Adolescence represents a sensitive developmental period characterized by heightened clinical vulnerability to tobacco, e-cigarettes, and antidepressants. While there are sociocultural influences, both clinical and preclinical data indicate that this adolescent sensitivity has strong neurobiological underpinnings. Although definitions of adolescence vary, the hallmark of this period is a profound reorganization of brain regions necessary for mature cognitive, executive, and emotional function, including serotonin (5-HT) and dopamine (DA) monoamine systems. However, as shown in this dissertation, perturbations of 5-HT systems during this time with nicotine or fluoxetine may have unique consequences on adolescent DA function.

In this dissertation, I highlight that the distinct neurobiology of adolescent 5-HT and DA systems create unique behavioral vulnerabilities to drugs of abuse. The present studies demonstrate that changes in adolescent 5-HT systems and 5-HT1A receptor activation during brief, low-dose pretreatment sensitize D2 receptor-mediated behaviors. Using a pharmacological approach, I have shown that increasing endogenous 5-HT signaling with fluoxetine enhances quinpirole-induced locomotion and acquisition of self-administration during adolescence via sensitization of D2 receptor responses. These effects are age-specific and analogous to those observed after adolescent nicotine exposure. Furthermore, antagonist studies indicate that this enhanced sensitivity to quinpirole and cocaine is mediated by increased activation of postsynaptic 5-HT1A receptors and persists even after pretreatment has ended. To characterize the lasting alterations in 5-HT1A receptor function, neurochemical and anatomical analysis suggest that nicotine and fluoxetine have both distinct and common effects on regional 5-HT1A receptor activity. While fluoxetine alone targets prefrontal cortical neurocircuitry and nicotine alone targets the amygdala, both drugs enhance adolescent 5-HT1A receptor activity in the primary motor cortex (M1). Thus, within the adolescent M1, maladaptive changes in 5-HT signaling and 5-HT1A activity after nicotine or fluoxetine exposure may potentiate D2 receptor responsiveness to dopaminergic drugs and prime adolescent vulnerability for future substance abuse.

While substance use disorders have been a topic of study in psychology and neurobiologyfor decades, they remain a major problem and only a few effective treatment methods have been developed. The reward, stress, and planning circuits which drive addiction behaviors have been identified and well-studied, uncovering the neurobiological basis for the similarity of addiction behaviors across a variety of substances. Some genetic, developmental, and psychosocial factors which contribute to addiction vulnerability have been identified, but these do not provide sufficient interventional opportunities for treatment or prevention, and do not fully explain the variability in addiction outcomes. Psychological and biological studies of resilience have demonstrated that resilience is an active process, with both overlapping and non-overlapping mechanisms with vulnerability. Here I explore mechanisms of resilience to addiction-like behavior across multiple substances. I use brain activity mapping, pharmacological and viral inhibition and excitation, behavioral measures, immunohistochemical anatomical analysis, and protein

quantification to identify and characterize differences in neuronal activity and circuitanatomy between resilient and susceptible animals. I identify a potential circuit mechanism for negative feedback during drug reward which develops after adolescence, which may contribute to the increased resilience of adult animals to addiction-like behavior compared to juvenile and adolescent animals. A preliminary study suggests that adolescent nicotine or nicotine + ethanol experience does not drive the same cellular response as adolescent high dose amphetamine experience, although similar behavioral outcomes have been observed. I also use a recently developed computational method to identify a population of neurons in the anterior cingulate cortex which both respond strongly to an animal’s first morphine experience and contribute to anxiety during morphine withdrawal. Together, these studies add to the developing literature focused on the compensatory mechanisms involved in resilience to addiction.

Smoking continues to be one of the leading causes of preventable death in the United States. Although the majority of smokers want to quit, most will not be successful at doing so. Despite extensive research and funding devoted to finding more efficacious pharmacotherapies for smoking cessation, current therapies are not very effective at keeping smokers abstinent for over a year. The goal of my dissertation was to establish a preclinical test that would have better face validity for the development of new pharmacological therapies for smoking cessation. Current preclinical tests of tobacco dependence use nicotine alone. Here I used an aqueous cigarette smoke extract (CSE), which contains nicotine and many other constituents of cigarette smoke, to examine its effects on cue- and drug-induced craving, factors known to influence relapse. The hypothesis that was tested was that CSE would induce greater craving and withdrawal than nicotine alone. Adult male rats that had self-administered CSE were found to reinstate to drug priming alone, unlike animals that had self-administered nicotine, which required the additional presentation of drug-associated cues. AT-1001, a functional α3β4 nicotinic acetylcholine receptor (nAChR) antagonist, attenuated drug-primed reinstatement of CSE- and nicotine-seeking behavior. However, AT-1001 was less potent in blocking drug-primed reinstatement in animals that had self-administered CSE than in those that had self-administered nicotine alone. This was the case even when nicotine alone was used to prime reinstatement in animals that had self-administered CSE, suggesting that prior CSE exposure had altered the functional role of this nAChR.

Another factor known to influence relapse is withdrawal to smoking. Teenagers are especially vulnerable to the effects of withdrawal. However, although adult rats show many of the same signs of withdrawal as humans following chronic nicotine exposure, adolescent rodents do not. In this study, adolescent and adult male rats were exposed for ten days to nicotine or CSE (1.5 mg/kg/day nicotine equivalent) by intravenous injection. Chronic CSE treatment resulted in greater spontaneous somatic and affective withdrawal symptoms in both adolescent and adult rats. Furthermore, adolescents treated chronically with CSE displayed major affective symptoms, as shown by an increase in anxiety-like behavior 30 days after drug withdrawal. Mecamylamine, a non-selective nAChR antagonist, was used to investigate if nAChRs are involved in the enhancement of withdrawal observed following chronic CSE treatment. Whereas mecamylamine precipitated greater somatic withdrawal in animals treated with CSE, it did not precipitate affective withdrawal.

Heavy smokers show an upregulation of nAChR through increased radioligand binding. Similarly, adult rodents show an upregulation of nAChR binding after chronic nicotine, adolescents do not. Since CSE exposure resulted in an increase in precipitated somatic withdrawal in adult and adolescent rats and also augmented nicotine-primed reinstatement, I hypothesized that chronic CSE exposure would result in enhanced nAChR binding in both adult and adolescent rats. Chronic CSE exposure results in enhanced α4β2, α3β4, and α7 nAChR binding regardless of age, in brain areas highly involved in negative aversive states of addiction.

Overall, this work provides evidence that the cigarette smoke constituents influence drug-primed craving, withdrawal, and changes in nAChR properties. Smoke constituents eliminated the protective effects that adolescent rodents display in preclinical tests of nicotine dependence, and enhanced both craving and withdrawal in adults. These findings suggest that use of CSE is a more valid way to study tobacco dependence than nicotine, and should be used in preclinical tests to assess tobacco cessation therapies.

Cigarette smoking is the leading preventable cause of death in the United States. Of those who smoke, 9 out of 10 report trying their first cigarette before the age of 18. Although most people who initiate tobacco use are teenagers, animal models for studying tobacco dependence have traditionally focused on how adult animals initiate, withdrawal from and relapse to cigarette smoking. Furthermore, cigarette smoke contains more than 7,000 constituents, including nicotine, yet pre-clinical research has focused on nicotine alone. Our lab began studying these constituents by creating cigarette smoke extract, CSE, a solution which contains the aqueous constituents present in cigarette smoke. Previous work from our lab found that CSE is more potent than nicotine alone and can enhance stress-induced reinstatement in adult male rats. In order to understand how the presence of tobacco smoke constituents may affect adolescents, I investigated the role of these constituents in models of smoking initiation and relapse. I found that the tobacco smoke constituents did not influence adolescent or adult acquisition of self-administration. Adolescents self-administered more low-dose nicotine than adults when their increased non-specific responding was corrected for. During reinstatement of drug seeking, I found that CSE enhanced stress- + cue-induced reinstatement of drug-seeking behavior with no effect of age. To investigate the role of tobacco smoke constituents on attenuation of adolescent self-administration, a novel smoking cessation pharmacotherapy, AT-1001, which is a selective α3β4 nAChR functional antagonist, was used. AT-1001 attenuated both CSE and nicotine self-administration in adolescent rats to a similar degree. My results suggest that both age and the presence of tobacco smoke constituents are important factors in establishing a proper model of tobacco dependence. Furthermore, my findings provide novel insights on adolescent initiation and relapse and offer an exciting potential for the development of a new tobacco dependence animal model that could help create innovative therapeutics to curb the addiction faced by many.

With the increasing use of e-cigarettes, teen nicotine exposure is becoming more widespread. This is a growing public health crisis, given findings from both clinical and preclinical studies that show adolescent brain to be uniquely sensitive to nicotine. Animal studies have shown that adolescent nicotine exposure results in increased sensitivity to cocaine and other drugs, although the mechanisms that underlie this are poorly understood. We now show microglia to be critical regulators of nicotine-induced increases in adolescent cocaine self-administration. Nicotine has age-dependent effects on microglial morphology and immune transcript profiles. Moreover, blocking D2 receptors eliminates both nicotine-induced increases in cocaine self-administration and microglial activation. Finally, we show nicotine causes microglial-dependent synaptic pruning and plasticity. Taken together, we demonstrate that adolescent microglia are uniquely sensitive to perturbations by drugs of abuse, crucial for nicotine-induced increases in cocaine-seeking, and D2 receptors play a mechanistic role in these phenomena.