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Variations in maternal behavior, either occurring naturally or in response to experimental manipulations, have been shown to exert long-lasting consequences on offspring behavior and physiology. Despite previous research examining the effects of developmental manipulations on drug-related phenotypes, few studies have specifically investigated the influence of strain-based differences in maternal behavior on drug responses in mice. The current experiments used reciprocal F1 hybrids of two inbred mouse strains (i.e. DBA/2J and C57BL/6J) that differ in both ethanol (EtOH) responses and maternal behavior to assess the effects of maternal environment on EtOH-related phenotypes. Male and female DBA/2J and C57BL/6J mice and their reciprocal F1 hybrids reared by either DBA/2J or C57BL/6J dams were tested in adulthood for EtOH intake (choice, forced), EtOH-induced hypothermia, EtOH-induced activity and EtOH-induced conditioned place preference (CPP). C57BL/6J and DBA/2J mice showed differences on all EtOH responses. Consistent with previous reports that maternal strain can influence EtOH intake, F1 hybrids reared by C57BL/6J dams consumed more EtOH during forced exposure than did F1 hybrids reared by DBA/2J dams. Maternal strain also influenced EtOH-induced hypothermic responses in F1 hybrids, producing differences in hybrid mice that paralleled those of the inbred strains. In contrast, maternal strain did not influence EtOH-induced activity or CPP in hybrid mice. The current findings indicate that maternal environment may contribute to variance in EtOH-induced hypothermia and EtOH intake, although effects on EtOH intake appear to be dependent upon the type of EtOH exposure.
As research progresses in clarifying the contribution of specific genes to individual differences in EtOH sensitivity, understanding the interplay of genetic and environmental factors becomes increasingly important. Early life experiences such as maternal separation produce a variety of neurochemical, hormonal and behavioral alterations (Huot et al. 2001; Jaworski et al. 2005; Moffett et al. 2007). The effects of maternal separation appear to be a result of changes in dam–pup interactions (Marmendal et al. 2004) as corroborated by reports that similar effects are observed when dams show natural differences in maternal care (Caldji et al. 2000; Liu et al. 1997, 2000). Importantly, variations in maternal behavior can be transmitted in a nongenomic fashion (Francis et al. 1999a) and early environment can influence animals in a genotype-specific manner (Barr et al. 2004; Roman et al. 2005), underscoring the difficulty of disentangling maternal environmental and genetic effects. Ova transfer and cross-fostering are among the laboratory techniques available to isolate maternal effects in animals. However, using such techniques may be complicated by independent effects of the procedures themselves (Bartolomucci et al. 2004; Randall & Lester 1975).
Reciprocal F1 hybrids provide an excellent tool to assess the effects of maternal environment on offspring with the same genotype (aside from sex chromosome-linked traits and genomic imprinting) while minimizing experimental intervention. Maternal effects on EtOH preference have been reported in reciprocal F1 hybrids of 129/J and C57BL/6ByJ strains (Bachmanov et al. 1996) but may be underestimated in many studies because males that are co-housed with their offspring engage in pup care (Priestnall & Young 1978), minimizing effects of maternal strain. As naturally occurring differences in maternal behaviors have been documented in B6 and D2 strains and substrains (Brown et al. 1999; Cohen-Salmon 1987), the current study used reciprocal F1 hybrids of B6 and D2 mice exposed only to the parental environment of their maternal strain as well as inbred B6 and D2 mice reared in our facility to investigate maternal effects on EtOH intake, EtOH-induced hypothermia, EtOH-stimulated locomotor activity and EtOH-induced CPP.
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The experiments presented here examined the effects of naturally occurring variations in maternal behavior between mouse strains on EtOH-related phenotypes in reciprocal F1 hybrids of B6 and D2 inbred strains. Consistent with previous research showing that B6 and D2 mice represent extremes of EtOH responsiveness, the current study found differences between inbred B6 and D2 mice on all EtOH phenotypes examined. Importantly, in hybrid mice, maternal strain influenced EtOH intake during forced exposure and EtOH-induced hypothermic responses, indicating that maternal strain contributed to individual variance in these traits. In contrast, maternal strain did not influence EtOH intake during choice exposure, EtOH-induced activity or EtOH-induced CPP in hybrid mice. In addition to showing maternal strain effects on some EtOH responses, the current findings underscore the sensitivity of D2 inbred mice to EtOH-induced CPP and show sex differences in EtOH intake and EtOH-induced activity.
Differences in maternal care have been shown to produce sustained alterations in gene expression in offspring, altering the epigenomic state of a gene in a reversible manner (Meaney & Szyf 2005; Weaver et al. 2005) and underscoring the possibility that phenotype differences among inbred strains and/or selected mouse lines may be mediated, at least in part, by variations in maternal behavior. In fact, naturally occurring strain-dependent differences in maternal care reportedly alter behavior and physiology in cross-fostered offspring of B6 and BALB/CJ mice (Caldji et al. 2004; Priebe et al. 2005). Reciprocal F1 hybrids were chosen in the current experiment because procedures typically used to examine maternal effects such as ova transfer and cross-fostering can exert lasting effects on the offspring independent of other manipulations (Bartolomucci et al. 2004; Randall & Lester 1975). Furthermore, maternal behavior was not directly measured in the current study because such observations are a departure from standard practices, requiring additional equipment and/or human traffic that may be disruptive in a vivarium. Instead, inbred strains were used that consistently show differences in maternal behaviors such as nest building, resting with, crouching over and nursing pups, and latency to pup retrieval (Broida & Svare 1982; Brown et al. 1999; Carlier et al. 1982; Cohen-Salmon 1987). Differences in pup care between B6 and D2 dams may be partly mediated by B6 females’ larger litters (Brown et al. 1999; Carlier et al. 1982) because as disparity in litter size declines over increasing parity so too do differences in maternal behavior (Brown et al. 1999; Cohen-Salmon 1987). In the current experiment, B6-reared litters were larger than D2-reared litters, and D2-reared hybrid pups weighed more than B6-reared hybrid pups, showing an effect of maternal strain on developmental outcome in hybrid mice.
Effects of maternal strain were also observed in adult hybrid mice. B6-reared hybrids consumed more EtOH than D2-reared hybrids during forced exposure. The current finding expands the existing literature showing maternal strain effects on EtOH intake (Bachmanov et al. 1996; Randall & Lester 1975), an understandable result given the influence dams have on taste reactivity in their offspring (Bronstein et al. 1975; Galef & Henderson 1972). Although maternal strain effects in previous studies were observed during preference tests, such effects were not observed in the choice procedure used here. For example, reciprocal F1 hybrids reared by 129/J dams showed lower preference for 10% EtOH than did F1 hybrids reared by C57BL/6ByJ dams (Bachmanov et al. 1996) and D2 mice from ova transferred into B6 dams drank more 7.5% EtOH in a preference test than D2 mice from ova transferred into D2 dams, although mice arising from transferred ova of both strains drank more EtOH than did nontransferred ova control groups complicating interpretation of those results (Randall & Lester 1975). Thus, despite the inclusion of EtOH concentrations similar to those used previously, maternal strain effects were not observed in the choice procedure used here.
Differences in animals’ ages may have contributed to the disparity in findings. Randall and Lester (1975) tested younger animals than those assessed here and taste preference varies with age (O’Callaghan et al. 2002). Alternatively, incremental increases in EtOH concentration during the choice procedure in the current experiment may have attenuated differences based on maternal strain in hybrid mice because it minimized stress associated with the introduction of a novel fluid. Variations in maternal care alter anxiety-like behavior and stress responsiveness (Caldji et al. 2000, 2004; Francis et al. 1999b; Liu et al. 1997; Priebe et al. 2005); therefore, maternal strain effects may be attenuated in tasks lacking stressful components. Likewise, effects of maternal strain may be accentuated in stressful tasks such as the forced procedure in which animals had alternating days of water deprivation, indicating that differences in EtOH intake in hybrid mice in the current experiment may have resulted from differences in offspring stress responsiveness rather than alterations in sensitivity to EtOH’s reinforcing properties. These findings emphasize that procedural variations may influence the appearance of maternal effects, altering heritability estimates for phenotypes that have stressful components.
Maternal strain also influenced EtOH-induced hypothermic responses. B6-reared hybrids showed more pronounced initial hypothermic responses and greater tolerance over days than did D2-reared hybrids, extending previous research showing maternal effects on hyperthermic responses to d-amphetamine (Jori & Rutczynski 1978). Dam–pup interactions help in establishing regulatory mechanisms for pup temperature control and, thus, may influence adult temperature regulation. Nest-building differences may also contribute to differences in thermal regulation. D2 females build larger and more complete nests than pregnant B6 females (Broida & Svare 1982), which may provide a warmer environment for D2-reared pups. In fact, alterations in pup temperature may modulate the effects of maternal separation, with pups separated to a warm environment showing reduced behavioral sensitivity to amphetamine compared with pups separated to a cold environment during maternal separation (Zimmerberg & Shartrand 1992).
Maternal effects on temperature regulation may be mediated by long-lasting changes in offspring glucocorticoid levels and/or receptors that have been shown to occur in response to variations in maternal care in rats (Caldji et al. 2000; Francis et al. 1999b; Liu et al. 1997). Glucocorticoids play a role in temperature regulation by modulating brown adipose tissue thermogenic activity (Strack et al. 1995), suggesting a possible mechanism of maternal effects on temperature regulation in the current study. To our knowledge, the current findings are the first to show maternal effects on EtOH-induced hypothermia and, if such effects are mediated by glucocorticoid activity, these results suggest that maternal behavior may influence other phenotypes involving glucocorticoid responses to environmental challenges such as exposure to stressors, drug administration and immune challenges.
Maternal strain did not influence EtOH-induced activity or EtOH-induced CPP in hybrid mice, suggesting that maternal behavior does not significantly contribute to individual variance on these traits. Hybrid mice showed dose-dependent increases in EtOH-induced activity and showed EtOH-induced CPP in response to the 3.0 g/kg but not the 1.5 g/kg EtOH dose, regardless of maternal strain. In contrast, D2 mice showed CPP in response to both 1.5 and 3.0 g/kg EtOH, underscoring the sensitivity of D2 mice to EtOH reinforcement as assessed by place conditioning, a procedure that minimizes the effects of preabsorptive and/or caloric as well as activity locomotor/sedative factors on the examination of drug reward. In the current experiment, D2 but not B6 or hybrid mice showed CPP in response to place conditioning with the 1.5 g/kg EtOH dose, a dose lower than those previously shown to result in EtOH-induced CPP in D2 mice (Cunningham 1995; Cunningham et al. 1992).
Because sex chromosome linkage of genes that contribute to EtOH traits would result in male hybrids behaving more like their maternal strain for genetic and not environmental reasons, male and female mice were examined for all phenotypes. Although sex effects were observed on some measures in hybrid mice, only one significant interaction of maternal strain and sex was found (i.e. forced intake of 2% EtOH); an interaction in which differences in hybrid animals were observed in females from different maternal strains but not in males and therefore not because of sex chromosome linkages.
Consistent with previous reports, female mice drank more EtOH (Juarez & Barrios de Tomasi 1999; Lancaster & Spiegel 1992), were more sensitive to the locomotor activating effects of EtOH (Dudek et al. 1991) and displayed greater EtOH-induced hypothermia (Crabbe et al. 1989) than did males, regardless of genotype. In particular, female mice altered their EtOH intake when choice procedures were reinstituted following forced EtOH exposure, which may have implications for procedures designed to promote excessive EtOH intake following short-term deprivation (Heyser et al. 1997). Interestingly, effects of replicate were genotype-specific with inbred B6 females decreasing intake and hybrid females increasing EtOH intake over replicates; results suggesting that the EtOH deprivation effect may occur differentially in male and female mice and be dependent upon genetic background.
The current experiment provides the first systematic investigation of maternal strain effects on EtOH responses, providing evidence that differences in maternal strain contribute to individual variance in EtOH-related phenotypes with high degrees of genetic influence. Although the appearance of maternal strain effects were dependent upon the trait assessed and procedure used, the current findings expand previous research showing long-lasting effects of differences in maternal care on offspring behavior and physiology.