We found that combined treatment with ethanol and nicotine throughout gestation results in minor deficits in maternal behavior onset, with ethanol/nicotine-exposed dams (E/N) less likely to be in the nest, more likely to rest away from their surrogate pups, and touch/sniff those pups less frequently on PPD one. There was no impact on retrieval, crouching, or licking behaviors, which are most essential for pup survival since they offer the opportunity for pups to feed and eliminate waste. This replicates findings that indicated the capacity to meet the basic physical demands of the pups is not altered by gestational ethanol alone [44
], unlike the effect of gestational cocaine on these behaviors [26
]. These results raise questions about whether alterations in non-pup directed behaviors, such as spending less time in contact with pups, can impact important aspects of development other than basic survival. The dam’s presence in the nest is important because separation from the dam is stressful for pups, and multiple periods of separation can alter stress reactivity [39
], anxiety and depressive-like behaviors [50
], as well as drug seeking behaviors [51
] in pups well into adulthood.
The effects we report here are similar to, though more moderate than, reports from previous studies following postpartum ethanol treatment alone, which more strongly impair maternal behaviors [48
] compared to gestational ethanol alone, which has a lesser affect on maternal behaviors in the rat [43
]. Maternal behavior following gestational and early postnatal nicotine treatment has not been thoroughly examined in a preclinical model, but in clinical populations, smoking alone has been shown to decrease breastfeeding, potentially through nicotine’s inhibition of prolactin release, though no effect has been observed in plasma oxytocin levels in response to nicotine [3
]. Given the paucity of data regarding the effects of nicotine in isolation on maternal behavior, it is impossible to draw any conclusions at this time. As our study employed both drugs, this exposure paradigm, specifically with respect to ethanol, may have resulted in behavioral tolerance to some of the effects of these drugs at the time of testing.
Ethanol/nicotine-exposed dams were exposed to forced ethanol consumption for 14 days, long enough to induce dependence and create withdrawal symptoms in virgin females [13
]. Though ethanol withdrawal can cause anxiety and decreases in locomotor activity [55
], withdrawal during the postpartum period has not been studied. Ethanol/nicotine-exposed dams are without ethanol access for approximately 24–36 hours prior to birth and testing. It is possible that this period was sufficient for ethanol withdrawal to pass. Additionally, high levels of central oxytocin, such as those seen immediately following parturition [61
], can relieve acute withdrawal symptoms [75
] and may have minimized the effects of withdrawal in the current study. Although no direct measurement of withdrawal was performed, the behaviors displayed by ethanol/nicotine-exposed dams were not indicative of withdrawal states. Animals displayed normal locomotor and exploratory behavior during the behavioral test, and no animal displayed abnormal behaviors such as ‘wet-dog shakes’. This lack of apparent severe withdrawal may also have been precipitated in part by the continuous presence of nicotine, which has been shown to alleviate many of the symptoms of ethanol withdrawal [36
]. Since there is very little impact of the experimental manipulation in the current study, the effect of withdrawal, if any, appears to be minimal.
The behavioral alterations we observed in the E/N-exposed dams are considered primarily a result of gestational drug treatment rather than any pup behavioral deficits as all dams were tested with surrogate unexposed pups. This is an important strength of this study, since prenatal exposure to ethanol can alter pup behaviors that play an important role in the maternal care these pups receive [4
]. The only gestational difference we found was lower weight gain in the E/N-exposed dams during the 21-day gestational period. Due to the anorectic effects of nicotine and perhaps lower liquid intake of ethanol, the pair-feeding paradigm was used to assure that control dams consumed only the amount of food consumed by the E/N dams, thus it is unlikely that these effects were due to differences in caloric intake. It is more likely that this difference is due to the significantly different litter birth weights and slightly different number of pups. Ethanol and nicotine have been independently shown to decrease birth weight in both animal and human models of prenatal exposure [21
], which would result in decreased gestational weight gain in these mothers. Furthermore, E/N-exposed offspring tended to gain less weight during the postpartum period, perhaps indicating that despite the intact crouching displayed by these surrogate dams, the pups may not have been receiving an adequate milk supply, due to poor solicitation of maternal care, which was not examined directly in this study.
Ethanol, a known positive modulator of GABAergic signaling, also has effects on glutamatergic, dopaminergic, serotonergic, and opioid systems, as well as several hypothalamic peptides [32
]. While nicotine acts primarily at nicotinic acetylcholine receptors, it can influence dopaminergic signaling as well [32
]. Dopamine, serotonin, norepinephrine, corticotrophin releasing hormone (CRH), and opioids, along with oxytocin, have all demonstrated important roles in the initiation and maintenance of maternal behavior [53
]. The onset of maternal behavior in the immediate postpartum period is precipitated by an increase in the expression and binding of oxytocin [58
]. It is possible that the interactions between ethanol and nicotine at these target systems could disrupt maternal behavior, potentially through oxytocinergic mechanisms, since it has been shown that gestational cocaine alters the receptor number and affinity of receptors. It is interesting that the behavioral results found in the present study differ in the magnitude and type of those seen following gestational cocaine treatment [26
], indicating that not all drugs of abuse effect maternal behavior similarly. Yet, despite these behavioral differences, there were reductions in oxytocin levels seen in the MPOA and VTA of E/N-exposed dams that are similar to some of those seen in cocaine-exposed dams [27
Deficits in oxytocin in the MPOA have been most strongly associated with disrupted crouching behavior [19
], and though we did not see a significant effect on crouching in the E/N dams, lower levels of oxytocin have also been associated with changes in other non-pup directed maternal behaviors, as was found in the present study. Had we examined maternal behavior later in the postpartum period, it is possible that we may have seen other pup-directed maternal behaviors affected by this treatment. Though it is unknown how ethanol affects central release of oxytocin, it has been shown to decrease suckling-induced release of oxytocin into the peripheral bloodstream [73
]. The direct action of nicotine on centrally released oxytocin or oxytocin receptor binding has not yet been determined and will be an interesting study for comparison.
Our finding that E/N treatment results in lower oxytocin levels in the VTA may suggest that E/N dams found pup contact less rewarding, though we did not directly test this theory. The VTA plays a prominent role in the dopaminergic-mesolimbic reward circuitry, and oxytocin has been shown to have an antagonistic effect on the dopamine systems associated with drug-seeking behaviors [34
]. Parvocellular projections from the oxytocin neurons in the paraventricular nucleus terminate in both the VTA and nucleus accumbens, and it is possible that the aspects of pup stimuli and the interactions with pups that are normally rewarding [37
] are mediated through these projections. Dopamine neurotransmission plays a role in specific aspects of maternal behavior [7
], and the rapid rise in oxytocin in the VTA may modulate which cues are the most rewarding during the early postpartum period. If this system has been altered due to prolonged drug exposure, it may not respond properly to cues that would normally induce and maintain maternal behavior. Both ethanol and nicotine can act to increase dopamine release from VTA neurons into the nucleus accumbens when given acutely [12
], an effect which does not appear to attenuate over repeated or chronic administrations of the drugs [12
]. Though studies indicate the two may interact synergistically [20
], and when given in combination, chronic exposure to these drugs may activate this system differentially.
With regard to the next generation ethanol preference, we did not see an overall increase in ethanol preference in the prenatal E/N exposure group as hypothesized (see ). However, we did observe that prenatal exposure to these drugs altered sex specific differences in ethanol consumption. At PND 30, a period in the rat similar to early adolescence in humans, control animals did not exhibit a sex difference in ethanol consumption overall, but animals prenatally exposed to E/N did, with females consuming more ethanol than males. This effect also appears to be phase-dependent. During the acquisition phase of two-bottle choice testing, both control and E/N females drank more than males, while during the forced consumption and subsequent two-bottle choice period, only E/N females drank more. Ethanol consumption in E/N-exposed males drops to almost nothing following forced ethanol consumption, a shift that is not apparent in other groups. Interestingly, the control subjects in both groups preferred ethanol on the first day following the end of forced choice phase, potentially indicating a slight relative aversion to ethanol in E/N offspring at this time.
Ethanol Preference in Adolescence and Adulthood (% total fluid consumption, mean ± SEM)
The few studies that have investigated sex differences in drinking behavior during adolescence in the rat have shown that females typically drink more than males [14
]. Though prenatal exposure to ethanol has previously been associated with increased ethanol drinking behavior in adolescence (for review see [14
]), it is possible that the forced ethanol consumption period used in the current study is more stressful, and adolescent E/N-exposed males react differently to that stress than females, thus resulting in a greater taste aversion to ethanol.
During early adulthood (PND60) control females consumed more ethanol than control males, a sex effect that is seen in most ethanol consumption studies and in Sprague-Dawley rats in particular [2
]. Interestingly, in the present study we found that prenatal exposure to ethanol combined with nicotine eliminated the typical sex difference at this age. Some literature suggests that prenatal exposure to ethanol or nicotine, especially in the late gestational period, “feminizes” male behavior. The presence of high levels of brain estradiol is important for masculinization of brains that occurs during this period, and prenatal exposure to either ethanol or nicotine has been shown to prevent this surge [40
]. This may be related to adult sex effects, since oxytocin is strongly regulated by estrogen [53
It has been reported that animals tested during the adolescent period will also consume higher amounts of ethanol and exhibit greater preference compared to adults [76
]. We could not directly compare the drinking patterns between adolescents and adults since the testing paradigms were different; however, the paradigm difference seems to have affected the ethanol intake primarily during the acquisition phase of testing, where adults consumed more than adolescents. The study designs used for adolescents and adults were dissimilar, although our original intent was to keep the study design consistent throughout both ages, with ethanol administered at a fixed 10% volume. However, after examining the data from the adolescents using this paradigm, the design was altered in an effort to increase consumption. Effects seen during the acquisition phase were not apparent during the forced consumption or two-bottle choice phases, where although most adults drank less than the adolescents did, this difference was not large and may not be biologically relevant. Other studies examining adolescent drinking compared to adult drinking do not employ a forced ethanol phase, which can dramatically affect behavioral responses [76
]. The forced consumption phase was included to ensure that each rat would consume enough ethanol to experience its pharmacological effects. We originally thought that this would overcome any barriers to consumption, such as its taste, the age of the animal, or the rat stain used; however, ethanol consumption in most groups was considerably lower following the forced consumption phase, especially in adult animals. Future studies should consider alternatives to this phase.
A common finding at PND 30 and PND 60 was that males with higher levels of oxytocin in the VTA consumed the least ethanol, while females with the highest levels of oxytocin in the VTA drank the most within their gender. Males also had consistently lower levels of oxytocin in the MPOA than did females. These results raise the possibility that oxytocin changes in the VTA are related to increased or decreased drinking in a sex-specific manner, which would be a major finding and perhaps have some clinical relevance for both alcohol research as well as for other dopaminergic reward associated drugs of abuse. Altered oxytocin system dynamics in brain regions associated with the reward system, including the VTA, nucleus accumbens, hippocampus, and amygdala, indicate a possible mechanism for the control of tolerance; however, oxytocin involvement in the behavioral intake of ethanol has not yet been investigated. Higher levels of oxytocin in cerebrospinal fluid have been associated with inhibitions in the development of tolerance to many drugs of abuse, including opiates [70
], cocaine [67
], and ethanol [74
]. Systemic and ICV injection of oxytocin can also inhibit the development of physiological and behavioral tolerance induced by cocaine, heroin and ethanol in rats (for review see [35
]). It is unknown if ethanol or nicotine effect more rapid changes in oxytocin system dynamics, such as receptors levels, but given the similarities in ethanol and nicotine’s targets, an interaction is likely.
High oxytocin levels in the VTA could also affect drinking behavior through the inhibition of dopaminergic signaling as discussed above. This may be explained by oxytocin’s modulatory role of dopaminergic transmission. Oxytocin pretreatment can attenuate dopamine utilization in the nucleus accumbens in response to cocaine [6
]. Chronic oxytocin injections are also able to inhibit the stimulated release of dopamine in the basal forebrain [33
]. It is possible that oxytocin receptor activation may be interfering with D2 receptors in the VTA, potentially leading to fewer incidences of burst firing within VTA projections to the nucleus accumbens, which have been shown to play an important role in the development of addiction [56
]. The finding may be one of the most important for the future elucidation of oxytocin’s role in the rewarding effects of a number of drugs of abuse.
It is important to note that Sprague-Dawley rats, though well established for the study of maternal behaviors and ethanol choice behaviors, do not spontaneously consume substantial quantities of ethanol. Based on similar ethanol liquid diet studies, we were expecting to see values around 130–150 mg% per deciliter [71
]; however, in our study, dams had lower than anticipated BECs. It is possible that these dams did not consume as much diet as those in other studies, though this is hard to determine from the literature. Acute nicotine can lower peak BECs in both adult female [57
] and neonatal rats [10
], although the effects of chronic nicotine are unknown. Future studies using methods and strains that induce higher levels of ethanol consumption may inform us better of any other effects of prenatal exposure to ethanol and nicotine on ethanol drinking. Additionally, if the interaction of a stimulant (nicotine) and depressant (ethanol) result in fewer behavioral or biological consequences than found with either drug alone, the mechanism of this interaction becomes very interesting in terms of potential interventions and our understanding of why different drugs are often co-administered by users.
Exposure to ethanol and nicotine during gestation affects the time spent in contact with pups and decreased oxytocin levels in both the MPOA and VTA of rat dams. Additionally, the effect of prenatal exposure to both ethanol and nicotine does not appear to increase drinking preference during either adolescence or early adulthood, though it does differentially affect normal sex differences in consumption, possibly involved with alterations in oxytocin interactions with the dopamine-reward system. This becomes particularly important when considering that early exposure to ethanol can affect responses to ethanol at distinct phases of development, and that adolescent drinking can lead to higher levels of adult drinking. The data we present here suggests the need for further research into questions of how the physiology of these drug interactions may affect important behaviors in this and other models.