Synaptic Structure and Transcriptomic Profiling of Reward and Sensory Brain Areas in Male Mice of Fentanyl Addiction

Abstract

Background: Opioid-based medications are powerful analgesics commonly prescribed for pain management, but they are also highly addictive. The over-prescription of opioids analgesics has triggered current opioid crisis, which now has expanded to heroin and illicit synthetic opioids like fentanyl and its analogues. The side effects of fentanyl abuse have been well recognized, yet the underlying molecular adaptations across brain regions upon fentanyl exposure remain elusive.

Methods: The transmission electron microscopy (TEM) and next-generation RNA-sequencing (RNA-seq) were used to investigate the ultrastructure synaptic alterations and transcriptional profiling changes of reward and sensory brain regions in mice after fentanyl exposure.

Results: The naloxone-precipitated acute withdrawal symptoms were observed in mice exposed to fentanyl. Results of TEM showed an increase in the number of synapses, widening of synaptic gaps, and thickening of postsynaptic density in the NAc of the fentanyl addiction mice, accompanied by obvious mitochondrial swelling. RNA-seq identified differentially expressed genes (DEGs) in prefrontal cortex of mice brains after fentanyl exposure, and the expression of some addiction-related genes such as Calm4, Cdh1, Drd1/2/3/4, F2rl2, Gabra6, Ht2cr, Oprk1 and Rxfp3 showed the most striking changes among experimental groups. KEGG enrichment analysis indicated that these DEGs were related to the development of addiction behavior, dopaminergic/GABAergic/serotonergic synapse, synapse assembly/synaptic plasticity/synaptic vesicle cycle, cAMP/MAPK signaling pathway, neuroactive ligand-receptor interactions. These transcriptomic changes may be correlated with the structural and behavioral changes observed in fentanyl-exposed mice.

Discussion: The findings of this study contribute to a better understanding of the molecular mechanism of addiction behavior, which is essential for the development of optimized therapy strategies for addicts.

Keywords: addiction; fentanyl; gene expression; synaptic plasticity; transcriptional profiles.

Figure 1

Synaptic ultrastructure of the nucleus accumbens of mice in SC and FA groups. (A) Schematic of areas within the nucleus accumbens core (AcbC; red squares) and shell (AcbSh; blue squares) analyzed at approximately Bregma 1.54 mm. (B) Representative TEM images of nucleus accumbens of SC and FA groups. Left, SC groups (×30,000, scale bars = 500 nm) and SC groups (×80,000, scale bars = 200 nm); Right, FA groups (×30,000, scale bars = 500 nm) and SC groups (×80,000, scale bars = 200 nm). The red arrows indicate synapses, while the green arrows indicate mitochondria.

Figure 2

Changes of the transcriptomic profiles in prefrontal cortex regions of mice in SC, FA and FA+NX groups. (A) the volcano plot of significant DEGs in the comparison of FA group vs SC group (fold change ≥ 2, P < 0.05); (B) the volcano plot of significant DEGs in the comparison of FA+NX group vs SC group (fold change ≥ 2, P < 0.05); (C) Bubble diagram of top 20 enriched GO terms for DEGs in the comparison of FA group vs SC group (P < 0.05); (D) Bubble diagram of top 20 enriched GO terms for DEGs in the comparison of FA+NX group vs SC group (P < 0.05); (E) Bubble diagram of top 20 enriched KEGG pathways for DEGs in the comparison of FA group vs.SC group (P < 0.05); (F) Bubble diagram of top 20 enriched KEGG pathways for DEGs in the comparison of FA+NX group vs SC group (P < 0.05).

Figure 3

Heatmap showing the significant correlations between the top 50 DEGs and synaptic changes. Spearman’s rho values are signed and color-coded according to their respective status: Red blocks indicate the positive correlation and the blue blocks indicate the negative correlation. The intensity of the color reflects the strength of the correlation, with darker shades indicating stronger associations. Significant correlation are denoted by *P < 0.05 or **P < 0.01.

Figure 4

Comparison of the relative expression levels of genes potentially associated with fentanyl addiction behavior in SC, FA and FA+NX groups by qRT-PCR. Genes were randomly selected, and their expression levels in SC group were set as 1, while their expression abundance in FA and FA+NX groups were relatively quantified to the SC group. GAPDH gene was used as the internal reference gene, and the results were mean ± standard deviation (SD) (n=3). Significant fold changes in expression levels between different groups were denoted by **P < 0.01 and *P < 0.05, whereas no significant changes were represent as ns.