ItemIncreased Excitability of Pyramidal Neurons in the Secondary Motor Cortex Enhances Cocaine-Seeking(2023-09) Huang, Donald; Atwood, Brady K.; Baucum, AJ; Hopf, Woody; Ma, Yao-Ying; Sheets, Patrick L.Cocaine addiction is a brain disorder characterized by chronic relapse. Although drug-seeking behaviors have been recognized to be associated with relapse, the role of the motor cortex, including the primary (M1) and secondary (M2) motor cortex, which are functionally important mediators of complex behaviors remains unclear in addiction. Here we use a rat cocaine intravenous self-administration (IVSA) model to investigate the intrinsic excitability of pyramidal neurons in the medial prefrontal cortices and motor cortices during withdrawal. Cocaine IVSA-trained rats performed a cocaine-seeking test on withdrawal day (WD) 1 or WD 45. Relative to WD 1 an increase in cocaineseeking was detected on WD 45. Whole-cell patch clamp recordings revealed an increase in intrinsic excitability in pyramidal neurons in Layer 2 of the secondary motor cortex (M2-L2) in cocaine trained rats on WD 45. Using a pharmacological approach, bath application of GABAA receptor agonist, muscimol, dosedependently (0.1 mM, 0.3 mM, and 1.0 mM) decreased the excitability of M2-L2 pyramidal neurons in cocaine IVSA-trained rats on WD 45. Pharmacological inactivation of M2-L2 by bilateral intra-M2 injection of muscimol (324 ng/1.0 μl) attenuated cocaine-seeking on WD 45. A chemogenetic approach was used to validate that M2-L2 pyramidal neurons play a contributing role in the increase in cocaine-seeking, a microinjection of rAAV5-CaMKIIa-hM4di-mCherry was performed to express Gi-DREADD receptors on M2-L2 pyramidal neurons. Activating Gi-DREADD with an intraperitoneal injection of compound 21 on WD 45 attenuated cocaine-seeking. To elucidate the mechanism that contributes to the increased excitability of M2-L2 pyramidal neurons, an analysis of the action potential kinetics revealed that calcium-activated small conductance potassium (SK) channel-mediated medium afterhyperpolarization amplitude decreased in cocaine vs. saline IVSA-trained rats on WD 45. SK channel activation by 1-EBIO (300 μm) increased the medium afterhyperpolarization amplitude and decreased the excitability of M2-L2 pyramidal neurons in cocaine IVSA-trained rats. Furthermore, intra-M2 injection of 1-EBIO on WD 45 attenuated cocaine-seeking. These experiments suggest that cocaine IVSA-training-induced persistent changes in M2-L2 pyramidal neurons' intrinsic excitability contributes to enhanced cocaine-seeking. Our results provide evidence targeting the SK channels in the superficial layer for M2 could be an important therapeutic approach for preventing cocaine relapse. ItemPropranolol Elicits Long Term Systemic Effects After Repetitive Mild Traumatic Brain Injury(2023-07) Smith, Jared Andre; Obukhov, Alexander; White, Fletcher; Hato, Takashi; Naugle, Kelly; Jin, Xiaoming; Truitt, William; Grimes, JaisonThere are almost 2 million new traumatic brain injuries (TBIs) every year in the US. Of these, 80% of these can be classified as mild TBI, also known as concussions, that can lead to pronounced long term symptoms months and years after injury. The presence of post traumatic headaches (PTH) is the most common chronic side effect with prevalence of 47-95% of mTBI patients within a week of injury. Though the mechanisms after TBI leading to these headaches and other post mTBI side effects are poorly understood, recent studies have suggested the role of the immune system after injury plays a causal role in this process. Peripheral immune cells can travel to the brain after mTBI as a result of blood brain barrier dysfunction, sympathetic nervous signaling, and the release of inflammatory mediators. Recent studies have shown sympathetic activation after injury can result in IL-10 dependent systemic immunosuppressive state after mTBI. In this study we sought to limit sympathetic dependent immune alterations after injury by injecting the beta blocker propranolol directly after injury and investigating the immune changes in the blood, brain, spleen, and bone marrow of mTBI animals. Together, these data show mTBI causes immune genetic and pathway level changes at least one month after injury and that propranolol alters genes important for metabolism, cytokine signaling, epigenetic modification, innate, and adaptive immunity. We also find that propranolol reduces the presence of Ly6C+ and increases the presence of Ly6C- monocytes in the blood one month after injury; however, it leads to increased Ly6C+ monocyte presence in the spleen of mTBI mice. In conclusion, propranolol administration directly after mTBI leads to immune changes that may lead to long-term improvement in post TBI symptomology. ItemWhite Matter Microstructural Changes in the Cingulum Bundle of the Hippocampus in Alzheimer's Disease(2023-07) Hirschfeld, Lauren Rose; Risacher, Shannon L.; Saykin, Andrew J.; Block, Michelle L.; Wu, Yu-Chien; Nho, Kwangsik; Cao, ShaAlzheimer’s disease (AD), the most common dementia, is the seventh leading cause of death for adults in the US. Recent FDA-approved therapeutics targeting hallmark amyloid pathology may slow but do not yet halt the disease. Thus, it is likely that other factors, such as neurodegeneration, play a role in disease development. Compromised white matter (WM) microstructural integrity may be a promising biomarker for disease progression, as it has been demonstrated in both preclinical and clinical AD. WM microstructural changes can be estimated using diffusion tensor imaging (DTI), a noninvasive imaging modality which measures the diffusion of water molecules within brain tissue. First, literature describing a biological relationship between signaling pathways involved in both insufficient myelin-repair mechanisms and AD pathology was assessed to establish a foundation for studying WM in the context of AD. Then, DTI was used to assess possible WM microstructural changes of the cingulum bundle of the hippocampus (CBH), a disease-relevant region, in the KBASE cohort of older Korean adults on the AD continuum. Similar to White cohorts of European ancestry, DTI values corresponding to worsening WM microstructure occurred in a stepwise fashion across diagnostic stages and were associated with both cognition and amyloid deposition. In a sample of older adults from the Indiana Memory and Aging Study (IMAS), CBH microstructure was a sensitive biomarker of preclinical conversion from cognitively unimpaired status to mild cognitive impairment. Finally, a novel extension of the T1/T2 weighted ratio imaging method, thought to estimate myelin- related tissue integrity, yielded values in IMAS subjects reflective of previously observed DTI patterns. The complex interplay between amyloid, tau, and neurodegeneration in AD is not yet fully characterized. This research shows decreased integrity of WM microstructure in the CBH, which may be useful as a biomarker of early disease conversion to MCI. ItemThe Anterior Insular Cortex Inputs to the Dorsolateral Striatum Govern Binge-Like Alcohol Intake(2023-07) Haggerty, David Leo; Oblak, Adrian L.; Atwood, Brady K.; Sheets, Patrick L.; McKinzie, David L.; Hopf, Frederic Woody; Lapish, Christopher C.Binge alcohol consumption, defined as consuming four to five or more drinks in about two hours, represents a large proportion of the deaths and economic costs associated with problematic alcohol use. The primary goal of the experiments presented in this dissertation is to uncover how binge drinking alcohol alters synaptic function, and if these changes maintain and perpetuate future binge consumption. The anterior insular cortex (AIC) and dorsolateral striatum (DLS) are brain regions both heavily implicated in alcohol use. In male mice, we found that binge drinking alcohol produced glutamatergic synaptic adaptations selective to AIC neurons that project to the DLS. Photoexciting AIC→DLS circuitry during binge drinking decreased future alcohol, but not water consumption and altered alcohol drinking mechanics. Further, using machine learning approaches we showed that drinking mechanics alone from a single drinking session predicted alcohol-related circuit changes. Additionally, we aimed to determine why female mice displayed different alcohol-induced neuroadaptations after binge alcohol consumption. We showed that female mice display different behaviors in-between binge drinking sessions that influence the amount of alcohol they drink when they binge. For male and female mice that drink similar amounts of alcohol, the mechanics in which they achieved those intakes also differed by sex. Further, we used in-vivo calcium imaging in AIC terminals as a proxy for AIC neurotransmission into the DLS of awake, behaving, and binge drinking mice. We found sex- and time-dependent changes in AIC activity for both water and alcohol groups that also showed strong lateralization effects between AIC inputs that project to the left DLS versus the right DLS. Together, these findings suggest that alcohol-mediated changes in AIC inputs govern behavioral sequences that establish and maintain binge drinking in a sex- and time-dependent fashion and these alterations may serve as circuit-based biomarkers for the development of alcohol use disorder. ItemThe Role of INPP5D in Microglial Function and Amyloid Pathogenesis(2023-07) Lin, Peter Bor-Chian; Block, Michelle L.; Oblak, Adrian L.; Landreth, Gary E.; Kim, Jungsu; Territo, Paul R.Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common cause of dementia. Genetic studies implicate the involvement of microglia-mediated immune responses during disease progression. Importantly, inositol polyphosphate-5-phosphatase D (INPP5D) serves as a regulator of microglial functions and its variants have been identified as risk of late-onset AD. The primary object of this thesis was to study the role of INPP5D in AD pathogenesis. First, increased levels of INPP5D were detected in brain regions of LOAD patients, and a positive association was noted between INPP5D expression and amyloid plaque density. Importantly, increased INPP5D expression was also observed in the amyloidogenic 5xFAD mouse model, with a similar pattern of elevated expression predominately in plaque-associated microglia. These results demonstrated that INPP5D plays an important role in AD. Second, we determined the effect of Inpp5d haplodeficiency on amyloid pathology and microglial functions in 5xFAD mice. The results revealed that Inpp5d haploinsufficiency reduced amyloid plaque burdens and reversed behavioral deficits in 5xFAD mice. Inpp5d haploinsufficiency enhanced microglial engagement to plaques while increasing amyloid plaque compaction in the brains. Furthermore, Inpp5d haploinsufficiency activates TREM2 signaling and suppresses proinflammatory cytokines release in cortical tissues. Spatial transcriptomic analysis highlights that Inpp5d haploinsufficiency modulated the functional pathways including immune cell activation, cytokines production, protein degradation, memory, and synaptic plasticity. Our study suggests that reducing INPP5D expression alters microglial responses and mitigates amyloid pathology during AD progression. Finally, we prepared primary microglial cultures from the wild-type and Inpp5d-haplodeficient mice. The microglial cultures were treated with fibrillar beta-amyloid (fAβ) to investigate the effect of INPP5D inhibition on microglial signaling. Our results demonstrate an increased fAβ uptake and decreased fAβ cytotoxicity in the Inpp5d-deficient microglia. Inpp5d haplodeficiency alters microglial functional pathways, including phagocytosis, apoptosis, cytokines production, and the complement system. Importantly, Inpp5d haplodeficiency elevates the expression of homeostatic microglia signatures. Furthermore, treatment of microglia with INPP5D antagonist (TAD32, 1 μM) showed similar effect as the Inpp5d deficiency in microglia. Collectively, our study validates the hypothesis that INPP5D inhibition may help protect against AD pathology. Treatments utilizing INPP5D antagonists to target microglia-mediated immune responses may be beneficial as an AD therapy. ItemTranscriptomic Profiling in Mild Cognitive Impairment and Alzheimer's Disease Using Neuroimaging Endophenotypes(2022-12) Bharthur Sanjay, Apoorva; Yoder, Karmen K.; Apostolova, Liana G.; Risacher, Shannon L.; Gao, Sujuan; Nudelman, KellyAlzheimer’s disease (AD) is a devastating neurodegenerative disease affecting more than 6 million Americans and 50 million people worldwide currently. It is an irreversible neurodegenerative disease which causes decline in memory, cognition, personality, and other functions which eventually lead to death due to complete brain failure. Recently there has been a lot of research that has focused on enabling early intervention and disease prevention in AD which could have a significant impact on this disease, be crucial for life management, assessment of risk for future generations, and assistance in end-of-life preparation. For a late-life complex multifactorial disease, such as AD, where both genetic and environmental factors are involved, integrating multiple layers of genetic, imaging, and other biomarker data is a critical step for therapeutic discovery and building predictive risk assessment tools. The multifactorial nature of AD suggests that multiple therapeutic targets need to be identified and tested together. Hence, we need a systems-level approach to build biomarker profiles which can be used for drug discovery and screening/risk assessment. The research presented in this dissertation focuses on utilizing a systems level approach to identify promising imaging genetics biomarkers that provide insight into dysregulated biological pathways in AD pathogenesis and identify critical mRNA measures that can be investigated further within the scope of novel therapeutics, as well as input variables in predictive models for AD risk, screening, and diagnosis. The overall research goal was the development of systems level, imaging genetics biomarker signatures to serve as tools for risk analysis and therapeutic discovery in AD. The specific outcomes of the analyses were characterization of patterns in gene expression at systems level using neuroimaging endophenotypes, and identification of specific driver genes and genotypic variants, which can inform predictive modeling for diagnosis, risk, and pathogenic profiling in AD. ItemDissecting the Effects of Different Pain Modalities and Oxycodone on Prodynorphin Expressing Neurons in the Mouse Prelimbic Cortex(2022-11) Zhou, Shudi; Atwood, Brady K.; Sheets, Patrick L.; McKinzie, David L.; Truitt, William A.; Jin, XiaomingCurrently, changes to endogenous opioid circuits in various pain modalities, including surgical and neuropathic pain, remain unclear. Dynorphin, which is released by prodynorphin-expressing neurons (Pdyn+ neurons), is the endogenous opioid ligand to kappa opioid receptors (KOR). Moreover, a recent study has shown an increase in prodynorphin (Pdyn) mRNA expression in the prelimbic cortex (PL) in a mouse model of chronic pain. However, alterations in the activity of PL Pdyn-expressing neurons (PLPdyn+ neurons) in postoperative and chronic pain have never been explored. Firstly, I found that the population of PLPdyn+ neurons consists of both pyramidal and inhibitory subtypes. Secondly, I found that one day after surgical incision of the mouse hind paw, the excitability of pyramidal PLPdyn+ neurons was increased in both male and female mice, while the excitability of inhibitory PLPdyn+ neurons was unchanged. However, when postoperative pain behavior subsided, inhibitory PLPdyn+ neurons were hyperexcitable in male mice, while pyramidal PLPdyn+ neurons were hypoexcitable in female mice. Lastly, I dissected electrophysiological changes to PLPdyn+ neurons in the spared nerve injury (SNI) model of chronic neuropathic pain. At both early and late stages of SNI pain development, increased excitability of pyramidal PLPdyn+ neurons was detected in both male and female mice. However, in both male and female mice, the excitability of inhibitory PLPdyn+ neurons decreased 3 days after SNI but was conversely increased when measured 14 days after SNI. My findings suggest that different subtypes of PLPdyn+ neurons manifest distinct alterations in the development of different pain modalities in a sex-specific manner. ItemElements of the Brain Network Regulating Social Behavior and Vocal Communication in Nf1+/- Mice: Relevance to Developmental Language Disorders and Autism Spectrum Disorders(2022-11) Karathanasis, Sotirios Ferris; Landreth, Gary E.; Clapp, D. Wade; Molosh, Andrei I.; Oblak, Adrian L.; Mosley, Amber L.; Shekhar, AnanthaCommunication is a vital tool used by humans to share information, coordinate behavior, and survive. However, the ability to communicate can become disrupted or remain absent in individuals with neurodevelopmental disorders: two prominent examples include autism spectrum disorders and developmental language disorders, found in nearly 2% and 10% of the population, respectively. Communication disorders are devastating to the autonomy and quality of life of affected individuals, but clinical solutions are limited due to the complex and often unknown neural etiology underlying these conditions. One known disorder with high incidence of disrupted communication is Neurofibromatosis type 1, the genetic disease caused by heterozygosity of the Ras GTPase-activating protein-coding gene NF1. Mice heterozygous for their ortholog of this gene (Nf1+/-) have been shown to recapitulate neuropsychiatric conditions seen in patients. Using a courtship trial paradigm as a model for testing communication, I have demonstrated that Nf1+/- male mice showed deficits in both courtship and non-courtship social behavior as well as a decrease in the number and duration of ultrasonic vocalizations (USVs). Immediate early gene (IEG) immunohistochemistry (IHC) in neurons of courtship-relevant brain regions revealed the Shell of the Nucleus Accumbens (NAcS) as a dysfunctional brain region in Nf1+/- mice compared to WT male mice following courtship trial. Optogenetic targeting of the Nucleus Accumbens (NAc) restored courtship social behaviors and USV number, but not USV duration or non-courtship gestural social behaviors, in Nf1+/- males. This study contributes to a preclinical foundation for understanding etiology of communication disorders in patients. ItemNovel Strategies for the Prevention of Post-Stroke Epilepsy and Sudden Unexpected Death in Epilepsy Patients(2022-10) Adhikari, Yadav Prasad; Truitt, William; Witkin, Jeffrey M.; Gupta, Kunal; Brutkiewicz, Randy; Jin, XiaomingStroke is the second leading cause of mortality worldwide, accounting for 5.5 million deaths annually. In addition to its high mortality rate, stroke is the most common cause of acquired epilepsy. Three to thirty percent of stroke survivors develop post-stroke epilepsy. Although currently available therapies such as thrombolytics and mechanical thrombectomy prevent immediate mortality by restoring blood flow after stroke, these treatments do not target the cellular and molecular mechanisms that lead to post-stroke epileptogenesis. With the increasing number of stroke survivors, there is an urgent need for therapies that prevent epilepsy development in this population. Here, we showed that homeostatic plasticity is involved in the development of hyperexcitability after stroke and can be targeted to prevent the development of post-stroke epilepsy. Using two-photon calcium imaging, we found that homeostatic regulation leads to cortical hyperexcitability after stroke. We also found that activity enhancement by optogenetic and pharmacological approaches can target homeostatic plasticity to prevent post-stroke epilepsy. This study demonstrates the high translational potential of activity enhancement as a novel strategy to prevent post-stroke epilepsy through regulating cortical homeostatic plasticity. Sudden premature death is a leading cause of death in patients with medically refractory epilepsy. This unanticipated death of a relatively healthy person with epilepsy in which no structural or toxicological cause of death can be identified after postmortem analysis is referred to as sudden unexpected death in epilepsy patients (SUDEP). Respiratory failure during seizures is an important underlying mechanism of SUDEP. Here, we showed that LPS-induced peripheral inflammation is protective against SUDEP. This protection is mediated at least in part via enhancing serotonergic function in the brain stem. To the best of our knowledge, this is the first study demonstrating the relationship between peripheral inflammation and SUDEP prevention. ItemImpact of PLCG2 Alzheimer's Disease Risk and Protective Variants on Microglial Biology and Disease Pathogenesis(2022-09) Tsai, Andy Po-Yi; Oblak, Adrian L.; Landreth, Gary E.; Lamb, Bruce T.; Liu, Yunlong; Mckinzie, David L.; Nho, KwangsikAlzheimer’s disease (AD) is typified by a robust microglial-mediated immune response. Genetic studies have demonstrated that many genes that alter AD risk are involved in the innate immune response and are primarily expressed in microglia. Among these genes is phospholipase C gamma 2 (PLCG2), a critical element for various immune receptors and a key regulatory hub for immune signaling. PLCG2 genetic variants are associated with altered AD risk. The primary objective of this thesis was to determine the role of PLCG2 in AD pathogenesis. We observed significant upregulation of PLCG2 expression in three brain regions of late-onset AD (LOAD) patients and a significant positive correlation of PLCG2 expression with amyloid plaque density. Furthermore, the differential gene expression analysis highlighted inflammatory response-related pathways. These results suggest that PLCG2 plays an important role in AD. We systematically investigated the impact of PLCG2 haploinsufficiency on the microglial response and amyloid pathology in the amyloidogenic 5xFAD mouse model. The results demonstrated that Plcg2 haploinsufficiency altered the phenotype of plaqueassociated microglia, suppressed cytokine levels, increased compact X34-positive plaque deposition, and downregulated the expression of microglial genes associated with immune cell activation and phagocytosis. Our study highlights the role of PLCG2 in immune responses; loss of function of PLCG2 exacerbates the amyloid pathology of AD. Genetic studies demonstrated that the hypermorphic P522R variant is protective and that the loss of function M28L variant confers an elevated risk for AD. Our results demonstrated that PLCG2 variants modulate disease pathologies through specific transcriptional programs. In the presence of amyloid pathology, the M28L risk variant impaired microglial response to plaques, suppressed cytokine release, downregulated disease-associated microglial genes, and increased plaque deposition. However, microglia harboring the P522R variant exhibit a transcriptional response endowing them with a protective immune response signature linked to their association with plaques and Aβ clearance, attenuating disease pathogenesis in an amyloidogenic mouse model of AD. Collectively, our study provides evidence that the M28L variant is associated with accelerated and exacerbated disease-related pathology, and conversely, the P522R variant appeared to attenuate disease severity and progression.