Basic brain mechanisms underlying drug addiction, craving, and relapse

在本报告所述期间，我们扩大了这一领域的研究，因为我们在上一个报告所述期间关于生长激素释放肽的工作非常有希望。因此，我们把我们的研究重点主要放在ghrelin和ghrelin受体上。生长激素释放肽是一种与进食和压力有关的胃源性激素，也可以调节奖励和成瘾行为，并通过与生长激素促分泌素受体（GHSR）结合而起作用。最初，我们对用CRISPR基因组编辑创建的新型GHSR敲除（KO）Wistar大鼠模型进行了开发、验证和初步表征。使用CRISPR/Cas9，我们在Wistar背景上开发了GHSR KO。使用RNAscope在野生型（WT; n=2）和KO（n=2）大鼠中对GHSR mRNA表达的丧失进行组织学验证。我们测试了腹腔注射酰基-ghrelin对WT（n=8）和KO（n=8）大鼠的摄食量和血浆生长激素（GH）浓度的影响。我们还分析了这些动物的运动，食物消耗和体脂组成。从早期发育到成年监测体重。RNAscope分析显示，在WT的下丘脑、中脑和海马中有丰富的GHSR mRNA表达，而在科斯中没有观察到探针结合。Ghrelin给药增加WT大鼠的血浆GH水平和摄食量，但不增加科斯。KO大鼠在基础条件下总体摄食量较少，在整个发育过程中体重显著低于WT。与WT相比，科斯表现出较高的棕色脂肪组织（BAT）浓度。我们使用组织学、生理学、神经内分泌学和行为学测量在我们的KO模型中验证了GHSR缺失。我们的研究结果表明，大鼠GHSR缺失不仅与缺乏对ghrelin的反应有关，而且与每日食物消耗和身体生长的减少以及BAT的增加有关。这种GHSR KO Wistar大鼠模型为研究生长激素释放肽系统在肥胖和广泛的医学和神经精神疾病中的作用提供了一种新的工具。此外，我们研究了多巴胺（DA）系统-对情绪和运动至关重要。DA系统可以通过两种方式激活：通过兴奋性输入引起爆发性放电和踩踏式学习，或者通过缓慢的兴奋性或抑制性输入--如瘦素、胰岛素、生长激素释放肽或皮质酮--减少或增加单棘波（起搏器）放电并调节动机。因此，我们监测了大鼠静脉注射可卡因或生理盐水自我给药之前和期间采集的血样。在服用可卡因期间，生长激素和乙酰化生长激素释放肽增加了10倍;胰高血糖素样肽-1（GLP-1）增加了一倍;非乙酰化生长激素释放肽、胰岛素样生长因子-1（IGF-1）和皮质酮增加了50%，脂联素增加了17%。在相同的血液样本中，瘦素、胰岛素、胃抑制多肽（GIP）和催乳素下降了40- 70%。在灭绝条件下测试的第一天-其中动物获得了意想不到的盐水而不是可卡因-生长激素和乙酰化生长激素释放肽增加了5倍，并且在除IGF-1（其以较慢的速率增加）之外的每种其他情况下观察到振幅和潜伏期的相等变化。单棘放电影响DA系统的强直激活水平，涉及与驱动突发放电的控制非常不同的控制;因此，我们的研究结果表明，药物可能用于药物戒断的早期阶段的有趣的新目标。我们还启动了一项研究的胃饥饿素受体在腹侧被盖区（VTA）使用RNAscope技术。该研究因COVID-19疫情导致BRC大楼被迫疏散而中断，但所有数据均已保存以供日后分析及发表。在另一个新的方向，我们研究了神经化学和行为比较的应急和非应急甲基苯丙胺（METH）暴露后，狂欢或轭长期访问自我管理。METH是一种精神兴奋剂，可对大脑单胺能系统造成长期损害，并与使用者的严重心理健康问题有关，包括持久的认知障碍。METH暴露的动物模型在剖析药物对认知的分子效应方面是有用的，但是许多研究使用急性的、非偶然的METH“狂欢”给药，这不足以近似人类的METH使用。已提出通过长期接触（LgA）自我给药的长期甲基溴化铵暴露更密切地反映了人类的使用，并诱导认知障碍。为了更好地了解METH诱导的认知障碍中的偶然性和暴露模式的作用，我们分析了成年雄性大鼠的行为和神经化学结果，比较了非偶然性“狂欢”METH给药与偶然性（LGA）METH自我给药和非偶然性轭动物。我们发现，狂饮METH（40 mg/kg，i. p.，超过1天）在药物暴露后75天显著改变纹状体和海马DA、DOPAC、5-HT、5-HIAA、BDNF和TrkB。相比之下，6小时LgA METH自我给药（累积24.8-48.9 mg METH，静脉内，超过16天）改变了特遣队和轭动物的海马BDNF，但仅减少了特遣队动物的纹状体5-HIAA。神经化学的改变后，狂欢METH管理并不伴随着认知缺陷，在莫里斯水迷宫，新的物体识别，或Y-迷宫测试。然而，偶然的LGA METH自我管理导致受损的空间记忆在水迷宫中。总体而言，METH暴露和自我管理模式之间的神经化学标志物的实质性差异并不一致地转化为认知任务的缺陷，突出了METH诱导的神经化学变化与认知结果相关的复杂性。

During the present reporting period, our research in this area expanded, as our work on ghrelin during the previous reporting period was so promising. Therefore, we focused our research attention primarily on ghrelin and the ghrelin receptor. Ghrelin, a stomach-derived hormone implicated in feeding and stress, also acts to regulate reward and addictive behaviors, and acts by binding to the growth hormone secretagogue receptor (GHSR). Initially, we carried out development, verification, and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing. Using CRISPR/Cas9, we developed a GHSR KO on a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type (WT; n=2) and KO (n=2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n=8) and KO (n=8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood. RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels and food consumption in WT rats but not KOs. KO rats consumed less food overall under basal conditions and weighed significantly less compared with WTs throughout development. Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT). We verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological, and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders. Further, we examined the dopamine (DA) system - essential for mood and movement. The DA system can be activated in two ways: by excitatory inputs that cause burst firing and stamp-in learning or by slow excitatory or inhibitory inputs - such as leptin, insulin, ghrelin, or corticosterone - that decrease or increase single-spike (pacemaker) firing and that modulate motivation. Therefore, we monitored blood samples taken prior to and during intravenous cocaine or saline self-administration in rats. During cocaine-taking, growth hormone and acetylated ghrelin increased 10-fold; glucagon-like peptide-1 (GLP-1) doubled; non-acetylated ghrelin, insulin-like growth factor-1 (IGF-1), and corticosterone increased by 50%, and adiponectin increased by 17%. In the same blood samples, leptin, insulin, gastric inhibitory polypeptide (GIP), and prolactin decreased by 40-70%. On the first day of testing under extinction conditions - where the animals earned unexpected saline instead of cocaine - 5-fold increases were seen for growth hormone and acetylated ghrelin and equal changes in amplitude and latency were seen in each of the other cases except for IGF-1 (which increased at a slower rate). Single-spike firing affects the tonic activation level of the DA system, involving very different controls than those that drive burst firing; thus, our findings suggest interesting new targets for medications that might be used in the early stages of drug abstinence. We also initiated a study of ghrelin receptors in the ventral tegmental area (VTA) using RNAscope technology. This study was interrupted by the forced evacuation of the BRC Building by the COVID-19 pandemic, but all data were saved for future analysis and publication. In another new direction, we looked at neurochemical and behavioral comparisons of contingent and non-contingent methamphetamine (METH) exposure following binge or yoked long-access self-administration. METH is a psychostimulant that can cause long-lasting damage to brain monoaminergic systems and is associated with profound mental health problems for users, including lasting cognitive impairments. Animal models of METH exposure have been useful in dissecting the molecular effects of the drug on cognition, but many studies use acute, non-contingent "binge" administrations of METH which do not adequately approximate human METH use. Long-term METH exposure via long-access (LgA) self-administration has been proposed to more closely reflect human use and to induce cognitive impairments. To better understand the role of contingency and patterns of exposure in METH-induced cognitive impairments, we analyzed behavioral and neurochemical outcomes in adult male rats, comparing non-contingent "binge" METH administration with contingent (LgA) METH self-administration and non-contingent yoked animals. We found that binge METH (40 mg/kg, i.p., over 1 day) dramatically altered striatal and hippocampal DA, DOPAC, 5-HT, 5-HIAA, BDNF, and TrkB 75 days after drug exposure. In contrast, 6-hour LgA METH self-administration (cumulative 24.8-48.9 mg METH, i.v., over 16 days) altered hippocampal BDNF in both contingent and yoked animals but reduced striatal 5-HIAA in only contingent animals. Neurochemical alterations following binge METH administration were not accompanied by cognitive deficits in the Morris water maze, novel object recognition, or Y-maze tests. However, contingent LgA METH self-administration resulted in impaired spatial memory in the water maze. Overall, substantial differences in neurochemical markers between METH exposure and self-administration paradigms did not consistently translate to deficits in cognitive tasks, highlighting the complexity of correlating METH-induced neurochemical changes with cognitive outcomes.