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Item Development of a primate model to evaluate the effects of ketamine and surgical stress on the neonatal brain(Sage, 2023) Wang, Cheng; Bhutta, Adnan; Zhang, Xuan; Liu, Fang; Liu, Shuliang; Latham, Leah E.; Talpos, John C.; Patterson, Tucker A.; Slikker, William, Jr.; Pediatrics, School of MedicineWith advances in pediatric and obstetric surgery, pediatric patients are subject to complex procedures under general anesthesia. The effects of anesthetic exposure on the developing brain may be confounded by several factors including pre-existing disorders and surgery-induced stress. Ketamine, a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, is routinely used as a pediatric general anesthetic. However, controversy remains about whether ketamine exposure may be neuroprotective or induce neuronal degeneration in the developing brain. Here, we report the effects of ketamine exposure on the neonatal nonhuman primate brain under surgical stress. Eight neonatal rhesus monkeys (postnatal days 5-7) were randomly assigned to each of two groups: Group A (n = 4) received 2 mg/kg ketamine via intravenous bolus prior to surgery and a 0.5 mg/kg/h ketamine infusion during surgery in the presence of a standardized pediatric anesthetic regimen; Group B (n = 4) received volumes of normal saline equivalent to those of ketamine given to Group A animals prior to and during surgery, also in the presence of a standardized pediatric anesthetic regimen. Under anesthesia, the surgery consisted of a thoracotomy followed by closing the pleural space and tissue in layers using standard surgical techniques. Vital signs were monitored to be within normal ranges throughout anesthesia. Elevated levels of cytokines interleukin (IL)-8, IL-15, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein (MIP)-1β at 6 and 24 h after surgery were detected in ketamine-exposed animals. Fluoro-Jade C staining revealed significantly higher neuronal degeneration in the frontal cortex of ketamine-exposed animals, compared with control animals. Intravenous ketamine administration prior to and throughout surgery in a clinically relevant neonatal primate model appears to elevate cytokine levels and increase neuronal degeneration. Consistent with previous data on the effects of ketamine on the developing brain, the results from the current randomized controlled study in neonatal monkeys undergoing simulated surgery show that ketamine does not provide neuroprotective or anti-inflammatory effects.Item Effects of Developmental Low-Level Lead Exposure on Voluntary Alcohol Consumption and Drug-Induced Behavioral Sensitization in Adulthood(2020-12) Hernández, Maribel; Boehm, Stephen; Filippelli, Gabriel; Graham, NicholasLead (Pb) is one of the most harmful and most abundant neurotoxins in the environment. Despite the extensive movement made to eradicate toxic levels of Pb in the environment, children, predominately in lower socioeconomic areas, are still exposed to varying levels of Pb. Human studies suggest that Pb exposure leads to altered drug consumption in adults by altering underlying neural mechanisms, specifically dopamine (DA) activity. However, there is limited research on this at blood Pb levels below 10 μg/dL, levels often seen in children growing up in neighborhoods located in old industrial and urban areas. To model how early-life low-level Pb exposure effects DA-dependent behaviors associated with addiction in adulthood, we used C57BL/6J mice. Litters were weaned at PND 21 and assigned to either a three-week history of 30 parts per million (ppm) Lead (IV) Acetate exposure or a control condition of 0 ppm Pb in DI drinking water. After the Pb exposure period, mice were switched to regular tap water until they reached adulthood. Afterward, separate animals were tested in one of three experiments: two-bottle choice alcohol preference drinking, alcohol-induced behavioral sensitization (EBS), and cocaine-induced behavioral sensitization (CBS). In experiment 1, our hypothesis was met, and both male and female mice with a prior Pb exposure displayed significantly higher alcohol intake and preference scores over the three-week period than control mice. In experiment 2, there were no differences in EBS and no evidence of EBS in any of the groups. However, there was an increased acute response to 2.0 g/kg EtOH in the Pb-exposed chronic group as compared to the control animals. Lastly, in experiment 3, Pb-exposed animals in the chronic cocaine group were more sensitive to the effects of cocaine (10 mg/kg) across days than the controls, both the acute cocaine groups and both saline control groups. Thus, with these experiments, we concluded that low levels of developmental Pb exposure might be targeting DA in the reward pathway, which is essential for alcohol intake and drug sensitization.Item Embryonic Methamphetamine Exposure Inhibits Methamphetamine Cue Conditioning and Reduces Dopamine Concentrations in Adult N2 Caenorhabditis elegans(Karger, 2016-05) Katner, S.K.; Neal-Beliveau, B.S.; Engleman, E.A.; Psychology, School of ScienceMethamphetamine (MAP) addiction is substantially prevalent in today's society, resulting in thousands of deaths and costing billions of dollars annually. Despite the potential deleterious consequences, few studies have examined the long-term effects of embryonic MAP exposure. Using the invertebrate nematode Caenorhabditis elegans allows for a controlled analysis of behavioral and neurochemical changes due to early developmental drug exposure. The objective of the current study was to determine the long-term behavioral and neurochemical effects of embryonic exposure to MAP in C. elegans. In addition, we sought to improve our conditioning and testing procedures by utilizing liquid filtration, as opposed to agar, and smaller, 6-well testing plates to increase throughput. Wild-type N2 C. elegans were embryonically exposed to 50 μM MAP. Using classical conditioning, adult-stage C. elegans were conditioned to MAP (17 and 500 μM) in the presence of either sodium ions (Na+) or chloride ions (Cl-) as conditioned stimuli (CS+/CS-). Following conditioning, a preference test was performed by placing worms in 6-well test plates spotted with the CS+ and CS- at opposite ends of each well. A preference index was determined by counting the number of worms in the CS+ target zone divided by the total number of worms in the CS+ and CS- target zones. A food conditioning experiment was also performed in order to determine whether embryonic MAP exposure affected food conditioning behavior. For the neurochemical experiments, adult worms that were embryonically exposed to MAP were analyzed for dopamine (DA) content using high-performance liquid chromatography. The liquid filtration conditioning procedure employed here in combination with the use of 6-well test plates significantly decreased the time required to perform these experiments and ultimately increased throughput. The MAP conditioning data found that pairing an ion with MAP at 17 or 500 μM significantly increased the preference for that ion (CS+) in worms that were not pre-exposed to MAP. However, worms embryonically exposed to MAP did not exhibit significant drug cue conditioning. The inability of MAP-exposed worms to condition to MAP was not associated with deficits in food conditioning, as MAP-exposed worms exhibited a significant cue preference associated with food. Furthermore, our results found that embryonic MAP exposure reduced DA levels in adult C. elegans, which could be a key mechanism contributing to the long-term effects of embryonic MAP exposure. It is possible that embryonic MAP exposure may be impairing the ability of C. elegans to learn associations between MAP and the CS+ or inhibiting the reinforcing properties of MAP. However, our food conditioning data suggest that MAP-exposed animals can form associations between cues and food. The depletion of DA levels during embryonic exposure to MAP could be responsible for driving either of these processes during adulthood.Item Micro-RNA regulation of hepatic drug metabolism : age-related changes in micro-RNA expression and genetic variants in micro-RNA target sites(2017-08-31) Burgess, Kimberly Sherrelle; Skaar, Todd C.; Arrizabalaga, Gustavo; Cummins, Theodore; Desta, Zeruesenay; Nass, Richard; Zhang, Jian-TianDevelopmental changes in the liver significantly impact drug disposition. Due to the emergence of microRNAs as important regulators of drug disposition, we hypothesize that age-dependent change in microRNA expression and genetic variants in microRNA target sites contribute to variability in drug disposition. In human liver tissues, expression of 533 microRNAs and over 14,000 genes were measured. In all, 114 microRNAs were upregulated and 72 downregulated from fetal to pediatric, and 2 and 3, respectively, from pediatric to adult. Among these microRNAs, 99 microRNA-mRNA interactions were predicted or have previously been validated to target drug disposition genes and over 1,000 significant negative correlations were observed between miRNA-mRNA pairs. We validated these interactions using various cell culture models. Genetic variants in the promoter and coding regions of drug disposition genes have also been shown to alter enzyme expression and/or activity. However, these variants do not account for all variability in enzyme activity. Emerging evidence has shown that variants in the 3’UTR may explain variable drug response by altering microRNA regulation. Five 3’UTR variants were associated with significantly altered CYP2B6 activity in healthy human volunteers. The rs70950385 (AG>CA) variant was associated with decreased CYP2B6 activity among normal metabolizers. In vitro luciferase assays confirmed that the CA allele altered miR 1275 targeting of CYP2B6 mRNA. Due to the large number of 3’UTR variants predicted to alter microRNA regulation, a high-throughput method, PASSPORT-seq, was developed to test over 100 3’UTR variants simultaneously in different cell lines. Thirty-eight variants resulted in FDR-significant altered expression between wild-type and variant sequences. Our data suggest a mechanism for the marked changes in hepatic gene expression between the fetal and pediatric developmental periods, support a role for these age dependent microRNAs in regulating drug disposition, and provide strong evidence that 3’UTR variants are also an important source of variability in drug disposition.Item The Collaborative Study on the Genetics of Alcoholism: Overview(Wiley, 2023) Agrawal, Arpana; Brislin, Sarah J.; Bucholz, Kathleen K.; Dick, Danielle; Hart, Ronald P.; Johnson, Emma C.; Meyers, Jacquelyn; Salvatore, Jessica; Slesinger, Paul; COGA Collaborators; Almasy, Laura; Foroud, Tatiana; Goate, Alison; Hesselbrock, Victor; Kramer, John; Kuperman, Samuel; Merikangas, Alison K.; Nurnberger, John I.; Tischfield, Jay; Edenberg, Howard J.; Porjesz, Bernice; Medical and Molecular Genetics, School of MedicineAlcohol use disorders (AUD) are commonly occurring, heritable and polygenic disorders with etiological origins in the brain and the environment. To outline the causes and consequences of alcohol-related milestones, including AUD, and their related psychiatric comorbidities, the Collaborative Study on the Genetics of Alcoholism (COGA) was launched in 1989 with a gene-brain-behavior framework. COGA is a family based, diverse (~25% self-identified African American, ~52% female) sample, including data on 17,878 individuals, ages 7-97 years, in 2246 families of which a proportion are densely affected for AUD. All participants responded to questionnaires (e.g., personality) and the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) which gathers information on psychiatric diagnoses, conditions and related behaviors (e.g., parental monitoring). In addition, 9871 individuals have brain function data from electroencephalogram (EEG) recordings while 12,009 individuals have been genotyped on genome-wide association study (GWAS) arrays. A series of functional genomics studies examine the specific cellular and molecular mechanisms underlying AUD. This overview provides the framework for the development of COGA as a scientific resource in the past three decades, with individual reviews providing in-depth descriptions of data on and discoveries from behavioral and clinical, brain function, genetic and functional genomics data. The value of COGA also resides in its data sharing policies, its efforts to communicate scientific findings to the broader community via a project website and its potential to nurture early career investigators and to generate independent research that has broadened the impact of gene-brain-behavior research into AUD.