Rats were trained to discriminate an injection of cocaine, 5.0 mg/kg, from an injection of saline, using a two-lever choice paradigm: one lever was correct after cocaine injection, the other lever was correct after a saline injection. After training, cocaine and methamphetamine were generalized to the cocaine lever, but phenethylamine (PEA) was only partially generalized. Cocaine was injected every 8 hrs, 20.0 mg/kg, and the discriminability of 5.0 mg/kg was tested every other day. Redetermination of the cocaine generalization curve after 6 days of chronic administration showed a shift to the right, from an ED50 of 4.1 mg/kg in the pre-chronic condition to 10.0 mg/kg. Tolerance did not develop to the behavioral effects of cocaine, measured by time to the first reinforcement and response rate. There was cross-tolerance to methamphetamine; however, no evidence for cross-tolerance to PEA was obtained. Following the acquisition of tolerance, chronic administration of cocaine was terminated, and the discriminability of 5.0 mg/kg was tested every other day for loss of tolerance. After 8 days the ED50 returned to 5.0 mg/kg.

Both genetic and environment factors influence susceptibility to substance use disorders. However, the genetic basis of these disorders is largely unknown. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene for reduced methamphetamine (MA) stimulant sensitivity. Mutation (heterozygous deletion of a small region in the first coding exon) in Hnrnph1 also decreased MA reinforcement, reward, and dopamine release. 5’UTR genetic variants in Hnrnph1 support reduced 5’UTR usage and hnRNP H protein expression as a molecular mechanism underlying the reduced MA-induced psychostimulant response. Interestingly, Hnrnph1 mutant mice show a two-fold increase in hnRNP H protein in the striatal synaptosome with no change in whole tissue level. Proteome profiling of the synaptosome identified an increase in mitochondrial complex I and V proteins that rapidly decreased with MA in Hnrnph1 mutants. In contrast, the much lower level of basal mitochondrial proteins in the wild-type mice showed a rapid, MA-induced increase. Altered mitochondrial proteins associated with the Hnrnph1 mutation may contribute to reductions in MA behaviors. hnRNP H1 is an abundant RNA-binding protein in the brain, involved in all aspect of post-transcriptional regulation. We examined both baseline and MA-induced changes in hnRNP H-RNA interactions to identify targets of hnRNP H that could comprise the neurobiological mechanisms of cellular adaptations occurring following MA exposure. hnRNP H post-transcriptionally regulates a set of mRNA transcripts in the striatum involved in psychostimulant-induced synaptic plasticity. MA treatment induced opposite changes in binding of hnRNP H to these mRNA transcripts between Hnrnph1 mutants versus wild-types. RNA-binding, transcriptome, and spliceome analyses triangulated on hnRNP H binding to the 3’UTR of Cacna2d2, an upregulation of Cacna2d2 transcript, and decreased 3’UTR usage of Cacna2d2 in response to MA in the Hnrnph1 mutants. Cacna2d2 codes for a presynaptic, voltage-gated calcium channel subunit that could plausibly regulate MA-induced dopamine release and behavior. The multi-omics datasets point to a dysregulation of mitochondrial function and interrelated calcium signaling as potential mechanisms underlying MA-induced dopamine release and behavior in Hnrnph1 mutants.

No description available.

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Indiana University-Purdue University Indianapolis (IUPUI) / Adolescent alcohol abuse is a global problem that initiates lifelong addiction. Alcohol use during adolescence is associated with subsequent Meth dependence in humans. Specifically, female adolescents are particularly vulnerable to serial alcohol and Meth use. However, it is unknown if prior voluntary alcohol drinking impacts subsequent Meth-taking in female adolescent rats. Both alcohol and Meth increase the prostaglandin synthesis enzyme cyclooxygenase-2 (COX-2) in the brain but the effect of serial exposure to alcohol and Meth on COX-2 has not been determined. The first study uses a novel method of serial voluntary alcohol drinking and Meth self-administration in female adolescent rats to model human patterns of co-abuse. Prior alcohol drinking did not affect subsequent Meth self-administration, but it reduced the cue-primed reinstatement of Methseeking after abstinence from Meth. Rats with a history of adolescent alcohol drinking also had increased COX-2 in the dorsal striatum, regardless of subsequent Meth selfadministration. These findings demonstrate that a history of adolescent alcohol drinking does not alter Meth self-administration but persistently reduces cue-primed Meth seeking and increases COX-2 after prolonged abstinence from alcohol. To further examine the role of COX-2 in alcohol drinking, the second study found that adolescent alcohol drinking not only increased COX-2 after four weeks of alcohol abstinence, but also increased endothelin-1 (ET-1) and prostaglandin E2 (PGE2) in the dorsal striatum. Furthermore, adolescent alcohol drinking increased alcohol drinking after abstinence, and this increase was attenuated by treatment with the COX-2 inhibitor nimesulide during abstinence. Antagonism of the interaction between PGE2 and its receptor 1 (EP1) also attenuated the increase in relapse drinking and restored alcohol drinking to the rate of alcohol naïve rats. Overall, these experiments identified a prostaglandin-mediated mechanism that is a putative target for the treatment of alcohol relapse following abstinence in individuals with a history of adolescent alcohol abuse.

Non-medical drug use in rural communities in the United States is a significant and growing public health threat. Understanding what motivates drug users in rural areas to seek substance abuse treatment may help in addressing the problem. Perceived need for treatment, a construct indicative of problem recognition and belief in problem solution, has been identified as an important predictor of help-seeking behavior. This cross-sectional study used data collected through face-to-face interviews to examine factors associated with perceived need for drug abuse treatment among not-in-treatment, adult, illicit stimulant drug users (n = 710) in rural areas of Ohio, Kentucky, and Arkansas. More than one-quarter of the sample perceived a need for treatment. Results from a stepwise multiple regression analysis showed that white users, users with better physical and mental health status, and occasional users of methamphetamine were significantly less likely to see a need for treatment. Users with higher Addiction Severity Index composite scores for family/social problems or legal problems, and users with prior drug abuse treatment experience were significantly more likely to perceive a need for treatment. These findings have practical implications for efforts addressing substance abuse in rural areas.

High-throughput liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methodology for the determination of methamphetamine (METH), amphetamine (AMP), 4-hydroxymethamphetamine (4-OH-METH), and 4-hydroxyamphetamine (4-OH-AMP) was developed and validated using simple trichloroacetic acid sample treatment. The method was validated in rat serum, brain, and testis. Lower limits-of-quantitation (LOQ) for METH and AMP were 1 ng·mL-1 using positive ion electrospray tandem mass spectrometry (MS/MS). The accuracy of the method was within 25% of the actual values over a wide range of analyte concentrations. The within-assay precision was better than 12% (coefficient of variation). The method was linear over a wide dynamic range (0.3-1000 ng·mL-1). Quantitation was possible in all 3 matrices using only serum standards because of minimal matrix-associated ion effects or the use of an internal standard. Finally, the LC-MS/MS method was used to determine serum, brain, and testis METH and AMP concentrations during a subcutaneous infusion (5.6 mg kg-1 day-1) of METH in rats. Concentrations of 4-OH-AMP and 4-OH-METH were below the LOQ in experimental samples. The bias introduced by using serum calibrators for the determination of METH and AMP concentrations in testis and brain was less than 8% and insignificant relative to the interanimal variability.

Addiction to psychostimulants such as methamphetamine (MA) is a significant public health issue in the United States with no FDA-approved pharmacological interventions. MA addiction is a heritable neuropsychiatric disorder, however, its genetic basis is almost entirely unknown. Available human genome-wide association studies (GWAS) lack sufficient power to detect the influence of common genetic variation on the risk of addiction. Mammalian model organisms offer an attractive alternative to more rapidly uncover novel genetic factors that contribute to addiction-relevant neurobehavioral traits. Using quantitative trait locus (QTL) mapping in mice, we identified a locus on chromosome 11 that contributed to a decrease in sensitivity to the locomotor stimulant properties of MA. To fine map this QTL, we generated interval-specific congenic lines and deduced a 206 kb critical interval on chromosome 11 that contained only two protein coding genes (Rufy1 and Hnrnph1). Replicate mouse lines heterozygous for Transcription Activator-like Effector Nucleases (TALENs)-induced frameshift deletions in Hnrnph1 (Hnrnph1+/-), but not in Rufy1 (Rufy1+/-), recapitulated the decrease in MA sensitivity observed in congenic mice; thus, identifying Hnrnph1 as a novel quantitative trait gene for MA sensitivity. Hnrnph1, an RNA-binding protein, has not previously been identified in human GWAS of neuropsychiatric disorders but has been implicated in mu-opioid receptor splicing associated with heroin dependence. The primary objectives of this dissertation is to (1) detail the forward genetic and reverse genetic approaches taken to identify Hnrnph1 as a quantitative trait gene for MA sensitivity; (2) assess the MA addiction-relevant behaviors presented by Hnrnph1+/- mice through conditioned place preference (CPP) and oral self-administration procedures; and (3) identify the neurobiological mechanisms through which Hnrnph1 affects behavior via transcriptome, immunohistochemical and neurochemical assessments of the mesocorticolimbic dopamine circuit. Overall, Hnrnph1+/- mice display increased dopaminergic innervation and MA dose-dependent dopamine release in nucleus accumbens, which could underlie reduced drug sensitivity, reward, and reinforcement. The results of this thesis provide substantial evidence to implicate Hnrnph1 in MA addiction.

The costs in terms of both monetary and human lives lost due to substance abuse in the United States is well documented and it is publicized that it is increasing. There has been a large amount of research completed that has examined methamphetamine users, heroin users, and the drug-crime nexus; however, there is a paucity of research that provides insight into these users’ arrest experiences. Using a phenomenological approach, this research examined methamphetamine and heroin users’ incidents of being arrested to gain a greater understanding of their lived experiences. The analysis was based on interviews that were conducted with five adults that had been regular users of methamphetamine and/or heroin and had been arrested for an offense that was either directly or indirectly a result of their drug use. The results revealed four general themes that indicated: (1) the users felt they were living self-destructive lifestyles at the time of their arrest; (2) they experienced shock and confusion at the time of the arrest and afterwards; (3) interactions with the police were commonplace and they each had mixed experiences dealing with the police; (4) each of the participants expressed directly or indirectly that they needed to be arrested, complete long term confinement, or the possibility of long-term confinement, after a charge to successfully achieve sobriety and positive changes in their lives. These findings were discussed, reviewing evaluations of drug court diversion, incarceration, or the concept of an individual hitting rock-bottom as a prerequisite for lasting positive change and rehabilitation. Future research comparing the success of these post-arrest outcomes is suggested.

Drug screening is widespread in contexts such as the criminal justice system, employment, and substance abuse treatment centers. Traditionally, drug testing methods have targeted urine matrices and extensive research is available regarding urine drug screening. Due to the nature of sample collection, urine specimen may be tampered or adulterated in efforts to invalidate or pass a drug test. Examples of this include substituting a sample with synthetic urine, diluting a sample with water, or adding a substance to the sample. The addition of a substance is referred to as adulteration and is done in an effort to mask drugs in the sample or to invalidate the test results. For various reasons, including the effects of adulteration, time, and costs associated with urine drug tests, oral fluid (OF) has become an increasingly important alternative matrix for screening drugs of abuse. It offers distinct advantages since tests can be administered noninvasively, quickly, and under observation, thus reducing the risk of tampering. Studies have also shown that drugs of abuse detected in OF may correlate better to user impairment at the time of collection, as compared to other matrices. In 2019, the Substance Abuse and Mental Health Service Administration (SAMHSA) published mandatory guidelines for oral fluid testing. Although these guidelines only directly impact federal spaces, they also serve as a blueprint for developing drug testing laws and policies in other organizations. Despite requirements and procedures controlling for specimen adulteration, it is recognized that manufacturers will continue to develop and market new products to avoid drug detection, just as with urine drug tests. The design of this experiment was to investigate the effects of the commercially available drug testing subversion products High Voltage Saliva Cleanse Mouthwash (High Voltage Detox, Las Vegas, Nevada, USA) and Stinger Detox Mouthwash (Stinger Detox, Phoenix, Arizona, USA) on immunoassay testing for tetrahydrocannabinol (THC) and methamphetamine (MET) in OF. The High Voltage Saliva Cleanse was designated adulterant “A”, and the Stinger Detox was designated adulterant “B”. Two separate immunoassay kits, Discover™ (American Screening Corporation, Inc., Shreveport, Louisiana, USA) and Orawell® (Jiangsu Well Biotech Co., Ltd, Changzhou, Jiangsu, China), were assessed to investigate the differences in performance of the current available test devices in addition to the effects of the subversion products. Using Discover™, samples were spiked according to 0.5 times (x), 1x, and 2x the cutoff concentrations of 50 ng/mL THC and 50 ng/mL MET without adulterant, with Adulterant A, and with Adulterant B. Testing with Orawell® devices was initially conducted at 1x and 2x the cutoff concentrations of 40 ng/mL THC and 50 ng/mL of MET. Based on the lack of response, testing at 0.5x was not conducted. Additional testing was conducted at 1.5x and 3x the cutoff concentrations without adulterant, with Adulterant A, and with Adulterant B. It was concluded that the adulterants affected the test results in the Orawell® device, by producing false positives for drugs of abuse not present in the sample for 17 (56.7%) of the 30 tests containing adulterants. Additionally, it was concluded that both immunoassay tests assessed were lacking in analytical sensitivity and reproducibility.