Rebecca L. Huot

Long-term behavioral and neuroendocrine adaptations to adverse early experience.

Adaptation of an organism to the environment occurs through numerous processes beginning in the prenatal period and continuing through the neonatal and early adolescent period. Environmental signals, through processes, such as activity dependent plasticity, interact with the concurrently unfolding genetic blueprint for the central nervous system giving rise to a stable, individual phenotype governing perception of, and responsiveness to, salient features of the environment. This process of adaptation may be viewed as “fine-tuning” or “environmental programming” of neural circuitry. Thus, the studies in this chapter have initially been focused on a comprehensive description of the phenotype resulting from exposure to neonatal handling or maternal separation at the level of behavior, neuroendocrine responsiveness, and the central nervous system circuitry. Seemingly beneficial adaptations in the short-term, may, under challenging environmental conditions, actually are maladaptive over the life span of the individual. Increasing basic, clinical, and epidemiological evidence supports the thesis that exposure to an adverse early environment may underlie vulnerability to, and later expression of, physio- and/or psychopathology.

Neonatal maternal separation reduces hippocampal mossy fiber density in adult Long Evans rats

Neonatal maternal separation of rat pups leads to a stable stress hyper-responsive phenotype characterized by increased basal levels of corticotropin releasing factor (CRF) mRNA in the hypothalamic and extra-hypothalamic nuclei, increased hypothalamic CRF release, and enhanced adrenocorticotrophin hormone (ACTH) and corticosterone (CORT) responses to psychological stressors. Stress and exposure to glucocorticoids either early in life or in adulthood have been associated with hippocampal atrophy and impairments in learning and memory. In this study, male Long Evans rat pups were exposed to daily 3-h (HMS180) or 15-min (HMS15) periods of maternal separation on postnatal days (PND) 2-14 or normal animal facility rearing. Maternal separation and subsequent reunion with the dam resulted in elevated plasma CORT levels versus HMS15 animals at PND7, a time when rat pups are normally hyporesponsive to stressors and show limited pituitary-adrenal responses. As adults, HMS180 rats exhibited elevated indices of anxiety, startle-induced pituitary-adrenal hyper-responsiveness, and slight, but significant impairment on acquisition in the Morris water maze task. In addition, HMS180 rats exhibited decreased mossy fiber density in the stratum oriens region of the hippocampus as measured by Timms staining, but no change in volume of the dentate gyrus. These changes may be the result of neonatal exposure to elevated glucocorticoids and/or changes in other signaling systems in response to maternal separation. Overall the results suggest that repeated, daily, 3-h maternal separations during critical periods of hippocampal development can disrupt hippocampal cytoarchitecture in a stable manner. The resulting change in morphology may contribute to the subtle, but consistent learning deficit and overall stress hyper-responsive phenotype observed in these animals.

Foster litters prevent hypothalamic-pituitary-adrenal axis sensitization mediated by neonatal maternal separation

Neonatal maternal separation of rat pups has been shown to produce long-term increases in hypothalamic-pituitary-adrenal (HPA) axis responsiveness, elevated levels of hypothalamic corticotropin releasing factor (CRF) mRNA in the hypothalamic paraventricular nucleus (PVN), and enhanced anxiety-like behavior. These effects appear to be at least partially mediated by subtle disruptions in the quality of maternal-pup interactions. This hypothesis was tested by providing half the dams with foster litters during the maternal separation paradigm, so that in those litters, only the pups and not the dams were experiencing a period of separation. The separation protocol took place daily from PND2-14 for either 15 min (HMS15, handled) or 180 min (HMS180, maternal separation). During the period of separation dams were either transferred to adjacent cages without any pups present (HMS15, HMS180) or to cages containing an age-matched foster litter (HMS15F, HMS180F). As adults, the HMS180 progeny exhibited the expected increased expression of CRF mRNA in the PVN, stress hyper-responsiveness to airpuff startle and evidence of impaired feedback both in the CORT response, as well as in response to the dexamethasone suppression test. The HMS180F rats, however, appeared to be resistant to these effects of maternal separation as they demonstrated CRF mRNA levels intermediate between HMS15 and HMS180 rats. Their stress responses and feedback regulation of the HPA axis was comparable to that of the HMS15 rats. GR mRNA was elevated in the cortex of HMS180F rats. Overall, these studies support the thesis that the long-term effects of neonatal maternal separation may largely result from alterations in the quality of maternal care rather than from direct effects of the separation per se on the pups.

Jugular vein catheterization for repeated blood sampling in the unrestrained conscious rat

The ability to obtain repeated, low-stress blood samples from adult rats enables the design of complex experiments in which time course information or evaluation of repeated treatments is necessary. Furthermore, it reduces the number of animals necessary to acquire such information and, thus, facilitates compliance with the animal use 3Rs (reduction, refinement and replacement). To this end, a microsurgical technique to collect blood samples from the right atrium through a catheter (cannula) implanted into the right external jugular vein of adult rats is described. Rats tolerate this simple and efficient vascular access technique as evidenced by the absence of overt morbidity or abnormal behaviors. Blood is easily sampled while the rats reside in their home cages. Because the sample volume is replaced, repeated sampling is possible without compromising blood volume. Successful adoption of this procedure by other investigators will be aided by the photographic illustrations accompanying this detailed description of the procedure. Application of this technique to monitor temporal changes in plasma stress hormones during stressor paradigms as well as after behavioral and pharmacological challenges is discussed.

Differential neuroendocrine responses to chronic variable stress in adult Long Evans rats exposed to handling-maternal separation as neonates

Burgeoning evidence supports a preeminent role for early- and late-life stressors in the development of physio- and psychopathology. Handling-maternal separation (HMS) in neonatal Long Evans hooded rats leads to stable phenotypes ranging from resilient to vulnerable to later stressor exposure. Handling with 180 min of maternal separation yields a phenotype of stress hyper-responsiveness associated with facilitation of regional CRF neurocircuits and glucocorticoid resistance. This study assessed whether or not prolonged HMS (180 min/day, HMS180) on post-natal days 2-14 sensitizes the adult limbic hypothalamo-pituitary-adrenal (LHPA) axis to chronic variable stress (CS) compared to brief HMS (15 min/day, HMS15). We examined regional mRNA densities of corticotropin-releasing factor (CRF), its receptor CRF1, glucocorticoid receptor (GR), and mineralocorticoid receptor (MR); regional CRF1 and CRF2alpha binding, and pituitary-adrenal responses to an acute air-puff startle (APS) stressor in four groups: HMS15, nonstressed; HMS15, stressed; HMS180, nonstressed; HMS180, stressed. As expected we observed exaggerated pituitary-adrenal responses to APS, increased regional CRF mRNA density, decreased regional CRF1 binding, and decreased cortical GR mRNA density in nonstressed HMS180 vs. HMS15 animals. However, in contrast to our hypothesis, CS decreased pituitary-adrenal reactivity and central amygdala CRF mRNA density in HMS180 rats, while increasing cortical GR mRNA density and CRF1 binding. CS had no effect on the pituitary-adrenal response to APS in HMS15 rats, despite tripling hypothalamic paraventricular CRF mRNA density. The data suggest that many effects of prolonged HMS are reversible in adulthood by CS, while the neuroendocrine adaptations imbued by brief HMS are sufficiently stable to restrain pituitary-adrenal stress responses even following CS.

Pubertal neuromaturation, stress sensitivity, and psychopathology

Normal adolescent development is often accompanied by transient emotional and behavioral problems. For most individuals with postpubertal-onset adjustment problems, there is a resolution by early adulthood and relative stability through the adult life span. But for a minority, adjustment problems escalate during adolescence and portend the development of serious mental illness in adulthood. In this article, we explore adolescent behavioral changes and neurodevelopmental processes that might contribute to stress sensitivity and vulnerability for the emergence of the mental disorders. Of particular interest is the role that hormonal changes might play in the expression of genetic vulnerabilities for psychopathology. Drawing on recent findings from clinical research and behavioral neuroscience, we describe the ways in which postpubertal hormones might alter brain function and, thereby, behavior. It is concluded that there are both activational and organization effects of hormones on the adolescent brain, and these contribute to developmental discontinuities in behavioral adjustment. Implications for adult psychopathology and preventive intervention are discussed.

Negative Affect in Offspring of Depressed Mothers Is Predicted by Infant Cortisol Levels at 6 Months and Maternal Depression during Pregnancy, but Not Postpartum

Abstract: This study tests the hypothesis that maternal depression during pregnancy predicts temperament in offspring aged 6 m to 5 y. Previous studies have shown that maternal depression is related to negative affect and that certain temperament factors, such as negative affect and behavioral inhibition, in children predict affective disorders. Here, maternal depression is divided into depression during pregnancy vs. depression postpartum. Maternal depression was determined by the Beck Depression Inventory (BDI) throughout pregnancy and postpartum (prospectively) and by a diagnostic interview (SCID) at 6 months postpartum. The data show that maternal depression during pregnancy, but not postpartum, predicted the ratings of negative affect in the offspring. Importantly, symptoms of depression in the mother (BDI) were used as a control variable in the analyses in order to control for potential bias related to the mothers mood. In addition, cortisol levels in response to a mild stressor at 6 months of age predicted negative affect in infants and toddlers. We conclude that the effects of maternal depression on behavioral problems and vulnerability to mental illness may be mediated by altered temperament and enhanced stress responsiveness.

Postnatal maternal separation elevates the expression of the postsynaptic protein kinase C substrate RC3, but not presynaptic GAP-43, in the developing rat hippocampus.

We have shown that exposure of rats to neonatal handling/maternal separation results in mossy fiber axon hypoplasia in field CA3 of the hippocampus. To better understand the molecular basis of this neuroanatomical alteration, the present study examined three developmentally regulated protein kinase C substrate mRNAs that are highly expressed in hippocampal granule cells during mossy fiber outgrowth: GAP-43, a presynaptic substrate implicated in axonal outgrowth, RC3 (neurogranin), a postsynaptic substrate implicated in calmodulin signaling, and MARCKS-like protein (MLP), which binds calmodulin and filamentous actin in neurons and glial cells. mRNA expression was examined by quantitative in situ hybridization in the developing [postnatal day 7 (P7), P13, P21, and P90] hippocampus (CA1, CA3, granule cells) in Long-Evans hooded rats: (1) reared under normal animal facility (AFR) conditions, (2) subjected to brief (15 min/day, HMS15), or (3) subjected to moderate (180 min/day) handling/maternal separation (HMS180) on P2–14. RC3 mRNA expression was consistently elevated in all of the hippocampal cell fields in HMS180 rats relative to HMS15 and/or AFR rats over postnatal development, but did not differ from HMS15 rats in adulthood. In contrast, neither GAP-43 mRNA nor MLP mRNA expression differed among AFR, HMS15, or HMS180 rats at any postnatal time point. Elevations in RC3 expression would be predicted to perturb calcium-calmodulin signaling that may, in turn, impair the formation and/or maintenance of mossy fiber-CA3 synapses during postnatal development.