Basic brain mechanisms underlying drug addiction, craving, and relapse

在本报告期间，我们在这一领域的研究扩大了，因为我们在上一个报告期间对胃饥饿素的研究非常有希望。因此，我们的研究重点主要集中在胃饥饿素和胃饥饿素受体。胃饥饿素是一种胃源性激素，与进食和应激有关，也通过与生长激素促分泌受体（GHSR）结合来调节奖励和成瘾行为。最初，我们利用CRISPR基因组编辑技术建立了一种新的GHSR敲除（KO） Wistar大鼠模型，并对其进行了开发、验证和初步表征。使用CRISPR/Cas9，我们在Wistar背景下开发了GHSR KO。在野生型（WT; n=2）和KO （n=2）大鼠中使用RNAscope进行组织学验证GHSR mRNA表达缺失。我们测试了WT （n=8）和KO （n=8）大鼠腹腔注射乙酰胃饥饿素对食物消耗和血浆生长激素（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。体重监测从早期发育到成年。RNAscope分析显示，在WTs的下丘脑、中脑和海马中有丰富的GHSR mRNA表达，而在KOs中未观察到探针结合。胃促生长素增加了WT大鼠的血浆生长激素水平和食物消耗，但对KOs没有影响。在基础条件下，KO大鼠消耗的食物总体较少，在整个发育过程中体重明显低于WTs。与对照组相比，ko组棕色脂肪组织（BAT）浓度较高。我们使用组织学、生理学、神经内分泌学和行为学方法验证了KO模型中的GHSR缺失。我们的研究结果表明，大鼠的GHSR缺失不仅与对ghrelin缺乏反应有关，还与日常食物消耗和身体生长减少以及BAT增加有关。这种GHSR KO Wistar大鼠模型为研究饥饿素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。进一步，我们检查了多巴胺（DA）系统——对情绪和运动至关重要。DA系统可以通过两种方式被激活：通过兴奋性输入，引起突发放电和学习戳痕；或者通过缓慢的兴奋性或抑制性输入，如瘦素、胰岛素、生长素或皮质酮，减少或增加单尖峰（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，生长激素和乙酰化胃饥饿素增加了10倍；胰高血糖素样肽-1 （GLP-1）加倍；非乙酰化胃饥饿素、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，动物们意外地获得了生理盐水而不是可卡因，生长激素和乙酰化胃饥饿素增加了5倍，除了IGF-1（增加速度较慢），其他情况下的幅度和潜伏期变化相同。单脉冲放电影响DA系统的强直激活水平，涉及的控制与驱动脉冲放电的控制非常不同；因此，我们的研究结果提出了有趣的新靶点，可能用于药物戒断的早期阶段。我们还利用RNAscope技术对腹侧被盖区（VTA）的胃饥饿素受体进行了研究。本研究因COVID-19大流行导致BRC大楼被迫疏散而中断，但所有数据均被保存以供将来分析和发表。在另一个新的方向上，我们研究了在暴饮暴食或长期自我给药后偶然和非偶然甲基苯丙胺暴露的神经化学和行为比较。甲基苯丙胺是一种精神兴奋剂，可对大脑单胺能系统造成长期损害，并与使用者严重的精神健康问题有关，包括持久的认知障碍。甲基苯丙胺暴露的动物模型在剖析甲基苯丙胺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“暴饮”甲基苯丙胺，这并不能充分接近人类对甲基苯丙胺的使用。长期甲基安非他明暴露通过长期获取（LgA）自我给药已被提出更密切地反映人类使用和诱导认知障碍。为了更好地理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“暴食”冰毒给药与偶然（LgA）冰毒自我给药和非偶然的枷锁动物。我们发现暴食冰毒（40 mg/kg, i.p.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冰毒自我给药（累计24.8-48.9 mg冰毒，静脉注射，超过16天）改变了偶发动物和对照动物的海马BDNF，但只在偶发动物中降低了纹状体5-HIAA。在莫里斯水迷宫、新物体识别或y迷宫测试中，暴食冰毒后的神经化学改变不伴有认知缺陷。然而，偶然的LgA自我给药导致水迷宫空间记忆受损。总的来说，冰毒暴露和自我给药范式之间的神经化学标志物的实质性差异并没有一致地转化为认知任务的缺陷，这突出了冰毒诱导的神经化学变化与认知结果之间的相关性的复杂性。

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.