Early life outcomes of prenatal exposure to alcohol and synthetic cannabinoids in mice
Department of Neuroscience and Experimental Therapeutics, Texas A&M College of Medicine, 8447 John Sharp Parkway, Bryan, TX 77807, United States
Abstract
Purpose
This study investigated the effects of prenatal co-exposure to alcohol and synthetic cannabinoids on offspring viability, physical development, and neurobehavioral outcomes in young adulthood. The goal of this investigation was to determine whether prenatal co-exposure produced distinct outcomes from single-drug exposures, including sex-specific vulnerabilities in motor coordination and exploratory behaviors.
Methods
Pregnant C57Bl/6 J mice were randomly assigned to one of four treatment groups: drug-free controls, alcohol (ALC)-exposed, cannabinoid (CP-55,940, CB)-exposed or ALC+CB-exposed, with drug exposure occurring between Gestational Days 12–15. Offspring viability, physical malformations, and developmental delays were first assessed at birth. Then, behavioral evaluations, including rotarod and open field tests, were conducted on young adult offspring (Postnatal Days 100–120).
Results
ALC+CB exposure significantly decreased litter survival (p = 0.006) and offspring viability compared to controls. Non-viable offspring exhibited craniofacial abnormalities, limb malformations, and developmental delays. Assessments of rotarod performance revealed that all exposures reduced motor coordination in males compared to controls (p < 0.05), while ALC and CB exposures alone produced this outcome in females. Open field tests indicated that ALC+CB exposure reduced time in the center of the arena in male offspring exclusively, while this same exposure increased hyperactivity compared to single-drug and control groups, independent of sex (p < 0.05).
Conclusions
Prenatal co-exposure to alcohol and synthetic cannabinoids exacerbated offspring mortality and induced sex-specific deficits in neurobehavioral motor outcomes. These findings highlight the distinct risks of polysubstance exposure during pregnancy and underscore the need for targeted interventions to mitigate the effects of prenatal polysubstance exposure on offspring health outcomes.
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Keywords: Prenatal, Alcohol, Cannabinoid, Motor behavior, Sex differences, Survival
Highlights
- •Alcohol and cannabinoid co-exposure significantly reduces offspring survival.
- •Reduced survival is associated with offspring craniofacial and limb malformations.
- •Prenatal exposures produce sex-specific impairments in motor coordination.
- •Co-exposed offspring display hyperactivity in young adulthood.
Article notes
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Received 2025 Jan 25; Revised 2025 Jun 18; Accepted 2025 Jun 26; Collection date 2025 Sep.
1.Introduction
Alcohol and cannabis are two of the most used substances by individuals of child-bearing age (Rouzer et al., 2023, Subbaraman and Kerr, 2015), and next to nicotine, are the most frequently co-used substances in human populations (Singh, 2019). Moreover, co-use of alcohol and cannabinoids occurs most often among young adults of reproductive age (Subbaraman and Kerr, 2015), the age group that is also most likely to experience unplanned pregnancy (Brown and Eisenberg, 1995). However, rates of prenatal alcohol and cannabinoid co-exposure are difficult to quantify, due to the societal stigma surrounding alcohol and cannabis use by pregnant individuals (Corrigan et al., 2017, Raifman et al., 2024). Conservative estimates place the prevalence of prenatal alcohol exposure (PAE) at ~10 % (Popova et al., 2017), while prenatal cannabis exposure (PCE) is reported among 3–35 % of pregnancies (Nashed et al., 2021). Given that half of young adults who use cannabis also engage in alcohol co-use (Yurasek et al., 2017), alcohol and cannabinoid co-exposure is likely a prevalent form of prenatal exposure.
Importantly, independent exposures to either alcohol or cannabinoids have been associated with compromised fetal and maternal health at the time of delivery. In humans, confirmed exposure to either substance has been associated with increased risk for adverse neonatal outcomes and pregnancy complications, including pre-term delivery and infants born small for gestational age (Addila et al., 2021, Ayonrinde et al., 2021, Bailey and Sokol, 2011; Bandoli et al., 2023; Petrangelo et al., 2019; Popova et al., 2021). However, reports of prenatal and early-postnatal complications associated with individual exposures are not consistent across all clinical assessments (as previously described in Rouzer et al. (2024)), indicating that the relationship between prenatal drug exposure and offspring health risk is likely moderated by other contributing factors. Given the prevalence of co-use of alcohol and cannabinoids among young adults, we propose that one of these moderating factors may be the influence of prenatal polysubstance exposure.
At present, there is minimal research comparing offspring outcomes from alcohol and cannabinoid co-exposures to single-drug exposures. Within this under-investigated research area are several independent studies demonstrating that co-exposure can produce distinct and sex-specific outcomes in offspring, affecting fetal intrauterine blood flow (Rouzer et al., 2024), viability at birth and birth weight, behavioral and cognitive performance, and underlying neurological systems, including those associated with memory, endocannabinoid signaling, and the Sonic Hedgehog gene (reviewed in detail in Rouzer et al. (2023)). However, select studies have also reported that co-exposure does not always augment outcomes from singular exposures, instead mimicking the outcomes of individual exposures (Ornelas et al., 2024, Reid et al., 2024). These conflicting reports further reflect the need to investigate and identify the contexts under which combined exposure may increase risk for offspring neurodevelopmental impairments throughout the lifespan.
Our study appends this limited body of research using gestational exposures to alcohol and/or synthetic cannabinoid CP-55940 in mice, controlling for the timing and quantities of drug exposures across pregnancies. Following characterization of birth outcomes, offspring aged into young adulthood and were assessed for motor deficits, a well-characterized consequence of offspring exposure to alcohol (Bhatara et al., 2006; Breit et al., 2019a; Cheng et al., 2018; Connor et al., 2006; Lucas et al., 2014; Mick et al., 2002) or cannabinoids (Breit et al., 2019b; Campolongo et al., 2011; de Salas-Quiroga et al., 2015; Hussain et al., 2022; Scheyer et al., 2019; Shabani et al., 2011). Our investigation reveals prenatal cannabinoid-driven effects, including the loss of offspring viability, which is associated with the expression of craniofacial malformations, underdeveloped limbs and digits, and immaturity for gestational age. In early adulthood, prenatal co-exposure reduces motor coordination and time spent in the center of an open field seen only in male offspring, while increases in hyperactivity were sex-independent in this group. Notably, all exposures produce deficits in motor behaviors, highlighting that this developmental domain is commonly targeted by intrauterine alcohol and cannabinoid exposures.
2.Methods
2.1.Generation of prenatally-exposed offspring
All breeding and prenatal drug exposure procedures are described in detail in our previous publication using this co-exposure model (Rouzer et al., 2024) and were performed with the oversight and approval of Texas A&M University’s Institutional Animal Care Committee. Briefly, breeding protocols involved overnight co-housing of one male with two female C57Bl/6 J mice (Jackson Laboratories, ME, Strain #000664) and successful mating was confirmed by the presence of a sperm plug (Gestational Day 1). Following pregnancy confirmation, females were assigned to one of four groups: control (CON), alcohol-only (ALC), cannabinoid-only (CB), or both substances (ALC + CB). From Gestational Days (G) 12–15, females received daily intraperitoneal injections of either CP-55,940 (750 µg/kg in 10 % DMSO/saline, Tocris Bioscience) or volume-equivalent 10 % DMSO/saline. This was followed by a 30-minute exposure to either ethanol vapor (95 % ethanol) or room air using a passive e-vape system (La Jolla Alcohol Research Inc., La Jolla, CA). This window of exposure corresponds with a critical period of cerebral cortical plate neurogenesis and angiogenesis (Caviness, 1982, Madelaine et al., 2017), and specifically corticostriatal neuron development, which regulates motor control (Sohur et al., 2014). As previously reported, this model of alcohol exposure produced blood alcohol levels of ~150 mg/dL in ALC dams, which were slightly elevated by CB co-exposure (Rouzer et al., 2024). CP-55940 was injected immediately before the 30-min ethanol-vapor session to facilitate collection of tail-blood alcohol samples the moment animals left the chamber, eliminating any post-vapor injection delay. Administering the cannabinoid in advance both captured its potential modulation of ethanol pharmacokinetics and minimized additional handling of the pregnant dams. Dams were then visually assessed daily to verify their well-being during the prenatal exposure period leading up to delivery. On days of CP-55940 administration, dams did not display overt changes in posture, but some demonstrated temporary increases in twitching and mobility compared to vehicle-treated controls, suggesting minimal acute maternal distress. Any pregnant dams that exhibited greater distress or experienced delivery complications were euthanized according to approved institutional protocols (IACUC 2021–0331).
2.2.Parameters for characterizing deceased offspring features
As previously described in our prenatal characterization of this co-exposure model, we observed instances of maternal and fetal fatality following, but not preceding, drug exposures (Rouzer et al., 2024), particularly among CB and ALC+CB litters. To investigate the effects of prenatal drug exposure on periconceptional offspring mortality, six non-viable litters were analyzed following euthanasia of the dam according to institutional protocol. Deceased offspring were photographed and these were independently reviewed by four researchers, who generated individual assessments of physical abnormalities and quantified resorptions. Analyses were conducted using defined parameters across multiple anatomical and developmental categories (described in Table 1), and ratings of deceased offspring physical malformations were reviewed for inter-rater reliability using intraclass correlation coefficient (ICC) analysis. Developmental delays were assessed using four criteria: skull shape, snout shape, pinna placement, and digit formation. Each feature was assigned to a Theiler stage based on visual appearance (Richardson et al., 2013), independent of gestational age at dissection. The discrepancy between the estimated Theiler stage and the actual Theiler stage was calculated as follows: Difference = Estimated Theiler stage – Actual Theiler stage. For example, if the fetal snout was estimated at Theiler Stage 22, but dissected at Theiler Stage 23, it would receive a score of −1.
| Region of Analysis | Evaluation Criteria | Scoring |
|---|---|---|
| Eyes | Observations were based on iris morphology until the development of eyelids obscured the iris, after which these cases were marked as “X.” | 0: Typical/Circular iris −1: Atypical/Moderately non-circular iris −2: Minor coloboma −3: Severe coloboma |
| Limbs | Evaluated for length, attachment, and presence. | 0: Typical −1: Abnormal length (too short/long) −2: Abnormal attachment or fused −3: Absent |
| Abdominal Region | Classification based on abdominal bleeding and wall closure. | 0: Typical −1: Wall open with bleeding −2: Wall open with organs visible through skin |
| Edema | - | 0: No edema −1: Presence of edema |
| Color | - | 0: Pink/Typical −1: Dark red or purple −2: Blue or purple (indicative of prolonged hypoxia) |
| Hemorrhage | - | 0: No visible hemorrhaging −1: Visible hemorrhaging |
| Resorptions | The number of resorptions was quantified based on fetal sacs. | 0: None identified −1: Initial signs of resorption or necrosis present −2: Blood clot or tissue remnants present |
2.2.1.Data processing and reporting
After individual scoring, numerical data were averaged across reviewers and compiled into a single summary table. If a value could not be confidently determined from an image or fetus, reviewers provided an “X” for that metric. Variables marked as “X” by more than one reviewer were excluded from numerical scoring and retained as “X” in the final dataset. Due to the selective impact of exposures on litter viability, only certain exposure groups were represented, and no statistical analyses were performed.
2.3.Assessments of offspring rotarod coordination
In young adulthood (Postnatal Day [PD]100–120), male and female offspring from all exposure groups (4–9 litters/group, 2–3 offspring/sex/litter, depending on litter survival) underwent rotarod testing (Rotarod Series 8, IITC Life Science Inc.) over three consecutive days. Each day, animals acclimated for 10 min in a red-light illuminated behavioral testing room, where all sessions were conducted under red-light conditions. On Day 1, animals completed a Habituation task, in which they were required to balance on the rotarod at 5 rotations-per-minute (RPM) for 120 sec. Animals failing to meet this threshold were given a 10 min break before undergoing up to two additional attempts. The number of attempts required to meet this threshold was recorded for each subject, and successful subjects advanced to subsequent testing days, which employed a new testing protocol. Each testing day was identical, and consisted of three trials with a 10 min inter-trial interval. Immediately after placement on the Rotarod, rod speed increased from 4 to 40 RPM over 108 sec. If subjects fell, their time to fall was recorded, and subjects were returned to their homecage for an inter-trial resting period. If subjects remained balanced on the rod for 150 s, demonstrating 30 + s of balance at the top speed, the trial was terminated, the maximum time of 150 s was recorded, and the subject was returned to their homecage. All subjects had ad libitum access to food and water in their homecages throughout the testing period.
2.4.Assessments of offspring open field activity
Within a week of completing Rotarod testing, subjects were evaluated for locomotor activity and exploratory behavior using an open field test in a 50 cm× 50 cm arena with 50 cm high walls, under dim lighting conditions. After 10 min to acclimate to the mouse behavioral testing room, each subject was placed at the center of the arena and allowed to explore freely for 10 min. An overhead camera connected to EthoVision XT software (Noldus Information Technology, The Netherlands) automatically recorded and analyzed subject movements, and provided heatmaps of subject activity based on designated exposure/sex. The software tracked and provided numerical outputs for 1) time spent in the center of the open field arena, 2) times entering the center from the periphery, 3) total distance travelled during testing, and 4) average speed travelled during testing.
2.5.Statistics
Litter mortality was analyzed using a Mann-Whitney test for independent variables of prenatal ALC exposure (Yes/No) and CB exposure (Yes/No), followed by Dunn’s multiple comparisons post-hoc tests to determine between-group differences. Offspring weights were analyzed using a mixed-effects ANOVA with independent variables of ALC exposure, CB exposure, and Age. Assessments of physical malformations in deceased offspring were reviewed for inter-rater reliability using Interclass correlation (ICC) analysis, with ICC estimates and their 95 % confident intervals calculated based on a mean-rating (k = 4), absolute-agreement, 2-way random-effects model. In behavioral measures on the Rotarod and Open Field, main effects of Sex, ALC exposure, and CB exposure were first assessed using analysis of variance (ANOVA), Assessments of litter effects on behavioral assessments determined that litter did significantly affect open field measures (all p’s < 0.05), but not Rotarod measures (all p’s > 0.05), and importantly, these findings were consistent across all exposure groups (Supplementary Table 29). To control for potential litter effects, ‘litter membership’ was included in each ANOVA model as a co-variate. In the event of a significant interaction between sex and one form of exposure (ALC, CB, or ALCxCB), behavioral analyses were performed within-sex to parse out differences in exposure-induced outcomes in males and females, respectively. In Rotarod assessments, additional independent variables of Testing Day (Day 1/Day 2), and Trial # (Trial 1/Trial 2/Trial 3) were also included ANOVAs. In all statistical assessments where sex was not a statistically significant main effect or interacting variable, data were collapsed to perform follow-up assessments investigating sex-independent effects of prenatal exposures.
For all assessments, in the event of a significant main effect or interaction between independent variables, post-hoc Tukey tests were used to determine significant differences between individual groups. All data were assessed for outliers using the ROUT method of regression with a false-discovery rate of 1 %, and identified outliers were removed from statistical analyses. Group differences were considered significant at p ≤ 0.05. ICC analysis and main effects ANOVAs were performed using SPSS (v28, IBM, NY). All other statistical tests were performed using Prism (v9, GraphPad Software, MA).
3.Results
3.1.Prenatal polysubstance exposure decreases offspring viability and litter sizes
In postnatal characterization of our ALC and CB co-exposure model, we found a significant main effect of CB exposure on litter survival [U = 112, p = 0.007] and no significant effect of ALC exposure on litter survival [U = 146.5, p = 0.105]. Post-hoc analyses revealed that this effect was driven by the ALC+CB group, which demonstrated significantly lower litter survival (~33 %) than the control group (p = 0.011; Supplementary Table 1; Fig. 1A). Furthermore, among viable litters, CB exposure significantly impacted the number of live pups/births [F(1, 20) = 13.88, p = 0.001], with ALC+CB litters bearing fewer offspring than control litters (p = 0.067) and ALC litters (p = 0.027; Supplementary Table 2; Fig. 1B). Among surviving offspring, there was a trend, though statistically non-significant, for an interaction between ALC and CB exposure on litter sex ratios [F(1, 19) = 3.706, p = 0.069], with no significant post-hoc comparisons (Supplementary Table 3; Fig. 1C). It is worth noting that the lack of statistical significance may reflect an insufficient sample size due to the significant loss of offspring viability associated with ALC+CB exposure, and given that twice as many male offspring survived among these litters as female offspring.
3.2.Non-viable offspring demonstrate physical malformations, including impaired craniofacial development and inappropriate limb and organ development
Deceased offspring from six CB and ALC+CB litters exhibited distinct physical and developmental abnormalities (Table 2), with the severity of observable deficits recorded as deviation from age-matched, appropriately developed fetuses (scored as 0; Fig. 2A). An ICC analysis was conducted to assess the inter-rater reliability of the ratings across four independent observers. Using a two-way random-effects model for absolute agreement with average measures, the ICC for observed physical deficits was 0.951, 95 % CI [0.932, 0.965], and the ICC for estimated Theiler stage was 0.868, 95 % CI [0.789, 0.922], indicating good-to-excellent reliability in assessments of deceased fetal metrics (Koo and Li, 2016).
| Exposure- Fetus ID | Dissection GD | Expected Theiler Stage | Complications | Appearance | Skull Shape | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Eyes | Limbs | Abdomen | Edema | Color | Hemorrhage | Skull Age | Development (Expected - Observed TS) | |||
| CB Litter 1- A | G16 | 24 | −1 | 0 | −1 | 0 | −1 | −1 | TS 22.5 | −1.5 |
| CB Litter 2- A | G16 | 24 | −1 | −1 | −1.25 | 0 | 0 | −1 | TS 22.5 | −1.5 |
| CB Litter 2- B | −0 | −1 | −0.75 | 0 | 0 | −1 | TS 22.5 | −1.5 | ||
| CB Litter 2- C | −2 | −1 | −1 | −1 | 0 | −1 | TS 21.75 | −2.25 | ||
| CB Litter 2- E | −2 | −1 | −0.25 | −0 | 0 | −1 | TS 23.25 | −0.75 | ||
| CB Litter 2- F | −1 | −1 | −0.25 | −0 | 0 | −1 | TS 23 | −1 | ||
| CB Litter 2- G | −1 | −1 | −1.75 | −0 | 0 | −1 | TS 22.75 | −1.25 | ||
| ALC+CB Litter 1- A | G16 | 24 | −3 | −1 | −1.25 | −1 | −1 | −1 | TS 22.5 | −1.5 |
| ALC+CB Litter 1- B | −1 | −3 | −1.25 | −1 | −1 | −1 | TS 22.75 | −1.25 | ||
| ALC+CB Litter 2- A | G17 | 25 | −2 | −0 | −1 | −1 | −1 | −1 | TS 23.5 | −1.5 |
| CB Litter 3- A | G24 | 26/27 | X | −1 | 0 | −1 | −2 | 0 | N.A | |
| CB Litter 3- B | X | −1 | 0 | −0 | −2 | 0 | ||||
| CB Litter 3- C | X | X | X | −0 | −2 | 0 | ||||
| CB Litter 3- D | X | −2 | 0 | −1 | −2 | 0 | ||||
| ALC+CB Litter 3- A | G24 | 26/27 | X | 0 | 0 | 0 | −2 | 0 | N.A | |
Offspring from ALC+CB litters demonstrated greater overall severity in physical abnormalities compared to CB litters. In ALC+CB litters, iris abnormalities ranged from moderate non-circular shapes to severe colobomas (Fig. 2C), with complication scores spanning −0.75 to −3, while CB litters showed milder iris defects, with scores ranging from −0.25 to −1.75. Limb abnormalities followed a similar trend, with ALC+CB litters displaying abnormal limb length and phocomelia, while CB-only litters were primarily affected by mild shortening/asymmetry (Fig. 2B). Abdominal wall deficits, including visible organ exposure and bleeding, were more consistent in ALC+CB litters (-1.00 to −1.25), whereas CB litters exhibited a broader range of scores, including cases closer to normal development (-0.25 to −1.75; Fig. 2D). ALC+CB litters also displayed higher frequencies of fetal resorptions, with up to eight resorptions in advanced stages (-2), compared to an average of < 2 resorptions in CB-only litters.
ALC+CB litters consistently exhibited skin discoloration, ranging from dark red to blue/purple (“-2” color scores). Notably, litters born after their anticipated delivery date demonstrated the most severe discoloration, potentially indicative of prolonged hypoxic distress. Edema was also more prevalent in ALC+CB litters, with ¾ offspring demonstrating at least moderate levels of swelling, while CB offspring occasionally exhibited no edema. Hemorrhagic features were prominent in both groups and exclusive to offspring born prior to their anticipated delivery date. Developmental delays were also prominent among offspring assessed prior to their anticipated delivery date. Skull development scores were relatively consistent between CB and ALC+CB offspring, with all premature litters demonstrating an average development delay of ~1.5 Theiler stages. Notably, snout development was disrupted more by ALC+CB exposure, with offspring displaying underdeveloped snouts by ~3 Theiler stages, while CB offspring averaged a lag of ~2 Theiler stages. Pinnas were undetectable among ~50 % of all assessed offspring, independent of exposure, although this absence was exclusive to fetuses born prematurely. Finally, there were select cases of underdeveloped digit formation following either exposure, including two separate cases of incomplete hindlimb formation. All offspring assessed following their anticipated delivery date demonstrated appropriate pinna and digit development.
3.3.Prenatal drug exposure contributes to increased early-life offspring mortality
In addition to periconceptual offspring mortality, postnatal offspring death was also observed only in drug-exposed litters. In two ALC litters and one ALC+CB litter, all offspring were cannibalized between PD0–2, which also prevented quantification of sex ratios, which were determined at PD2. After PD2, two CB litters demonstrated early neonatal mortality. From one litter, one male offspring died between PD2–4, while in a separate litter, one male offspring died between PD2–21 and two female offspring died between PD21–25. Control litters experienced no offspring mortality.
3.4.Juvenile weight gains following prenatal polysubstance exposure are not sustained into adulthood
On P2, offspring weights did not differ between exposure groups, although ALC+CB offspring exhibited the highest average weights across all exposures (mean = 1.55 g; Supplementary Figure 1). Sex-segregated offspring weights were first collected at weaning on PD21, and subsequently in adolescence (PD40) and young adulthood (PD80), prior to behavioral testing. Weights were then averaged among male and female offspring, respectively, per litter. At PD21, there were statistically significant interactions for ALC x CB exposure for both male offspring [F(1, 18) = 10.85, p = 0.004] and female offspring [F(1, 19) = 7.576, p = 0.013], with ALC+CB offspring demonstrating the highest average juvenile weights among all exposure groups (Supplementary Tables 4&5; Supplementary Figure 1). Notably, ALC+CB offspring did not sustain their increased weights into PD40 or PD80, indicating that initial weight gain was not maintained during the transition into adolescence.
3.5.Males and CB-exposed offspring require more trials to learn a Rotarod motor coordination task
During the first day of Rotarod testing, male and female mice were trained to perform the Rotarod task during a Habituation day, in which offspring were tasked with balancing on the Rotarod for 120 s at a speed of 5 rotations-per-minute (RPM). Animals who failed to balance for this long on the first trial were given two more opportunities to meet criteria, separated by a ten-minute inter-trial rest period. The number of trials required to meet the 120 sec threshold were recorded, and only animals who met this threshold advanced to testing days. Out of all offspring tested, only one (ALC female) failed to meet the threshold; this subject was removed from further testing. On Habituation Day, both sex [F(1, 97) = 6.385, p = 0.013] and CB exposure [F(1, 97) = 4.720, p = 0.032], but not ALC exposure, affected the number of trials required to learn the Rotarod task: specifically, female offspring habituated to the task faster than males, independent of exposure, while CB-exposed offspring required more trials overall to learn the task than non-CB-exposed offspring (Supplementary Table 6; Fig. 3A).
3.6.In control animals, females demonstrate consistently longer balance on the Rotarod than males
When progressing onto Rotarod testing days, we uncovered a significant main effect of sex [F(1, 97) = 27.583, p < 0.001], and significant interactions between Sex x ALC exposure x Testing Day [F(1, 194) = 6.377, p = 0.013] and Sex x ALC exposure x Trial [F(2, 194) = 4.826, p = 0.009]. These sex differences were observable even among drug-free controls, where there were also significant main effects of trial number [F(2, 96) = 4.297, p = 0.016] and testing day [F(1, 96) = 6.672, p = 0.011] on rotarod performance (Fig. 3B; Supplementary Table 7). While both sexes demonstrated significant improvement in coordination on the rotarod within and between testing days, female offspring demonstrated better overall balance than males across six total trials. Given the interactions of sex with ALC exposure on time balanced on the rotarod, the effects of prenatal exposure on rotarod performance were subsequently assessed within-sex.
3.7.In male offspring, all prenatal exposures reduce coordination across Rotarod testing days
In male offspring, there were significant main effects of ALC exposure [F(1, 52) = 4.894, p = 0.031], Testing Day [F(1, 104) = 8.275, p = 0.006], and Trial # [F(2, 104) = 28.797, p < 0.001]. There were also non-significant interactions between ALC exposure x CB exposure [F(1, 52) = 3.071, p = 0.086] and ALC exposure x CB exposure x Testing Day [F(2, 104) = 2.476, p = 0.089]. In follow-up comparisons with control offspring, all groups of drug-exposed offspring demonstrated significantly reduced time balanced on the Rotarod: ALC: [F(1, 84) = 21.340, p < 0.001], CB [F(1,84) = 7.946, p = 0.006], and ALC+CB [F(1, 78) = 14.180, p < 0.001] (Fig. 3C). However, offspring rotarod balance was not further impaired by ALC+CB exposure compared to single-drug exposure: ALC [F(1,72) = 0.890, p = 0.349] or CB [F(1,72) = 0.246, p = 0.621]. Notably, within-group, only control males demonstrate significant main effects of Trial [F(2,45) = 3.613, p = 0.035] and Testing Day [F(1,45) = 9.553, p = 0.003], indicating consistent improvement in Rotarod coordination within each testing day and between testing days (Supplementary Table 8). In contrast, there were no significant main effects of Trial and Testing Day in any drug-exposed group (all p’s > 0.05; Supplementary Tables 9–11), indicating that prenatal drug exposure prevented improvement of performance on the rotarod across trials, both within and between testing days.
This lack of improvement was emphasized by assessments of the first and final trial times on the Rotarod task (Fig. 3D). There was a significant main effect of ALC exposure [F(1,52) = 5.105, p = 0.028] on Rotarod coordination, a significant interaction between ALC x CB exposure [F(1,52) = 5.919, p = 0.019], and a significant three-way interaction between CB exposure x ALC exposure x Trial [F(1,52) = 4.362, p = 0.042] (Supplementary Table 12). During the first trial, post-hoc analyses revealed that there were no significant differences in time balanced on the rod, between control offspring and all exposure groups (all p’s > 0.05; Supplementary Table 13). However, on the final trial, control offspring balanced significantly longer than ALC offspring (p = 0.003), CB offspring (p = 0.007), and ALC+CB offspring (p = 0.023). There were no significant differences between single-drug exposed offspring and polysubstance-exposed offspring during the first or last trials (all p’s > 0.05; Supplementary Table 13).
3.8.In female offspring, prenatal single-drug exposure, but not polysubstance-exposure, reduces coordination on a Rotarod task
In female offspring, there were significant main effects of Testing Day [F(1, 90) = 11.433, p = 0.002] and Trial # [F(2, 90) = 10.050, p < 0.001], as well as significant interactions between ALC exposure x CB exposure [F(1, 45) = 7.149, p = 0.010], ALC exposure x Testing Day [F(1, 90) = 4.730, p = 0.035], and ALC exposure x Trial # [F(2, 90) = 3.657, p = 0.042]. In follow-up comparisons with control offspring, ALC-exposed females [F(1, 84) = 12.850, p < 0.001] and CB-exposed females [F(1, 87) = 13.100, p < 0.001] experienced significantly reduced coordination on the Rotarod (Fig. 3E). However, Rotarod balance was not significantly affected by polysubstance exposure when compared to control offspring [F(1, 66) = 0.601, p = 0.441]. In contrast, time balanced on the Rotarod was significantly longer in ALC+CB female offspring when compared to ALC [F(1, 48) = 10.590, p = 0.002] and CB [F(1, 51) = 9.010, p = 0.004] offspring. Within-group, no exposure produced a significant main effect of Trial (all p’s > 0.05; Supplementary Tables 14–17), indicating that performance within-day was relatively consistent for all groups. There was a significant main effect of Testing Day in ALC [F(1, 33) = 5.499, p = 0.025] and ALC+CB offspring [F(1, 15) = 16.350, p = 0.001], with offspring improving in their rotarod coordination between Days 1 and Day 2 of Testing. In contrast, there was no significant effect of ‘Testing Day’ in Control [F(1, 50) = 1.586, p = 0.214] or CB offspring [F(1, 34) = 1.754, p = 0.194], indicating no significant improvement in rotarod balance between testing days.
When comparing female offspring performance on the first and final trials of the Rotarod task (Fig. 3F), there was a significant interaction between ALC x CB exposure [F(1,45) = 5.220, p = 0.027], a significant interaction between ALC exposure x Trial [F(1,43) = 6.996, p = 0.011], and a significant three-way interaction between CB exposure x ALC exposure x Trial [F(1,43) = 4.301, p = 0.044] (Supplementary Table 18). During the first trial, post-hoc analyses revealed a significant reduction in time balanced on the Rotarod in ALC offspring (p = 0.043) and a non-significant reduction in CB offspring (p = 0.073), with no effect on performance in ALC+CB offspring (p = 0.215). In contrast, there were no significant differences in time balanced on the rotarod between any exposure groups during the final trial (all p’s > 0.05; Supplementary Table 19).
3.9.Prenatal polysubstance exposure decreases time exploring the center of the open field in males, while increasing hyperactivity measures
Offspring were next assessed in an open field assay, in which subjects could freely explore the apparatus for ten minutes, with their activity recorded for sex and exposure-induced changes in exploratory behaviors. When assessing time spent in the center of the open field across exposures, there was a significant interaction between CB x ALC Exposure [F(1,55) = 4.136, p = 0.044] and Sex x ALC Exposure [F(1,99) = 7.725, p = 0.007] (Fig. 4A). In post-hoc assessments, ALC+CB male offspring spent significantly less time in the center of the open field compared to control (p = 0.048) and CB males (p = 0.010) (Supplementary Table 21), whereas no effect of exposure was observed in any female offspring (Supplementary Table 22; Fig. 4C). In assessments of entries into the center of the open field, there was a significant interaction between CB x ALC Exposure [F(1,98) = 10.590, p = 0.002], with no influence of sex as a main effect or interacting variable (Supplementary Table 23). Data were subsequently collapsed by sex to perform post-hoc comparisons of exposure groups. ALC+CB offspring demonstrated significantly more entries into the center of the open field compared to ALC offspring (p = 0.027) and CB offspring (p = 0.019), but not control offspring (p = 0.406) (Supplementary Table 24; Fig. 4B).
Two measures within the open field were assessed as metrics of hyperactivity: average speed travelled during the testing session, and total distance travelled for the length of the session. For average speed, there was a statistically significant interaction between ALC x CB Exposure [F(1,99) = 7.540, p = 0.007], and no effect of sex as a main effect or interacting variable (Supplementary Table 25). This finding was similarly observed in distance travelled, with only a significant interaction between CB x ALC Exposure [F(1,77) = 7.544, p = 0.007] (Supplementary Table 26). Data were subsequently collapsed by sex to perform post-hoc comparisons of exposure groups. ALC+CB offspring demonstrated higher average speeds while traveling through the open field compared to ALC offspring (p = 0.031) and CB offspring (p = 0.058), but not control offspring (p = 0.255) (Supplementary Table 27). Similarly, ALC+CB offspring travelled farther distances during the open field test than ALC offspring (p = 0.031) and CB offspring (p = 0.058), but not control offspring (p = 0.254) (Supplementary Table 28; Fig. 4D).
4.Discussion
Recent studies in human populations have associated self-reported prenatal cannabis exposure with increased risk of offspring mortality (Bandoli et al., 2023, Bandoli et al., 2023; Crosland et al., 2024; Shi et al., 2021; Varner et al., 2014), pre-term delivery (Crosland et al., 2024, Luke et al., 2022, Petrangelo et al., 2019, Shi et al., 2021), and Sudden Infant Death Syndrome (SIDS; (Scragg et al., 2001)). Our data indicate a strong association between exposure, perinatal mortality and preterm birth. Importantly, we also observed elevated mortality through the pre-pubertal period, an outcome that needs to be assessed in human populations as well. We also found that the impact of prenatal cannabinoid exposure on offspring mortality was further exacerbated by simultaneous alcohol exposure, resulting in fewer live births and reduced litter sizes. This is significant, since polysubstance use in the context of risky alcohol consumption is increasingly common. There are a few preclinical studies on the combined effects of alcohol and cannabinoids, and our results are largely consistent with those studies. For example, an early investigation showed that combined prenatal exposure resulted in significant fetotoxicity among mice and rats compared to single-drug-exposed and drug-free offspring (Abel, 1985). More recently, Breit and colleagues (2019a) demonstrated greater lifetime mortality in offspring following prenatal co-exposure to alcohol and CP55940 compared to single-drug-exposed offspring. Notably, alcohol exposure alone in our model did not significantly affect offspring viability or litter size.
Prenatal exposure to alcohol and cannabinoids, both individually and in combination, can also result in significant growth deficits and developmental anomalies. Prenatal alcohol is well-documented to cause persistent growth deficits (Carter et al., 2013, Day et al., 1989, Day et al., 1994, Pielage et al., 2023; Day et al., 2002). Similarly, prenatal cannabinoid exposure has also been associated with infant growth restriction (Crosland et al., 2024, Huizink, 2014, Natale et al., 2020). In co-exposure models, more severe growth deficits have sometimes been observed in co-exposed offspring compared to single-drug-exposed offspring (Breit and Thomas, 2019b, Breit and Thomas, 2019a), although other studies have not replicated these findings (Breit et al., 2020). Interestingly, in our study, no growth restrictions were observed in viable offspring subjected to prenatal substance exposure. Instead, ALC+CB offspring demonstrated initial weight gains by PD 21, possibly because of smaller litter sizes and increased access to maternal care among surviving offspring. It is notable that these weight gains were only statistically greater than single-drug exposed offspring, indicating that ALC+CB offspring do not demonstrate a size improvement over drug-free controls, even with access to more maternal resources. Importantly, this early advantage did not persist; by PD 40, the weights of ALC+CB offspring were comparable to those of all other exposure groups.
A lack of weight differences among drug-exposed offspring may reflect a survival bias, as no offspring born prematurely survived to term. Underdeveloped animals may have died, skewing our results towards the most resilient litters. This possibility is supported by our observation that non-viable offspring across six cannabinoid or dual-exposed litters exhibited malformations including hypodevelopment of the face, abdomen, and limbs. Independently, PAE is well-documented to produce craniofacial dysmorphologies in humans, including midfacial anomalies (Muggli et al., 2017, Suttie et al., 2013), and these outcomes have been replicated in animal models as well [see review: Petrelli et al. (2018)]. Prenatal cannabinoid exposure has similarly contributed to the development of microphthalmia, iridial colobomas, facial clefts, and holoprosencephaly (Gilbert et al., 2016), as well as limb reduction, musculoskeletal deficiencies, and cardiovascular issues (Reece and Hulse, 2019). Moreover, limited studies on co-exposure suggest synergistic effects, such as enhanced craniofacial defects (Boa-Amponsem et al., 2019, Fish et al., 2019). Notably, non-viable cannabinoid-exposed offspring exhibited evidence of intra-abdominal hemorrhage, and preterm fetuses exhibited gastroschisis or failure of abdominal wall closure. One potential cause is prenatal inflammation (Ernst et al., 2010), which is strongly associated with preterm birth and fetal hemorrhaging. Unfortunately, as our morphometric analyses were limited to litters with complete mortality, and therefore, predominantly those exposed to cannabinoids, we could not evaluate whether combined exposure exacerbates outcomes relative to single-drug exposure in this cohort.
Significant sex differences emerged when assessing motor coordination in prenatally exposed young adult offspring. Male CB-exposed offspring required significantly more trials to habituate to the Rotarod task on the first day, aligning with prior findings (Breit et al., 2022). Among control offspring, females consistently outperformed male siblings, balancing longer on the Rotarod, although both sexes improved across testing trials, which was also consistent with previous studies (Ashworth et al., 2015, McFadyen et al., 2003). Prenatal exposures also resulted in distinct sex-specific outcomes. In males, all exposure groups showed reduced balancing time on the Rotarod compared to unexposed controls, with the most pronounced deficits occurring in the final trial. In contrast, females exhibited impairments exclusively in single-drug exposure groups, with the most significant deficits observed during the first trial, suggesting that drug-exposed females displayed "catch-up" performance with repeated testing. These findings are consistent with previous studies on prenatal single-drug exposure, which report impairments in motor learning and coordination (Breit et al., 2019a; Breit et al., 2022; Connor et al., 2006; Reekes et al., 2016; Thomas et al., 2000). Interestingly, the deficits observed in ALC males resemble a study of six-month-old male mice, where moderate prenatal alcohol exposure did not affect initial performance but reduced improvement across testing days (Reekes et al., 2016). However, some studies of prenatal alcohol or cannabinoid exposure report no significant coordination impairments following single-drug exposure (Breit et al., 2019a; Breit et al., 2022; Heaton et al., 2022).
These inconsistent outcomes in coordination deficits extend to preclinical investigations of ALC+CB exposure. For example, in a parallel bar test, co-exposure to CP-55940 and alcohol amplified adolescent coordination deficits compared to single-drug exposure, particularly in female offspring (Breit et al., 2019a). However, the same research group found that alcohol co-exposure did not exacerbate THC-induced deficits, with THC alone driving reduced coordination (Breit et al., 2022). In our study, co-exposure did not augment outcomes from single-drug exposure. Co-exposed offspring generally exhibited impairments comparable to single-drug exposed groups, except for ALC+CB females, who performed similarly to controls. Notably, ALC+CB females also had the lowest survival rate in our cohort, and their strong performance may reflect survival bias. When embedding our findings among existing literature, variability in reported outcomes likely arises from differences in the type of cannabinoid used, dosage, route of administration, and timing of gestational exposure, among other experimental variables.
In contrast to coordination assessments, sex differences were minimal in open field activity, a validated tool for assessing hyperactivity and anxiety-like behaviors in rodents (Kraeuter et al., 2019). Control male offspring did spend more time in the field center than control females. However, no additional sex differences were observed. Furthermore, unlike coordination measures, single-drug exposures did not alter open field behaviors. However, ALC+CB exposure significantly reduced center time in males compared to controls and CB-only offspring. Notably, ALC+CB offspring entered the center more frequently than ALC or CB groups, but this likely reflects polysubstance-induced hyperactivity, as co-exposed offspring also exhibited significantly increased speed and distance travelled. Overall, both male and female ALC+CB offspring displayed a thigmotaxic behavioral phenotype, characterized by faster movement along the periphery of the open field, as well as numerous, rapid crossings of the center from the periphery. Notably, within our study, prenatal ALC or CB individually did not alter open field behaviors, but simultaneous exposure produced a phenotype of hyperactivity and anxiety-like behavior consistent with FASD. Our findings align with prior research demonstrating augmented hyperactivity following co-exposure (Boa-Amponsem et al., 2019, Breit and Thomas, 2019a, Breit and Thomas, 2019b, Ornelas et al., 2024). Collectively, these studies highlight hyperactivity as a robust phenotype of prenatal co-exposure, supporting the need for further research into the distinct and synergistic effects of co-exposures compared to single-drug exposures.
4.1.Limitations
Our study employed a controlled model of prenatal alcohol and cannabinoid co-exposure to investigate offspring outcomes, building on prior assessments of how this exposure paradigm influences intrauterine fetal blood flow (Rouzer et al., 2024). As previously addressed, while this model provides valuable insights, it has several inherent limitations. First, our use of vaporized ethanol exposure from G12–15 allowed for precise control of maternal blood alcohol concentrations and exposure duration across pregnancies. This standardization minimizes variability, but may introduce maternal stress, potentially confounding offspring outcomes. Similarly, cannabinoid exposure relied on intraperitoneal injections of the synthetic cannabinoid CP-55,940, rather than natural phytocannabinoids such as THC or CBD. While this method ensured consistent dosing and pharmacokinetics compared to inhalation or oral routes, it limits translational applicability. CP-55940 models the use of synthetic cannabinoids in human populations, but not the complex agonist profiles of naturally-occurring cannabis products, which can contain upwards of 100 bioactive phytocannabinoids (Hopp et al., 2019). The dose included in this research (0.75 mg/kg) falls within attainable levels in humans, mimicking high cannabis exposure, and well within ranges from other preclinical dose-response studies, which include i.p doses from 0.01 to 3 mg/kg (Hamamoto et al., 2007). Furthermore, synthetic cannabinoids are growing in popularity for recreational use, particularly among individuals with a history of cannabis use (Palamar et al., 2025), both for their increased potency compared to THC products, as well as for their ability to remain undetected in routine drug tests. Future studies should therefore explore a broader range of exposure methods, doses, and cannabinoid compositions to enhance the translational relevance of these findings. Finally, although CP-55940 was administered before the ethanol vapor session to ensure timely assessment of blood alcohol levels, we cannot exclude the possibility that this specific order (and the faster pharmacokinetics of an intraperitoneal cannabinoid relative to inhaled ethanol) amplified some outcomes. Front-loading CP-55940 may have heightened acute maternal or fetal stress responses, thereby contributing to increased perinatal mortality. Future counter-balanced designs in which the timing and order of the two drugs are systematically varied will be necessary to disentangle true exposure interactions from sequence-dependent effects.
A notable limitation of this model was the significant mortality observed in offspring following co-exposure to alcohol and cannabinoids. Mortality was disproportionately high in co-exposed litters, underscoring the severe teratogenic risks of combined exposure. However, this reduction in offspring survival reduced sample sizes for subsequent behavioral testing, possibly compromising statistical power. Moreover, physiological adaptations that contributed to offspring survival in the co-exposure group may have also introduced ‘survivor’ biases in behavioral performance. We additionally acknowledge that DMSO, used here at 10 % as a vehicle for CP‑55940, can exert teratogenic effects at higher concentrations (Ferm, 1966); however, the dose and brief exposure employed in this study fall below reported developmental toxicity thresholds, and all groups received identical DMSO handling to control for any potential influence. Finally, our analyses of physical malformations were conducted post hoc due to the marked mortality observed in cannabinoid-exposed and co-exposed groups. The ALC and Control group did not exhibit similar loss of viability, which limited direct comparisons across all exposure conditions. To address this, future studies should incorporate planned, age-matched dissections and morphometric assessments across all experimental groups. for more comprehensive evaluations of fetal abnormalities due to prenatal drug exposures.
5.Conclusions
In this study, we demonstrated that prenatal alcohol and synthetic cannabinoid co-exposure significantly compromised offspring viability, with higher rates of pre- and postnatal mortality compared to single-drug exposures or controls. Behavioral assessments in surviving offspring revealed distinct sex-specific effects on motor coordination and activity levels, with co-exposed males and females displaying differing patterns of impairment or resilience. Notably, co-exposure augmented hyperactivity and anxiety-like behaviors in offspring, consistent with phenotypes observed in human populations with prenatal drug exposure. Despite the wealth of knowledge regarding the effects of individual alcohol or cannabinoid exposures, polysubstance exposure remains underexplored during pregnancy. Further investigation is needed to elucidate the neurobiological mechanisms underlying these outcomes, as well as factors like intrauterine inflammation, and vascular dysregulation, that are similarly targeted by individual exposures [as detailed in Rouzer et al. (2023)]. Such research is essential for advancing our understanding of the long-term consequences of prenatal co-exposure, and developing targeted interventions to mitigate its effects on offspring health.
Ethics declarations
All animal procedures and experiments were approved by Texas A&M’s Institutional Animal Care Committee. All animal experimental protocols were carried out in accordance with relevant guidelines and regulations. Experimental methods are described in this manuscript in accordance with ARRIVE guidelines for the reporting of animal experiments.
Contributions
SKR and RCM contributed to the conception/design of this research, and SKR was responsible for the acquisition and data curation. SKR, MD, AG, and AB contributed to the analysis of collected data, as well as the original manuscript preparation and revision. All authors read and approved the final version of the manuscript.
Funding Sources
This study was funded by grants from the National Institute of Alcohol Abuse and Alcoholism: F32 AA029866 (SKR), L40 AA030427 (SKR), K12 GM154716 (SKR) & R01 AA028406 (RCM).
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Siara Rouzer reports financial support was provided by National Institute on Alcohol Abuse and Alcoholism. Rajesh Miranda reports financial support was provided by National Institute on Alcohol Abuse and Alcoholism. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to thank Dr. Karienn Montgomery for providing training in the use of behavioral equipment. Furthermore, the authors would like to acknowledge the Neuroscience & Experimental Therapeutics Neurobehavioral Core for providing the space and resources to perform the behavioral assessments included in this manuscript.
Footnotes
Footnote Group
Contributor Information
Siara K. Rouzer, Email: srouzer@tamu.edu.
McKay Domen, Email: mckaydomen@tamu.edu.
Aisley George, Email: aisleyg@tamu.edu.
Abigail Bowring, Email: abigail.bowring@tamu.edu.
Rajesh C. Miranda, Email: rmiranda@tamu.edu.
Appendix A.Supplementary material
References
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