Decreased myelin-related gene expression in the nucleus accumbens during spontaneous neonatal opioid withdrawal in the absence of long-term behavioral effects in adult outbred CFW mice

Abstract

Prenatal opioid exposure is a major health concern in the United States, with the incidence of neonatal opioid withdrawal syndrome (NOWS) escalating in recent years. NOWS occurs upon cessation of in utero opioid exposure and is characterized by increased irritability, disrupted sleep patterns, high-pitched crying, and dysregulated feeding. The main pharmacological strategy for alleviating symptoms is treatment with replacement opioids. The neural mechanisms mediating NOWS and the long-term neurobehavioral effects are poorly understood. We used a third trimester-approximate model in which neonatal outbred pups (Carworth Farms White; CFW) were administered once-daily morphine (15 mg/kg, s.c.) from postnatal day (P) day 1 through P14 and were then assessed for behavioral and transcriptomic adaptations within the nucleus accumbens (NAc) on P15. We also investigated the long-term effects of perinatal morphine exposure on adult learning and reward sensitivity. We observed significant weight deficits, spontaneous thermal hyperalgesia, and altered ultrasonic vocalization (USV) profiles following repeated morphine and during spontaneous withdrawal. Transcriptome analysis of NAc from opioid-withdrawn P15 neonates via bulk mRNA sequencing identified an enrichment profile consistent with downregulation of myelin-associated transcripts. Despite the neonatal behavioral and molecular effects, there were no significant long-term effects of perinatal morphine exposure on adult spatial memory function in the Barnes Maze, emotional learning in fear conditioning, or in baseline or methamphetamine-potentiated reward sensitivity as measured via intracranial self-stimulation. Thus, the once daily third trimester-approximate exposure regimen, while inducing NOWS model traits and significant transcriptomic effects in neonates, had no significant long-term effects on adult behaviors.

Highlights: We replicated some NOWS model traits via 1x-daily morphine (P1-P14).We found a downregulation of myelination genes in nucleus accumbens on P15.There were no effects on learning/memory or reward sensitivity in adults.

Figure 1.. Reduced body weight from P2 through P14 following once daily morphine exposure (15 mg/kg; s.c.).

A. Schematic representing the split-litter design (top) and timeline of morphine administration, tissue collection, and behavioral phenotyping. B. Asterisks indicate differences between morphine and saline groups (sex-collapsed; n.s. = not significant, p < 0.01 = *, p < 0.001 = ***). All p-values reflect Bonferroni adjustment for multiple comparisons across days. Data are plotted as the mean ± SEM. Sample sizes by Treatment and Sex are listed next to the figure legend.

Figure 2:. Thermal hyperalgesia in the hot plate and tail withdrawal assays during spontaneous withdrawal on P7 and P14 following once daily morphine exposure (15 mg/kg, s.c.) from P1-P14.

Data are plotted as the mean ± SEM. A. Hot plate latencies: Main effects of Day (***p < 0.001) and Morphine Treatment (***p < 0.001), but no interactions. Morphine treated pups showed decreased latencies on P7 and P14 (*Tukey’s p < 0.05). P7 SAL: n = 123 (51 females, 72 males); SAL P14: n = 123 (51 females, 72 males); MOR P7: n = 92 (48 females, 44 males); MOR P14, n = 92 (48 females, 44 males). B. Locomotor velocities (cm/s) on the hot plate prior to nociceptive responses: No effect of Morphine Treatment but an effect of Day (p < 0.001). SAL P7 – P14: n = 60 (24 females, 36); MOR P7 – P14: n = 46 (27 females, 19 males). C. Tail withdrawal latencies: Effect of Morphine Treatment (***p < 0.001), Day (***p < 0.001), and interaction (**p = 0.004). Reduced latencies were observed in morphine treated pups on both P7 and P14 (*Tukeys; p < 0.05). SAL P7: n = 123 (51 females, 72 males); SAL P14: n = 119 (49 females, 70 males); MOR P7 – P14: n = 92 (48 females, 44 males).

Figure 3.. USV emissions and features during spontaneous withdrawal on P7 and P14 following once daily morphine exposure (15 mg/kg, s.c.) from P1-P14.

Data are plotted as the mean ± SEM. A-B. P7, P14 total USVs: There were no significant main effects of Morphine Treatment and Sex and no interactions. SAL P7: n = 77 (31 females, 46 males); SAL P14: n = 64 (25 females, 39 males); MOR P7: n = 59 (37 females, 22 males); MOR P14: n = 48 (30 females, 18 males). C. P7 peak frequency: No significant main effects nor interactions. D. P14 peak frequency: Main effect of Morphine Treatment (*p = 0.02; MOR > SAL) but no effect of Sex and no interaction. E. P7 peak amplitude: No effect of Morphine Treatment, Sex, or interaction. F. P14 peak amplitude: Main effect of Morphine Treatment (***p < 0.001; MOR < SAL) but no effect of Sex and nor interaction. SAL P7 – P14: n = 56 (22 females, 34 males); MOR P7 – P14: n = 43 (25 females, 18 males). G. Normalized P7 USVs per min: Main effect of Time (***p < 0.001) and a Morphine Treatment × Time interaction (*p = 0.047). Morphine treated pups emitted a greater number of USVs than saline treated pups during the second minute of testing (*p_adjusted = 0.03). H. Normalized P14 USVs per min: Main effect of Time (***p < 0.001) and a Sex × Time interaction (*p = 0.04). SAL P7: n = 77 (31 females, 46 males); SAL P14: n = 64 (25 females, 39 males); MOR P7: n = 59 (37 females, 22 males); MOR P14: n = 48 (30 females, 18 males).

Figure 4.. Locomotor activity during spontaneous withdrawal during USV recordings on P7 and P14.

Data are plotted as the mean ± SEM. A. P7 distance: Main effect of Morphine Treatment (***p < 0.001) but no effect of Sex and no interaction. SAL P7, n = 57 (24 females, 33 males); MOR P7, n = 46 (29 females, 17 males). B. P14 distance: No main effects or interactions. SAL P14: n = 65 (24 females, 41 males); MOR P14: n = 50 (30 females, 20 males).

Figure 5.. Bulk RNA-seq analysis in the nucleus accumbens (NAc) during spontaneous opioid withdrawal on P15 revealed significant down-regulation of myelin- and oligodendrocyte-related genes.

A. Volcano plot of gene expression in morphine mice [n = 14; 6 females, 8 males)] relative to saline mice [n = 14 (6 females, 8 males)]. The 13 genes with log₂FC ≤ −0.26 and adjusted p values < 0.05 are labeled. Genes (n = 90) with log₂FC ≤ −0.26 and unadjusted p < 0.01 were used as input for gene enrichment and network analysis. B. The largest subnetwork plot of significantly downregulated genes following neonatal morphine exposure is shown. Node color depicts gene log₂FC from −0.3 (purple) to −0.8 (green) relative to saline controls. Surrounding donut plots show the top 8 significantly enriched GO pathways. Connecting lines represent gene interactions, with stronger interactions depicted as thicker lines. See Supplementary Table 1 for the full DEG list with unadjusted p < 0.01, and Supplementary Table 2 for a list of significantly enriched GO Biological Process pathways associated with morphine treatment,

Figure 6.. No change in adult spatial learning as measured via the Barnes assay following once daily morphine exposure (15 mg/kg, s.c.) from P1-P14.

Data are plotted as the mean ± SEM. A-D. % time in the target zone, primary latency to identify the target hole, mean distance to the target hole, and entries to the target zone. No effect of Morphine Treatment, Sex, or interactions SAL: n = 28 (14 females, 14 males); MOR: n = 28 (16 females, 12 males).

Figure 7.. No change in on adult conditioned freezing behavior following daily morphine exposure (15 mg/kg, s.c.) from P1-P14.

Arrows in panel A indicate onset of the tone & subsequent foot-shock. In panel C, arrows indicate presentation of the conditioned stimulus (tone). A-C. No effect of Morphine Treatment or interactions with Day on % time freezing on Day 1, Day 2, or Day 3 of the fear conditioning tests. Main effect of time: (panel A). Main effect of time (panel B). Main effect of Day (panel C). Data are plotted as the mean ± SEM. SAL, n = 30 (14 females, 16 males); MOR, n = 30 (18 females, 12 males).

Figure 8.. No changes in adult methamphetamine potentiation of brain stimulation reward as measured via intracranial self-stimulation (ICSS) following once daily morphine (15 mg/kg, s.c.) from P1-P14. A-D.

Total rewards delivered, total rewards delivered as % vehicle, total responses for stimulation, total responses for stimulation as % vehicle, respectively. No effect of Morphine Treatment or interactions with Methamphetamine Dose for any of these measures. There was a significant effect of Methamphetamine Dose on total rewards (A: ***p = 0.001) and total responses (C: **p = 0.007), indicating methamphetamine-induced potentiation of brain stimulation reward. Data are plotted as the mean ± SEM. Sample sizes are shown in panel A next to the figure legend. Sample size by Sex: SAL: n = 10 (6 females, 4 males); MOR: n = 10 (7 females, 3 males).