VA Senior Research Career Scientist Identifying Genes that Impact Methamphetamine Use Disorder Tamara J. Phillips, Ph.D. Professor of Behavioral Neuroscience Director Scientific Director Methamphetamine (MA) • Speed, ice, crystal • Unpredictable and sometimes lethal • Remains a significant drug threat • 2017 – National Survey on Drug Use and Health (NSDUH): 1.6 million people in the U.S. reported using MA in the past year. • Average age of users is 23 y.o. • About 1 million had a MA use disorder • Highest availability in the western and Midwestern U.S. Pacific Northwest: Drug of Choice is MA The Columbian Newspaper, Vancouver WA Age-adjusted Rate of MA Overdose Deaths in 2017 SOURCE: NCHS National Vital Statistics System, Mortality files linked with death certificate literal text Adverse Health Effects • Weight loss • Severe dental problems • Intense itching, leading to skin sores • Anxiety, confusion, paranoia, hallucinations • Memory loss • Sleep problems • Changes in brain structure and function • Those using by injection have increased risk of contracting infectious diseases Individual Vulnerability • Not all initial MA users go on to use regularly or to develop MA addiction. • Genetic factors may influence sensitivity to MA. • Genetic factors may influence the amount of MA use. • Selective breeding provides a tool for examining genetic influences on behavior. Selective Breeding for Voluntary Methamphetamine Intake Frequency: 20 10 Originating Population (120 mice) 0 (a genetically heterogeneous sample) 20 10 1st selected generation (60/line) 0 20 10 2nd selected generation (60/line) 0 20 10 3rd selected generation (60/line) 0 MA Consumed (mg/kg): 0 1 2 3 4 5 6 7 Selection diagram credited to Dr. John K. Belknap Measurement of Voluntary Methamphetamine Drinking Habituation 20 mg/L MA vs. Water 40 mg/L MA vs. Water Days 1 & 2 Days 3-6 Days 7-10 Days (4+6)/2 and (8+10)/2= mean intake (mg/kg) for each concentration Day 3-10 daily timeline: water ------------water & methamphetamine------------ water 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Hours = water tube = MA tube Replicable Results F2 MALDR 8 MAHDR F2-2 MALDR-2 MAHDR-2 F2-3 ) l MALDR-3 / 6 MAHDR A MAHDR-3 M F2-4 Jeanna Wheeler n g MALDR-4 o i t m MAHDR-4 Zeni Shabani p 0 F2-5 m 4 MALDR-5 u ; 4 s s MAHDR-5 r n h o 8 C 1 / A g M k / 2 g m ( Harue Baba MALDR 0 Jason Erk S0 S1 S2 S3 S4 Selection Generation F2=Second filial cross of the D2 and B6 inbred strains MALDR=MA low drinking line; MAHDR=MA high drinking line Cheryl Reed Results for Several Phenotypes Compared in the MA Drinking Lines Phenotypes for which MAHDR > MALDR: MA drinking; 2-bc, 4-bc escalating concentration and lickometer MA blood levels AFTER MA drinking Operant ICV and operant oral MA SA MA-induced CPP, acute activation, sensitization and HYPERthermia Phenotypes for which MAHDR < MALDR: MA-induced CTA, CPA and HYPOthermia Spatial memory retention Morphine drinking and locomotor effects Phenotypes for which MAHDR = MALDR Quinine, saccharin and KCl drinking; 2-bc Basal locomotor activity and MA-induced locomotor stimulation MA clearance rate Novel object recognition, spatial memory learning and fear conditioning Ethanol-induced HYPOthermia COC-induced CPP, CTA and locomotor stimulation Conditioned Taste Aversion (CTA) Day 1-5 Day 6 Day 7, 9, 11, 13, and 15 Reduce access to Drink novel salty Drink salty water, then inject saline or MA. water so that water. the mice will be Thus, the effect of the injected solution is paired with the thirsty and will The purpose of this mostly novel taste. learn to drink at day is to introduce the a particular time novel taste and reduce If the effect of MA “feels good (or doesn’t feel bad)” the mice of day. neophobia. will continue to drink the salty water the NEXT day. If it “feels bad” the mice will reduce the amount of salty water they drink the next day. The MA Drinking Lines Differ in Sensitivity to MA- induced CTA Wheeler et al. (2009) Genes Brain Behavior 8:758; Shabani et al. (2012) Neuropharmacology 62:1134 The MA Drinking Line Mice do NOT Differ in CTA Response to Cocaine Gubner et al. (2013) Behav Brain Res 256:420-427 MA drinking Tube to Water Tube Ratio Impacts MA Intake Inspired by: Tordoff MG & Bachmanov AA (2003) Influence of the number of alcohol and water bottles on murine alcohol intake. Alcohol Clin Exp Res 27:600. Shabani et al. (2016) Front Neurosci 10:493 Measuring Sensitivity to Drug Reward (and Aversion) Place Conditioning set-up during testing Drug Absent Test: Are the cues that were previously paired with the drug preferred or avoided? Drug Present Test: What is the impact on preference/avoidance when the animals are in the drug-treated state? Hole floor Grid floor Image contributed by Dr. Chris Cunningham MA-induced CPP in MAHDR Mice in the Drug-Free Test Drug-Free CPP Test MAHDR MALDR *** MAHDR: Spent ~60% of their time on MA-paired floor MALDR: Spent ~50% of their time on the MA-paired floor Shabani et al. (2011) Gene Brain Behav 10:625 CPA in MALDR Mice in the Presence of MA Treatment MA-Present CPP Test *** *** MAHDR MALDR MAHDR: Spent ~66% of their time on the 0.5 mg/ kg MA-paired floor. MALDR: Spent ~33% of their time on the drug- paired floor. Shabani et al. (2011) Gene Brain Behav 10:625 Quantitative Trait Locus (QTL) Mapping D2 alleles increase MA intake B6 alleles increase MA intake Taar1 Belknap et al. (2013) Mamm Genome 24:446-58 MA Drinking is Associated withTaar1 Genotype (Chr 10: 23.92 Mb) = genotypes lacking TAAR1 receptor function Harkness et al. (2015) Neuropsychopharmacology 40:2175-2184 John Harkness Body Temperature Response to MA is Associated with Taar1 Genotype Response is to 2 mg/kg MA. There were no differences after saline treatment Harkness et al. (2015) Neuropsychopharmacology 40:2175-2184 Sensitivity to MA-induced CTA is Associated with Taar1 Genotype Harkness et al. (2015) Neuropsychopharmacology 40:2175-2184. Topology of Mouse Trace Amine-Associated Receptor 1 TAAR1 DBA/2J (D2) mice possess a unique non-synonymous SNP in Taar1 that is not present in 28 other strains of mice that have been genotyped. The mutation arose spontaneously. D2 is one of the progenitor strains of the MA drinking lines. Figure adapted from Lindemann et al., 2005, Genomics 85:372 The mouse SNP encodes a proline to threonine mutation that predicts a change in receptor conformation. This is not a deletion mutation, but we have determined that the receptor expressed by the mutant allele (Taar1m1J) is nonfunctional. The Mutant Allele Codes for a TAAR1 that is not Activated by MA in Transfected Cells GFP immunodetection indicates that both alleles express mTAAR1 in HEK293 cells. Human variants also have differential function. Confocal images of HEK293 cells transfected with GFP-tagged B6- or D2-like Taar1. Harkness et al. (2015) Neuropsychopharmacology 40:2175-2184 Trace Amine-Associated Receptor Genes Eyun et al. (2016) PLoS One 11:e0151023 Trace Amine-Associated Receptor 1: TAAR1 • All mouse TAARs except TAAR1 are expressed in the main olfactory epithelium. TAAR1 is expressed in reward-related brain areas. • TAAR1 is a cytosolic, stimulatory G-protein-coupled receptor that is responsive to trace amines, such as β-phenylethylamine, octopamine, and tyramine. • Methamphetamine is a TAAR1 agonist. • TAAR1 activation triggers accumulation of intracellular cAMP, and appears to modulate PKA and PKC signaling, and interfere with the β-arrestin2-dependent pathway via G protein-independent mechanisms. • TAAR1 appears to exert control over monoamine neuronal firing and release; unclear whether this is a direct or indirect effect. Greater dopamine in the absence of TAAR1 function has been reported. Knock-in Recovery of TAAR1-associated MA Traits Recovery of low MA intake No impact on fluid intake Alex Stafford Cheryl Reed Stafford, Reed et al. (2019) eLife 8:46472 Chronology for the Taar1m1J Allele X Reed et al. (2018) Front Pharmacol 8:993 Knock-in Recovery of TAAR1-associated MA Traits Recovery of low MA intake No impact on fluid intake Alex Stafford Cheryl Reed Stafford, Reed et al. (2019) eLife 8:46472 Range of Consumption Values in S4 Generation of the Replicate 3 MADR Lines 14 ) L / 12 A M n 10 o g i t m p 8 0 m 4 u ; s 6 s r n h o c 4 8 1 A / g 2 M k / g 0 m ( MAHDR F MALDR F MAHDR M MALDR M MADR Line and Sex Note that only those individual data points at locations outside of the boxes are shown Phillips and colleagues, unpublished Individual Variation and Patterns of MA Intake in MAHDR Mice These data are for mice that had daily access to 3 bottles of 80 mg/L MA and 1 bottle of water, which results in higher levels of intake. Shabani et al. (2016) Front Neurosci 10:493. Why is There Modest Variability in MA Intake in MALDR, and Considerable Variability in MAHDR Mice? • When TAAR1 function is absent, rewarding effects of MA can be experienced. - MAHDR exhibit MA reward and reinforcement as a group; MALDR do not • Did our selection not completely fix those genes that impact MA reward in the MAHDR line? • Are there modifiers in the genetic background that impact the Taar1m1J/ m1J effect on MA intake, resulting in lower levels of MA intake in some MAHDR individuals? • Are there epigenetic (environmental) effects that can modify the impact of the mutant allele? MA-induced CPP in MAHDR Mice in the Drug-Free Test Drug-Free CPP Test MAHDR MALDR *** MAHDR: Spent ~65% of their time on MA-paired floor MALDR: Spent ~50% of their time on the MA-paired floor NOT a TAAR1 effect, because Mixture of TAAR1 and non-TAAR1 TAAR1 is non-functional (e.g., dopamine) effects of MA? Shabani (2011) Gene Brain Behav 10:625 The TAAR1 Agonist, RO-5256390, Induces Conditioned Place Aversion (CPA) in MALDR Mice Drug-Free CPP Test Spent ~33% of their time on the drug-paired floor – clear avoidance.