Sex Differences in Behavioral and Brainstem Transcriptomic Neuroadaptations following Neonatal Opioid Exposure in Outbred Mice

Abstract

The opioid epidemic led to an increase in the number of Neonatal Opioid Withdrawal Syndrome (NOWS) cases in infants born to opioid-dependent mothers. Hallmark features of NOWS include weight loss, severe irritability, respiratory problems, and sleep fragmentation. Mouse models provide an opportunity to identify brain mechanisms that contribute to NOWS. Neonatal outbred Swiss Webster Cartworth Farms White (CFW) mice were administered morphine (15mg/kg, s.c.) twice daily for postnatal days (P) 1-14, an approximate of the third trimester of human gestation. Female and male mice underwent behavioral testing on P7 and P14 to determine the impact of opioid exposure on anxiety and pain sensitivity. Ultrasonic vocalizations (USVs) and daily body weights were also recorded. Brainstems containing pons and medulla were collected during morphine withdrawal on P14 for RNA-sequencing. Morphine induced weight loss from P2-14, which persisted during adolescence (P21) and adulthood (P50). USVs markedly increased at P7 in females, emerging earlier than males. On P7 and P14, both morphine-exposed female and male mice displayed hyperalgesia on the hot plate and tail flick assays, with females showing greater hyperalgesia than males. Morphine-exposed mice exhibited increased anxiety-like behavior in the open-field arena on P21. Transcriptome analysis of the brainstem, an area implicated in opioid withdrawal and NOWS, identified pathways enriched for noradrenergic signaling in females and males. We also found sex-specific pathways related to mitochondrial function and neurodevelopment in females and circadian entrainment in males. Sex-specific transcriptomic neuroadaptations implicate unique neurobiological mechanisms underlying NOWS-like behaviors. 3 SIGNIFICANCE STATEMENTNeonatal opioid withdrawal syndrome (NOWS) is a poorly understood condition that has both a genetic and environmental component and is thought to be mechanistically distinct from opioid withdrawal in adults. The development of murine models for measuring neurobehavioral responses is critical for informing the neurobiological adaptations underlying NOWS. Using outbred mice that more closely model human genetic variation, we discovered several sex differences in behavioral timing and severity of NOWS-model behaviors as well as transcriptomic adaptations in brain tissue that together suggest distinct mechanisms and sex-specific therapeutics for reversing withdrawal symptoms and restoring brain function.

Prefrontal Cortex Response to Prenatal Insult and Postnatal Opioid Exposure

“…Gene CDH1 participates in neurogenesis and cortical development and was under-expressed in the morphine-treated relative to the saline group ( Table 2 ). This pattern is aligned with reports of reduced expression in the brainstems of morphine-exposed female mice [ 57 ].…”

In utero exposure to morphine leads to sex-specific behavioral alterations that persist into adulthood in cross-fostered mice

“…Similar to our results, one study found that offspring from oxycodone-exposed dams have higher frequency USVs than control offspring (Zanni et al, 2021). Another study also found that neonate offspring injected with morphine from PND 1 -14 had increased USV frequency parameters (Borrelli et al, 2021).…”

Functional genomic mechanisms of opioid action and opioid use disorder: a systematic review of animal models and human studies

“…Seven articles investigated both sexes (n=7, 12%), using surprisingly low sample sizes (n=1-4 in male/female, and opioid-treated/control, groups; Fig.2D). Among these, 2 studies treated sex as a covariate [22,23], and 3 pooled both sexes for the analysis of opioid effects [24–26], therefore providing no direct description of male/female differences. This is concerning, considering that the 2 remaining studies found little overlap, from 5 to 35% (see [27] and [28]), among morphine-induced changes occurring in each sex.…”

“…Not surprisingly, a large majority focused on the mesocorticolimbic dopaminergic pathway: the NAc (21 articles [8,22,26,28–30,32,40–53]), frontal cortex (13 articles [8,25,27,29,51,54–61]), whole/unspecified striatal complex (11 articles [24,33,62–70]), dorsal striatum (7 articles [8,30,48–50,71,72]) and ventral tegmental area (VTA, 5 articles [28,51,73–75]). Other regions included the spinal cord (7 articles [62,76– 81]); hippocampus (5 articles [8,63,82–84]); amygdala (3 articles [8,85,86]); locus coeruleus [74, 87], ventral midbrain [42, 88], hypothalamus [89, 90], whole brain with [91] or without [92] cerebellum (1 each); periaqueductal gray matter [93], pituitary gland [90], arcuate nucleus [94], nucleus of the tractus solitarius [31], brainstem [23], cerebellum [84] or dorsal root ganglia [95] (DRG, 1 each). Below, we briefly describe the rationale for studying such diverse structures.…”