The study protocol was approved by the University of Kentucky Institutional Animal Care and Use Committee. Virgin female Sprague Dawley rats (Harlan, Indianapolis, IN, USA) weighing 194–223 g were individually housed at 22–25°C and maintained in a 14L:10D photoperiod (lights on at 0500 h) room with regulated 30–70% humidity. Rat chow and water were provided ad libitum.

Once released from quarantine, the females were fitted with a right atrial cannula connected to a subcutaneous (S.C.) access port implanted between the shoulder blades. The rats were allowed to recover for 1 week. To determine estrous cycles, vaginal lavages were obtained daily. Each female was group housed with a proven breeder male for breeding 1 week after cannulation. Gestational day (GD) 0 was designated as the day that sperms were detected in the vaginal smear, and the females were individually housed thereafter. Foster dams were bred at the same time and remained untreated throughout their gestation.

The cannulae were flushed daily via the S.C. port with sterile heparinized saline (0.4 cc, 100 IU/ml) until GD 8. From GD 8–21, the experimental dams were divided into three treatment groups that received either oxycodone (Mallinckrodt, St. Louis, MO, USA) at a low (OXY-L, 0.5 mg/kg/day, n = 5) or high dose (OXY-H, 2.0 mg/kg/day, n = 12) or an equivalent volume of vehicle [control (CON), normal saline solution (NSS, 1.0 ml/kg/day), n = 12] from GD 8–21. These solutions were slowly injected i.v. over 10 min via the S.C. access port manually.

On postnatal day (PD) 1, the pups were counted and weighed. Oxycodone or NSS was also administered on PD 1, 3, and 5 to the dams because brain development during early postnatal life overlaps the human “third trimester” brain growth spurt and to prevent maternal withdrawal symptoms that might affect maternal nursing behavior. On PD 2 all litters were adjusted to contain 10–11 pups with equal numbers of male and female pups when possible. At 1700 h on PD 5, all pups in each litter were transferred to an untreated foster dam to minimize exposure to altered maternal rearing behavior that has been described in rat dams after exposure to opiates (40). To preclude potential litter effects, only one male and one female from each litter were included in the behavioral studies (41). The pups were weighed daily and weaned at PD 25, when they were separated by sex.

Statistical analyses were considered significant if p < 0.05. Multilevel linear regression models or linear mixed effects models were used to analyze USVs, open field and water maze data. Such models accounted for potential litter effects and correlation among outcomes from the same pup over time. The models were fitted using restricted maximum likelihood to test for the impact of treatment group, gender, time, and any potential interactions these variables have on the mean values for the data. Due to skewness and outliers of the data, a natural log transformation was applied when appropriate. The Kenward and Roger (45) approximation was used to estimate standard error and degrees of freedom. Variables (trial days, sex, and treatment groups) were treated as categorical and backward elimination at the 5% significance level was utilized. Kruskal–Wallis tests were applied to compare litter sizes and number of males per litter between treatment groups. A linear mixed-effects model was applied to compare differences in body weight between groups with treatment group, gender, PD, and the interaction of gender and PD included as predictors of weight. Tests were two-sided and were conducted in SAS version 9.4 (SAS Institute, Cary, NC, USA).

Perinatal oxycodone exposure did not affect litter size or number of males and females per litter (Table 1).

Although the average maternal weight gain during pregnancy was different between groups (p = 0.005), perinatal oxycodone treatment did not affect mean birth weight of male or female pups weighed within 24 h after birth (Table 1). Dams in the OXY-H group gained less weight than CON dams (p = 0.01) and OXY-L dams (p = 0.023) (Table 1). Oxycodone treatment did not affect weight gain of the pups; body weights up to PD 50 were not different between groups (p = 0.61). However, there was an interaction between gender and postnatal age (p < 0.0001); males and females had different growth trajectories over time, as expected (Figure 1).

Body weights of both male and females were similar across treatment groups in the rats tested in the open field and water maze (p = 0.43), the females weighing less than the males as expected (p < 0.0001) (Table 1). The body weight of the pups at the time of USV testing (PD14) (g ± SEM) were 30.9 ± 1.8 in male and 29.1 ± 1.6 in female CON group; 30.2 ± 1.9 in male and 28.1 ± 1.9 in female OXY-L group; and 30.1 ± 1.4 in male and 28.7 ± 1.1 in female OXY-H group.

There were no significant sex differences so results were collapsed across sex for subsequent USV analyses. There was no significant difference in the latency to the first vocalization between treatment groups; however, there was a trend for longer latencies in OXY-exposed rats, Figure 2A. Latency to the first USV as plotted in Kaplan–Meier Plots is shown in Figure 2B, a point on the plot denotes the estimated probability of having a first USV after the given time point. Although there was a trend for prenatal oxycodone-exposed pups to display longer latencies to their first USV after isolation, there were no significant differences between treatment groups (p = 0.25), likely due to small sample sizes, Figure 2B.

Total USVs across time also did not differ among the three treatment groups (p = 0.85), Figure 2C. The number of USVs increased across time in all treatment groups (p = 0.0004); however, there was no significant effect of perinatal OXY exposure (p = 0.32), Figure 2D.

Using a multivariate repeated measures Gaussian linear model to analyze the data with trial days, treatment groups and sex as predictor categories, there was no effect of oxycodone or sex on the mean values of the average number of trials until criterion performance was reached on both days (p = 0.62). The expected decrease in the mean values of the average number of trials on day 2 was observed in all groups (p < 0.0001) (Figure 7).

Oxycodone low dose rats were hyperactive and traveled more in the inner zone of the open field compared to CON and OXY-H rats, resulting in a higher mean ratio of inner:outer zone distance traveled by the OXY-L males. Thus, perinatal exposure to oxycodone was associated with hyperactivity behavior in adolescence. In addition, the increase in the ratio of inner zone:total distance traveled suggests that perinatal oxycodone reduced anxiety-like behavior and decreased normal species-typical thigmotaxic behavior in a new environment. Alternatively, these differences might reflect overall hyperactivity with a failure to inhibit entries to the center. It remains to be determined whether the increased travel in the center is due to deficits in hyperactivity, impulse control, or reduction in anxiety.

Our finding that perinatal exposure to oxycodone was associated with hyperactivity in the offspring is in agreement with human studies that identify hyperactivity, impulsivity, and attention problems in children exposed to opiates in utero (33, 34). In animal models, however, the results are more conflicting due to differences in studied drugs, which were mainly morphine, as well as in paradigms, ages, instruments, and end points. For example, in one study, postnatal handling but not prenatal morphine exposure increased locomotor activities in the open field in adult male and female rats (31). In contrast, others reported that postnatal stress but not prenatal exposure to morphine increased locomotor activity in the open field (32). Thus, morphine yields different results than oxycodone in the open field. However, it should be noted that the effects of morphine are contradictory among studies and differ among tests. Thus, prenatal exposure to morphine was associated with increased anxiety-like behavior in an elevated plus maze (EPM) and reduction in time spent in the lit side of a light/dark box (L/D box) (47). Others reported that rats prenatally exposed to morphine exhibited decreased anxiety-like behavior in the EPM and L/D box with no differences in distance traveled over 30 min in the open field (48). Another study reported no significant anxiogenic effect of prenatal morphine exposure determined in the EPM (49). Finally, prenatal exposure to several opiates, such as methadone, buprenorphine, and most effectively morphine, increased anxiety-like behaviors in the light–dark transition test, with no effect on locomotor activity in an open field (50).

A possible mechanism by which prenatal exposure to opiates could result in hyperactivity may involve changes in multiple neurotransmitter signaling pathways such as dopaminergic pathways and the HPA system. It is well described that opioids have a significant role in controlling the release of dopamine and acetylcholine in the key reward regions of the brain including the ventral tegmental area (VTA) and the nucleus accumbens (51, 52, 53, 54). Long-term exposure to opiates leads to both structural and biochemical changes in the mesolimbic dopaminergic system; for example, a reduction in the cell size of dopaminergic neurons in the VTA (55), and increased levels of tyrosine hydroxylase, which is the rate-limiting enzyme in the synthesis of dopamine in the VTA (56). In addition, convincing evidence suggests that the impairment of dopamine-mediated development and the monitoring of motivated behavior and reward-related memory formation might be associated with ADHD symptoms (57, 58). Thus, taken together, it is possible that perinatal exposure to opiates may disrupt the normal development of dopaminergic reward-related circuits leading to hyperactive behavior. However, this speculation remains to be further elucidated.

Changes in the HPA system have also been linked to ADHD symptoms. In children, both reduced basal cortisol secretion and cortisol hyporeactivity have been associated with hyperactivity/impulsivity or a combined type ADHD (59). An abnormal diurnal rhythm and less effective negative feedback mechanisms after a dexamethasone suppression test were also identified more frequently in the children with ADHD that were severely hyperactive compared to those with milder symptoms (60). In agreement with many community-based studies, in which ADHD is more prevalent in males (61), in our study hyperactivity was more notable in the male OXY-L group. Interestingly, in adults with ADHD, although the basal salivary cortisol levels were not different, cortisol levels 20-min after a mental cognitive stress test were higher than those in healthy adult controls (62). These findings are similar to our observations that POE is associated with increased CRH-induced ACTH release (8) and with increased corticosterone reactivity to restraint stress in adult female rats (unpublished). However, we did not measure corticosterone, the major circulating glucocorticoid in rats, during the behavioral tests in the current studies. The discrepancy between hyperactivity in males and increased levels of corticosteroids in females could be due to the methods used to produce the stress in each study, relatively small sample sizes, and/or the gender of the participants. Referral biases and methodological difficulties were present in other human studies (63, 64). Thus, the relationship between POE, hyperactive behaviors, and abnormal HPA axis function that may interact with gender warrants further investigations, which should include the measurement of corticosterone during and after neurobehavioral testing. The abnormal HPA axis response to stress may be a promising candidate for use as a biomarker for early detection and treatment of ADHD in infants prenatally exposed to oxycodone/opiates.

Data from both animal and human studies suggest that early nutritional stress and malnourishment are associated with anxiety, high impulsivity, and attention problems (65). Although the weight gain of the OXY-H dams was significantly lower than that in the other groups, the weight gain of the OXY-L dams was actually comparable to that in the CON group. In addition, birth weights of the pups were comparable in all groups. Therefore, the hyperactivity in the open field of the OXY-L group could not solely be explained by poor maternal nutritional status.

Although there was a trend toward increased latency to first vocalization, oxycodone treatment had no effect on this end point or on the number of vocalizations per minute. The lack of differences in these USVs parameters may be due in part to the relatively low sample size in the OXY-L group. To our knowledge, there are no reports thus far on the effects of perinatal oxycodone on USVs In contrast to our results, however, perinatal cocaine treatment decreased the number of USVs on PD 1 and on PD 21, but this effect was not observed on PD 14 (66). Therefore, it is possible that the lack of an effect of oxycodone in our study occurred because testing was limited to PD 14. In fact, there are other types of USVs based on frequency and duration that are elicited as the rat becomes more mature (67). These include a 22 kHz USV emitted by juvenile and adult rats in response to predators and pain indicating a negative affective state (68, 69), and a 50 kHz USV emitted in adult rats in response to rewarding stimuli, expressing a positive affective state (70, 71). So it is also possible that POE may affect these other types of USVs that were not tested at an older age.

We did not find any effects of POE on spatial learning and/or memory in the water maze test as hypothesized; this may have resulted from not including rats perinatally exposed to a lower dose of oxycodone in this experiment due to the small sample size of this group. Previous studies using prenatal morphine exposure models report conflicting results; memory and learning in rodents are either impaired or enhanced. Namely, juvenile rats prenatally exposed to morphine had impaired spatial memory in the Morris water maze or Y-maze test (38, 72, 73), but aberrant memories such as morphine reward memory in the conditioned place preference or forced swim tests were enhanced (49, 74–76). In contrast to our study, Davis et al. (39) found that prenatal exposure to oxycodone impaired spatial learning and memory in a battery of spatial tasks; in the Morris water maze, rats prenatally exposed to OXY had increased latency and greater distance traveled to find the platform when the intertrial interval was long, not short. Rats prenatally exposed to oxycodone also had a decreased use of spatial strategies and more use of non-spatial strategies such as wall-hugging. In addition, the retention of learning memory in the T-maze, assessed 5 days after acquisition of the training, was impaired. This finding is actually consistent with our report that although POE male rats were able to discriminate between the stress and non-stress cues during a classical conditioning paradigm on PD 40, they had impaired discrimination ability when retested on PD 75 (77). Moreover, rats prenatally exposed to oxycodone have more reference memory errors in the radial arm maze (39). These differences in results could be due to many reasons. In their study, the dose and route of administration of OXY were different than those used in the current experiment with escalating doses of OXY (10 mg/kg/day up to 15 mg/kg/day) via gavage for 28 days prior to breeding. In addition, the pups were reared by their biological mothers in their study, while surrogate fostering was used in the current study. A number of previous studies have shown that opiate administration negatively alters maternal rearing behavior toward the pups including measures such as cleaning of the pups, delay of maternal behaviors, and maternal aversion to pup odor (78, 79, 80). Neonatal rearing condition and neonatal maternal interaction such as maternal separation had long-term effects on the stress response of the offspring including an increase in restraint stress-induced norepinephrine release in the PVN in adult rats (81) and changes in the HPA axis at multiple levels that could be linked to epigenetic modification (82). Variations in maternal care also influence learning and behaviors of the offspring (83, 84). The adverse effects of neonatal maternal separation on the HPA axis were lessened by fostering the litters (85). Thus, fostering the pups when the dams were exposed to opiates could alter the HPA axis and possibly the neurobehavioral outcomes of the offspring. In addition, even though signs of withdrawal in the dams were monitored in the study by Davis et al., body weights of the OXY pups were approximately 10% lower than those of the controls, indicating possible neonatal opiate withdrawal and poorer nutritional status that can also affect long-term outcomes. In contrast, the body weights of the pups in our study were comparable in all groups.

In humans, prenatal exposure to opiates results in impairments in cognitive function and learning. Bunikowski et al. also reported that when evaluated at 1 year of age, children prenatally exposed to opiates had a mild psychomotor developmental impairment compared to the control group; these included impairments in “hearing and speech” and “intellectual performance” subscales (36). Guo et al. found that in utero opiate exposure was associated with impairments in the Auditory Rare Event Monitoring task and the Sternberg Memory task in children 7–12 years of age (35). More recently, Hunt et al. reported from their case–control study that infants prenatally exposed to opiates are more likely to experience neurodevelopmental impairments compared to healthy control infants, when assessed at 18 months and 3 years of age (37). The deleterious effects of prenatal opiate exposure on cognitive function persisted and did not decrease over time after controlling for permanent home placement and heroin use in the mother when children prenatally exposed to opiates were retested on the Wechsler Intelligence Scale for Children from 1 year old up to 8.5 years of age (86). These data suggest that prenatal exposure to opiates is associated with impaired cognitive functions. Therefore, although we did not detect any effects of POE on cognitive function, learning, and memory using a water maze as a paradigm, further investigation is warranted with different testing paradigms, testing at different ages, and with a lower dose of oxycodone.

Interestingly, we found that exposure to the lower dose of oxycodone of 0.5 mg/kg/day but not the higher dose of 2.0 mg/kg/day was associated with hyperactivity in the offspring. This difference could be due to the development of tolerance to the higher dose of oxycodone. Opiate tolerance is characterized to be pharmacodynamic, time and dose-dependent, and opioid receptor specific (87). Tolerance can develop after exposure to a KOR agonist (88) even for as short as 5 days (17). Whether or not tolerance to the stimulatory effects on the HPA axis by KOR agonists is dose-dependent has not been well studied, but tolerance to MOR agonists was dose-related (89). It is possible that in our study, rat dams that were exposed to the higher dose of oxycodone (2 mg/kg/day) may have developed opioid tolerance leading to a decreased fetal CORT exposure, thus there were fewer effects on the developing HPA axis and the neurodevelopmental outcomes of the offspring.

In summary, POE was associated with hyperactivity in young adult rats. In these studies, perinatal oxycodone treatment had no effect on the responses of pups isolated from their dams, or in learning and memory deficits, which could be due to the small sample sizes, testing paradigms, or the doses of oxycodone tested. These issues remain to be resolved.