Addiction results in persistent neural and behavioral modifications characterized by compulsive drug use, compulsive drug seeking, and repeated relapse. The neural substrates associated with drug-use and addiction are believed to overlap with those involved in memory. Many protein kinases, transcription factors, and receptors are implicated in both processes; however, drug related and traditional associative memory may produce different neural modifications. One such addiction related memory is sensitization, a persistent increase in the response to a drug following repeated administration. The purpose of my dissertation is to examine whether certain post-translational mechanisms are similarly involved in associative forms of memory and sensitization. In my first experiment, I investigated the role of atypical protein kinase C (PKC) in the development and expression of locomotor sensitization. I used zeta inhibitory peptide (ZIP) to block atypical PKC activity. We found that the induction, but not maintenance, of locomotor sensitization was impaired when ZIP was administered both continuously and with a single infusion. In the second and third series of experiments, I examined the effects of disrupting the ubiquitin-proteasome system (UPS), a pathway that mediates protein degradation, on learning, memory, and addiction using two lines of novel knockin mice. Both mice had a mutation at a single amino acid residue, serine 120, a site on Rpt6, part of the proteasome’s regulatory component. This site is important as it is phosphorylated by CaMKII in an activity-dependent manner. In one line of mice the serine was switched to an aspartic acid to create a phospho-mimetic mutation (S120D), whereas in the second line of mice serine was switched to an alanine to create a phospho-dead (S120A) mutation. We found this mechanism to be a critical regulator of the UPS during addiction-related memory, as sensitization was completely blocked in phospho-mimetic mice. In contrast, both lines of knockin mice displayed intact associative memory, as assessed using Pavlovian fear conditioning and context discrimination . Therefore, while atypical PKCs and the UPS may be involved in both traditional, associative memory and addiction-related memory, the functional role of these mechanisms seems to differ between the two processes.

The classical monoamine neurotransmitters — dopamine, norepinephrine, and serotonin — are critically involved in a range of brain functions and their transporters (DAT, NET, and SERT) are targets for many psychoactive drugs. Some of the oldest and most widely prescribed psychotherapeutics (e.g., antidepressants, psychostimulants) are monoaminergic drugs, but their mechanisms remain poorly understood and many pose serious safety or efficacy challenges to patients. Still, there have been few meaningful advances in neuropsychiatric drug development over the last three decades. Growing evidence suggests that MDMA and other psychedelics may transform care for an array of poorly treated conditions. The purpose of my dissertation is to elucidate the mechanisms underlying the behavioral effects of monoaminergic drugs to inform the development of novel, optimized drugs that retain or lack specific therapeutic or adverse effects. We have taken a systematic approach to examining monoamine transporter inhibitors and reversers at both low and high doses on various behavioral outcomes in mice. In Chapter 2, we explore whether existing drugs may mimic the therapeutic, memory-enhancing effects of low-dose psychostimulants but lack the adverse, reinforcing effects of high-dose psychostimulants. Bupropion (a low affinity DAT inhibitor) and atomoxetine (a high affinity NET inhibitor) produced these desired effects in combination but not alone. In Chapter 3, we systematically analyze all preclinical findings on the cognitive effects of MDMA with a critical focus on dose. We found no evidence that low, clinically relevant doses (< 3 mg/kg MDMA) produce cognitive impairments. In Chapter 4, we further analyze the potential adverse effects of MDMA across a wide range of doses. High doses (≥ 3 mg/kg MDMA) produced memory impairments and some evidence of an addictive potential while low, clinically relevant doses (≤ 1 mg/kg MDMA) did not. In Chapter 5, we present a novel method for assessing prepulse inhibition of acoustic startle in rodents, which may be especially useful for antipsychotic drug screening. In Chapter 6, we discuss our findings in the context of the current “psychedelic renaissance” and provide a roadmap for systematically analyzing classical and novel monoaminergic compounds to advance drug development for the most critical unmet medical needs in neuropsychiatry.

Pavlovian fear conditioning, in which a neutral stimulus is paired with a shock, has been instrumental in uncovering the neural and molecular mechanisms underlying memory since the discovery that post-training lesions of the hippocampus disrupt contextual, but not cued, fear memory. Because of the paradigm’s relative simplicity, it has become the leading animal model of declarative memory. In contrast to post-training lesions, which produce severe amnesia of contextual fear, some studies have found that mice and rats with pre-training hippocampal lesions may acquire normal contextual memory, suggesting an alternative pathway for contextual fear learning. The prevailing theory proposes that the intact hippocampus uses a configural strategy, integrating environmental cues into a cohesive representation before it can be associated with shock, while a compromised hippocampus employs an elemental strategy, associating individual cues with shock. Despite the widespread use of contextual fear conditioning, it remains unclear what animals actually learn about static “elemental” environmental cues.This thesis introduces a novel within-subjects "context deconstruction" testing paradigm to systematically evaluate freezing responses to individual contextual components across three sensory modalities in mice. Chapter 1 reveals that intact mice exhibit an all-or-none response to the context, showing robust fear of the entire context, largely ignoring individual components, which aligns with the configural representation theory. This representation persists well into the systems consolidation period, despite repeated testing. Chapter 2 explores the protocol's utility by identifying distinct freezing patterns to contextual components following pharmacological manipulations known to impair context memory acquisition, highlighting the sensitivity of this approach. Under some dosing regimens, particularly those that produce amnesia, mice appear to use an elemental strategy. Chapter 3 directly tests whether hippocampal-lesioned mice acquire an elemental representation of the context. Results suggest an amalgamation of the configural and elemental hypotheses: there is evidence that mice respond to individual contextual cues, but freezing to the full training context remains more pronounced compared to freezing to partial contexts. This thesis emphasizes the need for more thorough fear conditioning protocols to elucidate subtle deficits in context learning, contributing to advancing our understanding of learning and memory processes in normal and compromised brain states.

This dissertation presents findings from two studies investigating the neuroscience of memory. Chapter 1 reports findings from a computational genomics study on Alzheimer's Disease (AD), a common neurodegenerative disease that causes memory loss and dementia. There is hope that genomic information can reveal insights to AD pathophysiology, along with aiding in risk assessment, screening, and diagnosis. This study involved using exome sequencing data from approximately 10,000 individuals (~5000 AD patients) to train neural net (NN) classifiers tasked with estimating the impact of single genomic variants on AD polygenic risk. Together, these chapters and the studies presented therein add to our corpus of knowledge on memory systems, and contribute to our quest of understanding and treating dementia. Chapter 2 covers a study that details a model (supported by experimental results) that can explain how memories can be formed and maintained within neural network synapses, despite continuous and complete molecular turnover of synaptic proteins. Memory formation is thought to involve acute changes to synaptic weights; the ability for synaptic weights to remain stable over long time-periods and undergo evoked change is considered fundamental to our brain’s information storage schema. Yet basic questions regarding synaptic plasticity remain unresolved, including a) how synaptic weights remain stable in the face of perpetual and complete molecular turnover; and b) how such weights can be modified to new stable levels by transient signals. Through a series of computational and biological experiments, we elucidate actin, a protein with unique polymer properties, as a potential central mediator of synaptic weights.

The ADHD drug methylphenidate improves long-term memory, but it is still unclear whether this is a direct effect of the drug or due to improvements in executive function. Other ADHD pharmacological treatments, such as atomoxetine, improve executive control in terms of impulse control, working memory, and attention, but not long-term memory. Moreover, methylphenidate improves long-term memory in tasks with little attentional requirements, such as tone fear conditioning. This suggests that methylphenidate improves long-term memory through a mechanism that does not necessarily require improvements in executive function. This led us to hypothesize that methylphenidate directly enhances long-term memory. Frontal cortex is required for executive function, while the medial temporal lobe, including the hippocampus, is required for long-term memory formation. Methylphenidate binds targets in both the prefrontal cortex and hippocampus, but which of these is responsible for the improvements in long-term memory has yet to be determined. This study was undertaken to determine whether the frontal areas implicated in executive function are recruited in long-term memory enhancement by methylphenidate. Executive function was disrupted by lesioning prefrontal cortex in mice. Sham lesions were performed to account for the effects of surgery. Mice were then subjected to classical Pavlovian fear conditioning following administration of either methylphenidate or saline, and tested for contextual and tone memory one week later. In this study, both frontal lesions and methylphenidate failed to influence long-term memory, which may be due to increased stress from surgery that altered the optimal dose of methylphenidate. Further studies are required to elucidate the role of frontal cortex in methylphenidate-mediated memory enhancement.