Cellular and network-level adaptations to in utero methadone exposure along the ventral respiratory column in the neonate rat.

Neonatal abstinence syndrome (NAS) occurs in babies chronically exposed to opioids during pregnancy. NAS shares features with opioid withdrawal symptoms seen in adults, including autonomic dysregulation. Here, the effect of low-dose in utero methadone (MTD) exposure on respiration-modulated networks along the ventral respiratory column (VRC) in ventrolateral medulla was investigated in the neonate Sprague-Dawley rat. MTD was administered via drinking water (3mg/kg/day in drinking water of the mother E7-E21). Lower expression levels of myelin-associated proteins phosphorylated axonal neurofilament subunit H (pNFH), 2',3'-Cyclicnucleotide 3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), in MTD-exposed pups compared to controls at P3, P6 and P10 indicated MTD transport across the placenta. We investigated whether in utero MTD exposure led to network-level excitability changes consistent with tolerance, and also probed for changes in endogenous opioid modulation of respiratory networks. To this end, high-speed (45.5Hz) optical recordings of respiratory network activity in control and MTD-exposed neonate (P0-P2) pups before and during administration of the μ-opioid receptor antagonist naloxone (NAL; 10μM) were carried out. Spike rate was estimated from optical traces via deconvolution, and coupling between all neuron pairs in recorded networks was quantified using the normalized transfer entropy (NTE). Recordings of local networks along the VRC, together with recordings of respiratory output from ventral root C1 did not reveal changes in respiratory activity at the system level, but cellular and network changes in MTD-exposed pups were consistent with the development of opioid tolerance. MTD-exposed pups were found to have i. higher neuronal firing rates; ii. higher covariance between neuronal activity and motor output; iii. more bidirectionally and unidirectionally coupled neurons, and fewer uncoupled neurons; iv. stronger coupling and shorter integration times between network constituents. The μ-opioid receptor antagonist NAL did not alter system-level function. The correlation between the activity of neurons caudal to -400μm and motor output was significantly reduced in control animals following NAL. In both control and MTD-exposed pups, the relative number of neurons whose correlation with motor output increased following NAL followed a rostrocaudal gradient along the VRC, with fewer neurons caudally, and more neurons rostrally. The up-regulation of coupling strength, firing rate and coefficient of variation between neurons and motor output following in utero opioid exposure suggests that these networks may contribute to NAS in infants born to opioid-dependent mothers.