Infants exposed to opioids in utero are an increasing clinical population and these infants are often diagnosed with Neonatal Abstinence Syndrome (NAS). Infants with NAS have diverse negative health consequences, including respiratory distress. However, many factors contribute to NAS, confounding the ability to understand how maternal opioids directly impact the neonatal respiratory system. Breathing is controlled centrally by respiratory networks in the brainstem and spinal cord, but the impact of maternal opioids on developing perinatal respiratory networks has not been studied. Using progressively more isolated respiratory network circuitry, we tested the hypothesis that maternal opioids directly impair neonatal central respiratory control networks. Fictive respiratory-related motor activity from isolated central respiratory networks was age-dependently impaired in neonates after maternal opioids within more complete respiratory networks (brainstem and spinal cords), but unaffected in more isolated networks (medullary slices containing the preBötzinger Complex). These deficits were due, in part, to lingering opioids within neonatal respiratory control networks immediately after birth and involved lasting impairments to respiratory pattern. Since opioids are routinely given to infants with NAS to curb withdrawal symptoms and our previous work demonstrated acute blunting of opioid-induced respiratory depression in neonatal breathing, we further tested the responses of isolated networks to exogenous opioids. Isolated respiratory control networks also demonstrated age-dependent blunted responses to exogenous opioids that correlated with changes in opioid receptor expression within a primary respiratory rhythm generating region, the preBötzinger Complex. Thus, maternal opioids age-dependently impair neonatal central respiratory control and responses to exogenous opioids, suggesting central respiratory impairments contribute to neonatal breathing destabilization after maternal opioids and likely contribute to respiratory distress in infants with NAS. These studies represent a significant advancement of our understanding of the complex effects of maternal opioids, even late in gestation, contributing to neonatal breathing deficits, necessary first steps in developing novel therapeutics to support breathing in infants with NAS.

Adults often encounter diverse inflammatory stimuli, yet only some adults are prone to breathing deficits after adult inflammation. The determinants of adult susceptibility to breathing deficits during inflammation is understudied and likely involves microglia, the resident immune cells of the central nervous system. In non‐respiratory control regions, neonatal inflammation primes adult microglia for exaggerated inflammatory responses to otherwise harmless inflammatory stimuli, such as adult subthreshold heterotypic inflammation (differing neonatal and adult inflammatory stimuli). Since male and female microglia have sexually dimorphic developmental trajectories, we hypothesized that neonatal inflammation would sex‐dependently prime adult microglia in respiratory control regions to subthreshold heterotypic inflammation, contributing to impaired adult breathing. Using flow cytometry to assess microglia number (%CD11bhighCD45low / homogenates) and priming, adult male medullary microglia after neonatal inflammation (LPS 1mg/kg, i.p.) and adult subthreshold heterotypic inflammation (polyIC 478ug/kg, i.p.) were primed (neonatal LPS + adult polyIC: 25±7% microglia n=9; neonatal LPS + adult saline: 18±2% microglia, n=9; neonatal saline + adult saline: 10±2% microglia, n=9, p<0.05; neonatal saline + adult polyIC: 11±2% microglia, n=8, p<0.0001). In females, neonatal inflammation increased, but did not prime, medullary microglia to adult heterotypic inflammation (neonatal LPS + adult polyIC: 17±3% microglia, n=6; neonatal saline + adult saline: 11±1% microglia, n=6; neonatal saline + adult polyIC: 11±2% microglia, n=6, p<0.005; neonatal LPS + saline: 14±2% microglia, n=8, p>0.9). To test whether these primed microglia in respiratory control regions contribute to adult breathing deficits, breathing was assessed using plethysmography in adults after neonatal and adult heterotypic inflammation. Contrary to our hypothesis, neonatal and adult heterotypic inflammation did not impair adult eupneic breathing, the hypercapnic ventilatory response, or the hypoxic ventilatory response in adult males (p>0.9) or females (p>0.9). Therefore, adults after neonatal and adult heterotypic inflammation are able maintain breathing in response to low levels of inflammation despite primed medullary microglia. Primed microglia, however, are prone to augmented inflammatory responses and may induce significant deficits to breathing when faced with more severe inflammatory challenges.

A population of adults are predisposed to breathing deficits during subsequent illness and disease, yet it is unknown what factor(s) contribute to this adult vulnerability. Since neonatal inflammation primes adult microglia in non‐respiratory control regions and augments peripheral immune responses, we hypothesized that neonatal and adult homotypic inflammation (same inflammatory stimulus at both ages) primes male and female microglia in respiratory control regions, contributing to impairments in adult breathing during subsequent inflammatory challenges. Using lipopolysaccharide (LPS) to induce homotypic inflammation in neonates (1mg/kg LPS, P4) and adults (24µg/kg LPS, i.p.), medullary microglia were primed in adult males (neonatal LPS + adult LPS: 25±4% microglia, n=9; neonatal LPS + adult saline: 18±2% microglia, n=9; neonatal saline + adult saline: 10±1% microglia, n=9; p<0.01) and adult females (neonatal LPS + adult LPS: 25±3% microglia, n=9; neonatal LPS + adult saline: 14±2% microglia, n=6; neonatal saline + adult saline: 10±2% microglia, n=8; p<0.0001). However, spinal cord microglia were only primed in adult males (neonatal LPS + adult LPS: 13±2% microglia, n=9; neonatal LPS + adult saline: 9±2% microglia, n=9; neonatal saline + adult saline: 10±2% microglia, n=9; p<0.01) and not adult females (neonatal LPS + adult LPS: 13±2% microglia, n=9; neonatal LPS + adult saline: 9±1% microglia, n=6; neonatal saline + adult saline: 9±1% microglia, n=8; p<0.01). Thus, neonatal and adult homotypic inflammation region‐ and sex‐dependently prime microglia in respiratory control regions. To test whether primed microglia in respiratory control regions contribute to deficits in adult breathing, breathing was assessed by plethysmography in adults at two time points: during the peak inflammatory response (3hrs) and peak microglial migration and proliferation (24hrs). Neither adult eupneic breathing nor chemoreflexes (hypercapnic ventilatory response, HCVR, and hypoxic ventilatory response, HVR) were primed in males or females by neonatal and adult homotypic inflammation at either time point. Interestingly, adult female chemoreflexes were time‐dependently increased by neonatal and adult homotypic inflammation. In adult females, neonatal and adult homotypic inflammation increased the peak HCVR at 3hrs (neonatal LPS + adult LPS: 298±112% ∆VE from baseline, n=6) compared to 24hrs (neonatal LPS + adult LPS: 169±41% ∆VE from baseline, n=6, p=0.04), and increased the peak HVR at 3hrs (neonatal LPS + adult LPS: 159±37% ∆VE from baseline) compared to 24hrs (neonatal LPS + adult LPS: 62±23% ∆VE from baseline, n=6, p=0.02). Thus, adult female chemoreflexes were time‐dependently impacted by the combination of neonatal and adult homotypic inflammation, suggesting time‐specific effects of adult inflammation. Despite no significant impact of primed microglia on baseline breathing and chemoreflexes, primed respiratory control microglia have the potential to significantly impair respiratory control during more severe inflammatory challenge...

An understudied population in the opioid crisis are infants exposed to in utero opioids experiencing severe respiratory deficits. Our recent work demonstrated maternal opioids destabilized neonatal breathing, increased apneas, and blunted opioid‐induced respiratory frequency depression; however, the mechanism(s) underlying these neonatal breathing deficits remains unclear. We hypothesized deficits in neonatal breathing after maternal opioids are due to downregulation of mu‐opioid receptors on key cell types (e.g. neurons and astrocytes), essential for respiratory rhythm generation and modulation in the preBötzinger Complex. While opioid receptor expression is downregulated in the whole brain after maternal opioids, it remains unclear if opioid receptor expression changes in the preBötzinger Complex after in utero opioids to induce neonatal breathing deficits. To assess the typical developmental changes in opioid receptors in the preBötzinger Complex, mu‐opioid receptor expression was first assessed using immunohistochemistry and confocal microscopy on neonatal neurons and astrocytes in the preBötzinger Complex at three ages: the day of birth (postnatal day 0, P0) when neonates must begin robust rhythmic breathing, an early neonatal age (postnatal day 4, P4), and during a critical developmental window (postnatal day 11, P11). At all ages, mu‐opioid receptors were expressed on both neurons and astrocytes in the preBötzinger Complex; however, mu‐opioid receptor expression decreased from P0 (n=6) to P4 (n=6) and was lowest at P11 (n=6). Preliminary data after maternal opioids suggest mu‐opioid receptor expression was lower in preBötzinger Complexes at P0 (n=2) and P4 (n=2), but returned to control levels by P11 (n=2). These results are further supported by in vitro brainstem‐spinal cord recordings of the cervical rootlets (C4) from neonates after maternal opioids, demonstrating decreased opioid sensitivity of isolated respiratory control circuitry after maternal opioids (n=2). Results from these studies will contribute to our understanding of the mechanisms contributing to impaired neonatal breathing after maternal opioids and may lead to better treatments for infants suffering breathing deficits after maternal opioids.