Neonatal Opioid Withdrawal Syndrome in Mice: Acute Molecular and Long-Term Behavioral Consequences.

Populations affected by the ongoing opioid epidemic include pregnant women and their offspring. Infants exposed to opioids in utero are at risk of developing Neonatal Opioid Withdrawal Syndrome (NOWS), a combination of somatic withdrawal symptoms including high pitched crying, sleeplessness, irritability, and potentially seizures. Confounding environmental factors and difficulty in conducting longitudinal clinical studies have made it challenging to elucidate the long-term consequences of NOWS. In order to investigate the molecular mechanisms and long-term effects of NOWS, we have developed a mouse model of opioid exposure and withdrawal that encompasses the developmental equivalent of all three trimesters of the human pregnancy. Female mice are implanted with morphine minipumps (10 mg/kg/day) before mating, ensuring that pups are exposed to morphine in utero for the entirety of embryonic development. Mice are then given twice daily morphine injections (10 mg/kg/day, SC) for post-natal days 1-14. Mice are tested for developmental milestones during this period, including eye opening, extinguishing pivoting, surface righting, and forelimb grasp. Following the final morphine injection withdrawal behaviors were measured by ultrasonic vocalizations, 55°C hotplate analgesia latency, and somatic withdrawal signs. After reaching adulthood, mice were tested for changes in affective behavior (elevated zero maze, tail suspension test and learned helplessness), opioid response (conditioned place preference and intravenous self-administration), social behavior (three-chamber social behavior test), and sleep patterns. Mice exposed to this paradigm show delayed latency to reach developmental milestones and acute withdrawal phenotypes. In adulthood, social behavior and sleep patterns were affected in a sex-dependent manner, while affective behavior and opioid response was unchanged. Bulk RNA sequencing of mice at post-natal day 1 revealed changes in gene expression related to synapse development and function, the GABAergic system, and mitochondrial function. These transcriptome changes may underlie the behavioral long-term effects of NOWS.