Ryan M. Bastle

Novel cues reinstate cocaine-seeking behavior and induce Fos protein expression as effectively as conditioned cues.

Cue reinstatement of extinguished cocaine-seeking behavior is a widely used model of cue-elicited craving in abstinent human addicts. This study examined Fos protein expression in response to cocaine cues or to novel cues as a control for activation produced by test novelty. Rats were trained to self-administer cocaine paired with either a light or a tone cue, or received yoked saline and cue presentations, and then underwent daily extinction training. They were then tested for reinstatement of extinguished cocaine-seeking behavior elicited by response-contingent presentations of either the cocaine-paired cue or a novel cue (that is, tone for those trained with a light or vice versa). Surprisingly, conditioned and novel cues both reinstated responding and increased Fos similarly in most brain regions. Exceptions included the anterior cingulate, which was sensitive to test cue modality in saline controls and the dorsomedial caudate-putamen, where Fos was correlated with responding in the novel, but not conditioned, cue groups. In subsequent experiments, we observed a similar pattern of reinstatement in rats trained and tested for sucrose-seeking behavior, whereas rats trained and tested with the cues only reinstated to a novel, and not a familiar, light or tone. The results suggest that novel cues reinstate responding to a similar extent as conditioned cues regardless of whether animals have a reinforcement history with cocaine or sucrose, and that both types of cues activate similar brain circuits. Several explanations as to why converging processes may drive drug and novel cue reinforcement and seeking behavior are discussed.

Pharmacological evidence for an abstinence-induced switch in 5-HT1B receptor modulation of cocaine self-administration and cocaine-seeking behavior.

Studies examining serotonin-1B (5-HT1B) receptor manipulations on cocaine self-administration and cocaine-seeking behavior initially seemed discrepant. However, we recently suggested based on viral-mediated 5-HT1B-receptor gene transfer that the discrepancies are likely due to differences in the length of abstinence from cocaine prior to testing. To further validate our findings pharmacologically, we examined the effects of the selective 5-HT1B receptor agonist CP 94,253 (5.6 mg/kg, s.c.) on cocaine self-administration during maintenance and after a period of protracted abstinence with or without daily extinction training. We also examined agonist effects on cocaine-seeking behavior at different time points during abstinence. During maintenance, CP 94,253 shifted the cocaine self-administration dose–effect function on an FR5 schedule of reinforcement to the left, whereas following 21 days of abstinence CP 94,253 downshifted the function and also decreased responding on a progressive ratio schedule of reinforcement regardless of extinction history. CP 94,253 also attenuated cue-elicited and cocaine-primed drug-seeking behavior following 5 days, but not 1 day, of forced abstinence. The attenuating effects of CP 94,253 on the descending limb of the cocaine dose–effect function were blocked by the selective 5-HT1B receptor antagonist SB 224289 (5 mg/kg, i.p.) at both time points, indicating 5-HT1B receptor mediation. The results support a switch in 5-HT1B receptor modulation of cocaine reinforcement from facilitatory during self-administration maintenance to inhibitory during protracted abstinence. These findings suggest that the 5-HT1B receptor may be a novel target for developing medication for treating cocaine dependence.

Interactions between Early Life Stress, Nucleus Accumbens MeCP2 Expression, and Methamphetamine Self-Administration in Male Rats.

Early life stress (ELS) is highly related to the development of psychiatric illnesses in adulthood, including substance use disorders. A recent body of literature suggests that long-lasting changes in the epigenome may be a mechanism by which experiences early in life can alter neurobiological and behavioral phenotypes in adulthood. In this study, we replicate our previous findings that ELS, in the form of prolonged maternal separation, increases adult methamphetamine self-administration (SA) in male rats as compared with handled controls. In addition, we show new evidence that both ELS and methamphetamine SA alter the expression of the epigenetic regulator methyl CpG-binding protein 2 (MeCP2) in key brain reward regions, particularly in the nucleus accumbens (NAc) core. In turn, viral-mediated knockdown of MeCP2 expression in the NAc core reduces methamphetamine SA, as well as saccharin intake. Furthermore, NAc core MeCP2 knockdown reduces methamphetamine, but not saccharin, SA on a progressive ratio schedule of reinforcement. These data suggest that NAc core MeCP2 may be recruited by both ELS and methamphetamine SA and promote the development of certain aspects of drug abuse-related behavior. Taken together, functional interactions between ELS, methamphetamine SA, and the expression of MeCP2 in the NAc may represent novel mechanisms that can ultimately be targeted for intervention in individuals with adverse early life experiences who are at risk for developing substance use disorders.

In silico identification and in vivo validation of miR-495 as a novel regulator of motivation for cocaine that targets multiple addiction-related networks in the nucleus accumbens

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and are implicated in the etiology of several neuropsychiatric disorders, including substance use disorders (SUDs). Using in silico genome-wide sequence analyses, we identified miR-495 as a miRNA whose predicted targets are significantly enriched in the Knowledgebase for Addiction Related Genes (ARG) database (KARG; http://karg.cbi.pku.edu.cn). This small non-coding RNA is also highly expressed within the nucleus accumbens (NAc), a pivotal brain region underlying reward and motivation. Using luciferase reporter assays, we found that miR-495 directly targeted the 3′UTRs of Bdnf, Camk2a and Arc. Furthermore, we measured miR-495 expression in response to acute cocaine in mice and found that it is downregulated rapidly and selectively in the NAc, along with concomitant increases in ARG expression. Lentiviral-mediated miR-495 overexpression in the NAc shell (NAcsh) not only reversed these cocaine-induced effects but also downregulated multiple ARG mRNAs in specific SUD-related biological pathways, including those that regulate synaptic plasticity. miR-495 expression was also downregulated in the NAcsh of rats following cocaine self-administration. Most importantly, we found that NAcsh miR-495 overexpression suppressed the motivation to self-administer and seek cocaine across progressive ratio, extinction and reinstatement testing, but had no effect on food reinforcement, suggesting that miR-495 selectively affects addiction-related behaviors. Overall, our in silico search for post-transcriptional regulators identified miR-495 as a novel regulator of multiple ARGs that have a role in modulating motivation for cocaine.

Immediate early gene expression reveals interactions between social and nicotine rewards on brain activity in adolescent male rats.

Smoking initiation predominantly occurs during adolescence, often in the presence of peers. Therefore, understanding the neural mechanisms underlying the rewarding effects of nicotine and social stimuli is vital. Using the conditioned place preference (CPP) procedure, we measured immediate early gene (IEG) expression in animals following exposure either to a reward-conditioned environment or to the unconditioned stimuli (US). Adolescent, male rats were assigned to the following CPP US conditions: (1) Saline+Isolated, (2) Nicotine+Isolated, (3) Saline+Social, or (4) Nicotine+Social. For Experiment 1, brain tissue was collected 90min following the CPP expression test and processed for Fos immunohistochemistry. We found that rats conditioned with nicotine with or without a social partner exhibited CPP; however, we found no group differences in Fos expression in any brain region analyzed, with the exception of the nucleus accumbens core that exhibited a social-induced attenuation in Fos expression. For Experiment 2, brain tissue was collected 90min following US exposure during the last conditioning session. We found social reward-induced increases in IEG expression in striatal and amydalar subregions. In contrast, nicotine reduced IEG expression in prefrontal and striatal subregions. Reward interactions were also found in the dorsolateral striatum, basolateral amygdala, and ventral tegmental area where nicotine alone attenuated IEG expression and social reward reversed this effect. These results suggest that in general social rewards enhance, whereas nicotine attenuates, activation of mesocorticolimbic regions; however, the rewards given together interact to enhance activation in some regions. The findings contribute to knowledge of how a social environment influences nicotine effects.

VTA BDNF enhances social stress-induced compulsive cocaine bingeing

Every day in the United States, 120 people die as a result of drug overdose, a rate that has more than doubled from 1999 to 2013 [1]. The degree of vulnerability to addiction is determined by the interaction between genetic and environmental factors. Social stress is an environmental factor some experience on a daily basis (e.g., domestic abuse, bullying, work-related issues, etc.). Since people under social stress are more vulnerable to become addictive to drugs of abuse [2], it is critical to understand the brain mechanisms underlying such vulnerability, thereby providing potential therapeutic targets. The rodent social defeat model has been used to mimic social stress in humans and to examine social stress-induced brain changes. In this procedure, a dominant rat psychologically threatens and physically attacks a subordinate rat intermittently over the course of 10 days. Defeated rats exhibit enhanced drug-taking behavior, as well as have increased expression of addiction-related molecules in critical reward-related brain regions [3, 4]. More specifically, defeated rats have increased expression of the transcription factor ∆FosB in the nucleus accumbens (NAc) and increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA) [3, 4]. The VTA is enriched with dopamine neurons that are activated in response to drugs of abuse and project to the NAc to signal attention and motivation towards rewarding stimuli. BDNF promotes neural growth and plasticity in response to environmental stimuli, whereas ΔFosB in the NAc regulates sensitivity to cocaine and susceptibility to stress [5]. Furthermore, increasing VTA BDNF expression enhances NAc ΔFosB levels [6]. Therefore, we [7] investigated whether increased VTA BDNF also enhances social stress-induced vulnerability to addiction. To test this hypothesis, VTA BDNF expression was artificially increased with an adeno-associated viral vector (AAV), where control groups only received an AAV carrying the gene for green fluorescent protein (GFP). One week later, rats were exposed to either intermittent social defeat stress or were briefly handled. To measure drug-taking behavior, rats were trained to lever press for cocaine infusions (i.e., self-administration). Initial acquisition and maintenance of cocaine self-administration was measured in daily 2-h sessions. Following maintenance, motivation for cocaine was measured by placing the rats on a high effort progressive ratio (PR) schedule of reinforcement, where the number of lever presses the animal needs to acquire a single infusion increases exponentially (often in the hundreds by the end of the session). Following PR testing, compulsive cocaine intake was measured by …

Epigenetics and Drug Abuse

Gene expression and inheritance are not only a function of the DNA code, but also epigenetic mechanisms that regulate DNA accessibility, transcription, and translation of the genetic code into a functional protein. Epigenetic mechanisms are invoked by life experiences, including stress and exposure to drugs of abuse, and the resulting changes in gene expression can be inherited by future generations. This chapter highlights recent research demonstrating epigenetic changes in response to drug exposure with a focus on three different mechanisms: DNA methylation, histone modification, and noncod‐ ing RNAs. We briefly describe each of these mechanisms and then provide key examples of drug-induced changes involving these mechanisms, as well as epigenetic manipula‐ tions that alter effects of drugs. We then review cutting-edge technologies, including viral-mediated gene transfer and gene editing, that are being used to manipulate epigenetic processes with temporal and cell-type specificity. We also describe and provide examples of intergenerational epigenetic modifications, a topic that has interesting implications for how addiction-related traits may be passed down across generations. Finally, we discuss how this research provides a greater understanding of drug addiction and may lead to novel molecular targets for preventions and interven‐ tions for drug abuse.

The effect of litter separation on methamphetamine-conditioned place preference in post-partum dams

Methamphetamine (METH) abuse among women has recently increased to levels comparable to those observed in men. Although studies using animal models of addiction have begun to include more female subjects, examination of the effects of drugs of abuse on post-partum females is currently lacking. This is especially important in light of the significant hormonal and neurobiological changes that accompany pregnancy and rearing experiences. Furthermore, stress in a known factor in addiction vulnerability and the post-partum experience in the clinical population can be highly stressful. Here, we utilized the conditioned place preference paradigm to investigate the conditioned rewarding effects of METH either in virgin rats or in dams exposed to brief separation (15 min) or long separation (180 min) from the litter. We found that females in the brief separation group showed significantly greater METH conditioned place preference compared with both the long separation and virgin groups. No differences were found in locomotor activity during the conditioning sessions. These findings suggest that peripartum experience and brief litter separation may enhance the rewarding effects of METH.