Janet L. Neisewander - US grants

The objective of the proposed research is to anatomically localize the dopamine receptor subtypes involved in the rewarding and stimulant effects of cocaine in rats. The rewarding effects of cocaine will be assessed using the conditioned place preference paradigm. Animals will be injected with cocaine and placed into a distinctive environment, and on alternate days they will be injected with saline and placed into a different environment. Following these drug-environment pairings, the rewarding properties of cocaine will be evident as an increase in the amount of time animals spend in the drug-paired environment relative to the saline-paired environment when given free-access to both simultaneously. The stimulant properties of cocaine will be assessed by measuring locomotor activity and stereotypy following acute and repeated administrations of cocaine. It is expected that the stimulant properties of cocaine will be enhanced by repeated administration of the drug. Subsequent behavioral tests will examine whether this sensitization response is due to conditioned or unconditioned factors. The first experiment will establish dose-response curves for the effects of cocaine on reward and psychomotor stimulation. The next series of experiments will assess the role of dopamine receptor subtypes in mediating these responses by pretreating the animals with selective D1, D2, D1 and D2, or atypical dopamine antagonists prior to cocaine administration. The last series of experiments will assess the role of dopamine receptor subtypes in specific regions of the mesocorticolimbic system by directly injecting the antagonists into either the ventral tegmental area, nucleus accumbens, or medial prefrontal cortex prior to systemic administration of cocaine. These experiments will help to elucidate the mechanisms of cocaine reward, and will provide crucial information for developing pharmacologic treatment strategies for cocaine abuse.

DESCRIPTION (provided by applicant): Drug dependence is considered a chronic disease due to the high incidence of relapse. Incentive motivational effects (e.g., craving) produced by consumption of drug or by exposure to stimuli present during the drug experience (e.g., paraphernalia or the environment) are thought to play a major role in relapse. Despite the importance of these phenomena to relapse, little is known about the neural mechanisms involved. Limbic-cortical circuits, which play a role in emotion, memory, and formation of associations between initially neutral stimuli and rewards, are likely involved in these effects. Furthermore, mounting evidence suggests that dopamine, serotonin (5-HT), and glutamate neurotransmitter systems may play a critical role in these effects as well. The objective of this research is to examine the role of limbic-cortical circuits in the incentive motivational effects produced by cocaine and cocaine-paired environmental stimuli. We hypothesize that the central and basolateral amygdala play predominant roles in the incentive motivational effects of cocaine and cocaine-paired stimuli, respectively, whereas the prelimbic and anterior cingulate cortex play a critical role in the incentive motivational effects of both types of stimuli. We will investigate these hypotheses by examining the effects of excitotoxic lesions of these regions on acquisition, expression, and cocaine reinstatement of cocaine-conditioned place preference (CPP). Expression of CPP is thought to reflect incentive motivational effects of cocaine-paired stimuli, whereas reinstatement of extinguished CPP by cocaine priming injections is thought to reflect the incentive motivational effects of cocaine itself. In addition, we will survey key regions of the brain that are involved in learning, memory, motivation, and sensorimotor processes for neuronal activation in response to exposure to cocaine-paired stimuli or to cocaine priming injections using induction of immediate early genes (IEGs), c-fos and zif/268, as markers. Furthermore, we will characterize the neurons that exhibit IEG induction to determine whether they co-express mRNAs for dopamine D1, D2, or D3 receptors, 5-HT1A, 5-HT2A, 5-HT2C, and 5-HT4 receptors, and glutamate AMPA receptor subunits GluRl, GluR2, and GluR3 using double-labeling in situ hybridization. Subsequently, we will build from these studies by examining the effects of lesions that disrupt acquisition of cocaine-CPP on IEG responses in brain regions interconnected with the lesion site. A disruption of IEG expression in regions interconnected with the lesion site would suggest functional connection between the regions that may be involved in acquisition of cocaine-CPP. These experiments will provide much new information on the role of the amygdala and cortical brain regions in incentive motivation for cocaine and will help elucidate the neural circuitry involved in relapse elicited by cocaine and cocaine-paired stimuli. The findings may be useful for developing behavioral and pharmacological treatments for cocaine dependence.

[unreadable] DESCRIPTION (provided by applicant): Cocaine dependence is a chronic condition because of the high incidence of relapse that occurs even after prolonged periods of abstinence. Thus, successful treatment must include relapse prevention. The objective of this grant is to elucidate the neural mechanisms underlying incentive motivational effects produced by sampling cocaine or by exposure to cocaine-associated cues (e.g., paraphernalia, cocaine- taking environment, etc), both of which are thought to play a major role in cocaine craving and relapse. Incentive motivational effects of cocaine and cocaine cues involve dopamine (DA), serotonin (5-HT), and glutamate systems. Furthermore, there is mounting evidence that suggests cocaine and cocaine cues activate different, but overlapping neural circuits and much attention has been given to understanding the role of DA and glutamate within these circuits. In contrast, little research has examined the role of 5-HT in these circuits. Recent research suggests that stimulation of 5-HT2C receptors (Rs) inhibits mesolimbic DA neurons and decreases the incentive motivational effects of both cocaine and cues, whereas stimulation of 5-HT2ARs may facilitate incentive motivational effects of these stimuli. The proposed research will examine specific hypotheses regarding the anatomical locus of these effects. Specifically, we hypothesize that increased stimulation of 5-HT2CRs in the amygdala inhibits incentive motivational effects of cocaine cues, whereas stimulation of these receptors in the ventral tegmental area inhibits incentive motivational effects of both cues and cocaine itself. Furthermore, we hypothesize that stimulation of 5-HT2CRs in the medial prefrontal cortex inhibits the incentive motivational effects of these stimuli, whereas stimulation of 5-HT2ARs in this region facilitates the effects. The hypotheses will be tested by examining the effects of localized infusions of 5-HT drugs on reinstatement of extinguished cocaine-seeking behavior (i.e., operant responding in the absence of cocaine reinforcement) following cocaine priming injections or exposure to cocaine cues. Contingent upon support for predicted effects, subsequent experiments will examine the anatomical and pharmacological specificity of the effects. This research will help to elucidate the role of 5-HT in the neural circuits underlying incentive motivational effects of cocaine and cocaine cues. Understanding these circuits will likely have important implications for developing treatments for cocaine dependence. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cocaine dependence is a chronic relapsing disorder for which there is currently no medication available. Due to the urgent need for anti-relapse medications, it is important to explore potential uses of FDA-approved drugs that would allow for rapid clinical translation. Minimally, potential treatments should reduce drug craving, since craving is often a precipitating factor in relapse. Another measure that is not often used, but has face validity for anti-relapse effects of treatment is resumption of cocaine self-administration after a period of abstinence. During previous funding periods, our research has made significant contributions toward understanding the neural mechanisms of motivation for cocaine that is elicited by cocaine priming injections or cocaine-associated cues, both of which elicit craving in cocaine abusers. In this new 3rd competing renewal, we propose to investigate an important lead from our work. Specifically, we discovered that although manipulations that increase serotonin (5-HT) 1B receptor (R) function enhance cocaine's reinforcing value during maintenance of self-administration in rats, these same manipulations given during protracted abstinence decrease cocaine's reinforcing value, as well as reinstatement of extinguished cocaine-seeking behavior induced by cocaine-associated cues or cocaine-priming injections. These findings suggest that 5-HT1BRs play a critical role in the pathological brain circuitry that underlies abstinence-induced increases in motivation for cocaine. More importantly, the findings suggest that 5-HT1BR agonists may be effective anti-relapse medications, which is exciting given that there are triptan-class 5-HT1BR agonists that are FDA-approved for the treatment of migraines. We have shown that the selective 5-HT1BR agonist CP94253 decreases resumption of cocaine self-administration and reinstatement of cocaine seeking behaviors after protracted abstinence and we have preliminary data suggesting that the FDA-approved, but less selective agonist zolmitriptan also decreases cocaine intake after a period of abstinence. In this proposal, we aim to compare the effects of CP94253 and zolmitriptan on cocaine self-administration and reinstatement of cocaine-seeking behavior during maintenance versus after a period of protracted abstinence. We also aim to examine whether inhibitory effects of the agonists are maintained after a lengthy abstinence period (60 days) and after 10 daily treatments. We plan to investigate whether the effects that we have observed with CP94253 in male rats that have self-administered cocaine generalize to female rats and generalize to male rats that have self- administered methamphetamine. Finally, we will investigate the neural mechanisms underlying the abstinence- induced switch in the direction of 5-HT1BR agonist effects using electrophysiological recording of dopamine neurons in the VTA. Our aims have the exciting potential to rapidly translate to new medications for cocaine dependence and will provide new knowledge regarding the mechanisms of cocaine addiction.

Brain abnormalities associated with substance use disorders (SUDs) involve long-term changes in gene expression that derail motivational circuits toward drug seeking and taking despite negative consequences. While the role of transcription factors in gene expression changes has been well established, the subsequent post-transcriptional events that result in functional change are far from understood. Recent breakthroughs suggest that circular RNAs (circRNAs) are critical in regulating post-transcriptional events. This new class of non-coding RNAs plays a critical role in brain development and synaptic function by acting as sponges for sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs), which regulate gene expression, often in a competitive manner. For instance, our laboratory has shown that the RBP, HuD, competes with miR-495 to regulate expression of addiction-related genes. Our new preliminary results demonstrate that HuD also binds to, and regulates, the expression and synaptic localization of 14 brain-specific circRNAs, including circHomer1. circHomer1 is generated from the same gene (Homer1) that generates linear Homer1b mRNA, which is also a HuD target. Importantly, Homer1 protein is involved in cocaine?induced plasticity and drug seeking via its role in regulating type I metabotropic glutamate receptor signaling and homeostatic synaptic downscaling after increased neuronal activity. We found that knockdown of circHomer1 levels increases synaptic activity and synaptic Homer1b mRNA levels. Using HuD overexpressing mice, we determined that this process requires binding of the circRNA to HuD. Furthermore, we found that circHomer1 levels in the nucleus accumbens (NAc) are decreased in mice displaying increased cocaine-seeking behavior in the conditioned place preference model. In contrast, the varying levels of operant cocaine-seeking behavior in rats living in an enriched environment (low levels) versus isolation (high levels) during forced abstinence after a history of cocaine self- administration inversely correlate with the ratios of circHomer1 to Homer1b mRNA levels in the NAc shell. Based on these results, we hypothesize that synaptic circHomer1 expression in the NAc shell regulates synaptic activity by competing with Homer1b mRNA for HuD binding, leading to downstream effects in Homer1b function that attenuate drug-seeking behavior. To test this hypothesis, we will first examine whether circHomer1 and Homer1b mRNA compete for HuD binding, transport to synapses, and the control of neuronal activity in neurons in culture. We will then evaluate the effects of lentiviral-mediated manipulations of circHomer1 levels on Homer1b mRNA synaptic levels and the modulation of operant cocaine-seeking behavior. This research will impact the field by elucidating the role of circHomer1 in the mechanisms underlying changes in cocaine-induced neuroplasticity and addiction-related behaviors. Ultimately, gaining an understanding of the molecular mechanisms underlying brain changes in SUDs will aid in developing novel treatment strategies for cocaine use disorders.