The effects of buprenorphine and morphine during pregnancy: Impact of exposure length on maternal brain, behavior, and offspring neurodevelopment

Abstract

The escalating incidence of opioid-related issues among pregnant women in the United States underscores the critical necessity to understand the effects of opioid use and Medication for Opioid Use Disorders (MOUDs) during pregnancy. This research employed a translational rodent model to examine the impact of gestational exposure to buprenorphine (BUP) or morphine on maternal behaviors and offspring well-being. Female rats received BUP or morphine before conception, representing established use, with exposure continuing until postnatal day 2 or discontinued on gestational day 19 to mimic treatment cessation before birth. Maternal behaviors - including care, pup retrieval, and preference - as well as hunting behaviors and brain neurotransmitter levels were assessed. Offspring were evaluated for mortality, weight, length, milk bands, surface righting latency, withdrawal symptoms, and brain neurotransmitter levels. Our results reveal that regardless of exposure length (i.e., continued or discontinued), BUP resulted in reduced maternal care in contrast to morphine-exposed and control dams. Opioid exposure altered brain monoamine levels in the dams and offspring, and was associated with increased neonatal mortality, reduced offspring weight, and elevated withdrawal symptoms compared to controls. These findings underscore BUP's potential disruption of maternal care, contributing to increased pup mortality and altered neurodevelopmental outcomes in the offspring. This study calls for more comprehensive research into prenatal BUP exposure effects on the maternal brain and infant development with the aim to mitigate adverse outcomes in humans exposed to opioids during pregnancy.

Keywords: Early life adversity; Gestation; Maternal care; Medication for opioid use disorders (MOUD); Opioids.

Fig 1.. Experimental timeline from Preconception (PC) through Postnatal Day (PND) 2.

Female rats received either vehicle (VEH), buprenorphine (BC, BD) or morphine (MC, MD) until Gestational day (GD) 19 (discontinued (D)) or until PND 2 (continued (C)). Rats underwent a series of behavioral tests during preconception and the postpartum. Rx: drug treatment; ↑Rx: increase drug dose for morphine; Sac: sacrifice.

Fig 2:. Overview of body weight changes across all groups from preconception (PC) to the postpartum (PND).

For a more detailed graph of each time period with all significant differences, please see Supplementary Figure S2. Only significant differences between each opioid group (i.e. BC(*), BD(#), MC($), MD (@) and VEH are indicated in this graph. There was no significant difference between treatment exposures before conception. During gestation, BC and BD dams weighed less than morphine dams on GD0, 7 and 14 and BUP dams weighed less than VEH dams on GD14 and 19. During late gestation (i.e. GD 20 and 21) BC, BD, and MD dams weighed less than MC and VEH dams. During the postpartum period (i.e. PND0-2) BC and BD weighed less than VEH dams on all days, while MC weighed less than VEH dams on PND1-2, MD weighed less than VEH dams on PND1, and BD weighed less than MD on PND1-2 (For more details see supplemental Fig S2D). Data are shown as mean ± SEM. *(BC), #(BD), $(MC), @(MD) <0.05 vs VEH, respectively. BC: buprenorphine continued, BD: buprenorphine discontinued, MC: morphine continued, MD: morphine discontinued, VEH: vehicle (saline), PC: Preconception, GD: Gestational Day, PND: Postnatal Day.

Figure 3:. Maternal behaviors and physiology.

A: Days to conceive. Shown are the effects of BUP, morphine and vehicle on days to conceive in milliparous female rats (reflected by the number of days between first mating exposure and positive spenn identification). There was a significant effect of Opioid (collapsed across treatment groups) demonstrating opioid exposure delayed time to conception. B: Serum levels of corticosterone (CORE, ng/ml) were significantly decreased in BUP dams on gestational day (GD) 7 compared to VEH dams (*p < 0.05) and morphine dams (#p < 0.05). C: Placentophagia was assessed by the total number of placentas the dam did not consume. BD dams consumed fewer placentas after birth than VEH dams (*p < 0.05). D: Pup directed maternal behavior (presented as percent of time the dam spent in the specified behavior) on postnatal day (PND) 0, 1 and 2. On PND1, BUP (BC, BD) dams spent significantly less time in pup-directed behaviors compared to VEH dams (*p < 0.05). There were no significant differences on PND0 or PND2. E: Nesting behavior was coded ordinally (See Suppl. Fig S1) every day from GD20-PND2. On GD20. BC & BD dam nest scores were lower compared to VEH (*), MC (#), and MD ($) scores. Similarly, MC & MD were also lower compared to VEH dams(*). BC and BD continued to display less evolved nests compared to VEH (*) until PND2 and compared to MC and MD at least until PND 0 (#,$) (*,#,$ p < 0.05). All data are shown as mean ± SEM and as individual data points. BUP: buprenorphine, BC: buprenorphine continued, BD: buprenorphine discontinued, MC: morphine continued, MD: morphine discontinued, VEH: vehicle (saline), GD: Gestational Day, PND: Postnatal Day.

Figure 4:. Pup retrieval.

Pup Retrieval Latency was scored as the time (in seconds) it took to retrieve the first pup (A) and the last pup (cut off time: 900-sec represented as the dotted line; B) back to the nest. BD dams took longer than VEH dams to retrieve the first and last pup (*p < 0.05). C: Pup Retrieval success was scored as a percent success rate (number of pups returned to the nest / total of pups included). There was a significant effect of drug with BUP dams retrieving fewer pups than VEH dams (*p < 0.05). Data are shown as mean ± SEM and each dam’s individual score. BLIP: buprenorphine, BC: buprenorphine continued, BD: buprenorphine discontinued, MC: morphine continued, MD: morphine discontinued, VEH: vehicle (saline).

Figure 5:. Monoamine and their metabolites concentrations in the PAG, VTA and NAc.

A: In the PAG, Morphine and BUP treatment were associated with reduced DOPAC and DOPAC turnover as compared to VEH dams (*). Morphine treatment was also associated with reduced levels of HIAA. B: In the VTA, MC treatment was associated with reduced levels of NE as compared to BD treatment (# p < 0.05). Morphine treatment was also associated with reduced levels of NE and HIAA as compared to BUP and VEH dams respectively. C: In the NAc, BD treatment was associated with reduced levels of HIAA as compared to VEII treatment (*p < 0.05). There were no significant effects of treatment in the medial preoptic area (data not shown). Data are shown as mean ± SEM plus each dam’s individual data point. PAG: periaqueductal gray, VTA: ventral tegmental area, NAcc: Nucleus Accumbens. DA: dopamine, 5-HT: serotonin, NE: norepinephrine, DOPAC: 3,4-Dihydroxyphenylacetic Acid, HVA: Homovanillic Acid, 3-MT: 3-Methoxytyramme, HIAA: 5-Hydroxymdoleacetic Acid, BC: buprenorphine continued, BD: buprenorphine discontinued, MC: morphine continued, MD: morphine discontinued, VEH: vehicle (saline).

Figure 6:. Offspring Mortality and physiology.

A: Mean number of deceased pups from PND0-PND2, Kruskal Wallis Test revealed a significant effect of Opioid Type on Total Mortality, with * BUP pups having a higher rate of morality than VEH (*p < 0.05). B: Presence of milk bands on PND2. BD pups had significantly less milk bands than VEH (*p < 0.05).C: Body weight on PND1 and PND2: BC, BD and MC pups weighed significantly less than VEH (*) and MD (#) pups on PND1 and 2 (*,# p < 0.05). D: Body length on PND1 and PND2. BD body length was significantly smaller than VEH on PND2 (*p < 0.05). Data are shown as mean ± SEM for each litter’s individual data point. PND: Postnatal Day, BUP: buprenorphine, BC: buprenorphine continued, BD: buprenorphine discontinued, MC: morphine continued, MD: morphine discontinued, VEH: vehicle (saline).

Figure 7:. Offspring Behavioral Tests.

A: Surface Righting Reflex. There was no treatment effect on the time it took for pups to right themselves, hut females took significantly longer to right themselves than males (B; *p < 0.05). Further, pups bom to dams that discontinued opioid treatment took significantly longer to right themselves as compared to pups from dams that continued their opioid treatment (C; *p < 0.05). D: Neonatal Opioid Withdrawal Symptoms (NOWS) scores. BC, BD, and MC exhibited significantly more withdrawal behaviors than VEII (*p < 0.05). There was also a significant sex effect (E), with males showing more withdrawal symptoms than females (*p < 0.05). Data are shown as mean ± SEM plus each pup’s or litter’s individual data point. BC: buprenorphine continued, BD: buprenorphine discontinued, MC: morphine continued, MD: morphine discontinued, VEH: vehicle (saline).

Figure 8:. DOPAC levels in the Prefrontal cortex (PFC) of neonatal offspring.

BC, BD and MC offspring exhibited lower DOPAC levels in the PFC on postnatal day (PND) 2 compared to VEH offspring (*p < 0.05). All other neurotransmitters and metabolites were not significantly different. BC: buprenorphine continued, BD: buprenorphine discontinued, MC: morphine continued, MD: morphine discontinued, VEH: vehicle (saline).