Basic brain mechanisms underlying drug addiction, craving, and relapse

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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.