*K. Gabriel, PhD, Department of Psychology, California State University, East Bay, 25800 Carlos Bee Boulevard, Hayward, CA 94542, USA. E-mail: firstname.lastname@example.org
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.
Four separate cohorts of B6 and D2 mice (7–8 weeks of age) were used as progenitors for reciprocal F1 hybrid and inbred B6 and D2 mice. Offspring from the first cohort were examined for EtOH-induced hypothermia, locomotor activity and CPP. Offspring from the second cohort were divided among all the behavioral measures. Offspring from the third cohort were used to complete testing of EtOH-induced hypothermia and offspring from the fourth cohort completed testing of EtOH-induced activity and CPP. Within each cohort, offspring from all the groups were represented in each behavioral measure examined but each individual mouse was tested on only one behaviour.
Breeding males were housed with one to two females in clear polypropylene cages (33 × 16 × 13 cm) with laboratory chow and water available ad libitum. After 1 week, males were removed and females were singly housed. Females were checked daily for the presence of pups with the day of birth designated postnatal (PN) 1. Pups were weighed on PN2, PN8, PN15 and PN22 and weaned on PN22. On PN29, animals were ear punched and housed three to four per cage in same sex, same genotype by maternal strain groups until testing in adulthood (7–8 weeks of age).
Because litter size, gender composition and parity have all been shown to influence offspring and maternal behavior (Alleva et al. 1989; Cohen-Salmon 1987; Egan & Royce 1973), only offspring from mixed-sex first litters (i.e. naive dams) with a minimum of three pups at weaning were included in these experiments. A maximum of one male and one female from each litter was assigned to an experimental group in each experiment to control for litter effects. All animals were maintained on a 12-h light:dark cycle at 22 ± 1°C with ad libitum access to food and water. All testing was conducted during the light phase. All protocols were approved by the Oregon Health & Science University Institutional Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals (National Research Council).
Mice were singly housed in standard wire hanging racks with ad libitum access to food in the cage and water available through a drinking tube. Following 7 days of acclimation, two drinking tubes (i.e. inverted 25-ml graduated cylinders), one with tap water and the other with an ascending series of EtOH concentrations (i.e. 3%, 6% and 10% v/v in tap water, 4 days at each concentration), were placed on each cage. Daily fluid consumption was recorded between 0830 and 0930 h (lights on 0700–1900 h). Body weight was measured and the positions of the drinking tubes were switched every 2 days. During the choice procedure, mean EtOH intake (g/kg), EtOH preference [EtOH intake (ml)/total fluid intake (ml)] and water intake (ml) at each EtOH concentration were calculated for each mouse.
After the choice procedure, one drinking tube was removed. Mice had 2 days exposure to a water-filled tube prior to initiation of single-bottle, forced EtOH exposure. Forced EtOH exposure consisted of alternating daily exposure to water and an ascending series of EtOH concentrations (i.e. 2–12%, increasing in 1% increments) for 22.5 h per day. Body weight and fluid consumption were measured every day between 0830 and 1000 h. During forced exposure, daily fluid intake [EtOH intake (g/kg) or water intake (ml)] was calculated for each mouse. After forced exposure was completed, mice had unlimited access to water through two drinking tubes for 2 days. The choice procedure was then repeated for the 6% and 10% EtOH concentrations.
On all days, intake measures were corrected for evaporation and spillage through EtOH and water tubes placed on empty cages. During the choice procedure, deletion of data as a result of excess spillage on more than 1 day per EtOH concentration for any mouse resulted in the removal of that mouse in calculations for that concentration. For cases of excess spillage during forced exposure, data were deleted from that day and from the following day because of possible dehydration effects.
Hypothermia procedures were consistent with those of previous reports (Crabbe et al. 1982). Mice were weighed and placed into individual, well-ventilated, nonrestraining, clear Plexiglas compartments (8 × 9 × 8 cm) with several 4-mm holes for ventilation. Approximately 90–120 min later, individual animals were removed from the chambers at 1-min intervals and placed into Plexiglas restraint tubes. Body temperatures were recorded with a TH-8 Thermalert Monitoring Thermometer with digital readout and resolution to 0.1°C (Physitemp Instruments, Clifton, NJ, USA) connected to a 1.8-cm, glycerol-lubricated metal probe that was inserted 0.8 cm into the rectum for a 5- to 10-second equilibration interval. Immediately after baseline temperature (T0) measurement, animals were removed from the restraint tube and injected with one of three EtOH doses (ip; 0, 1.5 or 3.0 g/kg). Mice were returned to their respective compartments and then similarly handled for temperature assessments at 30 (T30) and 60 (T60) min post-injection. Because of differences in T0 among the inbred and hybrid mice, EtOH-induced hypothermic responses were calculated as the mean difference between the pre-injection baseline (T0) and each of the two post-injection temperatures (T30, T60). On day 2, animals received injections with no temperature measurements. On day 3, T0, T30 and T60 were measured as on day 1 to examine the change in hypothermic response over days. Tolerance to the hypothermic effect of EtOH is calculated by comparing the mean change in hypothermic response between days 1 and 3. Increased tolerance is indicated by higher positive numbers that reflect an amelioration of the hypothermic response over days.
EtOH-induced activity and place conditioning
Place conditioning apparatus and procedures were as previously described (Cunningham 1995; Cunningham et al. 1992, 2006). Mice were run in 12 identical acrylic and aluminum chambers (30 × 15 × 15 cm) equipped with infrared photodetectors to measure activity and side position, and enclosed in separate light- and sound-attenuating enclosures (Model E10-20; Coulbourn Instruments, LeHigh Valley, PA, USA). Two sets of six photodetectors mounted at 5-cm intervals on the long walls of each box, 2.2 cm above the floor, measured general activity (10-msecond resolution). The floor of each chamber consisted of interchangeable halves made of one of two textures (grid, hole) and these tactile cues provided the conditioned stimuli (CS). Previous studies have shown that drug-naive mice spend about half their time on each floor type (Cunningham et al. 1992, 2003).
Place conditioning consisted of habituation, eight conditioning trials and a preference test, followed by an additional four conditioning trials and a second preference test. Place conditioning was extended because of reports indicating that optimal conditions for EtOH-induced CPP in B6 and D2 mice may differ (Cunningham 1995; Cunningham et al. 1992; Nocjar et al. 1999). Habituation was intended to reduce the novelty and stress associated with the procedure. During habituation, animals were weighed, injected with saline and placed on a smooth floor in the chambers for 5 min. Place conditioning sessions were conducted Monday through Friday with both preference tests occurring on a Friday.
During conditioning trials, two floor types were used (grid, hole), one of which was associated with EtOH treatment (CS+) and the other with saline (CS−). An unbiased procedure was used (Cunningham et al. 2003). Mice were randomly assigned to EtOH groups (0, 1.5, 3 g/kg; ip) and were then further randomly assigned to one of two conditioning groups (GRID+/HOLE−, GRID−/HOLE+). Mice in the GRID+ conditioning groups received EtOH prior to being placed on the grid floor (CS+ trials) and saline prior to being placed on the hole floor (CS− trials). These contingencies were reversed for mice in the GRID− groups. Animals in the 0 g/kg EtOH group received saline on both CS+ (nominal) and CS− trials to examine unconditioned floor preference. Order of exposure to the CS+ and CS− was counterbalanced within each group. During conditioning, subjects had access to the entire chamber, which contained one floor type. Activity data (counts per minute) were collected during CS+ and CS− conditioning trials and the preference tests.
During the preference tests, animals received a saline injection immediately before being placed in the chamber for 30 min. During preference tests, the chamber floor was half grid and half hole with the relative positions of each floor type counterbalanced in each conditioning group but with floor positions for each mouse the same on the two preference tests. Conditioning groups only differed in their floor–drug contingency; therefore, any differences between GRID+ and GRID− animals during the preference tests could be attributed to the development of a Pavlovian association between the CS+ floor and EtOH. Increased time on the CS+ paired floor is typically interpreted as formation of CPP (Cunningham et al. 2003, 2006).
Because of our a priori hypothesis that hybrid mice would differ depending upon their maternal strain, behavioral data in adult inbred and hybrid mice were separately analyzed by analyses of variance (anovas) for the factors of EtOH dose or concentration, maternal strain, and offspring sex. Analyses of EtOH intake in the choice procedure and CPP data included the factors of replicate and conditioning group (GRID+, GRID−), respectively. Whenever interactions involving EtOH dose or concentration complicated data interpretation, separate anovas were conducted at each dose or concentration for the factors of maternal strain and offspring sex (and additional factors as stated above). Main effects and/or interactions involving sex were further analyzed in an effort to isolate effects of sex chromosome linkages in hybrid mice. If the sexes did not differ statistically, sex was still included as a factor in any subsequent analyses but data were collapsed across sex in the figure presentation. Unless otherwise stated, data from males and females were not analyzed separately to maximize power to detect maternal effects. For all anovas, significant main effects and interactions were further analyzed by Bonferroni post hoc comparisons.
Separate anovas for maternal strain on breeding data in inbred and hybrid litters showed that B6 dams had more pups than D2 dams in both inbred [F(1, 66) = 17.86, P < 0.01] and hybrid litters [F(1, 65) = 53.15, P < 0.01]. Dam and pup weights were analyzed only if PN1 was known. In inbred litters, male:female pup ratios as well as maternal and pup body weights on PN2 and PN22 did not differ based on maternal strain. In hybrid litters, B6 dams weighed less than D2 dams on PN22 [F(1, 65) = 8.45, P < 0.01] and B6-reared pups weighed less than D2-reared pups on PN2 and PN22 [Fs(1, 65) = 30.98 and 100.62, Ps < 0.01] but male:female pup ratios did not differ (Table 1).
Table 1. Litter information (mean ± SEM)
F1 hybrid mice
B6 dam (B6 × B6)
D2 dam (D2 × D2)
B6 dam (B6 × D2)
D2 dam (D2 × B6)
Shared symbols in a row indicate groups significantly different from each other.
Two-bottle access occurred twice for each mouse, once before and once after single-bottle access. Visual inspection of Fig. 1 shows that, in inbred strains, B6 mice had higher EtOH intake and preference but lower water intake than D2 mice. Females achieved greater EtOH intake than males. Hybrid mice showed little difference based on maternal strain but female hybrids showed elevated EtOH intake and preference during the second exposure to two-bottle access. Data on EtOH intake, EtOH preference and water intake were analyzed separately for inbred and hybrid mice at each EtOH concentration for the factors of maternal strain and offspring sex. When EtOH concentration was repeated (6%, 10%), a repeated-measures factor of replicate was included in the analysis.
As expected, there were main effects and interactions involving maternal strain in inbred mice, indicating that inbred mice reared by B6 dams (i.e. B6 inbred mice) were significantly different from inbred mice reared by D2 dams (i.e. D2 inbred mice). Main effects of maternal strain showed that B6 mice consumed more EtOH [F(1, 26) = 52.93, P < 0.01] and had higher EtOH preference [F(1, 26) = 57.27, P < 0.01] than D2 mice during exposure to 3% EtOH. There was also a main effect of maternal strain on EtOH preference at 6% EtOH [F(1, 26) = 127.45, P < 0.01] with B6 mice showing higher EtOH preference than D2 mice, regardless of replicate. Main effects of maternal strain on EtOH intake during exposure to 6% and 10% EtOH [Fs(1, 26) = 122.98 and 237.29, respectively; Ps < 0.01] and on preference for 10% EtOH [F(1, 26) = 206.95, P < 0.01] were complicated by maternal strain × replicate interactions [Fs(1, 26) = 4.75, 5.98 and 7.95, respectively; Ps < 0.05] as well as by a maternal strain × sex interaction [F(1, 26) = 4.79, P < 0.05] on EtOH intake at 10% EtOH. Post hoc analyses of these interactions showed that, compared with D2 mice, B6 mice had higher EtOH intake during exposure to 6% and 10% EtOH and higher preference for 10% EtOH over both replicates (Ps < 0.05). B6 mice showed higher EtOH intake at 6% (P ≤ 0.06) and 10% EtOH (P < 0.05) and higher preference for 10% EtOH (Ps < 0.05) in their first compared with their second exposure to each EtOH concentration, whereas D2 mice showed no change over replicates. Furthermore, at 10% EtOH, female B6 mice showed elevated EtOH intake compared with male B6 mice (P < 0.05). There were main effects of maternal strain on water intake during exposure to 3% and 6% EtOH [Fs(1, 26) = 77.23 and 192.04, Ps < 0.01] and a maternal strain × replicate interaction on water intake during exposure to 10% EtOH [F(1, 26) = 27.79, P < 0.01]. D2 mice consumed more water than B6 mice at each EtOH concentration (Ps < 0.01). D2 mice also drank more water in their first than in their second exposure to 10% EtOH (P < 0.01).
In hybrid mice, there were no effects of maternal strain on EtOH intake, EtOH preference or water intake during exposure to 3% or 6% EtOH (Fs < 2.0), but there were main effects of maternal strain on EtOH preference and water intake during exposure to 10% EtOH [Fs(1, 20) = 4.39 and 6.43, respectively; Ps < 0.05]. D2-reared hybrids showed higher EtOH preference but lower water intake than B6-reared hybrids during exposure to 10% EtOH (Ps < 0.05), suggesting that higher EtOH preference was likely an artifact of lower water intake in D2-reared hybrids (see Fig. 1 for total fluid intake/day). There was also a main effect of replicate on water intake during exposure to 10% EtOH [F(1, 20) = 5.07, P < 0.05] with hybrid mice consuming less water in the second than in the first replicate (P < 0.05). During exposure to both 6% and 10% EtOH, there were replicate × sex interactions on EtOH intake [F(1, 21) = 7.14, F(1, 20) = 7.33, respectively; Ps < 0.05] and marginal replicate × sex interactions on EtOH preference [F(1, 21) = 3.83, F(1, 20) = 4.19, respectively; Ps ≤ 0.06]. Across maternal strains, female but not male hybrid mice had higher EtOH intake (Ps ≤ 0.06) and EtOH preference (Ps ≤ 0.08) in their second compared with their first exposure to each EtOH concentration. Female hybrids also had higher EtOH intake than males during their second exposure to each EtOH concentration (Ps ≤ 0.07).
EtOH intake: forced procedure
As shown in Fig. 2, EtOH intake increased in B6 but not in D2 mice as EtOH concentration increased. Hybrid B6-reared mice had higher EtOH intake than did hybrid D2-reared mice, indicating an effect of maternal strain on EtOH intake. In both inbred and hybrid mice, females had higher EtOH intake than males. Data on EtOH intake and water intake were analyzed separately for inbred and hybrid mice by repeated-measures anovas for the factors of EtOH concentration or water days, maternal strain and offspring sex.
In inbred animals, there were effects of maternal strain [F(1, 26) = 282.08, P < 0.01], sex [F(1, 26) = 35.72, P < 0.01] and EtOH concentration [F(10, 260) = 40.90, P < 0.01] as well as EtOH concentration × sex and EtOH concentration × maternal strain interactions [Fs(10, 260) > 3.04, Ps < 0.01] on EtOH intake. Further analyses of the interactions by separate two-way anovas for maternal strain and sex at each EtOH concentration showed that B6 mice had higher EtOH intake than D2 mice at all but the 2% EtOH concentration [Fs(1, 26) > 11.48, Ps < 0.01; 3% EtOH: F(1, 26) = 3.91, P = 0.05] and that females had higher EtOH intake than males at all EtOH concentrations [Fs(1, 26) > 4.86, Ps < 0.05]. At the lowest EtOH concentration (i.e. 2% EtOH), post hoc analysis of a maternal strain × sex interaction [F(1, 26) = 8.61, P < 0.01] showed that B6 females consumed more EtOH than B6 and D2 males and D2 females (Ps < 0.01). A repeated-measures anova on water intake across days showed main effects of maternal strain [F(1, 26) = 34.22, P < 0.01], sex [F(1, 26) = 5.31, P < 0.05], day [F(10, 260) = 6.46, P < 0.05] and a maternal strain × day interaction [F(10, 260) = 9.34, P < 0.01]. Overall, males drank more water than females. Further analysis of the interaction by separate maternal strain × sex anovas at each day showed that D2 mice consumed more water than B6 mice on all but the fourth day of alternating single-bottle access (i.e. the second day of water exposure, following 3% EtOH) [Fs(1, 26) > 4.42, Ps < 0.05].
In hybrid mice, there were main effects of maternal strain [F(1, 20) = 5.09, P < 0.05], sex [F(1, 20) = 28.80, P < 0.01], EtOH concentration [F(10, 200) = 152.85, P < 0.01] and an EtOH concentration × sex interaction [F(10, 200) = 3.37, P < 0.05] on EtOH intake. Overall, B6-reared hybrids drank more EtOH than D2-reared hybrids. Further analysis of the interactions by separate maternal strain × sex anovas at each EtOH concentration showed that female hybrids had higher EtOH intake than males at all EtOH concentrations [for most concentrations: Fs(1, 26) > 7.43, Ps < 0.05; 4% and 9% EtOH: Fs(1, 24) > 6.56, Ps < 0.05; 10% EtOH: F(1, 21) = 13.09, P < 0.01]. Analysis of water intake showed an effect of day [F(10, 200) = 7.79, P < 0.01] but no effects of maternal strain or sex. Water intake was higher on the sixth day of single-bottle access (i.e. the third day of water exposure, following 4% EtOH) than on all but the second day of single-bottle access (i.e. the first day of water exposure; Ps < 0.01). Water intake was also elevated on the 2nd day of single-bottle access compared with the 16th and 22nd day (Ps < 0.05).
In inbred mice, D2 mice had lower T0 than B6 mice [F(1, 89) = 23.20, P < 0.01]. In hybrid mice, separate analyses of maternal effects for each sex showed that males reared by D2 dams weighed more than males reared by B6 dams [F(1, 53) = 7.55, P < 0.01]; similar differences were not observed in either male or female inbred mice or in female hybrids (Table 2). Because of differences in T0 and body weight, hypothermic responses were calculated as a mean change from T0 for each mouse.
Table 2. Baseline body temperature and body weight [mean ± SEM (n)]
F1 hybrid mice
B6 dam (B6 × B6)
D2 dam (D2 × D2)
B6 dam (B6 × D2)
D2 dam(D2 × B6)
Shared symbols in a row indicate groups significantly different from each other.
As shown in Fig. 3, B6 mice had more pronounced EtOH-induced hypothermic responses than D2 mice on day 1. Compared with D2-reared hybrids, B6-reared hybrids showed more pronounced hypothermia responses to 3.0 g/kg EtOH on day 1 and had greater tolerance on day 3. Because of multiple EtOH dose interactions during initial EtOH dose × maternal strain × offspring sex anovas, data were subsequently analyzed by separate maternal strain × offspring sex anovas for inbred and hybrid mice at each EtOH dose. Neither inbred nor hybrid mice showed effects of maternal strain or sex on hypothermic responses to saline injection on day 1 or on tolerance to saline injection on day 3.
In inbred mice, there were main effects of maternal strain at 1.5 g/kg [F(1, 25) = 9.93, P < 0.05] and 3.0 g/kg EtOH [F(1, 27) = 14.01, P < 0.05]. D2 mice had diminished EtOH-induced hypothermic responses compared with B6 mice. In hybrid mice, there was a main effect of maternal strain on hypothermic responses to 3.0 g/kg [F(1, 34) = 5.18, P < 0.05] but not to 1.5 g/kg EtOH. D2-reared hybrids had diminished hypothermic responses to 3.0 g/kg EtOH compared with B6-reared hybrids. There was also a marginal effect of sex on hypothermic responses to 1.5 g/kg EtOH in hybrid mice [F(1, 33) = 3.82, P = 0.06]. Across maternal strains, female hybrids had more pronounced hypothermic responses to 1.5 g/kg EtOH than males (females: −0.77 ± 0.90°C; males: −0.19 ± 0.93°C).
Tolerance to the hypothermic effect of EtOH was measured by comparing responses on day 3 to those of day 1. Higher, positive numbers reflect an amelioration of the hypothermic response over days, an effect interpreted as greater tolerance to EtOH’s effect. Inbred mice showed no differences in the development of tolerance to EtOH’s hypothermic effects (Fs < 2.0). Hybrid mice showed no difference in tolerance to 1.5 g/kg EtOH, but there was a main effect of maternal strain on tolerance to 3.0 g/kg EtOH [F(1, 33) = 14.01, P < 0.01]. B6-reared hybrids showed more tolerance to the hypothermic effects of 3.0 g/kg EtOH than D2-reared hybrids.
Conditioning trial activity
Corresponding to previous reports (Phillips et al. 1994, 1995), D2 mice showed more pronounced EtOH-induced changes in activity than B6 mice (see Fig. 4). Maternal strain did not influence mean activity in the CS+ conditioning trials in hybrid mice, although female hybrids had higher CS+ trial activity than male hybrids. Data on mean conditioning activity for CS+ and CS− trials were analyzed by separate EtOH dose × maternal strain × offspring sex anovas for inbred and hybrid mice.
Analysis of mean activity counts over the six CS+ trials in inbred mice showed main effects of maternal strain [F(1, 141) = 147.25, P < 0.01] and EtOH dose [F(2, 141) = 188.62, P < 0.01] as well as maternal strain × EtOH dose [F(2, 141) = 28.49, P < 0.01] and EtOH dose × sex [F(2, 141) = 4.59, P < 0.01] interactions. Post hoc analyses of the interactions showed that both D2 and B6 mice increased activity as EtOH dose increased (0 < 1.5 < 3.0 g/kg EtOH; Ps < 0.05), and D2 mice had higher activity than B6 mice following 1.5 and 3 g/kg EtOH (Ps < 0.05). In addition, although female and male mice did not differ in response to each EtOH dose, female mice showed a substantial rise in activity in response to 3.0 g/kg EtOH, showing higher activity than either sex showed in response to 1.5 g/kg (Ps < 0.05). Separate analysis of mean activity over the six CS− trials showed a main effect of maternal strain [F(1, 141) = 17.41, P < 0.01] and EtOH dose [F(2, 141) = 18.27, P < 0.01]. D2 mice had higher activity than B6 mice in CS− trials (P < 0.01), and mice that received EtOH on CS+ days had higher activity in CS− trials than mice that received saline on CS+ days (Ps < 0.01).
In hybrid mice, there were no main effects or interactions involving maternal strain on conditioning activity but there was a main effect of sex [F(1, 154) = 4.47, P < 0.05] on mean activity in CS+ trials and main effects of EtOH dose in both CS+ and CS− trials [F(2, 154) = 346.32, F(2, 154) = 13.51; Ps < 0.01, respectively]. Across maternal strains, female hybrids had higher CS+ trial activity than male hybrids (P < 0.05). Activity in CS+ trials increased as EtOH dose increased, with 1.5 and 3.0 g/kg EtOH resulting in higher activity than saline (Ps < 0.01) and 3.0 g/kg EtOH producing higher activity than 1.5 g/kg EtOH (P < 0.01). In addition, hybrid mice that received EtOH on CS+ days had higher activity in CS− trials than mice that received saline on CS+ days (Ps < 0.01).
CPP test activity
Separate maternal strain × offspring sex × EtOH dose anovas on mean activity during the two preference tests (see Fig. 5) showed a main effect of maternal strain in inbred mice [F(1, 141) = 4.65, P < 0.05] and main effects of EtOH dose in both inbred and hybrid mice [F(2, 141) = 5.09, F(2, 154) = 5.90, respectively; Ps < 0.01]. In inbred mice, D2 mice had higher mean activity across the two preference tests than did B6 mice. In both inbred and hybrid mice, 1.5 g/kg EtOH on CS+ days resulted in higher preference test activity than did saline on CS+ days (Ps < 0.01). In hybrid mice, 1.5 g/kg EtOH on CS+ days also resulted in higher preference test activity than did 3.0 g/kg EtOH (P < 0.01).
Conditioned place preference
Differences between GRID+ and GRID− animals during preference tests are attributed to the development of a Pavlovian association between the CS+ floor and EtOH; therefore, main effects and/or interactions involving conditioning subgroup (GRID+/HOLE−, GRID−/HOLE+) are interpreted as reflecting the formation of EtOH-induced CPP (Cunningham et al. 2003, 2006). Because of multiple interactions involving EtOH dose during initial analyses, final analyses of mean time spent on the grid floor averaged across the two preference tests were conducted by separate conditioning subgroup (GRID+, GRID−) × maternal strain × offspring sex anovas for inbred and hybrid mice at each EtOH dose. During the preference tests, D2 mice showed more robust EtOH-induced CPP than B6 mice (see Fig. 5), showing CPP to 1.5 g/kg EtOH. Across strains, CPP was observed at the 3.0 g/kg EtOH dose. In hybrid mice, CPP was observed across maternal strains at the 3.0 g/kg but not at the 1.5 g/kg EtOH dose. Analyses of mean time spent on the grid floor in inbred and hybrid mice that had been conditioned with saline showed no significant effects or interactions of conditioning subgroup (Fs < 3.5), indicating an absence of unconditioned floor preference.
In inbred mice, the analysis of mean time spent on the grid floor on the two preference tests showed a main effect of conditioning subgroup [F(1, 43) = 8.49, P < 0.01] and a marginal maternal strain × conditioning subgroup interaction [F(1, 43) = 3.88, P = 0.06] at the 1.5 g/kg EtOH dose. Post hoc analysis of the interaction showed that GRID+ D2 mice spent more time on the grid floor than GRID− D2 mice (P < 0.01), indicating that D2 mice in the GRID+ and GRID− conditioning subgroups preferred their subgroups’ distinct CS+ floor type. This effect was not observed in B6 mice. At the 3.0 g/kg EtOH dose, there was a main effect of conditioning subgroup [F(1, 44) = 8.61, P < 0.01]. Regardless of maternal strain, GRID+ inbred mice spent more time on the grid floor than GRID− inbred mice, indicating the formation of EtOH-induced CPP at the 3.0 g/kg EtOH dose in both D2 and B6 mice.
In hybrid mice, there was a main effect of conditioning subgroup at the 3.0 g/kg EtOH dose [F(1, 49) = 6.37, P < 0.05] but no effects or interactions involving conditioning subgroup at the 1.5 g/kg EtOH dose (Fs < 3.0). GRID+ hybrid mice spent more time on the grid floor than did GRID− hybrid mice after conditioning with 3.0 g/kg EtOH, indicating a difference in the preferred floor type between conditioning subgroups in hybrid mice that was not influenced by maternal strain or sex.
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.
This work was supported by grants from the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health: AA13785, AA07468 and AA07702.