David H. Aitken

Neonatal Handling Alters Adrenocortical Negative Feedback Sensitivity and Hippocampal Type II Glucocorticoid Receptor Binding in the Rat

Adult rats handled (H) daily for the first 3 weeks of life show a dramatically altered adrenocortical response to stress. We found that H animals secreted less ACTH and corticosterone (B) during and following the termination of stress than did nonhandled (NH) controls. In contrast, H and NH animals did not differ in basal B secretion at any point in the diurnal cycle, nor in adrenocortical responses to exogenously administered oCRF or ACTH. Moreover, the clearance rate for B was similar in H and NH animals. H animals were more sensitive than NH animals to the inhibitory effects of either B or dexamethasone on stress-induced adrenocortical activity. In a dose-response study, both glucocorticoids administered 3 h prior to testing suppressed the adrenocortical response to a 20-min restraint stress to a greater extent in the H animals. Handling increased type II, glucocorticoid receptor binding capacity in the hippocampus of adult animals (approximately 50% increase in capacity, with no change in affinity). There were no handling-induced changes in type II receptor binding capacity in the hypothalamus or pituitary, nor in type I receptor binding capacity in the hippocampus. Following chronic (5 mg/kg/day) treatment with B, hippocampal type II receptor binding capacity was significantly reduced in the B-treated H animals, compared with saline-treated H animals, and indistinguishable from saline-treated NH animals. Down-regulated H animals, like NH animals, hypersecreted B following the termination of stress in comparison to the saline-treated H animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of neonatal handling on the development of the adrenocortical response to stress: Implications for neuropathology and cognitive deficits in later life

Several years ago Levine, Denenberg, Weininger, Ader, and others described the effects of postnatal handling on the development of behavioral and endocrine responses to stress. The handling procedure usually involved removing rat pups from their cages, placing the animals together in small containers, and 15-20 min later, returning the animals to their cages and their mothers. The manipulation was performed daily for the first 21 days of life. As adults, handled (H) rats exhibited attenuated fearfulness (e.g., decreased freezing, increased exploration) in novel environments and a less pronounced increase in the secretion of 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 on these earlier handling studies, convinced that this paradigm provides a marvelous opportunity to examine how subtle variations in the early environment alter the development of specific biochemical systems in the brain, leading to stable individual differences in biological responses to stimuli that threaten homeostasis. In this work we have shown how early handling influences the neurochemical development of certain brain regions that regulate the adrenocortical response to stress. Neonatal handling increases the efficiency of this endocrine response to stress, preventing excessive exposure to the highly catabolic adrenal steroids. In later life, this effect appears to protect the animal from potentially damaging effects of these steroids, ensuring the anatomical integrity of brain structures involved in cognitive functioning.

Early postnatal handling alters glucocorticoid receptor concentrations in selected brain regions.

Norway rat pups were either handled (H) or undisturbed (nonhandled, NH) in the period between birth and weaning on Day 21. Following weaning, half of the animals in each group were housed socially (Soc), and half were housed in isolation (Isol). At 120-150 days of age, all animals were sacrificed, and the following regions were dissected and frozen at -70 °C until the time of assay: frontal cortex, hippocampus, hypothalamus, amygdala, septum, and pituitary. [3H]Dexamethasone (3H Dex) binding in each region was examined by an in vitro, cytosol, receptor assay. 3H Dex binding was significantly higher in the hippocampus of both H-Soc and H-Isol than in NH groups. In the frontal cortex, 3H Dex binding was higher in the H-Soc animals than in the H-Isol and NH-Isol animals. There were no significant handling or housing effects found in the amygdala, hypothalamus, septum, or pituitary. Thus, early postnatal handling appears to influence the development of the glucocorticoid receptor system in the hippocampus and frontal cortex. These results are discussed as providing a possible mechanism for some of the previously reported effects of early handling on the development of the pituitary-adrenal response to stress.

The effects of chronic antidepressant treatment in an animal model of anxiety

We have examined the anxiolytic activity of acute and chronic antidepressant treatment in an animal model of anxiety involving novelty-suppressed feeding. Rats were food deprived for 48 h, placed into a novel environment containing food, and the latency to begin eating was recorded. Chronic (21 days), but not acute injections of desipramine (DMI; 10 mg/kg) and amitriptyline (AMI; 10 mg/kg) significantly reduced the latency to begin eating compared to controls, but the percentage decrease was not as great as that seen with either acute or chronic treatment with diazepam (2 mg/kg) or adinazolam (20 mg/kg). A time course study indicated that at least 2 weeks of treatment was necessary to observe a significant anxiolytic effect of antidepressants. The anxiolytic effect of the antidepressants was specific to the novel environment, as 2 weeks of treatment with either diazepam or DMI did not influence the latency to begin eating in the home cage. Finally, a single dose of the central benzodiazepine receptor antagonist, Ro15-1788 (20 mg/kg), given 15 min prior to testing, did not block the anxiolytic effects of chronic DMI, while it completely eliminated the effect of chronic diazepam treatment. These data suggest that antidepressants acquire anxiolytic properties following chronic administration and that this effect appears to be independent of the benzodiazepine receptor system.

POSTNATAL HANDLING ATTENUATES CERTAIN NEUROENDOCRINE, ANATOMICAL, AND COGNITIVE DYSFUNCTIONS ASSOCIATED WITH AGING IN FEMALE RATS

Hippocampal degeneration with aging is associated with increased hypothalamic-pituitary-adrenal (HPA) activity and, in male rats, both are attenuated by postnatal handling. Considering the important sex differences in the effects of handling and in HPA responses to stress in older rats, we have examined the effects of postnatal handling on aging in females. Female, Long-Evans rats were handled (H) during the first 3 weeks of life and later compared with nonhandled (NH) controls at various ages. Handling resulted in permanently increased hippocampal type II, glucocorticoid receptor binding. Relative to H females, NH females showed increased basal corticosterone levels in later life and hypersecreted corticosterone following stress at all ages examined. Both effects are similar to those reported in males. However, unlike males, H and NH females did not differ in corticosterone levels achieved during stress, a finding that may be related to sex-dependent effects of handling on pituitary transcortin receptors. There were no differences in hippocampal neuron density in 6-month-old animals. However, the older NH animals showed considerable neuron loss in the CA1 and CA3 hippocampal cellfields. There was little or no neuron loss in the H animals. Finally, the NH animals exhibited age-related spatial memory impairments, such that by 24 months of age the performance of the NH females was profoundly worse than that of the younger NHs and same-aged H animals. These data suggest that early handling permanently alters CNS systems that regulate hypothalamic-pituitary-adrenal (HPA) function, although the effect may depend on the gender of the animal. In both males and females, however, handling appears to prevent (or minimize) increased adrenal secretion in later life and to attenuate hippocampal cell loss and spatial memory impairments.

The effects of early postnatal handling on hippocampal glucocorticoid receptor concentrations: temporal parameters

Glucocorticoid receptor (Gr) concentrations in the rat brain are low at the time of birth and increase towards adult levels during the first two weeks of life. Brief, daily handling of pups for the first 21 days postnatally has been found to permanently increase Gr concentrations within the hippocampus. Thus, the development of this neural receptor system is modifiable by environmental stimulation. The work described in this paper indicates that the handling effect on hippocampal Gr concentrations is apparent as early as Day 7 of life. Moreover, handling on Days 1-7 is as effective in altering Gr concentrations as handling for the first 3 weeks of life; handling on Days 8-14 is somewhat less effective and handling on Days 15-21 is without effect. Thus, the sensitivity of the hippocampal Gr system to this early manipulation wanes through the first 3 weeks of life as Gr concentrations reach adult levels, suggesting that handling may directly alter the number of receptor sites per cell.

Basal ACTH, corticosterone and corticosterone-binding globulin levels over the diurnal cycle, and age-related changes in hippocampal type I and type II corticosteroid receptor binding capacity in young and aged, handled and nonhandled rats.

Basal corticosterone (B) levels increase with age in the rat, a result of decreased negative-feedback inhibition of hypothalamic-pituitary-adrenal (HPA) activity. Postnatal handling increases CNS negative-feedback sensitivity and appears to attenuate some of the changes occurring in the HPA axis in later life. In the experiments described here, we have examined basal HPA function in young (6-8 months) and old (22 months), handled (H) and nonhandled (NH) rats in relation to changes in corticosteroid receptor binding. Among young animals, there were no group differences in basal adrenocorticotropin (ACTH) or B levels at any point in the diurnal cycle. In contrast, plasma ACTH and B levels during the PM phase were significantly higher in old NH animals in comparison to old H animals and to both groups of young animals. The H and NH groups did not differ in in vivo adrenal responsiveness to exogenous ACTH. As expected, ACTH sensitivity was greater in all groups during the PM phase and in general, old animals showed a greater response to ACTH regardless of the treatment group. There were no differences across the groups in AM plasma corticosterone-binding globulin (CBG) levels. However, during the PM phase of the cycle, CBG levels were significantly lower and the percentage of B in the free form was significantly higher in the old NH animals. As expected, levels of free B during the PM phase of the cycle were significantly higher in the old NH animals. Thus, there is a significant increase in the PM corticoid signal in the old NH animals that occurs as a function of elevated B and decreased CBG levels; these age-related changes in basal HPA activity were not seen in the old H animals. Type I (mineralocorticoid-like) receptor binding in the hippocampus did not differ as a function of handling and was significantly reduced with age in both H and NH animals. Type II (glucocorticoid) receptor binding decreased as a function of age in both H and NH animals, but was consistently higher in the H animals. There were no differences in type II receptor binding in the hypothalamus or pituitary as a function of age or handling. These data suggest that the increase in basal HPA activity occurring in aged rats is largely restricted to the dark phase of the cycle and is attenuated by postnatal handling, a treatment that increases hippocampal type II corticosteroid receptor binding.

Thyroid Hormones Influence the Development of Hippocampal Glucocorticoid Receptors in the Rat: A Mechanism for the Effects of Postnatal Handling on the Development of the Adrenocortical Stress Response

The role of thyroid hormones on the development of intracellular glucocorticoid receptor concentrations was examined in the hippocampus, hypothalamus, and pituitary of the rat. Adult animals, administered triiodothyronine (T3; 1.0 micrograms/g body weight) on days 1, 2, and 4 of life or thyroxine (T4; 2.5 micrograms/g body weight) on days 1 and 2 of life, had significantly elevated glucocorticoid receptor concentrations in the hippocampus, but not in hypothalamus or pituitary. Adult animals treated with propylthiouracil (PTU; 0.2% in the mothers food), a thyroid hormone synthesis inhibitor, for the first 2 weeks of life showed decreased glucocorticoid receptor concentrations in hippocampus, but not in hypothalamus or pituitary. We then examined whether thyroid hormones might mediate the effects of early stimulation on the development of hippocampal glucocorticoid receptor concentrations. Animals that were handled for 15 min daily (Ha) for the first 2 weeks of life showed increased hippocampal glucocorticoid receptor concentrations as adults compared to nonhandled (NHa) controls. PTU administration blocked the effects of handling, such that Ha/PTU animals showed hippocampal glucocorticoid receptor concentrations that were indistinguishable from those of NHa animals. In contrast, corticosterone administration over the first 2 weeks of life had no effect on adult hippocampal glucocorticoid receptor concentrations. These data suggest that thyroid hormones mediate, in part at least, the development of glucocorticoid receptor concentrations in the hippocampus and that this effect occurs independently of their effects on corticosterone titers.

The effects of postnatal handling on the development of the glucocorticoid receptor systems and stress recovery in the rat

We have examined the effects of postnatal handling of rat pups from Days 1 to 21 on the development of the intracellular, glucocorticoid receptor in the hippocampus, and pituitary transcortin, and corticoid binding globulin in plasma. All animals were sacrificed 10-14h following adrenalectomy and the in vitro receptor assays were performed using 3H dexamethasone (intracellular receptors) or [3H] corticosterone (transcortin and corticoid binding globulin). Early handling resulted in a 30-40% increase in 3H dexamethasone binding (increase in Bmax with no change in Kd) in the hippocampus. In the pituitary handling was associated with a decrease in transcortin binding. There was no effect on plasma corticoid binding globulin. When tested as adults, nonhandled animals hypersecreted corticosterone following the termination of a stressor. This suggests a more efficient adrenocortical negative-feedback system in the handled animals and these data are consistent with previous work on the relationship between hippocampal glucocorticoid receptors and adrenocortical stress recovery.

[3H]Dexamethasone binding in rat frontal cortex.

The work described in this paper presents evidence for the existence of specific glucocorticoid receptors in the rat frontal cortex. Using [3H]dexamethasone we found a Kd approximately 6 nM and a Bmax approximately 270 fmol/mg protein, concentrations that were about 75% of those found in hippocampus. [3H]dexamethasone binding in the frontal cortex, like that in hippocampus, was regulated by the corticosterone: thus, one-week treatment with corticosterone results in a decrease and long-term adrenalectomy results in an increase in [3H]dexamethasone binding. Developmentally, as reported for other brain regions, [3H]dexamethasone binding in frontal cortex was low during the first week of life and then rose during the following 10 days to approximate adult levels. These results are discussed in terms of providing a possible mechanism for the influence of corticoids on catecholamine activity in the frontal cortex.