The present study indicates that exposure of female rats to morphine in early adolescence has transgenerational consequences, the nature of which are dependent upon the sex of the offspring. It was found that the effects of maternal adolescent morphine treatment on female offspring were associated with decreased anxiety-like behavior. These effects were estrous cycle-dependent, with MOR-F1 females exhibiting increased time in the center of the open field during diestrus and estrus. No effects of maternal adolescent morphine treatment on open field behavior were found in male offspring. In contrast, hot plate testing revealed increased sensitivity to acute morphine analgesia and more rapid development of morphine tolerance in MOR-F1 males, while no changes in morphine sensitivity in MOR-F1 females was observed. These findings indicate that exposure to morphine, even when confined to early adolescence, can significantly impact the phenotype of future progeny in a sex-specific manner.
Our previous work on the transgenerational effects of adolescent morphine exposure utilized a prolonged injection regimen with higher doses of morphine [7
]. The current findings indicate that transgenerational effects continue to be observed with a shorter duration of exposure, lower doses of morphine, and with an increased period of abstinence between the cessation of morphine administration and mating. It is unclear whether the age at the time of exposure (i.e. early-mid adolescence) is a key factor in modulating these offspring effects. It should be noted, however, that while the current results suggest that similar systems are affected by both exposure regimens, some differences in the nature of these offspring effects are evident, particularly with regard to anxiety-like behavior. For example, we previously reported a significant increase in anxiety-like behavior on the elevated plus maze in MOR-F1 females [7
]. In that study, all females were examined during estrus. Similar effects were not observed during estrus in the current study. Indeed, MOR-F1 females tended to demonstrate reduced anxiety-like behavior and increased locomotor activity when tested in the open field during estrus. Whether this represents a fundamental difference based on the duration of treatment or a difference based on the precise nature of the task (e.g. level of stress associated with the task) is unknown. Regardless, the results of the current study indicate that significant alterations in anxiety-like behavior as well as exploratory behavior are modulated by maternal adolescent treatment and that these effects are estrous cycle-dependent. Moreover, they support our previous findings that the transgenerational effects of anxiety-like behavior are observed in F1 female but not F1 males.
In MOR-F1 females, significantly reduced anxiety-like behavior was observed on diestrus but not on proestrus. Similar results have been reported in the adult female offspring of mice exposed to brief maternal separation and these effects were specific to the open field as similar results were not observed on the elevated plus maze [16
]. No significant differences in measures of maternal behavior were observed in that study. While we did not directly examine alterations in maternal care in the current study, our previous findings using a more prolonged morphine exposure during adolescence did not reveal any significant effects on gross measures of maternal behavior (retrieval and crouching latencies; [17
]). In addition, we observed no significant effects on bodyweight growth as a function of maternal adolescent exposure. Nonetheless, it is possible that subtle alterations in the maternal behavior of females exposed to morphine during early-mid adolescence may play a role in the effects observed in adult female offspring. Certainly, there are many studies that indicate a significant role of maternal behavior in adult emotionality [16
]. Moreover, studies have also revealed significant alterations in the endogenous opioid system following maternal separation paradigms [21
]. We are currently conducting studies examining the potential role of maternal behaviors in transgenerational effects of adolescent morphine exposure. These studies will utilize both home cage observation and cross-fostering designs to begin to dissociate maternal influences in this phenomenon.
In the current study, acute administration of low, threshold-doses of morphine significantly prolonged HPL in MOR-F1 males. In addition, MOR-F1 males also became tolerant to the analgesic effects of morphine more quickly. Acute analgesic responses to morphine and the development of tolerance are believed to reflect the sensitivity of brain opioid systems. Accordingly, the current study suggests enhanced opioid sensitivity in the offspring of females exposed to morphine during adolescence. There is a significant association with analgesic sensitivity and abuse liability [23
], with enhanced morphine sensitivity associated with increased risk of abuse. Together with our previous findings on morphine sensitization, these data strongly suggest that maternal adolescent morphine exposure may increase the risk of drug abuse vulnerability in her male offspring. The question of whether such transgenerational effects could be triggered by maternal drug exposure in early life is intriguing. Currently, the role of prior maternal drug use in the familial transmission of substance abuse liability is a relatively unexplored area of study.
With regard to differences in morphine sensitivity, no significant effects of maternal morphine exposure were observed in MOR-F1 females. Sex differences in morphine analgesia have been widely reported [25
] with males consistently demonstrating increased sensitivity to the analgesic properties of opiates [27
]. Similar effects were observed in the current study, with few analgesic effects observed in females with lower doses. A number of mechanisms may underlie these sex differences, including differential distribution of μ-opioid receptors, estrogenic effects on endogenous opioid function, and sex differences in brain dopamine and/or glutamate systems [25
]. Previous studies have also observed sex differences in the development of tolerance [32
] although the data are somewhat mixed with some demonstrating enhanced morphine tolerance in males [30
], others more rapid tolerance in females [35
], and still others indicating no sex differences [36
]. There is a rich body of literature detailing the impact of a variety of parameters on sex differences in morphine potency and tolerance, including the effects of the dosing regimen, the route of administration and the test used to measure analgesic response as well as the strain of the animal. Thus, it is possible that transgenerational effects of adolescent morphine exposure on morphine-induced analgesia may yet be revealed in MOR-F1 females under different test conditions.
The mechanism underlying the transmission of these effects remains unknown. It is possible that a modification in the postnatal environment induces the observed effects on both anxiety-like behavior and morphine analgesia in MOR-F1 animals. Indeed, maternal separation has been shown to induce significant alterations in both emotionality and morphine sensitization [38
]. On the other hand, the endogenous opioid system is a critical regulator of the prenatal environment [39
], and studies have shown that blocking endogenous opioids during pregnancy has significant consequences for offspring [43
]. Exposure of adolescent female rats to a similar increasing dose regimen of morphine results in significant long-term alterations in their endogenous opioid system [6
]. Such persistent changes could result in adaptations within the prenatal environment, including differences in the levels of circulating hormones. Interestingly, there is increasing evidence that prenatal stress can impact anxiety-like behavior and drug abuse liability. These effects are likely mediated via alterations in the HPA axis. Thus, one possible mechanism underlying the observed effects could be a shift in the HPA axis resulting in in utero
conditions that are similar to those observed in prenatally stressed mothers. Indeed, it is possible that both pre- and postnatal factors mediated by alterations in endogenous opioid regulation of the HPA and/or HPG axis may mediate these transgenerational effects. Finally, recent studies in the field of epigenetics suggest that changes in DNA methylation, via effects on gene transcription, may play a role in intergenerational transfer of behavioral phenotypes. However, to date there is no evidence to support direct germ line transmission of specific DNA methylation from mother to offspring.