Basic brain mechanisms underlying drug addiction, craving, and relapse

在本报告所述期间，我们在这方面的研究扩大了，因为我们在上一报告所述期间关于生长激素释放激素的工作非常有希望。因此，我们的研究重点主要集中在Ghrelin及其受体上。Ghrelin是一种胃源性激素，与进食和应激有关，也可以调节奖赏和成瘾行为，并通过与生长激素促分泌素受体(GHSR)结合来发挥作用。最初，我们对通过CRISPR基因组编辑创建的一种新的GHSR基因敲除(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背景下开发了一个GHSR KO。用RNAScope对野生型(WT；n=2)和KO(n=2)大鼠的GHSR mRNA表达缺失进行了组织学验证。在WT(n=8)和KO(n=8)大鼠模型上，观察了腹腔注射酰基-Ghrelin对大鼠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量、食物摄入量和身体脂肪组成。体重从发育早期到成年都受到了监测。RNAScope分析显示在WTS的下丘脑、中脑和海马区有丰富的GHSR mRNA表达，而在KOS中没有观察到探针结合。Ghrelin可增加WT大鼠的血浆GH水平和食物消耗量，但不增加KO。与WTS相比，KO大鼠在整个发育过程中总体上消耗了更少的食物，体重也明显减轻。与WTS相比，KOS表现出更高的棕色脂肪组织(BAT)浓度。我们用组织学、生理学、神经内分泌学和行为学方法验证了我们的KO模型中的GHSR缺失。我们的发现表明，大鼠的GHSR缺失不仅与对Ghrelin缺乏反应有关，而且与日常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Ghrelin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个新的工具。此外，我们还研究了对情绪和运动至关重要的多巴胺(DA)系统。DA系统可以通过两种方式被激活：通过引起突发性放电和记忆的兴奋性输入，或者通过缓慢的兴奋性或抑制性输入--如瘦素、胰岛素、Ghrelin或皮质酮--减少或增加单峰(起搏器)放电并调节动机。因此，我们监测了大鼠在静脉注射可卡因或生理盐水之前和期间采集的血液样本。在吸食可卡因期间，生长激素和乙酰化Ghrelin增加了10倍；胰高血糖素样肽-1(GLP-1)增加了一倍；非乙酰化Ghrelin、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--动物获得了意想不到的生理盐水而不是可卡因--生长激素和乙酰化Ghrelin增加了5倍，除了IGF-1(增加速度较慢)外，其他每一种情况下的幅度和潜伏期都有相同的变化。单峰放电影响DA系统的紧张激活水平，涉及的控制与驱动爆发放电的控制非常不同；因此，我们的发现为可能用于药物戒断早期阶段的药物提供了有趣的新靶点。我们还利用RNAScope技术对腹侧被盖区(VTA)中的Ghrelin受体进行了研究。这项研究因新冠肺炎疫情迫使BRC大楼疏散而中断，但所有数据都被保存，以供未来分析和发表。在另一个新的方向上，我们观察了暴饮暴食或束缚长时间自我给药后偶发和非偶发甲基苯丙胺(冰毒)暴露的神经化学和行为比较。冰毒是一种心理刺激剂，可对大脑单胺类系统造成长期损害，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。冰毒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，但许多研究使用的是急性的、非偶然的冰毒“狂欢”给药，这并不完全接近人类冰毒的使用。通过长期接触(LGA)自我给药的长期冰毒暴露已被提出，以更接近地反映人类的使用情况，并诱导认知障碍。为了更好地了解偶然性和暴露模式在冰毒诱导的认知障碍中的作用，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”冰毒注射与偶然性(LGA)冰毒自我管理和非偶然性轭动物。我们发现，暴饮暴食冰毒(40 mg/kg，ip，超过1天)在暴露75天后显著改变纹状体和海马区的DA、DOPAC、5-羟色胺、5-HIAA、BDNF和TrkB。相反，自我注射6小时LGA冰毒(持续16天，累积24.8-48.9 mg冰毒)改变了双侧动物和负重动物的海马区BDNF，但仅减少了双侧动物的纹状体5-HIAA。过量服用冰毒后的神经化学改变没有伴随着Morris水迷宫、新奇物体识别或Y迷宫测试中的认知缺陷。然而，应急LGA冰毒自我给药导致水迷宫空间记忆功能受损。总体而言，冰毒暴露和自我给药范例之间的神经化学标记物的实质性差异并不总是转化为认知任务中的缺陷，这突显了将冰毒诱导的神经化学变化与认知结果关联起来的复杂性。

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.