Mental illnesses like substance use and depression impact large portions of the population. Despite extensive research efforts, effective therapeutics for both substance use disorders and depression remain elusive. To fully address this crisis, we need to understand the complex nature of these disorders, which often involve networks that span the entire brain. This complexity underscores the importance of using comprehensive whole-brain imaging and analysis techniques in tandem with functional behavioral assays to develop therapeutic interventions and ultimately improve treatment outcomes. In the following studies, we investigate the relationship between depression- and drug-induced changes to whole-brain activity and resulting behavior. In the first two studies we identify networks of key structures that contribute to improved outcomes in treatment-resistant depression, and determine that these effects are likely modulated by direct input/output connectivity in identified network hubs. We next investigate how adolescent cannabis exposure impacts future withdrawal behaviors. We find that early life cannabis exposure reduces opioid-induced withdrawal symptoms but increases reinstatement of reward, and identify potential brain regions contributing to these results. In the final study, we demonstrate the ability to identify differentially activated populations during opioid withdrawal and specifically modulate them to improve behavioral outcomes. We apply whole-brain cFos imaging, immunohistochemical analysis, fiber photometry, and viral inhibition to demonstrate our ability to gain genetic access to key regions that go on to be important for opioid induced withdrawal behaviors. Together, these studies demonstrate the utility of employing whole-brain activity-based screens in behavioral studies that involve complex networks. These tools can be applied to a wide variety of questions related to drugs of abuse and neuropsychiatric conditions to identify novel target regions and customize treatment options. Future work applying these techniques could provide a key to developing improved therapeutics for a wide range of disorders.

Alzheimer’s disease (AD) is the most common neurodegenerative disease, often culminating in life-altering behavioral and cognitive changes. As current pharmacotherapies for the disease are limited, novel approaches to understanding the alterations in neuronal circuits prior to symptom onset are crucial for effective therapeutic intervention. Despite the critical need, the brain circuitry underlying AD pathophysiology is yet to be fully understood. Recent shifts towards early intervention strategies highlight the importance of understanding the preclinical stage of AD. By taking a circuit mapping approach, this body of work aimed to identify and probe the neuronal populations within entorhinal cortex circuitry that underlie these early stages of AD (Chapter 1). Here we used a cutting-edge viral-genetic method, rabies virus (RABV) tracing, which delineates the input connections of targeted starter cell populations. By implementing rabies virus tracing at different time points, we captured how these circuits look before the emergence of any overt behavioral or pathological deficits and during the later stages of the disease when both cognitive and pathological impairments are present. Our findings identified a critical circuit between the retrosplenial cortex (RSC) and entorhinal cortex (EC) that is disrupted related to the early development and progression of AD (Chapter 2). These results lay the groundwork for creating targeted therapies to mitigate or halt the development of AD.Parallel studies into the central mechanisms driving learning, memory, and cognitive decline are essential for advancing our understanding of AD progression. Long-term potentiation (LTP) is a fundamental cellular mechanism that underlies learning and memory processes, yet the dynamics of LTP stabilization or disruption over time remain to be fully understood. In this study, we assessed the effects of the small molecule macropinocytosis inhibitor amiloride on LTP stabilization and memory in the 5xFAD mouse model of Alzheimer’s disease (Chapter 3). Our findings indicated that amiloride administration into the basal lateral amygdala can enhance memory retention in these mice during the early stages of AD, as evidenced by increased freezing behaviors in an auditory fear conditioning task. These findings suggest that macropinocytosis inhibitors like amiloride may be a potential therapeutic agent for the treatment of AD-related memory deficits. Our final study shifted focus to the broader implications of brain changes in addiction-like behaviors in rodents, particularly examining the anterior cingulate cortex’s (ACC) role in the context of the opioid crisis—a pressing public health issue with no clear solution for opioid use disorders (Chapter 4). By exploring the biological differences between individuals susceptible to opioid addiction and those who are not, we employed an unbiased whole-brain activity method approach to investigate crucial networks and circuits related to opioid withdrawal behavior. The TRAP2 technique was used to permanently mark neurons activated by an initial opioid exposure, establishing a link between these activity patterns and subsequent opioid-induced behaviors. Utilizing network control theory to pinpoint critical brain areas contributing to resilience, we found a significant correlation between ACC activity and the development of resilience against addiction-like withdrawal behaviors. This dataset offers a promising platform for further explorations into the ACC's connection to morphine addiction, opening avenues for novel therapeutic interventions.

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that is growing inprevalence and is impactful on individuals and society as a whole. Treatment options are limited, and there is no known cure; a better understanding of this disease, particularly at its early stages, is essential for the betterment of human health. This dissertation investigates how neuronal communication is disrupted throughout disease progression in AD, how these changes may be explained molecularly, and how disease-associated changes appear to occur in a brain-region specific manner.

Chapter 1 of this work reviews prior AD research, and how methods we utilize such asneuronal tracing and transcriptomic analysis have previously been applied to the research of this disease. Following this summary of previous work, we explore a rabies-based viral tracing method, the results of which can reflect cellular excitability (Chapter 2). While this component of the dissertation did not focus on AD, the methodology and insights into cellular communication, communication between brain regions, and how gene expression can influence these factors informs the rest of this work, particularly Chapter 4.

In Chapter 3, we delve into the molecular changes present in the entorhinal cortex (ENT).We utilize a mouse model of AD, and noted cellular and neuronal communication pathways that were disrupted in disease. We saw some overlap of these changes with those observed in previously published human AD data, particularly in glutamatergic signaling.

Chapter 4 synthesizes and extends the work done in earlier chapters: we first utilize therabies-based viral tracing method characterized in Chapter 2, the results of which identified the retrospenial cortex (RSC) as an important input region to the ENT in AD mice. We followed up with multi-omic analysis of the RSC and the ENT over time in the AD mouse brain, as well as sequencing of tissue from patients with or without cognitive deficits. Glutamatergic neurons were once again seen to have gene expression changes implicating cellular excitability in disease.

Combined, these projects implicate cellular excitability in both the ENT and the RSC, butsuggest that changes in the RSC may precede those in the ENT. While further research is needed and questions remain (Chapter 5), this work has yielded insights into brain-region specific molecular changes in AD, and highlights the RSC as a promising target for future study.

Chronic pain and addiction are two prevalent and interconnected health challenges that significantly impact individuals' quality of life. This dissertation investigates the neurobiological underpinnings of these complex behaviors through two parallel research projects.The first part of the dissertation (Chapter 1) focuses on elucidating the neural circuits underlying chronic pain. Utilizing a combination of whole-brain screening techniques, classic pain assays, in vivo calcium imaging, and slice electrophysiology, key neural circuits implicated in chronic pain and comorbid mood disorders are identified. In particular, we identified a critical circuit projecting from the agranular insula (AI) to the basolateral amygdala (BLA) that emerges as a central hub orchestrating maladaptive nociceptive processing and emotional regulation in chronic pain states. This circuit not only contributes to sensory aspects of chronic pain but also mediates behaviors resembling emotional distress and anxiety commonly observed in chronic pain patients. In contrast, we identified a second projection from the paraventricular thalamus (PVT) to the lateral amygdala (LA) that contributes primarily to the sensation of pain, without altering anxiety-like behaviors. In the second half of the dissertation, we explored the relationship between early life adversity and nicotine addiction-related behaviors, such as addiction susceptibility and withdrawal symptoms. We found that both nicotine withdrawal and early life adversity induce increases in pain sensitivity (Chapter 2). However, animals with early life adversity that had undergone nicotine withdrawal display reduced pain sensitivity relative to control-reared counterparts (Chapter 3). Leveraging unbiased whole-brain screening methodologies, we identified the anterior cingulate area as a key brain region implicated in mediating the effects of early life adversity on these nicotine addiction-related behaviors (Chapter 4). Together, these projects offer complementary perspectives on the neural underpinnings of complex behaviors, highlighting common themes of circuit dysregulation and behavioral maladaptation across chronic pain and substance use disorders. By advancing our understanding of the brain's response to various challenges, this research contributes to a more holistic understanding of brain function and behavior and lays the groundwork for innovative approaches to treat neurological disease.