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Nongenomic Transmission Across Generations of Maternal Behavior and Stress Responses in the Rat

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Science  05 Nov 1999:
Vol. 286, Issue 5442, pp. 1155-1158
DOI: 10.1126/science.286.5442.1155

Abstract

In the rat, variations in maternal care appear to influence the development of behavioral and endocrine responses to stress in the offspring. The results of cross-fostering studies reported here provide evidence for (i) a causal relationship between maternal behavior and stress reactivity in the offspring and (ii) the transmission of such individual differences in maternal behavior from one generation of females to the next. Moreover, an environmental manipulation imposed during early development that alters maternal behavior can then affect the pattern of transmission in subsequent generations. Taken together, these findings indicate that variations in maternal care can serve as the basis for a nongenomic behavioral transmission of individual differences in stress reactivity across generations.

Individual differences in personality traits appear to be transmitted from parents to offspring. A critical question, however, concerns the mode of inheritance. Concordance studies with mono- and dizygotic twins have provided evidence for a genetic mechanism of transmission even of complex traits (1). In addition, parental behavior influences the development of the offspring (2) and could therefore serve as a mechanism for a nongenomic behavioral mode of inheritance. In the Norway rat (Rattus norvegicus), variations in maternal care are associated with the development of individual differences in behavioral and endocrine responses to stress in the offspring (3, 4). In the studies reported here we have examined the possibility that such variations in maternal care might be the mechanism for a behavioral transmission of individual differences across multiple generations.

Mother-pup contact in the rat occurs primarily within the context of a nest bout that begins when the mother approaches the litter, gathers the pups under her, licks/grooms her pups, and nurses her offspring while continuing to occasionally lick/groom the pups, and terminates when the mother leaves the nest (5). Naturally occurring variations in maternal licking/grooming and arched-back nursing (LG-ABN) have been associated with the development of individual differences in hypothalamic-pituitary-adrenal (HPA) and behavioral responses to stress in the offspring (3,4). As adults, the offspring of high LG-ABN mothers are behaviorally less fearful and show more modest HPA responses to stress than do the offspring of low LG-ABN mothers. The variation in maternal behavior may thus constitute a mechanism for the nongenomic behavioral transmission of fearfulness from parent to offspring. Alternatively, of course, the differences in fearfulness and those in maternal behavior may both be associated with a common genotype so that the observed continuity of individual differences from mother to offspring is mediated by a genomically based pattern of inheritance.

We found that the female offspring of high LG-ABN mothers showed significantly increased licking/grooming of pups in comparison with those of low LG-ABN mothers (12.9 ± 1.0 versus 6.9 ± 1.1; P < 0.001) (6), which suggests that individual differences in maternal behavior are transmitted across generations. To determine the mode of transmission we performed a cross-fostering study with the offspring of high and low LG-ABN mothers (7). A primary concern here was that the wholesale fostering of litters between mothers is known to affect maternal behavior (8). To avoid this problem and maintain the original character of the host litter, no more than 2 of 12 pups were fostered into or from any one litter (7). The control groups included (i) the offspring of low LG-ABN mothers fostered to other low LG-ABN mothers as well as offspring of high LG-ABN mothers fostered to other high LG-ABN mothers, (ii) sham-adoption animals, which were simply removed from the nest and fostered back to their biological mothers, and (iii) unmanipulated pups of high or low LG-ABN mothers. The limited cross-fostering design did not affect group differences in maternal behavior. The frequency of pup licking/grooming (Fig. 1A) and arched-back nursing across all groups of high LG-ABN mothers was significantly greater than that for any of the low LG-ABN dams, regardless of litter composition.

Figure 1

(A) Mean ± SEM percentage frequency of licking/grooming in high LG-ABN and low LG-ABN mothers (n = 6 to 8 per group), collapsed over the first 10 days postpartum in the adoption study (6, 7). The biological offspring of high LG-ABN or low LG-ABN mothers were (i) left undisturbed with their mothers, high/control (H/C) and low/control (L/C); (ii) cross-fostered back onto their own mothers, high/w (H/w) and low/w (L/w); (iii) cross-fostered to mothers of the same group, high-high (H-H) and low-low (L-L); and (iv) cross-fostered across groups, high-low (H-L) and low-high (L-H). No more than two pups were cross-fostered from any one litter. The ANOVA revealed a significant group effect (F = 12.67; P < 0.0001). Post-hoc analysis revealed that the frequency of licking/grooming was significantly higher in each of the high LG-ABN groups as compared to any one of the low LG-ABN groups (P < 0.05; differences are indicated by a solid horizontal line). (B) Mean ± SEM time in seconds spent in the inner area of a novel open field (exploration) (9) in the adult female offspring from the cross-fostering study (n = 6 to 8 per group). The ANOVA revealed a significant effect (F = 3.37; P < 0.05) of the mother, a significant effect of cross-fostering (F = 11.88; P < 0.0001) and a significant mother × cross-fostering interaction effect (F = 7.39; P < 0.001). Post-hoc analysis revealed that the time spent in inner area exploration was significantly (P < 0.01) higher in the biological offspring of low LG-ABN mothers reared by high LG-ABN mothers (L-H) than in the offspring of high LG-ABN mothers reared by low LG-ABN mothers (H-L). Groups lying below the solid line differ significantly from those above the line. (C) Mean ± SEM percentage frequency of licking/grooming, collapsed over the first 10 days postpartum in the adult female offspring from the cross-fostering study (n = 5 to 7 per group). The ANOVA revealed a significant effect (F = 26.28; P < 0.0001) of the mother, a significant effect of cross-fostering (F = 13.56; P < 0.0001) and a significant mother × cross-fostering interaction effect (F = 8.13; P < 0.001). Post-hoc analysis revealed that the frequency of maternal licking/grooming was significantly (P < 0.001; solid line) higher in the biological offspring of low LG-ABN mothers reared by high LG-ABN mothers (L-H) than in offspring of high LG-ABN mothers reared by low LG-ABN mothers (H-L).

The biological female offspring of low LG-ABN dams reared by high LG-ABN mothers were significantly less fearful under conditions of novelty (9) than were any of the female offspring reared by low LG-ABN mothers, including the biological offspring of high LG-ABN mothers (Fig. 1B). This was also observed for male offspring (10). A separate group of female offspring was then mated, allowed to give birth, and observed for differences in maternal behavior (6). The effect on maternal behavior followed the same pattern as that for differences in fearfulness. As adults, the female offspring of low LG-ABN dams reared by high LG-ABN mothers did not differ from normal, high LG-ABN offspring in the frequency of pup licking/grooming (Fig. 1C) or arched-back nursing (10). The frequency of licking/grooming and arched-back nursing in animals reared by high LG-ABN mothers was significantly higher than in any of the low LG-ABN groups, including female pups originally born to high LG-ABN mothers but reared by low LG-ABN dams.

Postnatal handling of pups is known to increase the frequency of maternal licking/grooming and arched-back nursing (11) and to decrease the response to stress in the offspring (12). Postnatal handling should alter the phenotype of the low LG-ABN offspring, and the behavioral transmission hypothesis would suggest that these effects should then be transmitted to the next generation. To see whether an experimental manipulation that alters maternal behavior would influence the transmission of these individual differences in behavior in subsequent generations, female offspring (F1) of high or low LG-ABN mothers were mated (6), and the pups (F2) in one-half of the litters in each group were exposed daily to brief sessions of handling (11). The female offspring of high LG-ABN mothers showed significantly more licking/grooming (Fig. 2A) and arched-back nursing than did the offspring of low LG-ABN mothers. Thus, as observed in our earlier study, individual differences in maternal behavior were transmitted across generations. The handling of the pups significantly increased the frequency of maternal licking/grooming and arched-back nursing in the offspring of low LG-ABN mothers but had no effect on the offspring of high LG-ABN mothers (Fig. 2A). Thus, the effects of maternal behavior of the low LG-ABN mothers with handled pups was indistinguishable from that of the high LG-ABN mothers. The maternal behavior of the adult female offspring (F2) showed the same pattern (Fig. 2A), and this result is consistent with the transmission of individual differences in maternal behavior across generations. As adults, the handled female offspring of low LG-ABN mothers did not differ from the offspring of high LG-ABN dams in the frequency of maternal licking/grooming and arched-back nursing.

Figure 2

(A) Mean ± SEM frequency (as a percentage of total observations) of licking/grooming, collapsed over the first 10 days postpartum in high and low LG-ABN mothers (F1), with handled (H) or nonhandled (NH) pups (n = 5 to 7 per group). The ANOVA revealed a significant group × pup treatment interaction effect (F = 7.67; df = 1, 19; P < 0.01). Post-hoc analysis showed that low LG-ABN mothers with nonhandled offspring showed significantly (∗, P < 0.01) less licking/grooming than any other group, including low LG-ABN mothers with handled offspring. The same group × pup treatment interaction effect (F = 9.78; df = 1, 24; P < 0.001) in pup licking/grooming was apparent in the lactating female offspring (F2) of these mothers. Open bar, High-NH; striped bar, High/H; black bar, Low/NH; gray bar, Low/H. (B) Mean ± SEM time in seconds spent in the inner area of a novel open field (exploration) (9) in the unmanipulated adult female offspring (F3) of H or NH, high or low LG-ABN (F2) mothers (n = 8 to 10 per group). The ANOVA revealed a significant group effect (F = 3.39; df = 3, 31; P < 0.05). Post-hoc analysis revealed that the time spent in inner area exploration was significantly lower in the offspring of the low LG-ABN/NH animals than in any other group (∗, P < 0.05). (C) A pseudocolor image of representative sections showing relevant brain regions from in situ hybridization studies examining GR mRNA expression in the dorsal hippocampus and CRF mRNA expression in the PVNh in the unmanipulated adult female offspring (F3) of high LG-ABN/NH, high LG-ABN/H, low LG-ABN/NH, and low LG-ABN/NH (F2) mothers (n = 4 per group). (D) Mean ± SEM levels of GR mRNA (arbitrary optical density units using [35S]-labeled standards) (14) in Ammon's Horn (CA1) and the dentate gyrus (DG) in the unmanipulated adult female offspring (F3) of high LG-ABN/NH, high LG-ABN/H, low LG-ABN/NH, and low LG-ABN/NH (F2) mothers (n = 4 per group). The two-way ANOVA (group × region) revealed a significant group effect (F = 7.74; df = 3, 12; P < 0.01). Post-hoc analysis showed that for both the DG (P < 0.05) and the CA1 (P < 0.002) regions, GR mRNA levels were significantly lower (∗) in the offspring of the low LG-ABN/NH animals than in any other group. Bar shading is the same as in (A). (E) Mean ± SEM levels of CRF mRNA (arbitrary optical density units using [35S]-labeled standards) (14) in the PVNh in the unmanipulated adult female offspring (F3) of high LG-ABN/NH, high LG-ABN/H, low LG-ABN/NH, and low LG-ABN/NH (F2) mothers (n = 4 to 5 per group). The ANOVA revealed a significant group effect (F = 4.11; df = 3, 15; P < 0.05). Post-hoc analysis revealed that in both regions mRNA levels were significantly highly in the offspring of the low LG-ABN/NH animals than in any other group (∗, P < 0.05). (F) Mean ± SEM levels of CBZ receptor binding (femtomoles per milligram protein) (15) in the basolateral nucleus (BLnA) and central nucleus (CnA) regions of the amygdala and the ventromedial nucleus of the hypothalamus (VMH) of unmanipulated, adult female offspring (F3) of high LG-ABN/NH, high LG-ABN/H, low LG-ABN/NH, and low LG-ABN/NH (F2) mothers (n = 4 per group). The two-way ANOVA (group × region) revealed a marginal group effect (F = 3.04; df = 3, 12; P < 0.10) and, more important, a significant group × region interactions effect (F = 3.18; df = 6, 24; P < 0.02). Post-hoc analysis showed that for both the basolateral (P < 0.05) and the central (P < 0.002) regions of the amygdala, CBZ receptor levels were significantly (∗, P < 0.05) lower in the offspring of the low LG-ABN/NH animals than in any other group. Bar shading is the same as in (A).

The next question concerned the effective transmission of the individual differences in behavioral responses to stress in the unmanipulated offspring (F3) of these females (F2). The level of fearfulness under conditions of novelty in the male or female offspring of handled, low LG-ABN mothers, which did not differ from high LG-ABN mothers in measures of maternal behavior, was comparable to that of the offspring of high LG-ABN mothers (Fig. 2B). The postnatal handling results suggest that environmental events that affect maternal behavior can alter the pattern of transmission of individual differences in stress reactivity and maternal behavior from one generation to the next.

The effects of variation in maternal care on the development of stress reactivity are mediated by changes in the levels of expression of specific genes in brain regions that regulate behavioral and endocrine responses to stress (3, 4, 13). In comparison to the offspring of low LG-ABN mothers, the adult offspring of high LG-ABN dams showed increased hippocampal glucocorticoid receptor (GR) mRNA expression, increased central benzodiazepine (CBZ) receptor levels in the central and basolateral nuclei of the amygdala, and decreased corticotropin-releasing factor (CRF) mRNA in the paraventricular nucleus of the hypothalamus (PVNh) (3, 4). As adults, the offspring of handled, low LG-ABN mothers showed hippocampal GR mRNA levels that were comparable to those observed in the offspring of either handled (H) or nonhandled (NH) high LG-ABN mothers and were significantly higher than those in the offspring of NH/LG-ABN females (Fig. 2, C and D) (14). Moreover, the offspring of the H/low LG-ABN females showed significantly reduced CRF mRNA levels in the paraventricular nucleus of the hypothalamus in comparison to the offspring of the NH/low LG-ABN mothers (Fig. 2, C and E) (14). CRF mRNA levels in these animals were comparable to those of the offspring of H or NH high LG-ABN mothers. In previous studies, we also found that the offspring of high LG-ABN mothers show increased CBZ receptor binding in the amygdala in comparison with the offspring of low LG-ABN mothers (3,4). As expected, the adult offspring of H/low LG-ABN mothers showed CBZ receptor levels in the central and basolateral nuclei of the amygdala that were comparable to those observed in the offspring of either H or NH high LG-ABN mothers and were significantly higher than those in the offspring of NH/LG-ABN females (Fig. 2F) (15).

These findings suggest that individual differences in the expression of genes in brain regions that regulate stress reactivity can be transmitted from one generation to the next through behavior. The studies of Denenberg (16) in rodents suggested that individual differences in behavioral fearfulness to novelty could be transmitted from parent to offspring through a nongenomic mechanism of inheritance. The results of the present study support this idea and suggest that the mechanism for this pattern of inheritance involves differences in maternal care during the first week of life. In humans, social, emotional, and economic contexts influence the quality of the relationship between parent and child (17) and can show continuity across generations (18). Our findings in rats may thus be relevant in understanding the importance of early intervention programs in humans.

  • * To whom correspondence should be addressed. E-mail: mdmm{at}musica.mcgill.ca

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