Staci D. Bilbo - US grants

DESCRIPTION (provided by applicant): Neonatal exposure to bacteria in rats leads to memory impairment following an immune challenge in adulthood. The proposed research will explore this phenomenon by examining 1) the role of the pro- inflammatory cytokine interleukin [IL]-1beta, 2) the mechanism(s) underlying cytokine-induced memory impairment, and 3) the generality of the phenomenon. To test the hypothesis that IL-1 produced in response to an immune challenge (LPS) in adulthood impairs memory, an IL-1 synthesis inhibitor will be administered prior to the learning experience and subsequent LPS, which normally impairs memory in neonatally-infected rats. To determine the mechanism(s) underlying cytokine-induced memory impairment, adult rats treated as neonates with bacteria or PBS will be injected with LPS or saline, and cytokine and glial cell marker gene expression will be measured in the hippocampus. LTP and BDNF expression will be measured in the hippocampus following LPS. To determine the generality of these effects, rats will be infected prenatally or neonatally with bacteria or virus, and behavioral consequences will be measured in adults. The duration of memory impairment will also be measured, and a number of memory paradigms will be tested.

[unreadable] DESCRIPTION (provided by applicant): An estimated 25% of infants in this country are exposed prenatally to drugs of abuse, a significant proportion of which are illicit drugs. Furthermore, a significant number of newborn infants, many of them premature, are exposed to opioids for pain relief following surgical or traumatic events, the long-term consequences for which are little understood. Beyond well documented developmental disabilities, the possibility that individuals exposed to drugs of abuse early in life are rendered more likely to abuse drugs as adults is a particularly alarming prospect, and has been suggested in a number of clinical studies. A critical role for glial cells (e.g., microglia, astrocytes) in addiction is becoming increasingly apparent. For instance, it has recently been demonstrated that morphine and cocaine directly activate glial cells within the CNS in a non-classical opioid receptor manner, via the innate immune system's pattern recognition receptor, toll-like receptor (TLR) 4, and, in the case of morphine, that this glial activation contributes strongly to their rewarding properties. Thus, glial inhibitors or selective TLR4 antagonists markedly reduce opioid-induced dependence, tolerance, and reward. In infants, glial cells are critical for many aspects of normal brain development, including apoptosis, axonal growth, angiogenesis, and synaptogenesis. As the primary "immuno-competent" cells of the brain, they also respond vigorously to virtually any disturbance of homeostasis, such as injury or infection, and this activation may cause long-term alterations in their function. This proposal has been designed to test the hypothesis that events occurring during the perinatal period of life often produce enduring effects on brain and behavior, including abuse liability later in life, via their influence on glial cell activity and subsequent neural development. This hypothesis will be tested by examining the following questions, using gene expression, protein expression, pharmacological and behavioral techniques: (1) What is the impact of perinatal exposure to morphine and cocaine on glial and [unreadable] neural cell development and function? (2) What is the role for alterations in glial cell development and/or function in later life abuse liability? and (3) Will attenuating glial activation early in life protect from increased abuse liability in adulthood? These collective data will provide novel insight into the role that early life noxious events, including drug exposure, have on neural and glial cell development within the CNS, as well as the role that glial cells play in substance abuse at a behavioral and pharmacological level. PUBLIC HEALTH RELEVANCE: An estimated 25% or more of infants in this country are exposed prenatally to drugs of abuse, and a significant number of newborn infants, many of them premature, are exposed to opioids for pain relief following surgical or traumatic events, the long-term consequences for which are little understood. A critical role for glial cells (e.g., microglia, astrocytes) in addiction is becoming increasingly apparent. The data collected from this proposal will provide novel insight into the role that early life noxious events, including drug exposure, have on neural and glial cell development within the CNS, the role that glial cells play in substance abuse at a behavioral and pharmacological level, and ultimately treatment decisions. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neuroendocrine or immune events occurring within the perinatal environment often produce effects on brain and behavior that endure throughout an organism's life span. An estimated 1/3 of pregnancies suffer complications involving infection or trauma of the uterus, fetus, or newborn, and one of the most common consequences of infection or inflammation during the perinatal period is cognitive dysfunction, including learning, memory, and attention disorders. Systemic infection with bacteria (Escherichia coli) on postnatal day 4 in rats is associated with dramatic memory impairments in conjunction with a peripheral immune challenge (lipopolysaccharide;LPS) in adulthood. The current proposal is designed to address two related questions: (1) What changes occur in the neonatal brain in response to the infection that render the brain vulnerable to a later challenge? and (2) What changes occur in the brains of neonatally-infected adult rats in response to the LPS challenge, which produce the memory impairments? The proposed experiments will test the hypothesis that long-term changes in brain microglia, the primary immune cells of the brain, occur in response to infection early in life, which then contribute to altered brain function (e.g., cytokine production, neurogenesis) and memory impairment in adulthood. This hypothesis will be tested by examining the following questions, using gene expression, protein expression, and behavioral techniques: (1) Does neonatal E. coli infection result in increased microglial reactivity in adulthood? (2) Do neonatal E. coli infection-induced changes in microglia underlie exaggerated brain cytokine responses and memory impairments in adulthood? and (3) Why does postnatal day 4 appear to be during a sensitive period for neonatal infection- induced vulnerabilities later in life? These collective data will provide novel insight into the influence of early immune activation on neural and immune system development, the role that the brain's immune response plays in cognition, and ultimate treatment decisions. PUBLIC HEALTH RELEVANCE: An estimated 1/3 of pregnancies suffer complications involving infection or trauma of the uterus, fetus, or newborn, and one of the most common consequences of infection or inflammation during the perinatal period is cognitive dysfunction, including learning, memory, and attention disorders. The data collected from this proposal will provide novel insight into the influence of early immune activation on neural and immune system development, the role that the brain's immune response plays in cognition, and ultimately treatment decisions aimed at preventing the negative consequences of early infection or trauma.

DESCRIPTION (provided by applicant): Many factors affect an individual's risk of addiction, including genetic, environmental, and physiological factors. Of these factors, the early-life environment of an individual, including maternal care, may be especially critical. Our research provides strong evidence that maternal care can permanently alter glial cell function within the brain, which has long-term consequences for neural function and behavior. Notably, glial cells (astrocytes and microglia) are activated by drugs of abuse, and their activation and subsequent release of cytokines and chemokines can markedly impact the physiological and addictive properties of drugs of abuse, including morphine. In this proposal, we describe our novel hypothesis that microglial-driven chemokine expression within the nucleus accumbens (NAc) underlies morphine-induced relapse in a model of addiction, and moreover that nurturing maternal care early in life induces resilience of the pups to drug relapse in adulthood by inducing an anti-inflammatory phenotype in microglia. Specifically, we show that: (1) Morphine profoundly activates glia within the adult rat NAc, inducing a rapid (minutes) increase in chemokines; (2) Inhibiting this morphine-induced chemokine response with a glial modulator, Ibudilast, completely prevents morphine- induced reinstatement of CPP assessed months later, without altering initial CPP which remains high; and (3) Neonatal handling mimics the glial modulator by completely preventing morphine-induced chemokine expression within the NAc in adulthood, and prevents the reinstatement of morphine CPP. Moreover, handled rats exhibit increased basal expression of the anti-inflammatory cytokine IL-10 within the brain compared to non-handled control rats, which is established early in life and maintained into adulthood via decreased methylation of the IL-10 gene specifically within microglia. Our central hypothesis is that this epigenetically induced constitutive increase in microglial IL-10 by enhanced maternal care prevents the morphine-induced chemokine response by glia and thereby prevents the neural plasticity changes underlying drug-induced reinstatement of morphine CPP, independent of changes in reward or stress reactivity. Thus, the overall objective of this application is to fist establish a causal relationship for microglial IL-10 in the resilience to the novel brain chemokine response to morphine and subsequent drug-induced reinstatement. Second, we will determine whether IL-10 impacts chemokine expression by inhibiting canonical proinflammatory gene transcription within microglia.

Project Summary Environmental toxins and microglia-synapse interactions in autism. It is increasingly evident that diverse genes and environmental exposure(s) combine or synergize to produce a spectrum of autism phenotypes dependent upon critical developmental windows. Multiple prenatal/maternal environmental toxins and exposures have been linked to human ASDs, but the associations of single agents have been relatively weak. This suggests it is the combination of multiple maternal exposures that increases vulnerability in offspring. We now recognize that non-chemical stressors, such as limited resources or social support of the mother, can increase vulnerability of the fetus to chemical stressor exposures (e.g., pollution or toxins), which could explain why a single exposure or risk factor in isolation is a modest predictor of autism risk. Models aimed at deciphering the mechanisms that contribute to ASD suffer from oversimplification, using single agents. We breach this gap by using a new model that employs the combined effects of an ethologically relevant maternal stressor and environmentally relevant pollutant, diesel exhaust, both of which have been implicated in autism. We show that maternal diesel exhaust particle (DEP) exposure combined with maternal stress (MS) (but neither in isolation) produces early-life communication deficits, and long-term cognitive deficits and strikingly increased anxiety in male but not female offspring. We show evidence that DEP exposure significantly alters microglial colonization of the male but not female embryonic brain, and combined prenatal DEP and MS exposure leads to persistent changes in the function of microglia of the same brain regions of males. Beyond their functions in innate immune defense of the brain, microglia are important regulators of experience-dependent synaptic remodeling during development. It is proposed that microglia prune inappropriate or weak synapses while sparing appropriate or strong connections. Autism has been well described as a disease of synaptic dysfunction, and functional network analyses have nearly all pointed out the importance of molecular pathways that control activity-dependent synaptic remodeling in the pathology of ASDs. Importantly, impaired microglia-mediated pruning in mice disrupts functional brain connectivity and social behavior, strongly suggesting that microglia-synapse interactions may contribute to autism?s pathophysiology. Thus, the specific hypothesis to be tested here is that microglial activation by combined environmental factors will cause aberrant synaptic pruning by these cells, leading to neural circuit dysfunction and ASD-like behaviors.

Gastrointestinal issues are extremely common in neurodevelopmental disorders like autism spectrum disorder (ASD), and alterations of the gut microbiome and intestinal epithelial barrier have been reported in recent studies. Environmental toxicant exposures early in life are increasingly implicated in neurodevelopmental disorders such as ASD, including air pollution. There is strong evidence that particulate matter (PM) in air pollution significantly impacts the gut microbiome and gut function of directly-exposed humans and rodents. Less characterized is if PM exposure to pregnant females alters the gut microbiome of offspring, though this is likely given evidence that the maternal gut microbiome sets the trajectory of the newborn microbiome, especially with a vaginal delivery. To study the impact of environmental pollutants on autism-like behaviors in mice, we developed a novel model combining prenatal diesel exhaust particle (DEP) exposure throughout pregnancy with maternal stress (MS) during the last trimester of gestation. Maternal stress is linked to autism in several recent studies, which may be most harmful for populations made vulnerable by other factors. We have demonstrated that combined prenatal DEP + MS produce striking communication and social deficits early in life, and persistent cognitive deficits and increased anxiety into adulthood, in male but not female offspring. Our preliminary data also show significant changes in the composition of gut bacteria and gut structural changes in male offspring exposed prenatally to DEP/MS compared to unexposed controls. Our goal is to test the hypothesis that gut microbiome changes in pregnant dams following combined environmental exposures are transmitted to newborn offspring and underlie the persistent behavioral abnormalities. Together these studies will: (1) fully characterize the impact of prenatal environmental toxicant (DEP) exposure on maternal and offspring microbiome development, (2) ascribe causality among microbiota changes, gut epithelial structure/function and inflammation, and behavioral abnormalities in offspring, and (3) establish the critical window(s) in which microbiome changes in offspring can be prevented or reversed using interventions at birth vs. post-weaning. If successful they will significantly advance our understanding of the emergence and causal link between gut dysbiosis and behavioral/brain dysfunction in devastating disorders such as autism, and the role of environmental toxins in inducing these changes, as well as suggest a potential therapeutic option and window for treatment.