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Maternal Care, Hippocampal Glucocorticoid Receptors, and Hypothalamic-Pituitary-Adrenal Responses to Stress

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Science  12 Sep 1997:
Vol. 277, Issue 5332, pp. 1659-1662
DOI: 10.1126/science.277.5332.1659

Abstract

Variations in maternal care affect the development of individual differences in neuroendocrine responses to stress in rats. As adults, the offspring of mothers that exhibited more licking and grooming of pups during the first 10 days of life showed reduced plasma adrenocorticotropic hormone and corticosterone responses to acute stress, increased hippocampal glucocorticoid receptor messenger RNA expression, enhanced glucocorticoid feedback sensitivity, and decreased levels of hypothalamic corticotropin-releasing hormone messenger RNA. Each measure was significantly correlated with the frequency of maternal licking and grooming (all r's > −0.6). These findings suggest that maternal behavior serves to “program” hypothalamic-pituitary-adrenal responses to stress in the offspring.

Several years ago Levine, Denenberg, and others (1) showed that the development of hypothalamic-pituitary-adrenal (HPA) responses to stress is modified by early environmental events, including infantile stimulation [or handling (2)]. As adults, animals exposed to brief periods of handling daily for the first weeks of life show reduced pituitary adrenocorticotropic hormone (ACTH) and adrenal corticosterone (the principal glucocorticoid in the rat) responses to stress compared with nonhandled animals (3). These differences are apparent as late as 24 to 26 months of age (4), indicating that the handling effect on HPA function persists throughout life.

Glucocorticoids act at a number of neural sites to exert an inhibitory, negative-feedback effect over the synthesis of hypothalamic releasing-factors for ACTH, notably corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) (5). Postnatally handled animals show enhanced glucocorticoid negative-feedback sensitivity compared with nonhandled rats (6) and therefore decreased hypothalamic CRH and AVP mRNA expression, as well as lower levels of both CRH and AVP immunoreactivity (7). The handling effect on feedback sensitivity is mediated by an increase in glucocorticoid receptor (GR) expression in the hippocampus (8, 9), a region that has been strongly implicated in glucocorticoid negative-feedback regulation (10). The increased hippocampal GR gene expression is therefore a central feature of the handling effect on HPA responsivity to stress, resulting in increased feedback inhibition of CRH and AVP synthesis and reduced pituitary ACTH release during stress.

A number of authors (11) have proposed that the effects of postnatal handling are mediated by changes in mother-pup interactions and that the handling manipulation itself might map onto naturally occurring individual differences in maternal care. Specifically, Levine proposed that handling of the pups altered the behavior of the mother and that these differences in mother-pup interactions then mediate the effect of handling on the development of endocrine and behavioral responses to stress. The question, then, is how this maternal mediation might occur and whether such factors might contribute to naturally occurring individual differences in HPA responses to stress.

In the Norway rat, mother-pup contact occurs primarily within the context of a nest-bout, which begins when the mother approaches the litter and gathers the pups under her; she then nurses her offspring, intermittently licking and grooming the pups (12, 13). Handling results in changes in mother-pup interactions (14). Mothers of handled pups spend the same amount of time with their litters as mothers of nonhandled pups; however, mothers of handled litters had shorter, but more frequent, nest-bouts (15).

We examined the behavior of mothers of handled or nonhandled litters over the first 10 days of life, a “critical” period for the handling effect on HPA development (16). Mothers of handled pups showed increased levels of licking and grooming of pups and arched-back nursing (LG-ABN) compared with mothers of nonhandled pups (Table 1). The frequency of these two behaviors was highly correlated (r = +0.91); over 90% of the instances of licking and grooming occurred while the mother was nursing her pups in the arched-back posture. Mothers of nonhandled pups nursed no less frequently than those of handled pups (17), but tended to more frequently adopt a “blanket” or passive posture when nursing, lying over or beside the pups. These differences in licking and grooming (and the accompanying arched-back nursing posture) were the only behaviors that served to reliably distinguish mothers of handled from those of nonhandled pups.

Table 1

Mean (±SEM) number of observations (from a total of 1200) of licking and grooming in the mothers of handled or nonhandled litters (Handling study) or high– or low–LG-ABN mothers (Maternal behavior study). Differences in maternal behavior were stable over the 10-day period of observation. In neither study were there group differences in the frequency with which dams nursed pups or in pup contact (16). *P < 0.01.

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To determine whether the increased maternal licking and grooming affects the development of HPA responses to stress, we examined the relation between naturally occurring individual differences in maternal care and HPA development (18). We detected pronounced and stable individual differences in maternal licking and grooming (which again was highly correlated with arched-back nursing; r= +0.94). The variability among the dams in licking and grooming was substantial and of sufficient range to meaningfully study the relation between variations in postnatal maternal care and the development of adult responses to stress (19).

As adults, the offspring of high–LG-ABN mothers that showed significantly reduced plasma ACTH and corticosterone responses to restraint stress (20) compared with the offspring of low–LG-ABN mothers (Fig. 1). There were no differences in basal hormone levels (Fig. 1). These findings parallel those observed in handled versus nonhandled rats, which differ in stress-induced, but not basal HPA, activity (3). Moreover, the frequency of maternal licking and grooming was significantly correlated with the magnitude of the plasma ACTH (r = −0.66, P < 0.01) and corticosterone (r = −0.65, P < 0.01) responses to stress in the adult offspring. Thus, the greater the frequency of maternal licking and grooming during infancy, the lower the HPA response to stress in adulthood.

Figure 1

Mean (±SEM) plasma ACTH (top) and corticosterone (middle) responses to a 20-min period of restraint stress (solid bar) in the offspring of high- versus low-licking and grooming and arched-back nursing (LG-ABN) mothers. (*) Significantly different at P < 0.05. Two animals from each of the nine litters were randomly choosen for testing, that is,n = 8 to 10 per group. (Bottom) Scattergram for the correlation between the frequency of maternal licking and grooming during the first 10 days of life and the integrated plasma corticosterone response to stress (calculated by use of the Trapezoidal rule).

We then examined glucocorticoid feedback sensitivity in the high– and low–LG-ABN offspring by administering a bolus injection of corticosterone 3 hours before acute restraint stress (21). Corticosterone treatment suppressed plasma ACTH responses to restraint stress to a significantly greater extent in the high–LG-ABN offspring compared with their low–LG-ABN counterparts (75 ± 5 versus 37 ± 12%, respectively; P < 0.01). These findings suggest that the offspring of the high–LG-ABN mothers, like the handled animals, show increased sensitivity to the inhibitory effects of glucocorticoids on stress-induced HPA activity.

Glucocorticoid inhibition of hypothalamic CRH gene expression represents a critical feature of feedback action (5). Thus, we examined CRH mRNA expression (22) in parvocellular neurons of the paraventricular nucleus of the hypothalamus (PVNh), which send projections to the median eminence and provide the neural signal for the stimulation of ACTH release (23). CRH mRNA expression in the PVNh was significantly decreased in the offspring of high–LG-ABN mothers compared with those of low LG-ABN mothers (Fig.2). Moreover, CRH mRNA expression in the PVNh was significantly correlated with the frequency of maternal licking and grooming during the first 10 days of life (Fig. 2).

Figure 2

(Top) Mean (±SEM) levels of CRH mRNA in the PVNh in the adult offspring of high– (n = 5) versus low–LG-ABN mothers (n = 7) from in situ hybridization studies of corticotropin-releasing hormone mRNA levels. CRH mRNA levels are expressed as arbitrary absorbance units.*P < 0.001. (Bottom) Scattergram of the correlation between the frequency of maternal licking and grooming during the first 10 days of life and CRH mRNA expression in PVNh neurons in adulthood.

Considering the importance of the hippocampal GR system for negative-feedback regulation of HPA activity (10), we examined GR mRNA expression in the hippocampus of the offspring of high– and low–LG-ABN mothers (24). Across each of the hippocampal cell fields there was increased GR mRNA expression in the offspring of the high– compared to low–LG-ABN mothers (Fig.3). Again, GR mRNA levels in each cell field of the hippocampus were significantly correlated with the frequency of maternal licking and grooming (r = +0.76,P < 0.002 for the dentate gyrus; r = +0.64, P < 0.02 for the CA1 region; r= +0.79, P < 0.001 for the CA3 region) (Fig. 3).

Figure 3

(Top) Mean (±SEM) grains over individual cells (as a function of cell area) in dentate gyrus (DG), CA1, and CA3 cell fields of the hippocampus in adult offspring of high– (n = 8) versus low–LG-ABN mothers (n = 6) ) from in situ hybridization studies of GR mRNA levels.* P < 0.01; ** P < 0.001; *** P < 0.0001. (Bottom) Scattergram of the correlation between the frequency of maternal licking and grooming during the first 10 days of life and GR mRNA expression in dentate gyrus neurons in adulthood.

These findings reveal a marked similarity between the HPA responses to stress in the offspring of high–LG-ABN mothers and those of handled animals. The offspring of high–LG-ABN mothers, like handled animals, show dampened plasma ACTH and corticosterone responses to stress, increased hippocampal GR expression, enhanced glucocorticoid feedback sensitivity, and decreased hypothalamic CRH expression. There is considerable evidence for the importance of the hippocampus as a critical site for glucocorticoid feedback inhibition over hypothalamic CRH synthesis (10). Indeed, hippocampal GR levels have been directly correlated with CRH concentrations in the portal system of the anterior pituitary as well as with pituitary-adrenal activity (25). The offspring of the high–LG-ABN mothers showed increased glucocorticoid feedback sensitivity coupled with decreased hypothalamic CRH mRNA expression and, as in the handled animals, the increased hippocampal GR expression appears likely to mediate these effects.

The magnitude of the HPA response to stress in adult animals was strongly correlated with maternal licking and grooming (Figs. to 3). These findings support the hypothesis of Levine that the effect of postnatal handling on HPA development is mediated by effects on mother-pup interactions. Thus, handling increases the frequency of licking and grooming (Table 1) and these maternal behaviors are, in turn, associated with dampened HPA responsivity to stress (Figs. to 3). Tactile stimulation derived from maternal licking and grooming regulates pup physiology and affects central nervous system (CNS) development (26). Variation among dams in this form of maternal behavior appears also to be associated with the development of individual differences in neuroendocrine responses to stress.

The results of the handling study suggest that the frequency of maternal licking and grooming can be regulated by stimuli associated with the pup. Thus, handling pups consistently increased maternal licking and grooming (Table 1), effectively ensuring a consistently high level of licking and grooming by the dam. This is consistent with earlier studies showing that handling increases ultrasonic vocalizations in pups which, in turn, serve to increase maternal care, including licking and grooming (14). However, it remains possible that the differences in maternal behavior observed here are associated with factors intrinsic to the mother—such as emotionality—in which case the data presented here may, in part, offer an example of a nongenomic mode of inheritance between parent and offspring.

We believe that the effects of early environment on the development of HPA responses to stress reflect a naturally occurring plasticity whereby factors such as maternal care are able to program rudimentary, biological responses to threatening stimuli. Like humans, the Norway rat inhabits a great variety of ecological niches, each with varied sets of environmental demands. Such plasticity could allow animals to adapt defensive systems to the unique demands of the environment. Since most mammals usually spend their adult life in an environment that is either the same as or similar to the one in which they were born, developmental “programming” of CNS responses to stress in early life is likely to be of adaptive value to the adult (12, 27). Such programming affords the animal an appropriate HPA response, minimizing the need for a long and perhaps unaffordable period of adaptation in adult life. Our results suggest that this neonatal programming occurs via the differentiation of the GR system in forebrain neurons that govern HPA activity in response to variations in maternal behavior.

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