Christian Caldji

Early environmental regulation of forebrain glucocorticoid receptor gene expression: implications for adrenocortical responses to stress.

The adrenal glucocorticoids and catecholamines comprise a frontline of defense for mammalian species under conditions which threaten homeostasis (conditions commonly referred to as stress). Glucocorticoids represent the end product of the hypothalamic-pituitary-adrenal (HPA) axis and along with the catecholamines serve to mobilize the production and distribution of energy substrates during stress. The increased secretion of pituitary-adrenal hormones in response to stress is stimulated by the release of corticotropin-releasing hormone (CRH) and/or arginine vasopressin (AVP) from neurons in the nucleus paraventricularis. In this way, a neural signal associated with the stressor is transduced into a set of endocrine and sympathetic responses. The development of the HPA response to stressful stimuli is altered by early environmental events. Animals exposed to short periods of infantile stimulation or handling show decreased HPA responsivity to stress, whereas maternal separation, physical trauma and endotoxin administration enhance HPA responsivity to stress. In all cases, these effects persist throughout the life of the animal and are accompanied by increased hypothalamic levels of the mRNAs for CRH and often AVP. The inhibitory regulation of the synthesis for these ACTH releasing factors is achieved, in part, through a negative feedback loop whereby circulating glucocorticoids act at various neural sites to decrease CRH and AVP gene expression. Such inhibitory effects are initiated via an interaction between the adrenal steroid and an intracellular receptor (either the mineralocorticoid or glucocorticoid receptor). We have found that these early environmental manipulations regulate glucocorticoid receptor gene expression in the hippocampus and frontal cortex, regions that have been strongly implicated as sites for negative-feedback regulation of CRH and AVP synthesis. When the differences in glucocorticoid receptor density are transiently reversed, so too are those in HPA responses to stress. Taken together, our findings indicate that the early postnatal environment alters the differentiation of hippocampal neurons. This effect involves an altered rate of glucocorticoid receptor gene expression, resulting in changes in the sensitivity of the system to the inhibitory effects of glucocorticoids on the synthesis of CRH and AVP in hypothalamic neurons. Changes in CRH and AVP levels, in turn, determine the responsivity of the axis to subsequent stressors; increased releasing factor production is associated with increased HPA responses to stress. Thus, the early environment can contribute substantially to the development of stable individual differences in HPA responsivity to stressful stimuli. These data provide examples of early environmental programming of neural systems. One major objective of our research is to understand how such programming occurs within the brain.

The Effects of Early Rearing Environment on the Development of GABAA and Central Benzodiazepine Receptor Levels and Novelty-Induced Fearfulness in the Rat

We compared the effects of handling or maternal separation from the day following birth until postnatal day 14 on behavioral responses to novelty and on GABAA and central benzodiazepine (CBZ) receptor levels in the rat. As adults, handled animals showed reduced startle responsivity, increased exploration in a novel open field, and decreased novelty-induced suppression of feeding relative to the handled (H) and/or maternal separation (MS) groups. As compared with handled animals, both nonhandled (NH) and MS animals displayed: (1) reduced GABAA receptor levels in the locus coeruleus (LC) and the n. tractus solitarius (NTS); (2) reduced CBZ receptor sites in the central and lateral n. of the amygdala, the frontal cortex, and in the LC and NTS; and (3) reduced levels of the mRNA for the γ2 subunit of the GABAA receptor complex, which confers high affinity BZ binding, in the amygdaloid nuclei as well as in the LC and NTS. Both the amygdala and the ascending noradrenergic systems have been considered as critical sites for the anxiolytic effects of benzodiazepines. These data suggest that early life events influence the development of the GABAA receptor system, thus altering the expression of fearfulness in adulthood.

Variations in maternal care in infancy regulate the development of stress reactivity.

Naturally occurring variations in maternal care in early postnatal life are associated with the development of individual differences in behavioral and hypothalamic-pituitary-adrenal responses to stress in the rat. These effects appear to be mediated by the influence of maternal licking/grooming on the development of central systems that serve to activate (corticotropin-releasing factor) or inhibit (gamma-aminobutyric acid) the expression of behavioral and endocrine responses to stress through effects on forebrain noradrenergic systems. Importantly, individual differences in maternal care are transmitted from mother to daughter, providing a mechanism for the behavioral transmission of individual differences in stress reactivity across generations.

Long-Term Consequences of Neonatal Rearing on Central Corticotropin-Releasing Factor Systems in Adult Male Rat Offspring

In a series of studies on the long-term consequences of neonatal rearing, we compared hypothalamic and extrahypothalamic central corticotropin-releasing factor (CRF) systems in male rats reared under conditions of animal facility rearing, nonhandling (HMS0), handling with brief maternal separation for 15 min (HMS15), or handling with moderate maternal separation for 180 min (HMS180) daily from postnatal days 2–14. CRF-like immunoreactivity (CRFir) was elevated in lumbar cerebrospinal fluid of adult HMS180 and HMS0 rats relative to the other groups. In the paraventricular nucleus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and locus coeruleus, CRFir and CRF mRNA levels were significantly elevated in HMS0 and HMS180 rats. Neonatal maternal separation was associated with regionally specific alterations in CRF receptor type 1 (CRF1) mRNA density in HMS180 rats. No rearing-associated differences in CRF2α binding were apparent in either the lateral septum or the ventromedial hypothalamus. These findings indicate that early rearing conditions can permanently alter the developmental set-point of central CRF systems, and potentially influence the expression of behavioral and endocrine responses to stress throughout life, thereby providing a possible neurobiological substrate for the relationship between early life events and increased vulnerability for hypothalamic–pituitary–adrenal axis and coping skill alterations and the frequency of mood disorders in patients with a history of such experiences.

The role of corticotropin-releasing factor–norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress

Naturally occurring variations in maternal care in early postnatal life are associated with the development of individual differences in behavioral and hypothalamic-pituitary-adrenal responses to stress in the rat. These effects appear to be mediated by the influence of maternal licking and grooming on the development of central corticotropin-releasing factor (CRF) systems, which regulate the expression of behavioral, endocrine, and autonomic responses to stress through activation of forebrain noradrenergic systems. These findings provide a neurobiologic basis for the observed relationship between early life events and health in adulthood. In more recent studies, we explored the behavioral transmission of individual differences in stress reactivity, and thus, vulnerability to stress-induced illness, across generations.

Epigenetic Programming of Stress Responses through Variations in Maternal Care

Abstract: Early life experiences shape an individuals physical and mental health across the lifespan. Not surprisingly, an upbringing that is associated with adversity can produce detrimental effects on health. A central theme that arises from studies in human and nonhuman species is that the effects of adversity are mediated by the interactions between a mother and her young. In this review we describe some of the long‐term effects of maternal care on the offspring and we focus on the impact of naturally occurring variations in the behavior of female rats. Of particular interest are mothers that engage in high or low amounts of licking/grooming (LG) and arched‐back nursing (ABN) of their pups, but do so within the normal range for this species. Such variations in LG‐ABN can alter the function of the hypothalamic‐pituitary‐adrenal (HPA) axis, and cognitive and emotional development by directly affecting the underlying neural mechanisms. At the heart of these mechanisms is gene expression. By studying the hippocampal glucocorticoid receptor gene, we have identified that maternal care regulates its expression by changing two processes: the acetylation of histones H3‐K9, and the methylation of the NGFI‐A consensus sequence on the exon 17 promoter. Sustained “maternal effects” appear elsewhere in biology, including plants, insects, and lizards, and may have evolved to program advantages in the environments that the offspring will likely face as adults. Given the importance of early life and parent‐child interactions to later behavior, prevention and intervention programs should target this critical phase of development.

Variations in maternal care alter GABA(A) receptor subunit expression in brain regions associated with fear.

Maternal care influences the development of stress reactivity in the offspring. These effects are accompanied by changes in corticotropin-releasing factor (CRF) expression in brain regions that regulate responses to stress. However, such effects appear secondary to those involving systems that normally serve to inhibit CRF expression and release. Thus, maternal care over the first week of life alters GABAA (gamma-aminobutyric acid)A receptor mRNA subunit expression. The adult offspring of mothers that exhibit increased levels of pup licking/grooming and arched back-nursing (high LG-ABN mothers) show increased α1 mRNA levels in the medial prefrontal cortex, the hippocampus as well as the basolateral and central regions, of the amygdala and increased γ2 mRNA in the amygdala. Western blot analyses confirm these effects at the level of protein. In contrast, the offspring of low LG-ABN mothers showed increased levels of α3 and α4 subunit mRNAs. The results of an adoption study showed that the biological offspring of low LG-ABN mothers fostered shortly after birth to high LG-ABN dams showed the increased levels of both α1 and γ2 mRNA expression in the amygdala in comparison to peers fostered to other low LG-ABN mothers (the reverse was true for the biological offspring of high LG-ABN mothers). These findings are consistent with earlier reports of the effects of maternal care on GABAA/benzodiazepine receptor binding and suggest that maternal care can permanently alter the subunit composition of the GABAA receptor complex in brain regions that regulate responses to stress.

Maternal behavior regulates benzodiazepine/GABAA receptor subunit expression in brain regions associated with fear in BALB/c and C57BL/6 mice.

Inbred strains of mice, such as BALB/cByJ and C57BL/6ByJ, have been used repeatedly to study genotype–phenotype relations. These strains differ on behavioral measures of fear. In novel environments, for example, BALB/c mice are substantially more neophobic than C57BL/6 animals. The benzodiazepine (BZ)/GABAA receptor system has been proposed as a regulator of behavioral responses to stress, and BALB/c and C57BL/6 mice differ in BZ/GABAA receptor binding. In the present study, we found increased BZ receptor levels in C57BL/6 mice in the central and basolateral nuclei of the amygdala as well as the locus coeruleus using either flunitrazepam (nonselective) or zolpidem (α1 subtype selective) as radioligands. Differences in receptor binding were most pronounced in the amygdala and locus coeruleus using [3H]zolpidem. C57BL/6 mice showed increased α1 mRNA levels in the locus coeuruleus compared to BALB/c mice. In addition, γ2 mRNA expression in BALB/c mice was decreased in the central nucleus of the amygdala to levels that were 2–2.5-fold lower than those of C57BL/6 mice. The results of an adoption study revealed that the biological offspring of C57BL/6 mothers fostered after birth to BALB/c dams showed decreased levels of γ2 mRNA expression in the central nucleus of the amygdala in comparison to peers fostered to other C57BL/6 mothers (the reverse was found for the biological offspring of BALB/c mothers). In a step-down exploration paradigm, BALB/cByJ mice crossfostered onto a C57BL/6ByJ dam expressed reduced anxiety responses. However, among C57BL/6ByJ mice, the relatively low levels of anxiety ordinarily evident were not increased when mice of this strain were reared by a BALB/cByJ dam. These preliminary findings suggest that the strain differences in the BZ/GABAA receptor system occur, at least in part, as a function of parental care. Such findings may reflect a mammalian example of an indirect genetic effect mediated by maternal care.

Environmental regulation of the neural epigenome

Parental effects are a major source of phenotypic plasticity. Moreover, there is evidence from studies with a wide range of species that the relevant parental signals are influenced by the quality of the parental environment. The link between the quality of the environment and the nature of the parental signal is consistent with the idea that parental effects, whether direct or indirect, might serve to influence the phenotype of the offspring in a manner that is consistent with the prevailing environmental demands. In this review we explore recent studies from the field of ‘environmental epigenetics’ that suggest that (1) DNA methylation states are far more variable than once thought and that, at least within specific regions of the genome, there is evidence for both demethylation and remethylation in post‐mitotic cells and (2) that such remodeling of DNA methylation can occur in response to environmentally‐driven, intracellular signaling pathways. Thus, studies of variation in mother–offspring interactions in rodents suggest that parental signals operate during pre‐ and/or post‐natal life to influence the DNA methylation state at specific regions of the genome leading to sustained changes in gene expression and function. We suggest that DNA methylation is a candidate mechanism for parental effects on phenotypic variation.

Maternal Care and Individual Differences in Defensive Responses

Familial transmission of mental illness is common. Recent studies in behavioral neuroscience and biological psychiatry reveal the importance of epigenetic mechanisms of transmission that center on the developmental consequences of variations in parental care. Studies with rats suggest that environmental adversity results in patterns of parent–offspring interactions that increase stress reactivity through sustained effects on gene expression in brain regions known to regulate behavioral, endocrine, and autonomic responses to stress. While such effects might be adaptive, the associated cost involves an increased risk for stress-related illness.