The present study investigated the impact of combined prenatal exposure to EtOH and THC, delivered via an e-cigarette, on spatial and working memory. The results revealed sex-specific effects of THC and EtOH exposure on cognitive and emotional domains. Cognitively, prenatal THC exposure adversely impacted spatial learning and memory in female offspring, whereas prenatal EtOH exposure impaired working memory. Notably, the combination of prenatal exposure to THC and EtOH did not intensify the cognitive effects of either substance.

Surprisingly, our findings showed that ethanol exposure during gestation did not produce deficits in spatial learning and memory, in contrast to other preclinical studies using prenatal models (Patten et al., 2014; Brancato et al., 2020; Aglawe et al., 2021; Mahdinia et al., 2021) and 3rd trimester equivalent models (Thomas et al., 2007; Ryan et al., 2008; Breit et al., 2019b). Variation in outcome may be related to route of administration and/or developmental timing. However, our failure to find effects may be related to the lower peak BACs compared to previous studies. Specifically, spatial learning deficits are more often observed when peak BACs are over 200 mg/dl, whereas the peak BACs in the current study were around 150 mg/dl (albeit see below).

Clinical studies have shown that prenatal alcohol exposure can have dose-dependent negative impacts on learning, memory, and academic attainment in children and adults. For example, frequent episodes of binge drinking during the first and second trimester predict learning and memory deficits, including deficits in spatial memory, in children (Richardson et al., 2002). A recent study suggests that heavy, but not moderate, prenatal EtOH exposure impairs spatial learning and memory (Dodge et al., 2019). Importantly, in the Dodge et al. (2019) study, there was a confound of age, as the moderately exposed group was older, so there may be a developmental delay in performance that was missed in the moderately exposed group. Nevertheless, the amount of alcohol consumed per occasion was related to spatial navigation; thus, the absence of prenatal alcohol effect in the current study could be attributed to the failure to attain a threshold BAC.

In contrast, prenatal EtOH did impair spatial working memory. Females exposed to prenatal EtOH showed memory impairments during the 0-s ITI sessions, whereas males showed only transient impairments during the early stages of learning, as evidenced by longer path lengths and larger heading angles on the initial days of testing. Previous preclinical studies have also reported that prenatal alcohol exposure impairs spatial working memory (Schambra et al., 2017; Lucia et al., 2019; Waddell et al., 2020; Gursky et al., 2021), even at low to moderate ethanol doses (Schambra et al., 2017; Waddell et al., 2020) and across different gestational periods (Schambra et al., 2017; Lucia et al., 2019; Waddell et al., 2020; Gursky et al., 2021). Similar working memory deficits have been reported in clinical populations (Kodituwakku et al., 1995; Moore et al., 2021; Chetty-Mhlanga et al., 2022). This deficit is likely related to dysfunction of the prefrontal cortex (Waddell et al., 2020; Gursky et al., 2021), and our data suggest that the alcohol levels achieved in the current study may disrupt development of this brain area.

Unlike prenatal EtOH, our study found that exposure to prenatal THC did alter spatial learning and memory, but only in females. This sex effect contrasts with previous studies reporting that prenatal THC exposure impairs spatial memory in males (de Salas-Quiroga et al., 2020; Castelli et al., 2023), but not females (de Salas-Quiroga et al., 2020). Prenatal cannabinoid exposure has also been found to impact CB1 receptors and GABAergic transmission in the hippocampus of adult male rats, potentially affecting long-lasting learning and memory performance (Castelli et al., 2007; Beggiato et al., 2017; de Salas-Quiroga et al., 2020), whereas others report that prenatal THC exposure may not alter hippocampal function in females (de Salas-Quiroga et al., 2020). Interestingly, previous research within our own lab using a synthetic cannabinoid found no significant effects on spatial learning (Breit et al., 2019b). However, it is important to note that exposure in that study occurred during postnatal days 4–9, equivalent to the human third trimester, rather than prenatal exposure used in the current study. Importantly, differential effects of prenatal THC and EtOH on memory are consistent with individual and opposing effects each drug has on the hippocampus (Reid et al., 2021).

Despite deficits in spatial learning and memory, there was no effect of prenatal THC on working memory. Research on the effects of prenatal THC on working memory has yielded mixed findings in both preclinical (Silva et al., 2012; Beggiato et al., 2020; Castelli et al., 2023) and clinical studies (Smith et al., 2016; Murnan et al., 2021; Cioffredi et al., 2022). Studies have reported poorer performance on learning and memory tasks in children exposed to marijuana in the first trimester (Richardson et al., 2002) and an increased risk of educational underachievement for children of mothers with cannabis use disorder (Betts et al., 2022). However, adjusting for socioeconomic status and other comorbidities attenuated this association, suggesting that additional environmental factors may contribute to learning and memory outcomes. The inconsistencies in findings may be attributed to various factors such as the form of administration (injections, gavage), type of drug (THC, synthetic cannabinoids), dose, timing of exposure, and age of outcome measurement.

Unexpectedly, the combination of prenatal THC and EtOH did not produce more severe cognitive deficits than either drug alone. This was particularly surprising given pharmacokinetic interactions of prenatal EtOH and THC, which led to higher BACs (EtOH + THC = 200 mg/dl vs. EtOH = 150 mg/dl) and transiently elevated plasma THC levels (Breit et al., 2020). Moreover, both prenatal THC and alcohol exposure can disrupt endocannabinoid function, making offspring more vulnerable to stressors and impairing memory and learning processes (Trezza et al., 2012; Richardson et al., 2016; Little et al., 2021). The ECS plays a crucial role in various developmental processes, including movement, memory, and emotions; changes in its activity during periods of high brain plasticity, such as prenatal development, can lead to long-term problems in behavior and brain function (Pinky et al., 2019; Navarrete et al., 2020; Little et al., 2021; De Genna et al., 2022). For example, prenatal alcohol exposure is associated with increased EC activity and increased CB1 receptor activation (Nagre et al., 2015; Subbanna et al., 2015; Rodrigues et al., 2017), and prenatal alcohol exposure is associated with long-lasting changes in the ECS, which may contribute to some of the behavioral deficits observed in FASD (Basavarajappa, 2015; Hungund, 2017). In contrast, prenatal cannabinoid exposure actually reduces CB1 activation (de Salas-Quiroga et al., 2015, 2020). However, when consumed together, combined exposure increases the bioavailability of each drug (Abel and Subramanian, 1990; Hartman et al., 2015, 2016), which may lead to greater alterations in the ECS.

Both the hippocampus and prefrontal cortex are rich in CB1 receptors and, in fact, combined exposure to THC and alcohol can lead to more severe neuronal degeneration and altered neuronal circuitry in the hippocampus (Hansen et al., 2008; Basavarajappa, 2015). The lack of combination effects on cognitive functioning are also surprising given findings in other behavioral domains. For example, the combination of EtOH and CP-55,940 produces more severe motor deficits and hyperactivity (Breit et al., 2019a), as well thigmotaxis (Breit et al., 2019b). Even littermates from the present study exposed to the combination illustrated more severe open field hyperactivity compared to either substance alone (Breit et al., 2022). Thus, the combination of ethanol and THC at the doses used in the present study affects behavioral domains outside of cognitive functioning.

In fact, the combination EtOH and THC did reduce thigmotaxis among female subjects compared to either prenatal EtOH or THC alone, suggesting that the combination may impact emotional processing, reducing anxiety and increasing risk-taking behavior. Interestingly, zebrafish exposed to a low-dose CB1R agonist during development exhibit increased risk-taking behavior, but only when combined with alcohol (Boa-Amponsem et al., 2019). Moreover, littermates from the current study exposed to the combination of prenatal EtOH and THC were less thigmotaxic in the open field, spending more time in the center (Breit et al., 2022), although this effect was seen in males, but not females. Together, these data suggest that the combination of prenatal EtOH and THC may produce unique effects on emotional development.

In contrast to the combination, exposure to either prenatal EtOH or THC alone increased thigmotaxis, suggesting increased anxiety. For example, female subjects exposed prenatally to THC exhibited increased thigmotaxis during both spatial and working memory tasks. The effects of prenatal cannabis exposure on emotional development have been diverse and dependent on various factors, including the age of testing, cannabinoid type, and sex of the subjects. For instance, prenatal exposure to whole-plant cannabis extract increased anxiety-like behavior in juveniles but not in adults (Weimar et al., 2020), whereas studies of prenatal exposure to synthetic cannabinoids have reported mixed results, including no effects (Manduca et al., 2020), decreased anxiety/increased risk-taking (Boa-Amponsem et al., 2019; Breit et al., 2019b) and elevated anxiety-like behaviors (Traccis et al., 2021; Lallai et al., 2022). Interestingly, in littermates prenatally exposed to THC alone, there were no effects on open field thigmotaxis, although subjects were tested during pre-adolescence (Breit et al., 2022). Clinically, cannabis exposure has been associated with increased frequency of depression and anxiety in children (Goldschmidt et al., 2004; Leech et al., 2006; Eiden et al., 2018; Grant et al., 2020); thus, prenatal cannabis may impact emotional development in complex ways.

Similarly, prenatal EtOH exposure produced modest increases in thigmotaxis among females. As with prenatal THC, the effects of prenatal EtOH exposure on emotional development are highly variable, with some reporting increased anxiety (Balaszczuk et al., 2019; Aglawe et al., 2021), while others report decreased anxiety/more risk-taking behavior (Breit et al., 2019b), and with variable sex-dependent effects (Weinberg et al., 2008; Muñoz-Villegas et al., 2017; Bake et al., 2021) related to alterations in the hypothalamic–pituitary–adrenal axis (Weinberg et al., 2008). Clinical studies have also reported mixed results (Goldschmidt et al., 2004; O’Leary et al., 2010).

Interestingly, the effects of prenatal EtOH and THC on cognitive and emotional functioning in the present study were more robust among females. However, prenatal THC did reduce swimming speed among males. We previously reported that prenatal THC exposure can delay sensorimotor development and impair motor function in adolescents, although we did not see sex-specific effects on those outcomes (Breit et al., 2022). Thus, motor coordination deficits induced by prenatal THC exposure may persist in males, but not females, as they age. Although clinical research has found varied effects of prenatal THC on motor development, including no impairments (Chandler et al., 1996; Richardson et al., 2002; Huizink, 2014), impairments (Richardson et al., 1995), and advanced motor skills (Fried and Watkinson, 1990; Huizink, 2014), further investigation is required to discern the impact of cannabis on motor development and coordination, and to identify any possible sex differences.

Notably, in addition to behavioral effects, offspring prenatally exposed to THC exhibited lower weight compared to those not exposed to THC. These results illustrate that even low levels of prenatal THC exposure can impair growth and that growth impairments extend beyond early life (Breit et al., 2022) into adolescence. Importantly, this growth impairment was seen in both sexes. Given the sex- and task-dependent behavioral effects of prenatal THC, body weight reductions do not relate to behavioral outcome. Nevertheless, long-lasting effects of prenatal THC on physical development represent another adverse outcome associated with prenatal cannabis exposure.

The current study illustrates that prenatal exposure to EtOH and THC can alter cognitive and emotional development, but there are several limitations to consider. First, the effects of only one dose of THC and EtOH were examined, and consequences of either drug alone or in combination are likely dose-dependent. This is particularly critical to investigate, as the lack of regulation of THC products and the dramatic variation in THC potencies (Giroud et al., 2015; Kahr et al., 2015) create challenges in establishing dose–response effects in clinical studies. Second, only THC was administered, but cannabis products include many constituents which may impact outcome. By solely focusing on one constituent of cannabis, THC, the potential interactive effects of other cannabinoids present in various cannabis products, such as CBD, are not considered.

Third, the study focused primarily on learning and memory during late adolescence, without follow-up testing as they transitioned into adulthood. The age range was chosen because rats have typically reached the ontological stage of hippocampal maturation and spatial learning ability (Morterá and Herculano-Houzel, 2012), and are capable of navigating and swimming efficiently in the maze (D’Hooge and De Deyn, 2001; Albani et al., 2014). It is possible that performance could be affected by factors such as pubertal hormones. For instance, the onset of puberty has been associated with improved cognitive performance in the Morris Water Maze, possibly due to prefrontal cortex maturation and changes in learning strategy (Willing et al., 2016). Performance in females can also be affected by differences in the estrous cycle, while alterations in sex hormones can impact spatial ability in males (D’Hooge and De Deyn, 2001; Moradpour et al., 2013). However, recent data suggest that hormonal factors may not necessarily induce large changes in behavior (Levy et al., 2023). Importantly, we do not know if prenatal alcohol or THC is affecting pubertal onset in the current study; thus, future studies should examine whether alterations in maturation contribute to behavioral outcomes.

Fourth, there is a possibility that the subjects’ performance on the initial Morris water maze task could affect subsequent performance on the working memory version of the task. For example, if the memory for the initial spatial location of the escape platform is strong, subjects may become biased towards a particular strategy or location and have more difficulty adapting to the changing demands of the working memory task. Importantly, subjects were tested on the working memory version 1 week after spatial memory testing to reduce carryover effects and all subjects showed improvement in performance. Moreover, even if prior spatial learning impacted performance on the working memory task, impaired performance would still reflect deficits in cognitive flexibility. Nevertheless, testing subjects on only the working memory version would determine whether interference influenced performance.

Finally, the control subjects in his study were exposed to propylene glycol vehicle, a well-known e-cigarette vehicle commonly used in e-liquids alongside various flavorings. The effects of inhaling e-cigarette vehicles by themselves, as well as the effects of mixing various commonly used flavorings, remain poorly understood. In fact, a recent study discovered that e-cigarette vehicles can dysregulate gene expression in the lungs and that adding flavorings can increase toxicity and alter immune lung cells, potentially leading to reduced lung function (Szafran et al., 2020).

In summary, our findings suggest that prenatal exposure to THC and EtOH can have differential effects on cognitive functions, with sex-specific and substance-specific patterns. Our results suggest that even low doses of THC delivered through e-cigarettes during pregnancy can lead to learning and memory deficits, whereas exposure to EtOH can impair working memory. Co-administration of THC and EtOH did not magnify the adverse effects of either substance on cognitive function, but may influence anxiety-like behavior and risk-taking tendencies. Overall, our findings underscore the potential harm of THC and EtOH on fetal development and reinforce the importance of educating pregnant individuals and promoting public health policies and interventions to minimize the risk of drug-related cognitive deficits in offspring.