Incubation of craving is a behavioral phenomenon in which patients experience a time-dependent increase in cue-elicited drug craving as they progress through abstinence. Currently, the precise neurobiological mechanisms which drive incubation of craving have not yet been confirmed. Furthermore, a large proportion of the literature that encompasses current knowledge on these mechanisms uses sucrose or food pre-training procedures prior to cocaine self-administration. Literature shows that sucrose/food self-administration induces both overlapping and opposing neurochemical changes in comparison to cocaine self-administration. Therefore, the use of sucrose/food pre-training to facilitate drug self-administration could lead to confounding results for an analysis of the neurobiological underpinnings of drug craving. The experiments detailed in this thesis serve to compare the ability of two different short access (2-hour) rat models of cocaine self-administration, to elicit an incubation of craving, without the use of sucrose/food pre-training. The purpose of using a short-access (2-hour) model for cocaine self-administration rather than classical long-access (6-hour) models is to use a higher throughput procedure to study incubation of cocaine craving in rats, for the purpose of a future biochemical analysis for changes in neurobiological correlates for incubation of cocaine-craving. Additionally, this study explores the use of lower-dose short-access procedures than those described in existing literature, without the use of food training to elicit incubation of craving. The results demonstrate that both cocaine self-administration models are able to elicit comparable incubation of craving, in spite of procedural differences. Thus, relatively simple models of cocaine self-administration can be employed to study the neurobiological underpinnings of the incubation of craving.

Highly addictive amphetamine-type stimulants, including methamphetamine (MA), pose significant health and socioeconomic issues, with an estimated 27 million users worldwide. Understanding the neurobiological changes underlying various stages of MA abuse is key to developing safe and effective therapies. While the majority of addiction research has implicated the dopamine system, the long-term neuroplasticity that maintains MA Use Disorder also involves drug-induced changes in glutamate signaling in reward regions of the brain like the nucleus accumbens (NAC) and prefrontal cortex (PFC). In order to further characterize the role of glutamate in MA reward and reinforcement, glutamate receptor scaffolding protein Homer2 expression in the NAC and activity in the glutamatergic projection PFC-NAC were manipulated prior to place- and operant-conditioning procedures. Together the results from these studies argue that while research of the functional neuroanatomy of addiction tends to focus on traditional dichotomies between the NAC core and shell or PFC prelimbic and infralimbic sub-regions, the behavioral outcomes of probing these areas were inconsistent with these models across variables measured. Other inputs upstream from our regions of interest may be able to compensate for our manipulations via neurobiological redundancy. This would suggest that as research technologies advance, targeting functional networks will be crucial for elucidating the neurobiological underpinnings of addiction and discovering potential therapeutics.

In alcohol-dependent humans, cessation of alcohol consumption produces a dysphoric, anxiogenic state that can persist into protracted withdrawal. This aversive withdrawal-induced affective state is theorized to contribute to the negative reinforcing properties of alcohol that maintains and/or precipitates relapse in those suffering with alcoholism. Despite this and evidence that alcohol withdrawal increases biochemical indices of stress, there are relatively few published studies examining the impact of cessation from binge drinking on behavioral measures of negative affect. To this end, 60 C57BL/6 mice were allowed to consume alcohol under modified Drinking-in-the-Dark (DID) procedures, which consisted of access to a sipper tube containing either 20% alcohol or water for 2hrs/day, 5 days/week, for 6 consecutive weeks. Half of the mice from each drinking group were subjected to a 2-day behavioral test battery consisting of conventional assays of anxiety and depression either 24hrs (n=30) or 21 days (n=30) into withdrawal. A combined analysis revealed higher indices of anxiety assessed in the light/dark shuttle box, novel object encounter, elevated plus maze, and marble burying tests in alcohol-drinking (AD) compared to water-drinking (WD) controls, independent of withdrawal duration. AD mice also showed a shorter latency to float on the first day of the forced swim test, indicating a higher prevalence of behavioral despair; however, these mice also showed an increase in swimming behavior on a re-exposure session, suggesting impaired emotional memory. The present results indicate that increases in anxiety and depression emerge very early after cessation of drinking and persist into protracted withdrawal, suggesting that a history of heavy binge drinking produces enduring neuroadaptations within brain circuits mediating emotional arousal.

The majority of addiction research examining the brain has centered on neuronal adaptations both resulting from cocaine use and contributing to the development of addiction. However, neurons constitute a small minority of the total cells within the central nervous system (CNS) and the role of glial cell types, which make up the vast majority of CNS cells, in cocaine addiction is largely unexplored. Imaging studies have revealed white matter deficits within the corpus callosum and post-mortem tissue analysis has revealed decreased oligodendrocyte-specific protein expression within the brains of chronic cocaine users. The extended access (6h/day) cocaine self-administration paradigm conducted in rodents is used to model the escalation of cocaine intake seen within human cocaine users and is critical for gaining insight to the molecular adaptations that result from excessive cocaine use. The series of experiments detailed in this dissertation utilize this model to address three specific aims: (1) characterize changes in glial-specific proteins and mRNA expression in cortical and limbic brain regions of rats with a history of extended access cocaine self-administration, (2) characterize the behavioral consequences of decreased myelin basic protein on a variety of prefrontal cortex-dependent tasks that are impaired following a history of cocaine use, and (3) to determine the functional significance of decreased myelin basic protein expression on behavioral measures in an animal model of cocaine addiction, including the acquisition and maintenance of intravenous cocaine, dose sensitivity, as well as the extinction and reinstatement of drug-seeking. Taken together, the experiments detailed herein provide further support for an enduring effect of cocaine on the expression of glial-specific proteins within the forebrain and highlight a role for myelin basic protein within the ventromedial prefrontal cortex in regulating impulsive choice, but not other cocaine addiction- related behaviors.

This dissertation investigates how age of drinking onset and biological sex influence the affective and cognitive consequences that follow from a history of alcohol binge-drinking. Using a rodent model of voluntary binge-drinking, this research examines how a history of binge- drinking impacts withdrawal-induced negative affect, spatial and working memory, and protein expression through its effects on key brain regions involved in these processes. The findings suggest that binge-drinking during adolescence leads both to long-term cognitive deficits and increases in anxiety and depression-like behaviors in adulthood. As in the human population, female rodents in the present data exhibit earlier and more severe behavioral disturbances during alcohol withdrawal relative to males—underlining the urgent need for sex-specific approaches in managing alcohol use disorder. Additionally, this research explores how sex-specific chemosensory stimuli influence anxiety-related behaviors, providing insights into environmental and procedural factors modulating the manifestation of negative affect. A key finding includes the identification of sex-dependent variations in the modulation of marble-burying behavior in response to sex-related odors. Biochemical analyses reveal significant alterations in glutamate receptor expression and other neuropathological markers in the prefrontal cortex, entorhinal cortex, and amygdala, which may be implicated in the animals’ observed behavioral anomalies. These results suggest that early alcohol exposure disrupts normal brain development, resulting in long-lasting effects that may manifest in later life. Taken together, this research offers key insights into the complex effects of age and sex on the neurobiological and behavioral impacts of alcohol binge-drinking. More broadly, these results highlight the benefits of creating distinct interventions across age groups and sexes both for treating alcohol use disorder and managing its cognitive and affective consequences.

Incubation of craving is the phenomenon in which craving intensifies over the course of abstinence. Although understanding craving may be key in understanding why relapse occurs, not much is known about craving, especially the underlying mechanisms that occur in the brain. Clinical and preclinical models have recently implicated the ventromedial prefrontal cortex as one of the key regions in this phenomenon. In order to further characterize this region’s role in incubation, a variety of behavioral, pharmacological, and molecular techniques are used to examine how withdrawal from long access cocaine self-administration directly affects the glutamatergic system. The series of experiments detailed in this dissertation aim to: (1) characterize extracellular glutamate fluctuations in the vmPFC during the incubation of cocaine-seeking, (2) characterize metabotropic glutamate receptor (mGlu) 2/3 changes during both short term and long term withdrawal, and (3) to determine the functional relevance of endogenous glutamate in the two subregions of the vmPFC. Taken together, these experiments suggest that although there seem to be no changes in mGlu2/3 at either point of withdrawal, glutamate in the prelimbic cortex of the vmPFC is functionally relevant in the incubation of cocaine seeking. Together, these results suggest pharmacotherapeutic strategies geared toward damping excitatory glutamate drive within corticofugal projections from the prelimbic cortex may be effective to prevent incubated cue-induced craving during protracted abstinence.

The research presented in this dissertation characterizes the manifestation and neurobiological underpinnings of negative affect during alcohol withdrawal in adult and adolescent mice using an animal model of voluntary binge drinking. A history of binge drinking elicits changes in glutamate-related protein expression within regions of the extended amygdala relevant to emotional regulation. Studying withdrawal-induced changes in protein expression may provide insight into the mechanisms mediating age-dependent differences in the negative affective consequences of alcohol abuse. The results of this work demonstrate that, similar to the human population, adult and adolescent mice differ both in their pattern of alcohol consumption and also in the consequences of that consumption. Although adolescent animals typically consume larger quantities of alcohol, adolescents are less sensitive than adults to negative affect during early withdrawal. However, adolescents binge drinkers show an emergence of negative affect later in adulthood, as well as elevated alcohol consumption. Moreover, these studies identify specific changes in protein expression within two brain regions critically involved in both emotion and addiction neurobiology- the central nucleus of the amygdala (CEA) and the nucleus accumbens shell (AcbSh). Withdrawal from binge drinking is associated with increased neuronal activity within the AcbSh, concomitant with increased glutamate-related protein expression in adult animals. Conversely, decreased expression of protein indices of Homer2-dependent mGluR signaling was observed within the CEA of both adult and adolescent animals. Reversing these changes using neuropharmacological or virus-mediated transgene delivery approaches reduced behavioral signs of withdrawal-induced anxiety. Together, these studies identify neurobiological substrates and pathophysiology of relevance to the negative reinforcing properties of alcohol and characterize ontogenetic differences in the behavioral and neurobiological consequences of alcohol abuse.

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