Evaluation of inflammation in the locus coeruleus during physical withdrawal symptoms and cognitive development in a rat model of neonatal opioid withdrawal syndrome (NOWS)

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Abstract In the past decade, opioid use disorders have significantly increased in pregnant women and women of childbearing age. In parallel, we have seen a drastic rise in neonatal opioid withdrawal syndrome (NOWS). Despite having well-defined, short-term phenotypic symptoms for NOWS, molecular effects and long-term consequences are incomprehensible. This is of particular concern given that opioid exposure alone, as well as withdrawal, can induce inflammation in the brain, and recent work has shown an increase in pro-inflammatory cytokines and chemokines, which may be promoted by reactive glia as a result of in utero opioid exposure. Moreover, cognitive functioning and memory deficits have been observed in preclinical models of in utero opioid exposure, and intellectual impairments and increased attention disorders have been observed in clinical populations previously diagnosed with NOWS. While much of the research has focused on inflammation and in utero opioid exposure in brain regions associated with reward and cognition, less is known about the brain regions associated with withdrawal symptoms in NOWS, and how this may relate to cognitive function later in life. Understanding the pro-inflammatory effects of in utero opioid exposure and withdrawal may allow for novel targets for the treatment of NOWS, and better understanding of long-term consequences. Therefore, by using an established rat model of NOWS that has previously shown an upregulation of inflammatory markers due to in utero opioid exposure, we will investigate inflammation in a withdrawal-associated brain region, the locus coeruleus, and determine effects on behavioral indices of withdrawal and subsequent cognitive function. More specifically, in a rat model of NOWS, we will utilize immunohistochemistry, qPCR, and multiplex assays to evaluate inflammatory markers including reactive glial cells, toll-like receptor 4 expression, and levels of pro- inflammatory cytokines and associated neuropeptides, and their respective associations with quantified behavioral withdrawal symptoms. In addition, we will test the specific role of reactive glia on physical withdrawal symptoms in the rat neonate by systemically administering the glia-inhibiting agent, minocycline. We will assess the resultant cognitive function at PN21 and PN60, when rats are considered preadolescent and early adulthood, respectively. We will test spatial learning and memory in heroin- or saline-exposed rat pups who experienced precipitated withdrawal at PN10. Additional experiments will also include the use of neonatal, systemic minocycline administration to mitigate cognitive deficits previously observed in preclinical and clinical populations of NOWS. Together, these experiments will allow us to better understand the effects of inflammation on physical withdrawal symptoms, as well as the relationship between physical withdrawal symptoms and subsequent cognitive function, as a result of in utero opioid exposure and precipitated withdrawal, thereby establishing novel targets for the treatment of NOWS and laying the foundation for future studies evaluating its long-term consequences.