Development disturbed: sex-specific transcriptional alterations in adolescent mice after perinatal fentanyl exposure

The opioid crisis has escalated over the last decade and during this decade potent synthetic opioids such as fentanyl became prevalent in the illicit drug market. Opioid use often continues during pregnancy. However, the long-term effects of fentanyl on offspring are unknown.

In this issue of Neuropsychopharmacology, Olusakin et al. [1] address this question by examining the effect of perinatal fentanyl exposure on the transcriptional landscape within three reward and two sensory brain regions of male and female juvenile mice. They used several complementary approaches and analyzes to show large scale sex-specific changes in gene expression within both reward and sensory-related brain regions of juvenile mice after perinatal fentanyl exposure. Furthermore, differential analysis suggests that multiple processes, including mitochondrial respiration, are disturbed in multiple brain regions.

Developing preclinical models to test the impact of prenatal drug exposure requires careful consideration due to neurodevelopmental differences between humans and rodents. The rodent brain at birth more closely matches the human brain during the second trimester of human gestation. If the experimental goal is to match the human neurodevelopmental trajectory, drug exposure in rodents should be perinatal, meaning exposure should be start at embryonic day 0 and last through weaning (postnatal day 21 in mice). In a previous study by the same group [2], they developed such a model of fentanyl exposure via drinking water. They found that mice exposed to fentanyl had low birth rates, mirroring what is observed in humans. While measures of maternal behavior were not different between fentanyl exposed and control dams, fentanyl exposed mice exhibited somatic withdrawal symptoms after weaning. In adolescence (postnatal day 35), there were sex-specific differences in anxiety-like behaviors. In adulthood (postnatal day 55), fentanyl-exposed mice exhibited impaired discrimination in a two-tone auditory discrimination task. Together, this previous study [2] established the preclinical model and demonstrated lasting effects of perinatal fentanyl exposure.

The current study by Olusakin et al. [1] builds on this by conducting a multi-region, unbiased transcriptomic screen of the adolescent brain after perinatal exposure in male and female mice from the previous study. On postnatal day 35 after the perinatal fentanyl exposure procedure, bulk tissue punches were taken from multiple brain regions of female and male mice. Regions isolated included three reward-related regions (ventral tegmental area [VTA], nucleus accumbens [NAc], and prelimbic cortex [PrL]) and two sensory-related regions (primary somatosensory cortex [S1] and ventrobasal thalamus [VBT]).

After tissue isolation and RNA-sequencing, the authors analyzed the data using multiple, complementary methods to address different aspects of the impact of perinatal fentanyl exposure on the developing mouse brain. First, they performed differential gene expression analysis with a multiple comparison correction threshold. Surprisingly, the authors found very few differentially expressed genes (DEGs) in PrL and S1 that only occurred in only females. The VTA and NAc had the largest changes in gene expression (in both sexes), and gene ontology analysis of DEGs revealed that multiple processes are likely affected, including neuronal migration, synaptic function, and mitochondrial function. This is an intriguing result and agrees with a previous study by the same group [3] showing that perinatal fentanyl exposure affects mitochondrial copy number in blood cells and mitochondria-related gene expression in the NAc, which is an aspect reflected in clinical populations.

Comparing DEG lists between groups (such as across sexes) with traditional differential expression analysis that requires a threshold setpoint can be vulnerable to false negatives. To circumvent this issue, the authors performed a rank-rank hypergeometric overlap analysis (a threshold free method) to look at gene set concordance. This analysis revealed that across sexes, the transcriptional signatures of perinatal fentanyl exposure is concordant in the PrL, VTA, and S1, while the NAc and S1 are discordant. The authors then used weighted gene co-expression network analysis to understand gene expression programs or networks and found converging ontology results. Together, this study maps region and sex-specific changes and provides a novel resource for studying perinatal opioid exposure.

Multiple lines of investigation are possible based on the hypotheses generated from this dataset and previous studies by the group. A previous study from the group found that fentanyl-exposed offspring have impaired stimuli adaptation and found functional impairment in S1 cortex [4], in line with clinical data that in utero opioid exposure in humans is associated with somatosensory dysfunction through adolescence. Mechanistic studies that bi-directionally modulate gene expression within S1 cortex could help understand if and how the gene expression changes observed here might underly functional deficits observed in the previous study. In a separate study, the group found that environmental enrichment could reverse these functional deficits [5], but it remains unknown if fentanyl-induced molecular changes can be prevented or reversed by implementing this practical intervention in early development.

Additionally, while neuronal-specific processes were identified in some of the analyses, it is currently unknown whether these effects are region-wide or involve specific cell-type/circuits in or across these regions. Follow up studies combining single cell methods and circuit-specific genetic labeling strategies could help further isolate the role of specific cell types and circuits following perinatal fentanyl exposure.

Overall, the study of Olusakin et al. [1] is novel and important given the continuing opioid epidemic, and prevalence of fentanyl misuse. The datasets generated (and accompanying analysis scripts) can serve as the foundation for follow-up studies to identify molecular, cellular, and circuit-level mechanisms of both short- and long-term effects of perinatal fentanyl exposure on the brain and body. This unbiased, broad screen expands our understanding of the transcriptional changes induced by fetal opioid exposure.

Competing interests

The author declares no competing interests.