Plasticity in the adrenocortical response of a free-living vertebrate: the role of pre- and post-natal developmental stress

Optimal functioning of the hypothalamo–pituitary–adrenal (HPA) axis is paramount to maximizing fitness in vertebrates. Research in laboratory mammals has suggested that maternally-induced stress can cause significant variation in the responsiveness of an offspring's HPA axis involving both pre- and post-natal developmental mechanisms. However, very little is known regarding effects of maternal stress on the variability of offspring adrenocortical functioning in free-living vertebrates. Here we use an experimental approach that independently lowers the quality of both the pre- and post-natal developmental environment to examine programming and plasticity in the responsiveness of the HPA axis in fledglings of a free-living passerine, the European starling (Sturnus vulgaris). We found that mimicking a hormonal signal of poor maternal condition via an experimental pre-natal increase in yolk corticosterone decreased the subsequent responsiveness of the HPA axis in fledglings. Conversely, decreasing the quality of the post-natal developmental environment (by decreasing maternal provisioning capability via a maternal feather-clipping manipulation) increased subsequent responsiveness of the HPA axis in fledglings, apparently through direct effects on nestling body condition. The plasticity of these responses was sex-specific with smaller female offspring showing the largest increase in HPA reactivity. We suggest that pre-natal, corticosterone-induced, plasticity in the HPA axis may be a ‘predictive adaptive response’ (PAR): a form of adaptive developmental plasticity where the advantage of the induced phenotype is manifested in a future life-history stage. Further, we introduce a new term to define the condition-driven post-natal plasticity of the HPA axis to an unpredictable post-natal environment, namely a ‘reactive adaptive response’ (RAR). This study confirms that the quality of both the pre- and post-natal developmental environment can be a significant source of variation in the responsiveness of the HPA axis, and provides a frame-work for examining ecologically-relevant sources of stress-induced programming and plasticity in this endocrine system in a free-living vertebrate, respectively.     

Developmental responses to early‐life adversity: Evolutionary and mechanistic perspectives

Adverse ecological and social conditions during early life are known to influence development, with rippling effects that may explain variation in adult health and fitness. The adaptive function of such developmental plasticity, however, remains relatively untested in long‐lived animals, resulting in much debate over which evolutionary models are most applicable. Furthermore, despite the promise of clinical interventions that might alleviate the health consequences of early‐life adversity, research on the proximate mechanisms governing phenotypic responses to adversity have been largely limited to studies on glucocorticoids. Here, we synthesize the current state of research on developmental plasticity, discussing both ultimate and proximate mechanisms. First, we evaluate the utility of adaptive models proposed to explain developmental responses to early‐life adversity, particularly for long‐lived mammals such as humans. In doing so, we highlight how parent‐offspring conflict complicates our understanding of whether mothers or offspring benefit from these responses. Second, we discuss the role of glucocorticoids and a second physiological system—the gut microbiome—that has emerged as an additional, clinically relevant mechanism by which early‐life adversity can influence development. Finally, we suggest ways in which nonhuman primates can serve as models to study the effects of early‐life adversity, both from evolutionary and clinical perspectives.     

Energetics of embryonic development: effects of temperature on egg and hatchling composition in a butterfly

Phenotypic plasticity may allow an organism to adjust its phenotype to environmental needs. However, little is known about environmental effects on offspring biochemical composition and turnover rates, including energy budgets and developmental costs. Using the tropical butterfly Bicyclus anynana and employing a full-factorial design with two oviposition and two developmental temperatures, we explore the consequences of temperature variation on egg and hatchling composition, and the associated use and turnover of energy and egg compounds. At the lower temperature, larger but fewer eggs were produced. Larger egg sizes were achieved by provisioning these eggs with larger quantities of all compounds investigated (and thus more energy), whilst relative egg composition was rather similar to that of smaller eggs laid at the higher temperature. Turnover rates during embryonic development differed across developmental temperatures, suggesting an emphasis on hatchling quality (i.e. protein content) at the more stressful lower temperature, but on storage reserves (i.e. lipids) at the higher temperature. These differences may represent adaptive maternal effects. Embryonic development was much more efficient at the lower temperature, providing a possible mechanism underlying the temperature-size rule.     

Psychological stressors as a model of maternal adversity: diurnal modulation of corticosterone responses and changes in maternal behavior

Maternal adversity is associated with long-lasting consequences on cognitive development, behavior and physiological responses in rat offspring. Few studies have examined whether repeated maternal stress produces repeated activation of the hypothalamus-pituitary-adrenal (HPA) axis in mothers and whether it modifies maternal behavior. Here, we tested a novel model of perinatal stress using repeated exposure to "purely" psychological stressors throughout the gestation and lactation periods in rats. We first tested the diurnal influences of repeated 1-h strobe light exposure on maternal corticosterone secretion. Despite the hyporesponsiveness to stress documented in late pregnant and lactating mothers, we observed an enhanced response to strobe light in the afternoon compared to the morning in stressed mothers during lactation. Next, dams were exposed to 24-h forced foraging followed by 10-h wet bedding during the diurnal peak of corticosterone secretion. Although no corticosterone responses to forced foraging and wet bedding were observed, the combination of both stressors had a significant effect on maternal behavior. Mother-pup interactions were significantly altered during the first 8 days of lactation. Taken together, these findings suggest that lactating mothers maintain responsiveness to specific and repeated psychological stressors, in particular at the time of the diurnal peak in corticosterone secretion. Depending on the stressor applied, either neuroendocrine activation or changes in maternal behavior might be important determinants of the long-term consequences in the offspring. The combination of forced foraging, wet bedding and strobe light might represent a novel model of mild maternal adversity using "purely" psychological stressors.     

Maternal investment in egg size: environment- and population-specific effects on offspring performance

Geographic variation in maternal investment in offspring size can be adaptive if differences in investment translate into improved offspring performance in the given environments. We compared two moor frog, Rana arvalis, populations in the laboratory to test the hypothesis that investment in large eggs in populations originating from stressful (acid) environments improves offspring performance when reared in stressful (acid) conditions. We found that large initial size (hatchling mass) had moderate to strong, environment-dependent positive effects on larval and metamorphic traits in the acidic origin population, but only weak effects in the neutral origin population. Our results suggest that interactions between environmental conditions and initial size can be important determinants of individual performance, and that investment in large eggs is adaptive in acid environments. These findings emphasize the role of maternal effects as adaptations to environmental stress.     

Cellular mechanisms underlying the development and expression of individual differences in the hypothalamic-pituitary-adrenal stress response

Several years ago Levine, Denenberg, Ader, and others described the effects of postnatal "handling" on the development of behavioral and endocrine responses to stress. As adults, handled rats exhibited attenuated fearfulness in novel environments and a less pronounced increase in the secretion of the adrenal glucocorticoids in response to a variety of stressors. These findings clearly demonstrated that the development of rudimentary, adaptive responses to stress could be modified by environmental events. We have followed these earlier studies, convinced that this paradigm provides a marvellous opportunity to examine how subtle variations in the early environment alter the development of specific neurochemical systems, leading to stable individual differences in biological responses to stimuli that threaten homeostasis. In this work we have shown how early handling influences the development of certain brain regions that regulate glucocorticoid negative-feedback inhibition over hypothalamic-pituitary-adrenal (HPA) activity. Specifically, handling increases glucocorticoid (type II corticosteroid) receptor density in the hippocampus and frontal cortex, enhancing the sensitivity of these structures to the negative-feedback effects of elevated circulating glucocorticoids, and increasing the efficacy of neural inhibition over ACTH secretion. These effects are reflected in the differential secretory pattern of ACTH and corticosterone in handled and nonhandled animals under conditions of stress. In more recent years, using a hippocampal cell culture system, we have provided evidence for the importance of serotonin-induced changes in cAMP levels in mediating the effect of postnatal handling on hippocampal glucocorticoid receptor density. The results of these studies are consistent with the idea that environmental events in early life can permanently alter glucocorticoid receptor gene expression in the hippocampus, providing evidence for a neural mechanism for the development of individual differences in HPA function.     

The fitness costs of developmental canalization and plasticity

Organisms are capable of an astonishing repertoire of phenotypic responses to the environment, and these often define important adaptive solutions to heterogeneous and unpredictable conditions. The terms ‘phenotypic plasticity’ and ‘canalization’ indicate whether environmental variation has a large or small effect on the phenotype. The evolution of canalization and plasticity is influenced by optimizing selection‐targeting traits within environments, but inherent fitness costs of plasticity may also be important. We present a meta‐analysis of 27 studies (of 16 species of plant and 7 animals) that have measured selection on the degree of plasticity independent of the characters expressed within environments. Costs of plasticity and canalization were equally frequent and usually mild; large costs were observed only in studies with low sample size. We tested the importance of several covariates, but only the degree of environmental stress was marginally positively related to the cost of plasticity. These findings suggest that costs of plasticity are often weak, and may influence phenotypic evolution only under stressful conditions.     

Plasticity to light cues and resources in Arabidopsis thaliana: testing for adaptive value and costs

Plants shaded by neighbors or overhead foliage experience both a reduction in the ratio of red to far red light (R:FR), a specific cue perceived by phytochrome, and reduced photosynthetically active radiation (PAR), an essential resource. We tested the adaptive value of plasticity to crowding and to the cue and resource components of foliage shade in the annual plant Arabidopsis thaliana by exposing 36 inbred families from four natural populations to four experimental treatments: (1) high density, full sun; (2) low density, full sun; (3) low density, neutral shade; and (4) low density, low R:FR-simulated foliage shade. Genotypic selection analysis within each treatment revealed strong environmental differences in selection on plastic life-history traits. We used specific contrasts to measure plasticity to density and foliage shade, to partition responses to foliage shade into phytochrome-mediated responses to the R:FR cue and responses to PAR, and to test whether plasticity was adaptive (i.e., in the same direction as selection in each environment). Contrary to expectation, we found no evidence for adaptive plasticity to density. However, we observed both adaptive and maladaptive responses to foliage shade. In general, phytochrome-mediated plasticity to the R:FR cue of foliage shade was adaptive and counteracted maladaptive growth responses to reduced PAR. These results support the prediction that active developmental responses to environmental cues are more likely to be adaptive than are passive resource-mediated responses. Multiple regression analysis detected a few costs of adaptive plasticity and adaptive homeostasis, but such costs were infrequent and their expression depended on the environment. Thus, costs of plasticity may occasionally constrain the evolution of adaptive responses to foliage shade in Arabidopsis, but this constraint may differ among environments and is far from ubiquitous.     

Fearfulness Affects Quail Maternal Care and Subsequent Offspring Development

Our study investigated relationships between a precocial bird’s fearfulness and maternal care, and the implication of maternal care as a vector for non-genomic transmission of fearfulness to chicks. We compared care given to chicks between two sets of female Japanese quail selected to present either high (LTI) or low fearfulness (STI). Chicks, from a broiler line, were adopted by these females following a sensitization procedure. Chicks’ fearfulness after separation from their mother was assessed by well-established procedures. LTIs took longer to present maternal responses, pecked chicks more during the first days post-hatch, presented impaired maternal vocal behaviour and were globally less active than STI females. Chicks mothered by LTIs presented more fearful reactions than did chicks mothered by STIs, supporting the hypothesis of a non-genetic maternal transmission of fearfulness. We suggest that the longer latencies required by LTIs to become maternal are a consequence of their greater fear of chicks, and that their lower general and vocal activity could be components of a heightened antipredatory strategy. We discuss the transmission of maternal fearfulness to fostered chicks, taking into account the possible implication of several well-known mechanisms underlying maternal effects.     

Population size and identity influence the reaction norm of the rare,endemic plant Cochlearia bavarica across a gradient of environmental stress

Habitat degradation and loss can result in population decline and genetic erosion, limiting the ability of organisms to cope with environmental change, whether this is through evolutionary genetic response (requiring genetic variation) or through phenotypic plasticity (i.e., the ability of a given genotype to express a variable phenotype across environments). Here we address the question whether plants from small populations are less plastic or more susceptible to environmental stress than plants from large populations. We collected seed families from small (<100) versus large natural populations (>1,000 flowering plants) of the rare, endemic plant Cochlearia bavarica (Brassicaceae). We exposed the seedlings to a range of environments, created by manipulating water supply and light intensity in a 2 x 2 factorial design in the greenhouse. We monitored plant growth and survival for 300 days. Significant effects of offspring environment on offspring characters demonstrated that there is phenotypic plasticity in the responses to environmental stress in this species. Significant effects of population size group, but mainly of population identity within the population size groups, and of maternal plant identity within populations indicated variation due to genetic (plus potentially maternal) variation for offspring traits. The environment x maternal plant identity interaction was rarely significant, providing little evidence for genetically- (plus potentially maternally-) based variation in plasticity within populations. However, significant environment x population-size-group and environment x population-identity interactions suggested that populations differed in the amount of plasticity, the mean amount being smaller in small populations than in large populations. Whereas on day 210 the differences between small and large populations were largest in the environment in which plants grew biggest (i.e., under benign conditions), on day 270 the difference was largest in stressful environments. These results show that population size and population identity can affect growth and survival differently across environmental stress gradients. Moreover, these effects can themselves be modified by time-dependent variation in the interaction between plants and their environment.     

Prenatal exposure to maternal depression,neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses

Background: In animal models, variations in early maternal care are associated with differences in hypothalamic-pituitary-adrenal (HPA) stress response in the offspring, mediated via changes in the epigenetic regulation of glucocorticoid receptor (GR) gene (Nr3c1) expression. Objective: To study this in humans, relationships between prenatal exposure to maternal mood and the methylation status of a CpG-rich region in the promoter and exon 1F of the human GR gene (NR3C1) in newborns and HPA stress reactivity at age 3 months were examined. Methods: The methylation status of a CpG-rich region of the NR3C1 gene, including exon 1F, in genomic DNA from cord blood mononuclear cells was quantified by bisulfite pyrosequencing in infants of depressed mothers treated with a serotonin reuptake inhibitor antidepressant (SRI) (n=33), infants of depressed non treated mothers (n=13) and infants of non depressed/non treated mothers (n=36). To study the functional implications of the newborn methylation status of NR3C1 in newborns, HPA function was assessed at 3 months using salivary cortisol obtained before and following a non noxious stressor and at a late afternoon basal time. Results: Prenatal exposure to increased third trimester maternal depressed/anxious mood was associated with increased methylation of NR3C1 at a predicted NGFI-A binding site. Increased NR3C1 methylation at this site was also associated with increased salivary cortisol stress responses at 3 months, controlling for prenatal SRI exposure, postnatal age, and pre and postnatal maternal mood. Conclusions: Methylation status of the human NR3C1 gene in newborns is sensitive to prenatal maternal mood and may offer a potential epigenetic process that links antenatal maternal mood and altered HPA stress reactivity during infancy.     

Resilience and vulnerability are dose-dependently related to neonatal stressors in mice

Early life experiences have been shown to adjust cognitive abilities, stress reactivity, fear responses and immune activity in adult mammals of many species. However, whereas severe stressors have been generally associated with the emergence of hypothalamic pituitary adreno-cortical (HPA)-mediated pathology, mild neonatal stressful experiences have been traditionally associated with ‘positive’ effects or resilience. External stressors stimulate the HPA axis to induce a corticosterone secretion in mouse dams, which, in turn is directly transmitted to the progeny through lactation. Such corticosteroid transfer may offer a unitary mechanism whereby early low corticosterone exposure may favor resilience in the offspring and high corticosterone increase vulnerability to pathology. In this study we further investigated this hypothesis by evaluating the long-term effects of a neonatal exposure to low (33 mg/l) and high (100 mg/l) doses of corticosterone during the first 10 days of life in outbred CD-1 mice through supplementation in the maternal drinking water. Offspring attentional set-shifting abilities, central neurotrophic regulation and levels of natural auto-antibodies (na-Abs) directed to serotonin (SERT) and dopamine (DAT) transporters were assessed in adulthood. While low levels of neonatal corticosterone improved adult cognitive abilities and increased na-Abs levels directed to SERT, high doses of neonatal corticosterone reduced hippocampal BDNF levels and na-Abs directed to DAT. These findings confirm and extend our previous findings, supporting the view that both adaptive plasticity and pathological outcomes in adulthood may depend on circulating neonatal corticosterone levels and that these effects follow a U-shaped profile.     

Epigenetic mechanisms of perinatal programming of hypothalamic-pituitary-adrenal function and health

Environmental effects on the materno-foetal interaction determine birth outcomes that predict health over the lifespan. Thus, maternal undernutrition or stress associate with low birth weight, leading to an increased risk of metabolic and cardiovascular illness in the offspring. We argue that these effects are, in part, mediated by direct and indirect effects on the hypothalamic-pituitary-adrenal (HPA) axis such that (i) the effect of maternal adversity on foetal growth is mediated by adrenal glucocorticoids and (ii) environmental adversity alters maternal physiology and behaviour, which then programs HPA activity in the offspring.     

下丘脑-垂体-肾上腺皮质轴应激反应的中枢控制

应激反应是所有生物对紧张性事件的适应性反应,对生物的存活具有十分重要的意义。应激反应的主要特征是下丘脑－垂体－肾上腺皮质（ＨＰＡ）轴激活。ＨＰＨ轴激活的呆区控制十分复杂。海马参与整合感知的信息、解释环境信息的意义及定调行为反应和神经内分泌反应。杏仁核是应激性行为反应以及自主神经和神经内分泌反应的行旅地部位。下丘脑室６ 有直接激活ＨＰＡ轴的作用。负反馈机制、下丘脑局部回路和细胞因子也可能参与了调节Ｈ     

Does phenotypic plasticity initiate developmental bias?

The generation of variation is paramount for the action of natural selection. Although biologists are now moving beyond the idea that random mutation provides the sole source of variation for adaptive evolution, we still assume that variation occurs randomly. In this review, we discuss an alternative view for how phenotypic plasticity, which has become well accepted as a source of phenotypic variation within evolutionary biology, can generate nonrandom variation. Although phenotypic plasticity is often defined as a property of a genotype, we argue that it needs to be considered more explicitly as a property of developmental systems involving more than the genotype. We provide examples of where plasticity could be initiating developmental bias, either through direct active responses to similar stimuli across populations or as the result of programmed variation within developmental systems. Such biased variation can echo past adaptations that reflect the evolutionary history of a lineage but can also serve to initiate evolution when environments change. Such adaptive programs can remain latent for millions of years and allow development to harbor an array of complex adaptations that can initiate new bouts of evolution. Specifically, we address how ideas such as the flexible stem hypothesis and cryptic genetic variation overlap, how modularity among traits can direct the outcomes of plasticity, and how the structure of developmental signaling pathways is limited to a few outcomes. We highlight key questions throughout and conclude by providing suggestions for future research that can address how plasticity initiates and harbors developmental bias.     

Constraints on the evolution of adaptive phenotypic plasticity in plants

The high potential fitness benefit of phenotypic plasticity tempts us to expect phenotypic plasticity as a frequent adaptation to environmental heterogeneity. Examples of proven adaptive plasticity in plants, however, are scarce and most plastic responses actually may be 'passive' rather than adaptive. This suggests that frequently requirements for the evolution of adaptive plasticity are not met or that such evolution is impeded by constraints. Here we outline requirements and potential constraints for the evolution of adaptive phenotypic plasticity, identify open questions, and propose new research approaches. Important open questions concern the genetic background of plasticity, genetic variation in plasticity, selection for plasticity in natural habitats, and the nature and occurrence of costs and limits of plasticity. Especially promising tools to address these questions are selection gradient analysis, meta-analysis of studies on genotype-by-environment interactions, QTL analysis, cDNA-microarray scanning and quantitative PCR to quantify gene expression, and two-dimensional gel electrophoresis to quantify protein expression. Studying plasticity along the pathway from gene expression to the phenotype and its relationship with fitness will help us to better understand why adaptive plasticity is not more universal, and to more realistically predict the evolution of plastic responses to environmental change.     

Inbreeding and adaptive plasticity: an experimental analysis on predator‐induced responses in the water flea Daphnia

Several studies have emphasized that inbreeding depression (ID) is enhanced under stressful conditions. Additionally, one might imagine a loss of adaptively plastic responses which may further contribute to a reduction in fitness under environmental stress. Here, we quantified ID in inbred families of the cyclical parthenogen Daphnia magna in the absence and presence of fish predation risk. We test whether predator stress affects the degree of ID and if inbred families have a reduced capacity to respond to predator stress by adaptive phenotypic plasticity. We obtained two inbred families through clonal selfing within clones isolated from a fish pond. After mild purging under standardized conditions, we compared life history traits and adaptive plasticity between inbred and outbred lineages (directly hatched from the natural dormant egg bank of the same pond). Initial purging of lineages under standardized conditions differed among inbred families and exceeded that in outbreds. The least purged inbred family exhibited strong ID for most life history traits. Predator‐induced stress hardly affected the severity of ID, but the degree to which the capacity for adaptive phenotypic plasticity was retained varied strongly among the inbred families. The least purged family overall lacked the capacity for adaptive phenotypic plasticity, whereas the family that suffered only mild purging exhibited a potential for adaptive plasticity that was comparable to the outbred population. We thus found that inbred offspring may retain the capacity to respond to the presence of fish by adaptive phenotypic plasticity, but this strongly depends on the parental clone engaging in selfing.     

Effects of early life adversity on cortisol/salivary alpha-amylase symmetry in free-ranging juvenile rhesus macaques

Early life adversity (ELA) affects physiological and behavioral development. One key component is the relationship between the developing Hypothalamic-Pituitary-Adrenal (HPA) axis and the Sympathetic Nervous System (SNS). Recent studies suggest a relationship between early life adversity and asymmetry in cortisol (a measure of HPA activation) and salivary alpha-amylase (sAA: a correlate of SNS activation) responses to stress among human children, but to our knowledge there have been no comparable studies in nonhumans. Here, we investigate the responses of these two analytes in “low stress” and “high stress” situations in free-ranging juvenile rhesus macaques (Macaca mulatta) on Cayo Santiago, Puerto Rico. Behavioral data on maternal maltreatment were collected during the first 3 months of life to determine individual rates of ELA, and saliva samples were collected from subjects noninvasively during juvenility. Irrespective of ELA, salivary alpha-amylase levels were lower in low stress situations and higher in high stress situations. For cortisol however, high ELA subjects exhibited higher low stress concentrations and blunted acute responses during high stress situations compared to moderate and low ELA subjects. Cortisol and sAA values were positively correlated among low ELA subjects, suggesting symmetry, but were uncorrelated or negatively correlated among moderate and high ELA subjects, suggesting asymmetry in these individuals. These findings indicate dysregulation of the stress response among juveniles maltreated during infancy: specifically, attenuated cortisol reactivity coupled with typical sAA reactivity characterize the stress response profiles of juveniles exposed to higher rates of ELA during the first 3 months of life.     

Animal learning as a source of developmental bias

As a form of adaptive plasticity that allows organisms to shift their phenotype toward the optimum, learning is inherently a source of developmental bias. Learning may be of particular significance to the evolutionary biology community because it allows animals to generate adaptively biased novel behavior tuned to the environment and, through social learning, to propagate behavioral traits to other individuals, also in an adaptively biased manner. We describe several types of developmental bias manifest in learning, including an adaptive bias, historical bias, origination bias, and transmission bias, stressing that these can influence evolutionary dynamics through generating nonrandom phenotypic variation and/or nonrandom environmental states. Theoretical models and empirical data have established that learning can impose direction on adaptive evolution, affect evolutionary rates (both speeding up and slowing down responses to selection under different conditions) and outcomes, influence the probability of populations reaching global optimum, and affect evolvability. Learning is characterized by highly specific, path‐dependent interactions with the (social and physical) environment, often resulting in new phenotypic outcomes. Consequently, learning regularly introduces novelty into phenotype space. These considerations imply that learning may commonly generate plasticity first evolution.     

Neonatal handling enhances contextual fear conditioning and alters corticosterone stress responses in young rats

Previous studies have indicated that neonatal handling influences development of hypothalamic-pituitary-adrenal (HPA) control of corticosterone. In addition, corticosterone influences memory consolidation processes in contextual fear conditioning. Therefore, neonatal handling may affect hippocampal-dependent memory processes present in contextual fear conditioning by influencing the development of HPA control of corticosterone. To investigate the effects of neonatal handling on early learning, rat pups were either handled (15-min removal from home cage) on the first 15 days after birth or left undisturbed in their home cage. Handled rats and nonhandled rats were fear conditioned at 18, 21, or 30 days of age and then tested at two time points--24 h following conditioning and at postnatal day 45. Subsequently, at approximately postnatal day 60, rats were exposed to restraint stress and corticosterone levels were assessed during restraint and recovery. Handled and nonhandled rats did not differ significantly in their freezing response immediately following footshock on the conditioning day. However, when tested for contextual fear conditioning at 24 h following conditioning and at postnatal day 45, handled rats showed more freezing behavior than nonhandled rats. When exposed to restraint stress, handled rats had a more rapid return of corticosterone to basal levels than nonhandled rats. These results indicate that neonatal handling enhances developmentally early memory processes involved in contextual fear conditioning and confirms previously reported effects of neonatal handling on HPA control of corticosterone.