多巴胺末端兴奋性降低。

，

. The expression of both

. 两者的表达

DRD2

and

和

OPRK1

in the VTA was surprisingly not correlated with dopamine release in the NAc in controls, but showed a positive correlation in drinkers (Fig.

在VTA中，与NAc中的多巴胺释放的相关性在对照组中令人惊讶地不存在，但在饮酒者中显示出正相关（图。

4D–E

）。

DRD2

expression was also not correlated with V

表达式也与V不相关

max

最大值

in controls but showed a trend toward a positive correlation in drinkers (Fig.

在对照组中，但在饮酒者中显示出正相关的趋势（图。

4小时

) and neither group showed a relationship between V

）并且两组之间均未显示出与V的关系

max

最大值

and

和

OPRK1

expression (Fig.

表达式（图。

). The transporters encoded by

）。编码的转运蛋白

DAT

数据

and

和

VMAT2

are enriched in dopamine-releasing neurons in the VTA and are responsible for the reuptake through the cytoplasmic membrane and the vesicular packaging of dopamine, respectively (Fig.

在VTA中富含释放多巴胺的神经元，并分别负责通过细胞质膜的再摄取和多巴胺的囊泡包装（图。

)

，

. Once again, only in drinkers, expression of the

. 再次强调，只有在饮酒者中，表达

DAT

数据

and

和

VMAT2

transporter encoding genes in the VTA showed a trend toward a positive correlation with dopamine release (Fig.

VTA中编码转运蛋白的基因显示出与多巴胺释放呈正相关的趋势（图。

4F, G

4楼，G

) and were positively correlated with V

）并与V呈正相关关系

max

最大值

(Fig.

（图。

4J, K

4J，K

). Thus, across these a priori genes of interest, variance stabilized read counts from the VTA and dopamine dynamics in the NAc did not correlate in control subjects, contrary to the canonical assumption that transcript expression is tightly related to protein expression and thereby function.

）。因此，在这些预先关注的基因中，VTA 的方差稳定读数与 NAc 中的多巴胺动态在对照组中并未相关，这与转录表达紧密关联蛋白表达从而影响功能的经典假设相反。

The a priori aim of the analyses described above was to assess how increased transcriptional expression might be driving persistent changes in dopamine reuptake throughout abstinence. Unexpectedly, these analyses revealed a striking pattern which was apparent across comparisons whereby the expression of individual genes appeared decorrelated with dopamine release and reuptake in controls, but displayed synchrony between expression and function in animals with a history of alcohol use.

上述分析的先验目的是评估转录表达的增加如何在戒断期间驱动多巴胺再摄取的持续变化。然而，这些分析揭示了一个显著的模式，在不同比较中均明显可见：在对照组中，单个基因的表达似乎与多巴胺释放和再摄取不相关，但在有酒精使用史的动物中，表达与功能之间显示出同步性。

Indeed, a comparison of the slopes of the linear regressions for each group revealed that a history of alcohol intake and abstinence increased the mean slope compared to controls (Fig. .

确实，对各组线性回归斜率的比较表明，与对照组相比，有饮酒和戒酒历史的组平均斜率增加（图。

4升

). Further, comparison within each group revealed that drinkers showed a positive mean slope, whereas the mean slope for controls did not differ from zero (Fig.

). 此外，各组内的比较显示，饮酒者表现出正的平均斜率，而对照组的平均斜率与零无差异（图。

4升

). This pattern raises the intriguing possibility that cycles of alcohol use and abstinence may alter the fundamental relationship between somatic transcriptional activity and axonal function independent of changes, or lack thereof, in transcript expression levels. To explore this possibility, data were transformed to a within-group rank order for each dependent variable.

这种模式引发了一个有趣的可能性，即酒精使用和戒酒的周期可能会改变体细胞转录活动与轴突功能之间的基本关系，而与转录表达水平的变化与否无关。为了探索这种可能性，数据被转换为每个因变量的组内秩次排序。

Thus, each subject was assigned a value from 1 to 8 denoting lowest to highest expression for each of the 4 transcripts, paired with a 1 to 8 value indicating rank order of V.

因此，每个受试者被分配了一个从1到8的值，表示4种转录本中每种的最低到最高表达量，并配有一个从1到8的值，表示V的排序。

max

最大值

and a 1 to 8 value indicating rank order of dopamine release magnitude. This allowed all 8 correlations (64 total x-y pairs per group) to be plotted and analyzed in a single coordinate plane with rank-expression on the y-axis and rank-function on the x-axis. Overall, there was no discernable relationship between gene expression and function in controls, with an .

以及一个表示多巴胺释放量排名顺序的1到8的值。这使得所有8个相关性（每组总共64个x-y对）可以在一个坐标平面上绘制和分析，其中y轴为排名-表达，x轴为排名-功能。总体而言，在对照组中基因表达与功能之间没有可辨别的关系。

-value approaching zero (Fig.

-值接近零（图。

4米

). On the other hand, there was a strong positive relationship, indicating tight synchrony between gene expression and terminal function, after chronic alcohol consumption and abstinence (Fig.

). 另一方面，存在一种强烈的正相关关系，表明在长期饮酒和戒酒后基因表达与终末功能之间有紧密的同步性（图。

）。

Given the surprising, experience-dependent induction of intercompartmental synchrony between transcription and function, and the striking degree of similarity across the correlations in drinkers, we next sought to probe the veracity of these findings and test whether these relationships showed selectivity for the genes of interest.

鉴于在转录和功能之间观察到的令人惊讶的、依赖于经验的跨区室同步性，以及饮酒者中相关性表现出的高度相似性，我们接下来试图验证这些发现的真实性，并测试这些关系是否对目标基因具有选择性。

Thus, we next performed a secondary analysis on a set of genes selected .

因此，我们接下来对一组选定的基因进行了二次分析。

a posteriori

后验的

. This set was matched in size (4 transcripts) and type of encoded protein (2 GPCR encoding genes, and 2 solute carrier-family transporter encoding genes), but were selected based on known enrichment in non-dopamine releasing neurons within the VTA

这个集合在大小（4个转录本）和编码蛋白的类型（2个GPCR编码基因和2个溶质载体家族转运蛋白编码基因）上进行了匹配，但选择依据是它们在VTA内非多巴胺能神经元中的已知富集情况。

. Expression of the genes encoding for dopamine receptor 1 (

. 编码多巴胺受体1的基因表达 (

DRD1

; ENSMMUG00000005147), dopamine receptor 5 (

；ENSMMUG00000005147），多巴胺受体 5 (

DRD5

; ENSMMUG00000002783), vesicular glutamate transporter 2 (

；ENSMMUG00000002783），囊泡谷氨酸转运体2（

SLC17A6;

ENSMMUG00000020368; referred to throughout by the gene alias

ENSMMUG00000020368；全文中使用基因别名指代

VGLUT2

), and vesicular GABA transporter (

），以及囊泡GABA转运体（

SLC32A1;

ENSMMUG00000003376; referred to throughout by the gene alias

ENSMMUG00000003376；全文中以基因别名指代

VGAT

) was thus compared with dopamine release and reuptake using the same analyses as described above (Fig.

）因此与使用上述相同分析方法的多巴胺释放和再摄取进行了比较（图。

S3A-H

）。

Importantly, we found that transcriptional expression of these

重要的是，我们发现这些的转录表达

a posteriori

后验的

-selected genes within the VTA were poor predictors of downstream dopaminergic function within the NAc in both controls and drinkers, corroborating the relationships observed in drinkers for dopamine-regulating transcripts are unlikely to be epiphenomenological. Across the

-VTA内选定的基因在对照组和饮酒者中对NAc下游多巴胺功能的预测能力较差，这证实了在饮酒者中观察到的多巴胺调节转录本的关系不太可能是表观现象。在整个

a posteriori

后验的

-selected gene set, there was only one positive correlation found: that between

-选定的基因集中，仅发现一个正相关：即

DRD5

expression and dopamine release in controls (Fig.

对照组中的表达和多巴胺释放（图。

S3B

) that was lost in drinkers. Further, the mean slope of the linear regressions did not differ from zero in both controls and drinkers (Fig.

) 在饮酒者中丢失了。此外，线性回归的平均斜率在对照组和饮酒者中均未与零不同（图。

S3I

). Likewise, when gene expression and terminal function were transformed to a within-group rank order, as described above, there was no correlation between rank expression and rank function in drinkers or controls (Fig.

同样，当基因表达和终端功能被转换为上述的组内秩次时，饮酒者或对照组中秩次表达与秩次功能之间不存在相关性（图。

S3J-K

). In the dopamine-regulating transcripts, the apparent induction of synchrony between transcription and function occurred in the absence of differential expression of these transcripts between groups (Fig.

）。在调节多巴胺的转录本中，转录和功能之间的同步性明显增强，而组间这些转录本的表达并未出现差异（图。

). Of interest, neither dopamine release, dopamine reuptake, nor transcriptional expression levels correlated with lifetime alcohol intake in the drinkers (Figs.

有趣的是，饮酒者的多巴胺释放、多巴胺再摄取以及转录表达水平均与终生酒精摄入量无相关性（图。

，

). Together, these data show that a history of alcohol consumption induces synchrony between the transcriptional activity of autoregulatory dopaminergic modulators during protracted abstinence. Broadly, these results also call into question long-held assumptions regarding transcriptional control of synaptic function..

这些数据共同表明，饮酒史会在长期戒断期间诱导自调节多巴胺调节因子的转录活动同步性。更广泛地说，这些结果也对长期以来关于突触功能转录控制的假设提出了质疑。

Supersensitivity of axonal kappa opioid receptors in the accumbens persists into protracted abstinence

伏隔核轴突κ阿片受体的超敏性持续到长期戒断期

One of the inhibitory regulators of terminal dopamine release, the kappa opioid receptor, has been shown to play a causal role in behaviors characteristic of AUD and may serve as a potential therapeutic target. A large body of work across species and methods of alcohol administration has shown that sensitivity of the kappa opioid receptor is augmented by chronic, ongoing alcohol exposure, resulting in hyperpolarization of dopamine terminals and thereby likely contributing to the hypodopaminergic state seen in AUD.

κ阿片受体是终末多巴胺释放的抑制性调节因子之一，已被证明在AUD（酒精使用障碍）特征行为中起因果作用，并可能成为潜在的治疗靶点。大量跨物种和酒精给药方式的研究表明，κ阿片受体的敏感性会因长期持续的酒精暴露而增强，导致多巴胺终端超极化，从而可能促成AUD中出现的低多巴胺能状态。

，

. The importance of alcohol-induced plasticity of the kappa opioid receptor system is underscored by findings highlighting a role for selective kappa opioid receptor antagonists in the treatment of AUD

酒精诱导的κ阿片受体系统可塑性的重要性通过研究结果得以凸显，这些结果强调了选择性κ阿片受体拮抗剂在AUD治疗中的作用。

，

. Despite the apparent importance of kappa opioid receptor activity in ongoing drinking behaviors, the precise signaling mechanisms underlying kappa opioid receptor inhibition of dopamine release remains unclear, and whether drinking-induced plasticity in this system persists beyond the acute withdrawal phase remains untested..

尽管κ阿片受体活动在持续的饮酒行为中显然具有重要性，但κ阿片受体抑制多巴胺释放的确切信号机制仍不清楚，且该系统中由饮酒引发的可塑性是否在急性戒断阶段之后仍然持续也尚未得到验证。

To functionally assess the sensitivity of kappa opioid receptor regulation of presynaptic dopamine release, a selective, unbiased kappa opioid receptor agonist, U50,488 was bath applied to ex vivo slices and 1 pulse dopamine release was recorded and compared to pre-drug baseline values (Fig.

为了从功能上评估κ阿片受体对突触前多巴胺释放的调节敏感性，将一种选择性、无偏倚的κ阿片受体激动剂U50,488浴应用于离体切片，并记录1脉冲多巴胺释放，与给药前的基线值进行比较（图。

). Dopamine release was inhibited to a greater degree across concentrations of U50,488 in drinkers compared to controls, demonstrating that supersensitivity of the kappa opioid receptor persists through long-term periods of abstinence (Fig.

). 在饮酒者中，与对照组相比，U50,488的各种浓度对多巴胺释放的抑制程度更大，这表明κ阿片受体的超敏性在长期戒断期间仍然存在（图。

). In separate slices, a non-selective inhibitor, 500 µM barium chloride (BaCl

）。在单独的切片中，使用非选择性抑制剂500 µM氯化钡（BaCl

)

, was used to block G protein-coupled inwardly rectifying K

，被用来阻断G蛋白偶联的向内整流K通道

加号

channels (GIRKs)—a downstream effector activated by kappa opioid receptors among others

通道（GIRKs）——由κ阿片受体等激活的下游效应器

，

, which act to reduce membrane excitability. Bath application of BaCl

，这些作用会降低膜的兴奋性。氯化钡的浴液应用

induced a greater increase in evoked dopamine release in drinkers compared to controls, suggesting augmented basal GPCR-mediated inhibitory regulation of dopamine terminals in the NAc core (Fig.

与对照组相比，饮酒者诱发的多巴胺释放增加更为显著，这表明NAc核心区域多巴胺终端的基底GPCR介导的抑制性调节增强（图。

）。

Fig. 5: Chronic drinking drove persistent upregulation of kappa opioid receptor control of dopamine release and altered function-transcription relationships.

图5：长期饮酒导致κ阿片受体对多巴胺释放的持续上调，并改变了功能-转录关系。

Representative dopamine release (1 µM U50,488).

代表性的多巴胺释放（1 µM U50,488）。

U50,488 decreased dopamine release to a greater extent in drinkers (repeated measures two-way ANOVA; dose: F

U50,488在饮酒者中减少多巴胺释放的程度更大（重复测量双因素方差分析；剂量：F

1, 14

= 79.28,

< 0.0001; group: F

< 0.0001；组别：F

1, 14

= 4.81,

= 4.81，

< 0.05; dose x group: F

< 0.05；剂量 x 组别：F

1, 14

= 0.01,

= 0.91).

= 0.91)。

C语言

BaCl

氯化钡

increased dopamine release to a greater extent in drinkers (unpaired

饮酒者多巴胺释放增加的程度更大（不成对

-test;

-测试；

= 3.41,

= 0.02; controls:

= 0.02；对照组：

= 3, drinkers:

= 3，饮酒者：

= 4).

= 4)。

Nalfurafine decreased dopamine release in both groups (two-way ANOVA; concentration: F

Nalfurafine 降低了两组中的多巴胺释放（双因素方差分析；浓度：F

1, 6

= 30.76,

= 30.76，

= 0.0015; group: F

= 0.0015；组别：F

1, 6

= 0.10,

= 0.76; concentration x group: F

= 0.76；浓度 x 组别：F

1, 6

= 0.75,

= 0.42; Šídák’s test: baseline vs. nalfurafine; controls:

= 0.42；Šídák检验：基线 vs. 那呋拉非；对照组：

= 4.06,

= 4.06，

= 0.01,

= 3; drinkers:

= 3；饮酒者：

= 3.82,

= 0.02,

= 5).

= 5)。

–

Best-fit linear regression. Inset: Pearson’s

最佳拟合线性回归。插图：皮尔逊

and

和

-values.

-值。

Change in dopamine release by U50,488 (300 nM) was correlated with

U50,488（300 nM）引起的多巴胺释放变化与

OPRK1

expression in drinkers, not controls.

饮酒者而非对照组中的表达。

U50,488 potency was not correlated with

U50,488 的效力与

PDYN

, (

，（

)

ARRB1

, or (

，或者（

)

ARRB2

expression in either group.

任意一组中的表达式。

我

There was no association between

之间没有关联

OPRK1

expression and efficacy of 1 µM U50,488 in controls.

对照组中1 µM U50,488的表达和功效。

Efficacy was not associated with

疗效未与之相关联

PDYN

or (

或 (

)

ARRB1

in either group.

在任一组中。

ARRB2

and efficacy were not correlated in controls, but negatively correlated in drinkers.

在对照组中，两者之间没有相关性，但在饮酒者中呈负相关。

Mean slope of the regressions was greater in controls (unpaired

对照组回归的平均斜率更大（未配对

-test;

-测试；

= 3.08,

= 3.08，

= 0.008). The mean slope for controls (one sample

= 0.008)。对照组的平均斜率（一个样本

-test;

-测试；

= 3.45,

= 3.45，

= 0.01), but not drinkers (one sample

= 0.01)，但饮酒者不（单样本

-test;

-测试；

= 1.52,

= 1.52，

= 0.17), was greater than zero.

= 0.17)，大于零。

北

In controls, ranked expression was positively correlated with rank sensitivity.

在对照组中，排名表达与排名敏感性呈正相关。

This association was reversed in drinkers. All statistical tests were two-tailed. Unless otherwise indicated, values indicate mean ± SEM. (*

在饮酒者中，这种关联是相反的。所有统计测试均为双尾检验。除非另有说明，数值表示平均值±标准误。(*

≤ 0.05, **

≤ 0.01, ***

≤ 0.001, ****

≤ 0.0001,

小于等于0.0001，

†

≤ 0.05 vs 0) (unless otherwise noted, controls:

≤ 0.05 vs 0)（除非另有说明，对照组：

= 8; drinkers:

= 8；饮酒者：

= 8).

Full size image

全尺寸图像

G protein activity is necessary and sufficient for kappa opioid receptor inhibition of axonal dopamine release, but does not account for alcohol-induced supersensitivity

G蛋白活性对于κ阿片受体抑制轴突多巴胺释放是必要且充分的，但不能解释酒精诱导的超敏性。

We next sought to determine which pathway was involved in the supersensitization of the receptor seen with a history of alcohol intake. Many agonists of the kappa opioid receptor are biased toward either the G protein or β-arrestin signaling cascades downstream of the receptor

我们接下来试图确定哪条通路参与了酒精摄入历史中受体的超敏化。κ阿片受体的许多激动剂倾向于受体下游的G蛋白或β-抑制蛋白信号级联之一。

，

. Each of these pathways has been shown to drive differential cellular and behavioral outcomes of kappa opioid receptor activation

每条通路都被证明能够驱动κ阿片受体激活的差异性细胞和行为结果。

，

. Thus, we aimed to elucidate whether kappa opioid receptor control of dopamine release in the NAc was mediated via the G protein or the β-arrestin pathway. To achieve this goal, we utilized various pharmacological agents with specificity towards targets involved in the G protein or β-arrestin signaling cascades (Fig. .

因此，我们的目标是阐明 kappa 阿片受体对 NAc 中多巴胺释放的控制是通过 G 蛋白还是 β-arrestin 途径介导的。为实现这一目标，我们利用了各种对 G 蛋白或 β-arrestin 信号级联中涉及的靶点具有特异性的药理学试剂（图。

S7A

). An unbiased agonist, such as U50,488, activates both pathways to a similar degree and, in combination with inhibitors of the β-arrestin and G protein pathways, can be used to dissociate the necessity of each in the induction of kappa opioid receptor-mediated dopamine inhibition. Compound 101 (CMPD101) is a GRK2/3 inhibitor, which results in inhibition of β-arrestin signaling by preventing phosphorylation necessary for its recruitment.

). 一种无偏激动剂，如 U50,488，能够以相似的程度激活两条通路，并且结合 β-arrestin 和 G 蛋白通路的抑制剂，可以用来区分每条通路在 kappa 阿片受体介导的多巴胺抑制中的必要性。化合物 101（CMPD101）是一种 GRK2/3 抑制剂，通过阻止其招募所需的磷酸化来抑制 β-arrestin 信号传导。

Thus, in the presence of CMPD101, application of U50,488 would result in preferential recruitment of effectors in the G protein pathway, thereby testing the necessity of the β-arrestin cascade.

因此，在CMPD101存在的情况下，应用U50,488会导致G蛋白通路中效应器的优先招募，从而检验β-arrestin级联反应的必要性。

. N-ethylmaleimide (NEM), in contrast, is a sulfhydryl alkylating agent shown to inhibit G protein signaling, therefore in combination with U50,488, the β-arrestin pathway would be preferentially activated and G protein necessity revealed

相比之下，N-乙基马来酰亚胺（NEM）是一种巯基烷基化剂，已被证明可以抑制G蛋白信号传导，因此与U50,488联合使用时，β-arrestin通路会被优先激活，并揭示G蛋白的必要性。

。

On separate slices from both drinkers and controls, either NEM (50 µM) or CMPD101 (30 µM) was bath applied and evoked dopamine was allowed to re-stabilize. Subsequently, U50,488 (1 µM) was added to the bath to determine agonist-induced effects on dopamine release in the presence of each inhibitor. When normalized to pre-drug baseline dopamine release, there was a differential effect of NEM and CMPD101 through consecutive wash-ons with U50,488 (Fig. .

在来自饮酒者和对照组的不同切片上，分别浴应用NEM（50 µM）或CMPD101（30 µM），并允许激发的多巴胺重新稳定。随后，将U50,488（1 µM）添加到浴中，以确定在每种抑制剂存在下激动剂对多巴胺释放的影响。当将多巴胺释放标准化为药物前的基础水平时，通过连续使用U50,488冲洗，观察到NEM和CMPD101的不同效果（图。

S7B

). When NEM was present, there was no change in dopamine release with the application of U50,488, suggesting that the G protein pathway is necessary for the effect, but there was a near significant decrease in dopamine release in the presence of CMPD101 (Fig.

当存在NEM时，应用U50,488后多巴胺释放没有变化，这表明G蛋白通路对该效应是必要的，但在CMPD101存在的情况下，多巴胺释放有近乎显著的减少（图。

S7C

). The ability of NEM to block kappa opioid receptor inhibition of dopamine release supports the idea that kappa opioid receptor-mediated dopamine downregulation is mediated through G protein downstream signaling cascades.

). NEM阻断κ阿片受体抑制多巴胺释放的能力支持了κ阿片受体介导的多巴胺下调是通过G蛋白下游信号级联介导的观点。

Given the necessity of G protein signaling in kappa opioid receptor-mediated dopamine inhibition, we next wanted to determine whether this signaling cascade was sufficient to regulate release. To this end, nalfurafine, a biased kappa opioid receptor agonist which preferentially activates G protein signaling over the β-arrestin pathway.

鉴于G蛋白信号在κ阿片受体介导的多巴胺抑制中的必要性，我们接下来希望确定这一信号级联是否足以调节释放。为此，我们使用了纳呋拉啡，一种偏向性κ阿片受体激动剂，它优先激活G蛋白信号而非β-抑制蛋白通路。

, was used on separate slices. Indeed, nalfurafine was sufficient to inhibit release in both drinkers and controls; however, in contrast to U50,488, there were no group differences in the ability of nalfurafine to decrease release magnitude (Fig.

，分别用于不同的切片。事实上，纳尔呋拉芬足以抑制饮酒者和对照组的释放；然而，与U50,488不同，纳尔呋拉芬在减少释放量方面没有组间差异（图。

). Together, these pharmacological results suggest that G protein signaling may be crucial to kappa opioid receptor-mediated dopamine inhibition, but increased receptor sensitivity induced by chronic drinking may be due to alterations in other signaling mechanisms such as β-arrestin activity.

）。这些药理学结果共同表明，G蛋白信号传导可能对κ阿片受体介导的多巴胺抑制至关重要，但长期饮酒引起的受体敏感性增加可能是由于其他信号机制（如β-抑制蛋白活性）的改变所致。

Chronic drinking reverses the intercompartmental relationship between somatic transcription and axonal kappa opioid receptor regulation

慢性饮酒逆转了体细胞转录与轴突κ阿片受体调节之间的区室间关系

To determine whether this altered function in the NAc core was associated with upstream VTA transcriptional changes, the magnitude of U50,488-induced decrease in dopamine release in the NAc was correlated with the expression of genes in the VTA encoding the kappa opioid receptor (

为了确定NAc核心中这一改变的功能是否与上游VTA的转录变化相关，U50,488诱导的NAc中多巴胺释放减少的程度与VTA中编码κ阿片受体的基因表达进行了关联分析。

OPRK1

),the precursor to its primary endogenous ligand, prodynorphin (

)，其主要内源性配体前体，强啡肽原（

PDYN

; ENSMMUG00000009984), β-arrestin 1 (

；ENSMMUG00000009984），β-抑制蛋白 1 (

ARRB1

; ENSMMUG00000017529), and β-arrestin 2 (

；ENSMMUG00000017529），以及β-arrestin 2（

ARRB2

; ENSMMUG00000017654). Both β-arrestins are important effectors of kappa opioid receptor, contributing to ligand-gated and constitutive activity

；ENSMMUG00000017654）。两种β-抑制蛋白是κ阿片受体的重要效应因子，有助于配体门控和组成性活性。

，

. The concentrations of U50,488 used, 300 nM, and 1 µM, allowed us to probe two facets of the kappa opioid receptor’s control over dopamine release: sensitivity (moderate concentration, 300 nM), and efficacy (saturating concentration, 1 µM)

使用的U50,488浓度为300 nM和1 µM，这使我们能够探究κ阿片受体对多巴胺释放控制的两个方面：敏感性（中等浓度，300 nM）和效能（饱和浓度，1 µM）。

. When comparing

。当比较

OPRK1

expression and the decrease in dopamine release at 300 nM U50,488, a linear association was only observed in drinkers, such that the greater the

在300 nM U50,488浓度下，表达和多巴胺释放减少之间，只有饮酒者观察到了线性关联，即

OPRK1

expression in the VTA, the lower the sensitivity of the kappa opioid receptor (Fig.

在VTA中的表达，kappa阿片受体的敏感性越低（图。

). There was also a trend toward this negative correlation in drinkers with

）。在饮酒者中也存在这种负相关的趋势，

PDYN

前动力蛋白

expression and potency of U50,488, respectively (Fig.

U50,488 的表达和效价（图。

). There was no significant relationship between

）。两者之间没有显著的关系

ARRB1

或者

ARRB2

expression in the VTA and the effect of U50,488 at 300 nM in the NAc, (Fig.

VTA中的表达和U50,488在300 nM时在NAc中的效果，（图。

5G, H

5G，H

). At the high concentration (1 µM) of U50,488,

). 在高浓度（1 µM）的U50,488时，

OPRK1

and

和

PDYN

showed a trend toward a positive correlation in controls alone (Fig.

在单独的对照组中显示出正相关的趋势（图。

5I, J

5我，J

）。

ARRB1

expression in the VTA was not significantly correlated with kappa opioid receptor-mediated inhibition of accumbal dopamine release in drinkers or controls (Fig.

在饮酒者或对照组中，VTA中的表达与κ阿片受体介导的伏隔核多巴胺释放抑制无显著相关性（图。

). However, in drinkers only, there was a correlation between the ability of the high dose of U50,488 to inhibit dopamine release and

）。然而，仅在饮酒者中，高剂量U50,488抑制多巴胺释放的能力之间存在相关性，且

ARRB2

expression, whereby greater inhibition was associated with lower transcriptional expression (Fig.

表达式，其中更强的抑制作用与较低的转录表达相关（图。

5升

). Qualitatively similar to the alterations in expression × function relationships for dopamine release dynamics reported above, the slope of the best-fit linear regressions between

）。与上述多巴胺释放动力学中表达×功能关系的变化在性质上相似，最佳拟合线性回归的斜率介于

OPRK1

，

PDYN

，

ARRB1

, and

，以及

ARRB2

expression in the VTA and kappa opioid receptor agonist-induced inhibition of accumbal dopamine release displayed an alcohol-induced sign change.

VTA中的表达和κ阿片受体激动剂诱导的伏隔核多巴胺释放抑制显示出酒精诱导的符号变化。

To probe the expression × function relationship pattern, we directly compared the slopes between drinkers and controls and found that controls showed a positive mean slope that was greater than that seen in drinkers (Fig.

为了探讨表达×功能关系模式，我们直接比较了饮酒者和对照组之间的斜率，发现对照组呈现出比饮酒者更大的正平均斜率（图。

5米

). Similar to the effect seen with homosynaptic regulators of dopamine release, this effect on the association between kappa opioid receptor-mediated regulation and expression of kappa opioid receptor-associated proteins supports the hypothesis that a history of alcohol intake and protracted abstinence modulates the relationship of transcriptional activity in the somatic compartment (VTA) with downstream functional outcomes (NAc core).

）。与在多巴胺释放的同突触调节剂中观察到的效果相似，这种对κ阿片受体介导的调节与κ阿片受体相关蛋白表达之间关联的影响支持了以下假设：饮酒史和长期戒断会调节躯体区室（VTA）中的转录活性与下游功能结果（NAc核心）之间的关系。

To further probe the modulation of this relationship by alcohol history, dependent variables were again rank ordered. The lowest expression of .

为了进一步探究酒精史对这种关系的调节作用，因变量再次被排序。最低的表达。

OPRK1, PDYN, ARRB1

OPRK1，PDYN，ARRB1

, and

，以及

ARRB2

, and the lowest potency and efficacy of kappa opioid receptor control of dopamine release to the highest expression and greatest action of the receptor were assigned a value of 1 to 8, respectively. This allowed for all 8 of the correlations to be plotted and analyzed in a single coordinate plane with rank-expression on the y-axis and rank-function on the x-axis.

，并且将κ阿片受体对多巴胺释放的最低效力和效能到最高表达和最强作用分别赋予1到8的值。这使得所有8个相关性可以在单一坐标平面上绘制和分析，其中y轴为排名-表达，x轴为排名-功能。

In control subjects, there was a strong positive correlation between kappa opioid receptor transcript expression and the ability of U50,488 to inhibit dopamine release, suggesting synchrony between VTA transcription and functional sensitivity of axonal kappa opioid receptors in the NAc (Fig. .

在对照受试者中，κ阿片受体转录表达与U50,488抑制多巴胺释放的能力之间存在很强的正相关，这表明VTA转录和NAc中轴突κ阿片受体的功能敏感性之间存在同步性（图。

5牛

). A history of chronic alcohol self-administration and abstinence reverses this effect such that in drinkers, more VTA expression of

). 慢性酒精自我给药和戒酒的历史逆转了这种效应，使得饮酒者中更多的VTA表达

OPRK1, PDYN, ARRB1

OPRK1，PDYN，ARRB1

, and

，以及

ARRB2

is correlated with less kappa opioid receptor control over accumbal dopamine release (Fig.

与对伏隔核多巴胺释放的κ阿片受体控制减少相关（图。

). Thus, voluntary alcohol intake and protracted abstinence can reverse the relationship between expression of genes encoding elements of the kappa opioid receptor system and its ability to inhibit dopamine release in the NAc.

因此，自愿摄入酒精和长期戒酒可以逆转编码κ阿片受体系统元素的基因表达与其抑制NAc中多巴胺释放能力之间的关系。

These effects were once again independent of differential gene expression between drinkers and controls (Fig.

这些影响再次与饮酒者和对照组之间的基因表达差异无关（图。

S4B

，

S4I-K

) and did not correlate with lifetime alcohol intake (Fig.

）且与终生酒精摄入量无关（图。

S5B

，

S5I-K

), demonstrating that alcohol self-administration, and abstinence in particular, modulates the relationship between transcription and function independent of overall transcript expression levels in the VTA. We also assessed whether the ratio of ligand to receptor (

），证明了酒精自我给药，尤其是戒酒，调节了转录与功能之间的关系，而这种调节与VTA中整体转录表达水平无关。我们还评估了配体与受体的比率（

PDYN/OPRK1

) expression in the VTA was correlated with functional measures in the NAc core, given the close relationship between endogenous ligand levels and receptor sensitivity and previous work showing alcohol experience-dependent relationships with this ratio

）VTA中的表达与NAc核心中的功能测量相关，鉴于内源性配体水平和受体敏感性之间的密切关系，以及之前的研究表明酒精体验依赖性与该比率的关系。

. There was no relationship between

。两者之间没有关系

PDYN/OPRK1

VTA expression and accumbal dopamine regulation in either the drinkers or controls (Fig.

饮酒者或对照组中VTA表达和伏隔核多巴胺调节（图。

). Ultimately, this demonstrates that the supersensitivity of the kappa opioid receptor which is induced by chronic alcohol self-administration

）。最终，这表明由慢性酒精自我给药引起的κ阿片受体的超敏性

，

persists at least one month into abstinence and is associated with an inversion of the relationship between expression of

持续至少一个月的戒断，并且与表达之间的关系反转相关联

OPRK1

，

PDYN, ARRB1

PDYN，ARRB1

, and

，以及

ARRB2

and the ability of kappa opioid receptors to inhibit dopamine release.

κ阿片受体抑制多巴胺释放的能力。

Kappa opioid receptor activation is necessary for CRF-mediated dopamine inhibition

激活κ阿片受体是CRF介导的多巴胺抑制所必需的。

Given that responses to stressors are altered during abstinence and in light of evidence suggesting that CRF can affect the function of kappa opioid receptors

鉴于在戒断期间对压力源的反应会发生改变，并且有证据表明CRF可以影响κ阿片受体的功能

，

, we also probed potential interactions between the CRF and kappa opioid receptor systems in regulating dopamine release in the NAc. Urocortin, an endogenous CRF receptor agonist, had no effect on dopamine release when bath applied alone in control and alcohol slices (Fig.

我们还探讨了CRF和κ阿片受体系统在调节NAc中多巴胺释放方面的潜在相互作用。尿皮素是一种内源性的CRF受体激动剂，在对照组和酒精切片中单独浴液应用时对多巴胺释放没有影响（图。

S9A

). However, the application of U50,488 in the presence of urocortin decreased dopamine release in both controls and drinkers similarly (Fig.

). 然而，U50,488 在尿皮质素存在下的应用同样减少了对照组和饮酒者的多巴胺释放（图。

S9B

). When urocortin was applied after U50,488, CRF receptor activation was able to further decrease dopamine release in both groups (Fig.

）。当在U50,488之后应用尿皮素时，CRF受体的激活能够进一步减少两组中的多巴胺释放（图。

S9C

). Urocortin had no effect on dopamine release in the presence of a kappa opioid receptor antagonist, norbinaltorphimine (NorBNI) (Fig.

）。在κ阿片受体拮抗剂诺宾那托非明（NorBNI）存在的情况下，尿皮素对多巴胺释放没有影响（图。

S9D

). Together, this suggests that kappa opioid receptor activation is necessary for CRF-mediated dopamine inhibition, and that activation of the CRF receptor is able to occlude the supersensitization of the kappa opioid receptor system seen at this abstinence timepoint.

）。总之，这表明κ阿片受体的激活是CRF介导的多巴胺抑制所必需的，并且CRF受体的激活能够掩盖在这一戒断时间点看到的κ阿片受体系统的超敏化。

Alcohol history increases dynorphin release probability

酒精历史增加强啡肽释放概率

Prior work as well as the results presented up to this point have examined the effect of exogenous activation of the kappa opioid receptor system on dopamine release dynamics in the NAc. Here, we sought to stimulate the release of endogenous ligands of the kappa opioid receptor and infer the effects of kappa opioid receptor activation by examining dopamine release before and after application of a kappa opioid receptor antagonist.

以往的研究以及到目前为止所呈现的结果已经考察了κ阿片受体系统的外源性激活对NAc中多巴胺释放动力学的影响。在此，我们试图刺激κ阿片受体内源性配体的释放，并通过在应用κ阿片受体拮抗剂前后检测多巴胺释放来推断κ阿片受体激活的效果。

To measure endogenous activation of the kappa opioid receptor, the highly selective kappa opioid receptor antagonist NorBNI (10 nM) was used; any change in dopamine release magnitude due to the application of NorBNI is attributable to the blockade of endogenous activation of kappa opioid receptors, putatively by dynorphin, the primary known endogenous ligand of kappa opioid receptors.

为了测量κ阿片受体的内源性激活，使用了高选择性的κ阿片受体拮抗剂NorBNI（10 nM）；由于应用NorBNI而导致的多巴胺释放量的任何变化都归因于κ阿片受体内源性激活的阻断，据推测这是由强啡肽引起的，强啡肽是κ阿片受体的主要已知内源性配体。

This pharmacological strategy was employed such that NorBNI-induced disinhibition of dopamine release evoked by multi-pulse stimulations was used as a proxy for dynorphin release probability. In both drinkers and controls, dopamine release was measured in response to 5 pulse stimulations delivered at 5 to 100 Hz to establish a pre-drug baseline, then these stimulations were repeated in the presence of the kappa opioid receptor antagonist NorBNI..

采用这种药理学策略，将多脉冲刺激引起的NorBNI诱导的多巴胺释放去抑制作为强啡肽释放概率的替代指标。在饮酒者和对照组中，通过以5到100 Hz的频率进行5脉冲刺激来测量多巴胺释放，以建立用药前的基线，然后在κ阿片受体拮抗剂NorBNI存在的情况下重复这些刺激。

In controls, NorBNI did not modulate release magnitude across the stimulation frequency curve (Fig.

在对照组中，NorBNI 并未调节整个刺激频率曲线的释放幅度（图。

6A–C

), indicating no measurable impact of endogenous dynorphin release evoked by the stimulations. In drinkers on the other hand, application of NorBNI produced a clear disinhibition of dopamine release (Fig.

），表明刺激引发的内源性强啡肽释放没有可测量的影响。另一方面，在饮酒者中，应用NorBNI明显解除了多巴胺释放的抑制（图。

). A comparison of release over stimulation frequencies before and after NorBNI application in drinkers showed a significant effect of NorBNI on dopamine release such that, predominantly at high frequency stimulations, NorBNI increased dopamine release (Fig.

）。在饮酒者中，对比诺RBNI应用前后不同刺激频率下的释放情况，结果显示诺RBNI对多巴胺释放有显著影响，尤其是在高频刺激下，诺RBNI增加了多巴胺的释放（图。

). NorBNI also increased the area under the curve (AUC) of dopamine over stimulation intensities in subjects with a history of alcohol self-administration (Fig.

). NorBNI还增加了有酒精自我给药史的受试者在刺激强度下多巴胺的曲线下面积（AUC）（图。

6楼

). The increase of dopamine release with NorBNI seen in drinkers suggests that kappa opioid receptors on terminals were endogenously activated by high frequency stimulations after chronic alcohol intake and protracted abstinence, most likely via increased release probability of dynorphin.

）。在饮酒者中观察到的NorBNI引起的多巴胺释放增加表明，慢性饮酒和长期戒断后，末梢上的κ阿片受体很可能通过增加强啡肽的释放概率，被高频刺激内源性激活。

Fig. 6: Chronic alcohol consumption increased dynorphin release probability in long-term abstinence.

图6：长期戒酒后，慢性酒精摄入增加了强啡肽的释放概率。

Representative traces indicating dopamine release in controls at 10 Hz and 60 Hz normalized to 1 pulse dopamine release before and after NorBNI.

代表对照组在10 Hz和60 Hz下多巴胺释放的轨迹，归一化为NorBNI前后的单脉冲多巴胺释放。

Dopamine release in controls with and without NorBNI across stimulation frequencies.

不同刺激频率下，使用和不使用NorBNI的对照组多巴胺释放情况。

There was no effect of a history of ethanol intake, but there was an effect of NorBNI, on the area under the curve (AUC) of dopamine release across frequencies (mixed model two-way ANOVA: ethanol history: F

乙醇摄入历史对多巴胺释放频率曲线下的面积（AUC）没有影响，但NorBNI有影响（混合模型双因素方差分析：乙醇历史：F

1, 14

= 0.03,

= 0.88; drug: F

= 0.88；药物：F

1, 14

= 6.152,

= 6.152，

= 0.03). NorBNI had no effect on AUC of dopamine release in controls (Šídák’s test: baseline vs. NorBNI in controls:

= 0.03)。NorBNI 对对照组多巴胺释放的 AUC 没有影响 (Šídák’s test：对照组基线 vs. NorBNI：

= 0.66,

= 0.66，

= 0.77).

= 0.77)。

Representative traces indicating dopamine release in controls at 10 Hz and 60 Hz normalized to 1 pulse dopamine release with and without NorBNI.

在10 Hz和60 Hz下，有无NorBNI时，代表性的轨迹显示了对照组中多巴胺释放的情况，并归一化为单脉冲多巴胺释放。

Dopamine release in drinkers with and without application of NorBNI across stimulation frequencies. AUC of dopamine release is visually represented by figure shading.

在不同刺激频率下，饮酒者应用NorBNI和未应用NorBNI的多巴胺释放情况。多巴胺释放的AUC通过图形阴影直观表示。

NorBNI administration increased the AUC of normalized dopamine release in drinkers suggesting significant dynorphin release at these stimulation intensities after chronic alcohol self-administration (mixed model two-way ANOVA: ethanol history: F

NorBNI的施用增加了饮酒者在慢性酒精自我给药后这些刺激强度下的归一化多巴胺释放的AUC，表明此时强啡肽的显著释放（混合模型双因素方差分析：乙醇史：F）。

1, 14

= 0.03,

= 0.88; drug: F

= 0.88；药物：F

1, 14

= 6.152,

= 0.03; Šídák’s test: baseline vs. NorBNI in drinkers:

= 0.03；Šídák检验：饮酒者基线与NorBNI对比：

= 2.85,

= 0.03).

= 0.03)。

，

Upstream expression of

上游表达

OPRK1

and

和

PDYN

were correlated within-subject with the proportion of change in AUC of dopamine release. The best-fit linear regression is shown for each group. Inset: Pearson’s

在受试者内部与多巴胺释放的AUC变化比例相关。每组的最佳拟合线性回归如图所示。插图：皮尔逊

and

和

-values.

-值。

Expression of

表达

OPRK1

in the ventral tegmental area was positively correlated with the change in dopamine release with NorBNI administration in controls, not drinkers.

在腹侧被盖区，与对照组中使用NorBNI后多巴胺释放的变化呈正相关，而非饮酒者则不然。

PDYN

expression was also only correlated with the dynorphin release probability in controls, not drinkers. All statistical tests were two-tailed. Unless otherwise indicated, values indicate mean ± SEM. (*

表达也仅与对照组中的强啡肽释放概率相关，而与饮酒者无关。所有统计检验均为双尾检验。除非另有说明，数值表示平均值±标准误。(*

≤ 0.05, **

≤ 0.01, ***

≤ 0.001, ****

≤ 0.0001) (controls:

≤ 0.0001)（对照组：

= 8; drinkers:

= 8；饮酒者：

= 8).

= 8）。

Full size image

全尺寸图像

Interestingly, dynorphin release probability, quantified as the percent change in pre- and post-drug AUC for each group, was positively correlated with upstream

有趣的是，强啡肽的释放概率（量化为每组药物前后AUC的百分比变化）与上游因素呈正相关。

OPRK1

and

和

PDYN

RNA levels in controls, but not drinkers (Fig.

对照组中的RNA水平，但不是饮酒者（图。

6G, H

6G，H

). These effects were once again not associated with previous lifetime alcohol intake and occurred in the absence of group differences in expression of these genes (Figs.

这些影响再次与之前的终生饮酒量无关，并且在这些基因表达没有组间差异的情况下发生（图。

，

). In sum, this suggests that chronic alcohol intake and long-term abstinence augments kappa opioid receptor control over dopamine release through the increase in release probability of dynorphin in the NAc and via a mechanism that disrupts the synchrony between functional release and upstream transcription..

总之，这表明慢性酒精摄入和长期戒酒通过增加NAc中强啡肽的释放概率，并通过一种破坏功能释放与上游转录之间同步性的机制，增强了κ阿片受体对多巴胺释放的控制。

Discussion

讨论

The mesolimbic dopamine system undergoes dramatic neuroplasticity after chronic alcohol consumption and is a target for existing and promising therapeutics for AUD. A wealth of literature has suggested that the dopamine system is hypofunctional after chronic alcohol exposure, creating a hypodopaminergic state that is implicated in impaired decision-making associated with AUD.

中脑边缘多巴胺系统在长期饮酒后会发生显著的神经可塑性变化，并且是现有和潜在酒精使用障碍（AUD）治疗药物的靶点。大量文献表明，长期暴露于酒精后，多巴胺系统功能减退，形成低多巴胺能状态，这与AUD相关的决策能力受损有关。

Here, we used RNA-seq and fast-scan cyclic voltammetry to assess upstream transcription in the VTA and its relationship to downstream dopamine terminal function, respectively, at the end of a one-month abstinence period in drinkers with a history of alcohol intake (and calorically-yoked and housing controls).

在这里，我们使用RNA-seq和快速扫描循环伏安法分别评估了在一个月戒酒期结束时，有饮酒史的饮酒者（以及热量匹配和住房对照组）VTA区上游转录情况及其与下游多巴胺末端功能的关系。

Though no genes were differentially expressed between drinkers and controls, and most expression networks were unchanged by a history of alcohol self-administration and repeated episodes of abstinence, clear functional changes at the dopamine terminal were seen after protracted abstinence. The maximal rate of dopamine reuptake, kappa opioid receptor sensitivity, and dynorphin release probability were upregulated in drinkers compared to controls, supporting the hypothesis of a hypodopaminergic state during abstinence.

尽管饮酒者和对照组之间没有基因表达差异，且大多数表达网络并未因酒精自我给药史和反复的戒断发作而改变，但在长期戒断后，多巴胺终端出现了明显的功能变化。与对照组相比，饮酒者的多巴胺再摄取最大速率、κ阿片受体敏感性和强啡肽释放概率均上调，这支持了戒断期间低多巴胺能状态的假设。

To determine potential mechanisms by which these long-lasting changes were maintained, measures of dopamine terminal function were correlated with VTA expression of genes encoding for regulators of these pathways. In the case of dopamine release dynamics, synchrony between function and transcription was only present in drinkers, while there was a reversal in the relationship between kappa opioid receptor/dynorphin transcription and receptor sensitivity in these subjects.

为了确定维持这些长期变化的潜在机制，将多巴胺终端功能的测量值与编码这些通路调节因子的基因在VTA中的表达进行了关联。在多巴胺释放动态方面，功能和转录之间的同步性仅在饮酒者中存在，而κ阿片受体/强啡肽转录与受体敏感性之间的关系在这些受试者中发生了逆转。

Together, this work demonstrates that abstinence is characterized by persistent modulation of dopamine terminal.

总之，这项工作表明，戒断的特征是多巴胺终端的持续调节。

Clinical literature has shown that AUD is associated with marked hypofunction of dopaminergic activity in the ventral striatum which can be observed into abstinence

临床文献表明，AUD与腹侧纹状体多巴胺能活动的显著低功能有关，这种低功能在戒断期间仍可观察到。

，

. Here we demonstrate that multiple inhibitory regulators of dopaminergic activity, dopamine transporters and kappa opioid receptors, are functionally upregulated in protracted abstinence from alcohol, providing a putative mechanism for the lasting hypodopaminergic state observed in AUD. Minimal work has assessed the persistence of plasticity at specific receptors and transporters into protracted abstinence and the molecular mechanisms that underly them.

在这里，我们证明了多巴胺活性的多个抑制性调节因子——多巴胺转运体和κ阿片受体——在长期戒酒后功能上调，这为AUD中观察到的持续低多巴胺能状态提供了一个推定机制。极少有研究评估特定受体和转运体在长期戒断期间的可塑性持久性及其潜在的分子机制。

By demonstrating augmented dopamine reuptake, kappa opioid receptor sensitivity, and dynorphin release probability that exists after protracted abstinence, we highlight functional processes that are disrupted with chronic alcohol consumption and offer evidence supporting the potential utility of these pathways as therapeutic targets during the critical abstinence intervention point.

通过展示长期戒酒后存在的多巴胺再摄取增强、κ阿片受体敏感性和强啡肽释放概率，我们强调了慢性酒精摄入所扰乱的功能过程，并提供了支持这些通路作为关键戒酒干预点治疗靶点的潜在效用的证据。

Though more work needs to be done, the initial findings suggest that kappa opioid receptor control of dopamine release is G protein mediated while alcohol-induced upregulation may be related to altered β-arrestin. Together, this suggests that biased kappa opioid receptor ligands may be a beneficial approach to developing therapeutics for AUD and limiting off-target effects..

尽管还需要做更多的工作，但初步研究结果表明，κ阿片受体对多巴胺释放的控制是通过G蛋白介导的，而酒精诱导的上调可能与改变的β-抑制蛋白有关。总之，这表明偏向性的κ阿片受体配体可能是开发AUD治疗药物并限制脱靶效应的一种有益方法。

Initially, we hypothesized that plasticity that persisted into abstinence might be explained by alcohol-induced changes in gene expression. Surprisingly, there were no differentially expressed genes and few altered gene networks between subjects with a history of alcohol self-administration and controls.

最初，我们假设持续到戒酒期的可塑性可能由酒精诱导的基因表达变化来解释。令人惊讶的是，在有酒精自我给药史的受试者和对照组之间，没有差异表达的基因，且改变的基因网络也很少。

With an approximately 93% sequence identity homology between humans and rhesus macaques.

人类与恒河猴之间大约有93%的序列同一性同源性。

, the dataset generated from this work may still inform future investigations into the mechanisms underlying AUD and therapeutic targets. Importantly, there was no significant differential expression of genes encoding for proteins that make up the dopamine and kappa opioid receptor systems (e.g.,

，这项工作生成的数据集仍可能为未来对AUD机制和治疗靶点的研究提供信息。重要的是，编码组成多巴胺和κ阿片受体系统的蛋白质的基因没有显著的差异表达（例如，

VMAT2

，

DRD2

，

OPRK1

). This is in line with previous post-mortem human work demonstrating no difference in overall expression of

）。这与之前的人体解剖后研究一致，表明总体表达没有差异

OPRK1

或

PDYN

in the nucleus accumbens of individuals with a history of AUD, but that AUD is associated with complex co-expression and transcriptional coordination of dopamine related genes

在有酒精使用障碍（AUD）病史的个体的伏隔核中，但AUD与多巴胺相关基因的复杂共表达和转录协调有关。

. When we correlated upstream transcription of these genes with downstream function of dopamine terminals, we found that, although they were not differentially expressed, transcription of these genes of interest was closely associated with differential function. Understanding the complex relationship between transcription and function is critical to the interpretation of RNA-seq data which is often used to draw conclusions regarding circuit and synaptic activity.

当我们把这些基因的上游转录与多巴胺终端的下游功能相关联时，我们发现，尽管它们没有差异表达，但这些目标基因的转录与功能差异密切相关。理解转录和功能之间的复杂关系对于解读RNA-seq数据至关重要，RNA-seq数据常用于得出有关神经回路和突触活动的结论。

Typically, gene expression is assumed to be indicative of protein expression, despite reports that protein and transcript levels are often inversely correlated.

通常，基因表达被认为可以指示蛋白质表达，尽管有报道称蛋白质和转录本水平常常呈负相关。

. Previous work has also demonstrated that the ratio between RNA and protein levels is dependent on tissue type

以前的研究还表明，RNA和蛋白质水平之间的比率取决于组织类型。

or aging

或老化

, but here, we go a step further by comparing transcript expression with measures of protein function and showed that this relationship, or lack thereof, is dynamic and experience-dependent. For example, the correlation between transcription and function, which is often assumed to exist, in some cases is not even present in controls but rather only induced with long term alcohol exposure and abstinence.

，但在这里，我们通过比较转录表达与蛋白质功能的测量值，进一步深入了一步，并展示了这种关系（或缺乏这种关系）是动态的且依赖于经验的。例如，转录与功能之间的相关性（通常被认为存在）在某些情况下甚至在对照组中都不存在，而只是在长期酒精暴露和戒断后才被诱导出来。

For other genes and functions, a history of alcohol intake eliminates or even reverses the correlation between transcription and terminal activity that is seen in controls. Ultimately, altered transcription does not imply directionality or the presence of changes in function and vice versa; rather the relationship between transcriptional expression and function should not be stated or implied without quantitative assessments supporting any given claim.

对于其他基因和功能，饮酒历史会消除甚至逆转在对照组中观察到的转录与终末活动之间的相关性。最终，转录的改变并不意味着方向性或功能变化的存在，反之亦然；转录表达与功能之间的关系不应被陈述或暗示，除非有定量评估支持特定主张。

While the current dataset cannot speak to causality between the expression and function of any given transcript, we show that across dopamine-related transcripts a history of alcohol drinking and abstinence causally alters the likelihood that expression × function correlations emerge..

虽然当前的数据集无法说明任何给定转录本的表达和功能之间的因果关系，但我们展示了在多巴胺相关转录本中，饮酒和戒酒的历史会因果性地改变表达与功能相关性出现的可能性。

More research will certainly be needed to elucidate the mechanisms underlying the observed changes in synchrony between transcript expression and function. Experience-dependent shifts in a plethora of biological processes such as alterations in rate of protein translation, post-translational modifications, and trafficking could potentially explain these results.

当然需要更多的研究来阐明观察到的转录表达与功能同步性变化背后的机制。依赖经验的生物过程变化，如蛋白质翻译速率的改变、翻译后修饰以及运输，可能可以解释这些结果。

For example, a higher level of DNA transcription may not be reflected in RNA read counts if the RNA is being translated to protein at a faster rate, resulting in a decorrelation between RNA counts and protein expression. This could occur through a range of mechanisms including rate of 5’ capping, mRNA stability, or localization.

例如，如果 RNA 正以更快的速度被翻译成蛋白质，则较高的 DNA 转录水平可能不会反映在 RNA 读数中，从而导致 RNA 计数与蛋白质表达之间的去相关性。这种现象可能通过多种机制发生，包括 5' 帽形成速率、mRNA 稳定性或定位。

Even in cases where a given gene is primarily regulated at the level of transcription and RNA levels are correlated with protein expression, if post-transcriptional alterations, such as RNA editing or alternative splicing, dictates protein activity, transcript expression × function relationships may not be present.

即使在某个基因主要在转录水平受到调控且RNA水平与蛋白质表达相关的情况下，如果转录后修饰（如RNA编辑或可变剪接）决定了蛋白质活性，转录表达与功能之间的关系可能并不存在。

Likewise, any number of post-translational modifications could alter functional outputs independent of transcript expression, through differential trafficking, affinity states, and rates of protein degradation. These examples only constitute a small number of possible explanations..

同样，任何数量的翻译后修饰可以通过不同的运输、亲和状态和蛋白质降解速率来改变功能输出，而与转录表达无关。这些例子仅构成了可能解释的一小部分。

Surprisingly, not only did we observe that experience-dependent plasticity in the presence or directionality of transcript expression × function relationships appear to be the rule rather than the exception, but the control condition was most associated with a lack of a correlation between transcript expression and functional output.

令人惊讶的是，我们不仅观察到经验依赖的可塑性在转录表达与功能关系的存在或方向性上似乎是普遍规律而非例外，而且对照条件下转录表达与功能输出之间缺乏相关性的情况最为明显。

It is intriguing to speculate that in an alcohol-naïve system, dopamine release, reuptake and regulation may be primarily through fast, local mechanisms at the presynaptic terminal, such that a full range of activity can be achieved with a minority of the available receptors and transporters (i.e. operating in a ‘spare receptor reserve’ mode); on the other hand, the emergence of expression × function relationships after chronic alcohol and protracted abstinence, despite no change in transcript levels, may indicate that the system is operating under conditions where the range of cellular activity is dictated by the availability of protein present at synaptic sites at a given time (i.e.

有趣的是，可以推测，在一个未接触过酒精的系统中，多巴胺的释放、再摄取和调节可能主要通过突触前终端快速的局部机制进行，从而在只使用少数可用受体和转运体的情况下实现全部活动范围（即以“备用受体储备”模式运作）；另一方面，在慢性酒精暴露和长期戒断后出现的表达×功能关系，尽管转录水平没有变化，这可能表明系统正在一种细胞活动范围由特定时刻突触位点上存在的蛋白质数量所决定的条件下运作（即...

in a ‘spare receptor depleted’ mode). If this is the case, it would likely produce a restricted range of function (e.g. Fig. .

在“备用受体耗尽”模式下）。如果是这种情况，它可能会产生有限的功能范围（例如，图。 .

) and a much less plastic system, even though there may be no observable change in transcript expression or function in a cross-sectional measurement. Given the presence of wide-spread alterations in transcript expression × function relationships observed across protein classes in protracted abstinence, it is important to consider whether these changes in synchrony may contribute to inflexibility of dopaminergic signaling to precipitate relapse-related behaviors..

）和一个更不易变化的系统，即使在横截面测量中可能观察不到转录表达或功能的变化。鉴于在长期戒断期间，在各类蛋白质中观察到的转录表达×功能关系的广泛变化，有必要考虑这些同步性的变化是否可能导致多巴胺信号传导的不灵活性，从而引发与复发相关的行为。

Finally, this work was exclusively conducted in male subjects, and it will thus be critical to perform parallel investigations in female subjects to determine whether this plasticity and modulation of transcription × function relationship is consistent across sexes. Nonetheless, here we show that alcohol intake induces long-lasting functional changes that are present after protracted abstinence and are characterized by a reorganization of the relationship between gene expression and functional measures of dopamine terminal activity.

最后，这项工作仅在男性受试者中进行，因此在女性受试者中开展平行研究将至关重要，以确定这种可塑性及转录×功能关系的调节是否在两性之间一致。尽管如此，我们在此展示，酒精摄入会诱导在长期戒断后仍然存在的持久功能变化，并且这些变化的特征是基因表达与多巴胺终端活动功能指标之间的关系发生了重组。

In an animal and experimental model with great translational relevance, this work offers insights into the biological changes during abstinence, a crucial timepoint for therapeutic intervention for AUD, and highlights the complexity of a biological relationship often taken for granted..

在一个具有重要转化意义的动物和实验模型中，这项工作提供了关于戒断期间生物变化的见解，这是针对酒精使用障碍（AUD）进行治疗干预的关键时间点，并强调了通常被视为理所当然的生物关系的复杂性。

Methods

方法

Subjects

主题

Subjects were 28 male rhesus macaques (

受试者为28只雄性恒河猴 (

Macaca mulatta

猕猴

) between the ages of 7 and 8.3 years across two cohorts (“Rhesus 10” [alias: Cohort 1] and “Rhesus 14” [alias: Cohort 2], cohort details at

）在7至8.3岁之间的两个队列（“恒河猴10”[别名：队列1]和“恒河猴14”[别名：队列2]，队列详情见

www.matrr.com

). Weights of the subjects ranged from 7 to 12.6 kg. Animals were individually housed in quadrant cages (0.8 × 0.8 × 0.9 m) with constant temperature (20–22 °C) and humidity (65%) and an 11 h light cycle (lights on at 8:00 AM). Animals had visual, auditory, and olfactory contact with other conspecifics, in addition to 2 h of pair housing each day.

). 受试者的体重范围为7至12.6千克。动物被单独饲养在四边形笼子（0.8 × 0.8 × 0.9米）中，环境温度恒定（20-22°C），湿度为65%，并保持11小时的光照周期（早上8:00开灯）。动物除了每天有2小时的配对饲养外，还能通过视觉、听觉和嗅觉与其他同类保持接触。

Body weights were measured weekly. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Oregon National Primate Research Center Institutional Animal Care and Use Committee..

体重每周测量一次。所有程序均按照《实验动物护理和使用指南》进行，并经俄勒冈国家灵长类研究中心机构动物护理和使用委员会批准。

Drinking procedure

饮酒程序

Monkeys (17 alcohol drinkers and 11 calorically yoked or housing controls) were trained to obtain fluids and their meals from an operant panel that replaced one of the walls of their home cage, as described previously

猴子（17只饮酒者和11只热量匹配或住房对照）被训练从一个操作面板上获取液体和食物，该面板取代了它们家笼子的一侧墙，如前所述。

，

. Briefly, the panels had two spouts, one to each side of a 15-inch video display screen. Near each spout, the display showed a set of three stimulus lights (white, red, and green) that indicated an active session or food or fluid availability, respectively. A centrally located recessed dowel activated the fluid spouts, and an infrared finger poke activated the pellet dispenser (env-203-1000; Med Associates).

简而言之，面板有两个出水口，分别位于15英寸视频显示屏的两侧。每个出水口附近，显示屏上显示一组三个刺激灯（白色、红色和绿色），分别表示活动会话或食物或液体的可用性。一个中央凹陷的圆木棒激活液体出水口，红外手指插入激活颗粒分配器（env-203-1000；Med Associates）。

Each spout was connected via Nalgene tubing to a 1 L fluid reservoir set on a digital scale (Adventurer Pro Balances AV4101C; Ohaus). Dowel pulls, finger pokes, and fluid consumption were recorded every 500 ms via a computerized system (Dell Optiplex) using custom hardware and programming using a National Instruments interface and Labview software.

每个喷口通过Nalgene管连接到一个放置在数字秤（Adventurer Pro Balances AV4101C；Ohaus）上的1升液体储液器。通过使用定制硬件和编程的计算机化系统（Dell Optiplex），利用National Instruments接口和Labview软件，每500毫秒记录一次木栓拉扯、手指戳动和液体消耗。

Schedule-induced polydipsia, as described previously.

如前所述，时间表诱导的多饮症。

，

, was used to induce alcohol self-administration in daily 16 h sessions. Briefly, a 1 g banana food pellet (Research Diets) was dispensed every 300 s (fixed time, 300 s) until a water volume equivalent to 1.5 g/kg of 4% (w/v) ethanol was consumed. Following at least 30 days of water induction, 4% ethanol replaced water.

，在每天16小时的实验中用于诱导酒精自我摄入。简而言之，每300秒（固定时间，300秒）投放一粒1克的香蕉食物颗粒（Research Diets），直到摄入相当于1.5克/千克体重的4%（重量/体积）乙醇的水量。在至少30天的水诱导后，4%乙醇取代了水。

In 30-day increments, each animal consumed increasing doses of 4% ethanol: 0.5 g/kg/d, 1.0 g/kg/d, then 1.5 g/kg/d. Following consumption of the programmed volume, water was immediately available, and any remaining pellets were available on a fixed-ratio 1 (FR-1) schedule after a 2 h time-out. Following completion of ethanol induction, daily 22 h open access sessions were performed, during which water and ethanol were concurrently available.

每只动物以30天为增量，逐步增加摄入4%乙醇的剂量：0.5克/千克/天、1.0克/千克/天，然后是1.5克/千克/天。在消耗完预定体积后，立即提供水，并且在2小时暂停后，剩余的颗粒物按照固定比率1（FR-1）的时间表提供。完成乙醇诱导后，进行每日22小时的开放获取阶段，在此期间水和乙醇同时可用。

Food pellets were available on a FR-1 schedule in at least three daily meals in 2 h intervals starting at the session onset. Data were downloaded, husbandry tasks were performed, food and fluids were replenished, and fresh fruit was provided each day by technicians during the 2 h break..

食物颗粒在每次会议开始时，以2小时为间隔，至少提供三餐，按照FR-1计划供应。数据被下载，饲养任务得以执行，食物和液体得到补充，并且技术人员在2小时的休息时间内每天提供新鲜水果。

After drinking was initiated, subjects had 12 months of open access to alcohol for 22 hours a day. 6 months into this period, an endocrine profile was collected. After 12 months of alcohol access, subjects underwent a one-month abstinence period. Following this abstinence period, subjects returned to open access alcohol exposure for three months, then underwent another one-month withdrawal period, returned to three months of open access alcohol exposure, and finally underwent one month of withdrawal at the end of which subjects were necropsied.

在开始饮酒后，实验对象每天有22小时、为期12个月的酒精开放获取期。在此期间的6个月时，收集了内分泌特征。在12个月的酒精获取期结束后，实验对象经历了一个月的戒断期。此戒断期结束后，实验对象重新回到三个月的酒精开放获取暴露，然后经历了另一个月的戒断期，再回到三个月的酒精开放获取暴露，最后在一个月的戒断期结束时，对实验对象进行了尸检。

During open access, blood was collected weekly for blood ethanol concentration measurements. Behavioral data collection and analysis was blind to voltammetry and RNA-seq results..

在开放获取期间，每周收集血液以测量血乙醇浓度。行为数据的收集和分析对伏安法和RNA测序结果保持盲态。

Tissue preparation

组织制备

Tissue preparation was described previously

组织制备方法如前所述。

，

. Briefly, monkeys were anesthetized with ketamine (10 mg/kg), maintained on isoflurane, and perfused with ice-cold oxygenated monkey perfusion solution [containing (in mM) 124 NaCl, 23 NaHCO3, 3 NaH2PO4, 5 KCl, 2 MgSO4, 10 d-glucose, 2 CaCl2]. Brains were quickly removed and 4-6 mm sections were made along the coronal plane using a brain matrix (Electron Microscopy Sciences), with the brain knife position guided by each individual’s MRI.

简而言之，猴子用氯胺酮（10毫克/千克）麻醉，用异氟醚维持，并用冰冷的充氧猴灌注液灌注 [含（单位为mM）124 NaCl、23 NaHCO3、3 NaH2PO4、5 KCl、2 MgSO4、10 d-葡萄糖、2 CaCl2]。迅速取出大脑，使用脑矩阵（电子显微镜科学公司）沿冠状面制作4-6毫米的切片，脑刀位置由每个个体的核磁共振成像指导。

An isolated tissue block containing only the striatum (caudate, putamen, and nucleus accumbens) was placed in ice-cold oxygenated monkey perfusion solution and transported on ice for slicing..

一个仅包含纹状体（尾状核、壳核和伏隔核）的孤立组织块被放置在冰冷却的氧气饱和的猴灌注液中，并在冰上运输以进行切片。

In vitro voltammetry

体外伏安法

Fast-scan cyclic voltammetry was then used to characterize presynaptic dopamine release and uptake as well as the ability of kappa opioid receptors to decrease dopamine release, or kappa opioid receptor sensitivity, in the NAc core. Voltammetric detection of dopamine in brain slices has been used by the authors and others to examine receptor regulation of dopamine release and uptake kinetics.

随后使用快速扫描循环伏安法来表征突触前多巴胺的释放和摄取，以及κ阿片受体减少多巴胺释放的能力或κ阿片受体敏感性在伏隔核核心中的作用。作者及其他研究者已使用伏安法检测脑切片中的多巴胺，以研究受体对多巴胺释放和摄取动力学的调节。

，

. A ceramic blade attached to a vibrating tissue slicer was used to prepare 250-μm-thick coronal brain sections containing the NAc core. The tissue was immersed in oxygenated artificial CSF (aCSF) containing the following (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.2 MgCl2, 25 NaHCO3, 11 glucose, and 0.4 l-ascorbic acid, pH adjusted to 7.4.

使用连接在振动组织切片机上的陶瓷刀片制备包含NAc核心的250微米厚的冠状脑切片。组织浸没在含氧的人工脑脊液（aCSF）中，其组成如下（单位：mM）：126 NaCl、2.5 KCl、1.2 NaH2PO4、2.4 CaCl2、1.2 MgCl2、25 NaHCO3、11葡萄糖和0.4 l-抗坏血酸，pH调整为7.4。

Once sliced, the tissue was transferred to testing chambers containing bath aCSF (32 °C), which flowed at 2 ml/min. A carbon fiber microelectrode (50–150 μm length, 7 μm diameter) and bipolar stimulating electrode were placed in close proximity on the tissue. Extracellular dopamine was recorded by applying a triangular waveform (−0.4 to +1.2 to −0.4 V vs Ag/AgCl, 400 V/s) to the recording electrode and scanning every 100 ms.

一旦切片，组织被转移到含有浴液aCSF（32°C）的测试室中，流速为2 ml/min。将一根碳纤维微电极（长度50-150微米，直径7微米）和双极刺激电极近距离放置在组织上。通过向记录电极施加三角波形（相对于Ag/AgCl，-0.4到+1.2到-0.4 V，400 V/s）并每100毫秒扫描一次，记录细胞外多巴胺。

This waveform allows for the assessment of oxidation and reduction peaks for dopamine, and has been used extensively to detect dopamine in brain.

该波形允许评估多巴胺的氧化和还原峰，并已广泛用于检测大脑中的多巴胺。

. Dopamine release was evoked by 1 pulse stimulations (350 μA, 4 ms, monophasic) applied to the tissue every 5 min, based on the shared EA

. 每隔5分钟，通过共享的EA，对组织施加1个脉冲刺激（350 μA，4 ms，单相）来诱发多巴胺释放。

between drinkers and controls. When a stable baseline was established (three collections within 10% variability) and predrug measures were taken, the selective kappa opioid receptor agonist U50,488 (0.3 μm and 1 μm, cumulatively) was bath applied to the slice, and stimulations continued until stability was reached at each concentration..

在饮酒者和对照组之间。当建立了一个稳定的基线（三次收集在10%的可变性范围内）并进行了药物前测量后，将选择性κ阿片受体激动剂U50,488（0.3 μm和1 μm，累积浓度）浴应用于切片，并持续进行刺激，直到在每个浓度下达到稳定。

Voltammetry analysis

电化学分析

For all acquisition and analysis of FSCV data, Demon voltammetry and analysis software was used

对于所有FSCV数据的采集和分析，均使用了Demon伏安法及分析软件。

. Recording electrodes were calibrated by recording responses (in electrical current; in nanoamperes) to a known concentration of dopamine (3 μm) using a flow-injection system. This was used to convert electrical current to dopamine concentration. Baseline recordings (i.e., after stabilization criteria were met but prior to drug application) FCSV data were modeled using Michaelis–Menten kinetics (K.

记录电极通过使用流动注射系统记录已知浓度多巴胺（3 μm）的响应（以电流表示，单位为纳安）进行校准。这被用于将电流转换为多巴胺浓度。基线记录（即，在达到稳定标准后但药物应用之前）的FCSV数据使用米氏动力学（K）进行建模。

set to 160 nM), which allows for the determination of evoked dopamine release and the maximal rate of dopamine uptake (V

设置为160 nM），这使得可以测定诱发的多巴胺释放和多巴胺摄取的最大速率（V

max

最大值

). Investigators were blind to group assignment during data collection and analysis of voltammetry data.

). 在数据收集和伏安数据分析期间，调查人员对组别分配保持盲态。

Tissue preparation and RNA-seq

组织准备和RNA测序

RNA was extracted from frozen tissue biopsies using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen Sciences, Germantown, MD, USA) following manufacture’s protocol by the ONPRC Primate Genetics Core. RNA integrity was confirmed with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto). Samples were sequenced on Illumina NovaSeq 6000.

使用AllPrep DNA/RNA/miRNA Universal Kit（Qiagen Sciences，美国马里兰州杰曼敦）按照制造商的协议，由ONPRC灵长类遗传学核心设施从冷冻组织活检中提取RNA。使用2100生物分析仪（安捷伦科技，帕洛阿尔托）确认RNA完整性。样品在Illumina NovaSeq 6000上进行测序。

The Vanderbilt Creative Data Solutions Shared Resource (RRID:SCR_022366) assisted with bulk RNAseq preprocessing and analysis. Paired-end RNA sequencing reads (150 bp long) were trimmed and filtered for quality using Trimgalore v0.6.7 (Krueger et al. 2021). Trimmed reads were quantified using Salmon.

范德比尔特创意数据解决方案共享资源（RRID:SCR_022366）协助进行了批量RNAseq的预处理和分析。使用Trimgalore v0.6.7（Krueger等人，2021）对配对末端RNA测序读数（150 bp长）进行修剪和质量过滤。修剪后的读数使用Salmon进行量化。

v1.9.0 with the Mmul10

v1.9.0 版本搭配 Mmul10

Macaca mulatta

猕猴

genome. Sample read counts were normalized using DESeq2 v1.36.0

基因组。样本读取计数使用DESeq2 v1.36.0进行了标准化。

. Features counted fewer than 5 times across at least 3 samples were filtered. For pairwise comparisons aligned read counts were analyzed for differential gene expression using the Biojupies analysis package with default settings

在至少3个样本中计数少于5次的特征被过滤。对于两两比较，使用Biojupies分析包和默认设置对齐读取计数进行差异基因表达分析。

。

Weighted gene correlation network analysis (WGCNA)

加权基因相关网络分析 (WGCNA)

We performed weighted correlation with individual sample weights determined with the ‘signed hybrid’ network (where negatively correlated genes are assumed not connected)

我们使用“符号混合”网络确定的个体样本权重进行了加权相关性分析（其中假设负相关的基因未连接）。

. The soft thresholding power was determined using scale-free topology of each sample as a fit index. From the determined scale of independence and mean connectivity calculated, we used a soft thresholding power of eight to perform WGCNA. To identify unique modules, a one-step network was constructed using blockwise modules constructed with unsigned topological overlap matrices.

软阈值功率是通过每个样本的无尺度拓扑作为拟合指数来确定的。根据所确定的独立性尺度和计算出的平均连通性，我们使用了软阈值功率八来进行WGCNA分析。为了识别独特的模块，我们使用无符号拓扑重叠矩阵构建的块状模块构建了一个单步网络。

To identify distinct modules, we utilized the Dynamic Tree Cut method. Of note, only module eigengenes that reached a threshold of 0.99 or above were included for subsequent network analysis. The edge and node data for all modules were exported to the external R package cytoHubba.

为了识别不同的模块，我们使用了动态树切割方法。值得注意的是，只有达到0.99或以上阈值的模块特征基因被纳入后续的网络分析。所有模块的边和节点数据被导出到外部R包cytoHubba。

to determine significant hub objects via the topological analysis method, Maximal Clique Centrality (MCC). The top 25 eigengenes with the (MCC) scores for a specific module were visualized using Cytoscape software to highlight important hubs and for ease of visualization

通过拓扑分析方法最大团中心性（MCC）确定重要的枢纽对象。使用Cytoscape软件对特定模块中具有MCC分数的前25个特征基因进行可视化，以突出重要的枢纽并方便可视化。

。

To determine the relationship between modules, the Pearson correlation coefficients between module eigengenes was calculated. Similarly, the relationship between individual eigengenes and with treatment (‘controls’ versus ‘drinkers’) were calculated using a Pearson correlation. Heatmaps and pathway analyses for WGCNA figures included all genes assigned to a specific module..

为了确定模块之间的关系，计算了模块特征基因之间的皮尔逊相关系数。同样，使用皮尔逊相关系数计算了单个特征基因与处理（“对照组”与“饮酒者”）之间的关系。WGCNA图的热图和通路分析包括分配给特定模块的所有基因。

Drugs

药物

CMPD101 was obtained from Hellobio. NEM, BaCl

CMPD101 购自 Hellobio。NEM、BaCl

, U50,488, and NorBNI were received from Sigma Aldrich, and nalfurafine was received from Fisher. Each drug was made fresh as a stock solution at 1 mM for NorBNI, or 10 mM for U50,488, CMPD101, NEM, and BaCl

，U50,488 和 NorBNI 从 Sigma Aldrich 获得，nalfurafine 从 Fisher 获得。每种药物都新鲜配制成储备溶液，NorBNI 为 1 mM，U50,488、CMPD101、NEM 和 BaCl 为 10 mM。

. Stock solutions were then added to the aCSF reservoir to reach the final concentration.

然后将储备溶液加入到aCSF储液器中以达到最终浓度。

Statistics

统计学

Statistical analyses were performed using GraphPad Prism (V10). For all pairwise comparisons between two conditions or groups, we utilized paired or unpaired

统计分析使用GraphPad Prism（V10）进行。对于所有两组或两种条件之间的成对比较，我们采用了配对或非配对方法。

-tests, respectively. Comparisons across three or more variables were made using one-way ANOVAs or two-way ANOVAs (followed by Šídák’s multiple comparisons when planned comparisons were made or interactions were detected). For correlation analyses, Pearson’s correlation coefficient was used for continuous variables and Spearman’s correlation coefficient was used for ordinal variables.

-测试，分别进行。对三个或更多变量的比较使用单因素方差分析或双因素方差分析（在进行计划比较或检测到交互作用时，随后使用Šídák多重比较）。对于相关性分析，连续变量采用皮尔逊相关系数，有序变量采用斯皮尔曼相关系数。

In both cases, correlations were performed within-group only, as required to meet assumptions of bivariate normality in correlative analyses.

在两种情况下，相关性分析都仅在组内进行，这是为了满足双变量正态性在相关性分析中的假设要求。

，

. Primary fast-scan cyclic voltammetry measures were collected from Cohort 1, thus all voltammetry x gene expression correlations only include that subset of subjects. Cohort 2 was used for follow-up fast-scan cyclic voltammetry experiments and therefore did not have the correlated measured recorded.

主要的快速扫描循环伏安测量数据是从队列1中收集的，因此所有伏安图与基因表达的相关性仅包括该子集的受试者。队列2用于后续的快速扫描循环伏安实验，因此未记录相关测量值。

Bulk gene expression and drinking behavior x gene expression analyses included both cohorts. All tests were two-tailed and .

批量基因表达和饮酒行为 x 基因表达分析包括两个队列。所有测试均为双尾。

values <0.05 were considered to be statistically significant.

值小于0.05被认为具有统计学意义。

Reporting summary

报告摘要

Further information on research design is available in the

有关研究设计的更多信息，请参见

Nature Portfolio Reporting Summary

自然组合报告摘要

linked to this article.

与本文相关联。

Data availability

数据可用性

The sequencing data generated in this study have been deposited in the GEO database under accession code

本研究中生成的测序数据已存入GEO数据库，登录号为

GSE244557

。

Source data

源数据

are provided with this paper.

随本文提供。

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Acknowledgements

致谢

This work was supported by NIH grants R00 DA04510 (C.A.S), R01 AA030115 (C.A.S.), U01 AA029971 (C.A.S.), U01 AA013510 (K.A.G.), R24 AA019431 (K.A.G.), U24 AA013641 (K.A.G.), P51 OD0119092 (K.A.G.), U01 AA014091 (S.R.J., K.M.H.), P50 AA026117 (S.R.J), T32AA007565 (K.M.H, S.C.F), and T32DA041349 (S.R.J.) as well as through the Alkermes Pathways Research Award (C.A.S.), the Brain Research Foundation (C.A.S.), and the Whitehall Foundation (C.A.S).

本研究得到了美国国立卫生研究院（NIH）的资助，包括R00 DA04510（C.A.S.）、R01 AA030115（C.A.S.）、U01 AA029971（C.A.S.）、U01 AA013510（K.A.G.）、R24 AA019431（K.A.G.）、U24 AA013641（K.A.G.）、P51 OD0119092（K.A.G.）、U01 AA014091（S.R.J.、K.M.H.）、P50 AA026117（S.R.J.）、T32AA007565（K.M.H.、S.C.F.）和T32DA041349（S.R.J.），以及Alkermes路径研究奖（C.A.S.）、大脑研究基金会（C.A.S.）和Whitehall基金会（C.A.S.）的支持。

Z.Z.F was supported by an NIH fellowship (DA056196)..

Z.Z.F. 得到了 NIH 奖学金 (DA056196) 的支持。

Author information

作者信息

Authors and Affiliations

作者与所属机构

Department of Pharmacology, Vanderbilt Brain Institute, Vanderbilt Center for Addiction Research, Vanderbilt University, Nashville, TN, 37232, USA

美国田纳西州纳什维尔范德比尔特大学药理学系，范德比尔特脑研究所，范德比尔特成瘾研究中心，邮编37232

Zahra Z. Farahbakhsh, Alberto J. López & Cody A. Siciliano

法拉赫巴赫什·扎赫拉，洛佩兹·阿尔贝托，西西利亚诺·科迪

Wake Forest University School of Medicine, Department of Physiology and Pharmacology, Winston-Salem, NC, 27157, USA

维克森林大学医学院，生理学与药理学系，北卡罗来纳州温斯顿-塞勒姆，邮编27157，美国

Katherine M. Holleran, Jonathon P. Sens, Madelyn I. Mauterer, Drew D. Kiraly & Sara R. Jones

凯瑟琳·M·霍勒兰，乔纳森·P·森斯，马德琳·I·毛特雷尔，德鲁·D·基拉利，莎拉·R·琼斯

The University of North Carolina at Greensboro, The Department of Nutrition, Greensboro, NC, 27412, USA

美国北卡罗来纳州格林斯伯勒市格林斯伯勒分校营养学系，邮编27412

Steve C. Fordahl

史蒂夫·C·福特哈尔

Oregon National Primate Research Center, Oregon Health & Science University, Division of Neuroscience, Portland, OR, USA

俄勒冈国家灵长类动物研究中心，俄勒冈健康与科学大学，神经科学部，波特兰，俄勒冈州，美国

Verginia C. Cuzon Carlson & Kathleen A. Grant

弗吉尼亚·C·库松·卡尔森和凯瑟琳·A·格兰特

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Zahra Z. Farahbakhsh

法拉赫巴赫什·扎赫拉·Z

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Drew D. Kiraly

德鲁·D·基拉利

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Kathleen A. Grant

凯瑟琳·A·格兰特

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Sara R. Jones

萨拉·R·琼斯

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Cody A. Siciliano

科迪·A·西西利亚诺

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Contributions

贡献

Conceptualization: Z.Z.F., S.C.F., K.A.G., S.R.J., C.A.S.; Data curation: Z.Z.F., K.A.G., C.A.S.; Formal analysis: Z.Z.F., K.M.H., J.P.S., D.D.K., K.A.G., C.A.S.; Funding acquisition: K.M.H., V.C.C.C., K.A.G., S.R.J., C.A.S.; Investigation: K.M.H., S.C.F., M.I.M., A.J.L., V.C.C.C., K.A.G., C.A.S.; Methodology: K.A.G., S.R.J., C.A.S.; Project administration: V.C.C.C., K.A.G., S.R.J., C.A.S.; Resources: V.C.C.C., K.A.G., S.R.J., C.A.S.; Supervision: K.A.G., S.R.J., C.A.S.; Validation: Z.Z.F., J.P.S., D.D.K., K.A.G., C.A.S.; Visualization: Z.Z.F., J.P.S., D.D.K., C.A.S.; Writing—original draft: Z.Z.F., J.P.S., D.D.K., C.A.S.; Writing—review & editing: Z.Z.F., K.M.H., J.P.S., S.C.F., M.I.M., A.J.L., V.C.C.C., D.D.K., K.A.G., S.R.J., C.A.S..

概念化：Z.Z.F.、S.C.F.、K.A.G.、S.R.J.、C.A.S.；数据管理：Z.Z.F.、K.A.G.、C.A.S.；形式分析：Z.Z.F.、K.M.H.、J.P.S.、D.D.K.、K.A.G.、C.A.S.；资金获取：K.M.H.、V.C.C.C.、K.A.G.、S.R.J.、C.A.S.；调查：K.M.H.、S.C.F.、M.I.M.、A.J.L.、V.C.C.C.、K.A.G.、C.A.S.；方法论：K.A.G.、S.R.J.、C.A.S.；项目管理：V.C.C.C.、K.A.G.、S.R.J.、C.A.S.；资源：V.C.C.C.、K.A.G.、S.R.J.、C.A.S.；监督：K.A.G.、S.R.J.、C.A.S.；验证：Z.Z.F.、J.P.S.、D.D.K.、K.A.G.、C.A.S.；可视化：Z.Z.F.、J.P.S.、D.D.K.、C.A.S.；撰写—原始草稿：Z.Z.F.、J.P.S.、D.D.K.、C.A.S.；撰写—审阅与编辑：Z.Z.F.、K.M.H.、J.P.S.、S.C.F.、M.I.M.、A.J.L.、V.C.C.C.、D.D.K.、K.A.G.、S.R.J.、C.A.S.。

Corresponding author

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Correspondence to

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Cody A. Siciliano

科迪·A·西西利亚诺

。

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Competing interests

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The authors declare no competing interests.

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Nature Communications

自然通讯

thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

感谢匿名评审员对该工作的同行评审所做出的贡献。同行评审文件可供查阅。

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Supplementary information

补充信息

Supplementary Information

补充信息

Supplementary Dataset 1

补充数据集1

Reporting Summary

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Transparent Peer Review file

透明同行评审文件

Source data

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Source Data

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Farahbakhsh, Z.Z., Holleran, K.M., Sens, J.P.