James W. Smythe

Sex-specific effects of prenatal stress on hypothalamic-pituitary-adrenal responses to stress and brain glucocorticoid receptor density in adult rats

Previous research indicates that the offspring of dams exposed to stress during late gestation show altered hypothalamic-pituitary-adrenal (HPA) responses to stress. However, the results are inconsistent and a review of the literature suggests that the effects may differ depending upon the gender of the offspring. In the present study, we measured plasma adrenocorticotropin (ACTH) and corticosterone (B) levels prior to, and at 0, 20, 40 and 70 min following restraint stress in catheterized adult male and female offspring of dams stressed in the last week of gestation (i.e. days 15-19 of gestation). Prenatal stress significantly increased both plasma ACTH and B levels in response to restraint, but only in females; male offspring were largely unaffected. In addition, plasma corticosteroid-binding globulin (CBG) levels were significantly increased in prenatally-stressed females, but not in males. Despite these differences in plasma CBG, estimated free B levels following restraint were also significantly elevated in prenatally-stressed females. We then examined glucocorticoid receptor binding in a variety of forebrain structures. Prenatal stress had no effect on glucocorticoid receptor density in the hypothalamus or hippocampus in either males or females. Differences in glucocorticoid receptor density across groups were observed in the septum, frontal cortex, and amygdala. However, the pattern of observed differences across the groups was not consistent with the pattern of hormonal differences. In summary, the effect of prenatal stress on HPA function is substantially more marked in females than in males. Interestingly, a similar pattern of effects on HPA activity has been reported for prenatal alcohol exposure.

Thyroid hormones, brain function and cognition: a brief review.

In addition to their role in cellular metabolic activity, thyroid hormones (THs), also regulate neural development; the central nervous system is particularly dependent on TH for normal maturation and function. Specifically, there appears to be extensive inter-reliance between TH and acetylcholine (Ach), nerve growth factor and hippocampal function. These associations led us to investigate the possible effects of thyroxine (L-T4) on performance of a spatial learning task, where cholinergic activity and hippocampal function are known to be important. Groups of rats (n=20) received saline (controls) or L-T4 at 2.5 or 5mg/kg daily for 4 days as a sub-chronic treatment, or 0, 5 or 10mg/kg doses administered every third day for 28 days prior to testing as a chronic regimen. Rats were assessed in a water maze for their ability to find a submerged or visible platform. Forty minutes prior to water maze testing, half the animals in each group received 1mg/kg scopolamine to elicit a cognitive deficit. Following testing, rats were decapitated, blood samples taken, and the frontal cortex and hippocampus were dissected out for acetylcholinesterase (AChE) assay. The results showed that L-T4 treatment, administered both sub-chronically and chronically, significantly enhanced the ability of rats to learn a spatial memory task, compared with controls. Moreover, both short-term and long-term L-T4 treatment reduced the cognitive-impairing effects of scopolamine. Improvements in performance were shown to occur alongside significantly increased cholinergic activity in frontal cortex and in the hippocampus of treated animals. These findings demonstrate an augmentative effect of L-T4 upon cognitive function, possibly mediated by an enhancement of cholinergic activity. The results support previous findings of a relationship between L-T4 and acetylcholine, and underscore possible mechanisms by which disorders of thyroid function may be associated with cognitive decline.

Neonatal handling alters serotonin (5-HT) turnover and 5-HT2 receptor binding in selected brain regions: relationship to the handling effect on glucocorticoid receptor expression.

Neonatal handling permanently alters hypothalamic-pituitary-adrenal responses to stress. This effect is, in part, mediated by a handling-induced increase in forebrain glucocorticoid receptor gene expression. The effect of postnatal handling on glucocorticoid receptor expression appears to be mediated by an increase in serotonin (5-HT) activity, acting via a 5-HT2 receptor with a high affinity for 5-HT (i.e. the 5-HT2H receptor). In the present study we examined the nature of the effects of handling on the relevant 5-HT systems. We found that: (1) handling increases 5-HT turnover in regions of the neonatal rat brain where glucocorticoid receptor expression is altered (i.e. the hippocampus and frontal cortex), but not in regions where glucocorticoid receptor expression in unaffected (e.g. hypothalamus and amygdala); (2) handling has no long-term effects on hippocampal or frontal cortex 5-HT turnover, and is actually associated with a decrease in 5-HT concentrations; and (3) handling does not alter 5-HT2 receptor density in the hippocampus or frontal cortex in neonates (although there are surprising effects on 5-HT2 receptor density in the frontal cortex of adult animals). Taken together these data provide further evidence for the importance of 5-HT in mediating the effects of handling on the development of glucocorticoid receptor expression, but suggest that the role of 5-HT is unique to early development; differences in glucocorticoid receptor expression in adult handled and non-handled animals are not associated with long-term differences in either 5-HT levels or 5-HT2 receptors.

Molecular basis for the development of individual differences in the hypothalamic-pituitary-adrenal stress response

Summary1.Several years ago, investigators described the effects of infantile handling on the development of hypothalamic-pituitary-adrenal (HPA) responses to stress in the rat. Rat pups exposed to brief periods of innocuous handling early in life showed reduced HPA responses to a wide variety of stressors, and the effect persists throughout the life of the animal. These effects are robust and provide an excellent model for understanding how early environmental stimuli, which are external to the organism, alter neural differentiation and, thus, neuroendocrine responsivity to stress.2.This paper reviews the endocrine mechanisms affected by early handling and our current understanding of the neural transduction of environmental events and their effects at the level of the target neurons (in the hippocampus and frontal cortex).3.In brief, handling serves to increase glucocorticoid receptor gene transcription, increasing sensitivity to glucocorticoid negative feedback regulation and, thus, altering the activity within hypothalamic corticotropin-releasing factor/vasopressin neurons. Together these changes serve to determine neuroendocrine responsivity to stress.

Hippocampal Mineralocorticoid, but Not Glucocorticoid, Receptors Modulate Anxiety-Like Behavior in Rats

Stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis is regulated by negative-feedback mechanisms in the form of cytosolic and nuclear steroid receptors, sensitive to levels of circulating corticosterone (CORT). There are two types of steroid binding sites found in the brain: (i) mineralocorticoid receptors (MR); and (ii) glucocorticoid receptors (GR). The hippocampus expresses the highest density of both MR and GR relative to other brain regions, and has long been recognized as a principal component controlling HPA axis inhibition. Because hippocampal cholinergic blockade produced anxiety-like behaviour, and affected HPA axis function, we explored if the induction of anxiety might be attributable to changes in CORT. CORT also produced anxiety, although in a qualitatively unique manner than that produced by cholinergic blockade. In the present study, we have examined if CORT-induced anxiety occurs through an interaction with hippocampal MR or GR. Adult, male Lister Hooded rats were implanted bilaterally with hippocampal cannulae, and received infusions of either the MR antagonist, spironolactone (150 ng), or the GR antagonist, RU38486 (150 ng), either 10 min or 3 h prior to being tested in the Black-White box. MR blockade, 10 min prior to testing, led to a pronounced anxiolytic effect as revealed by the increased amount of time spent in the white compartment, and increased amount of intercompartmental exploration. There was no effect of MR blockade 3 h prior to testing, and GR antagonism produced no effects at either pretreatment time. These data are the first to show that hippocampal MR are directly involved in anxiety; moreover, the time course of the effect demonstrates that a non-genomic mechanism probably underlies this response. Stress may be an important predisposing factor in the development and expression of anxiety.

Environmental regulation of the development of glucocorticoid receptor systems in the rat forebrain. The role of serotonin.

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 reduced adrenal glucocorticoid response to a variety of stressors. These findings demonstrated that the development of rudimentary, adaptive responses to stress could be modified by environmental events. In addition the handling paradigm provided a marvelous 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. The effects of handling and other environmental manipulations, such as maternal separation or early immune challenge, on the development of HPA responses to stress have been reviewed recently in detail.@ In this paper the focus is on how these early environmental events alter the differentiation of the forebrain neurons which regulate HPA function, and thus we will present only a brief summary of the neuroendocrine differences between adult animals that were handled vs nonhandled early in life. The important point to be gleaned here is that the handling effect on pituitary-adrenal responses to stress is mediated, in part at least, by changes in hippocampal glucocorticoid receptor levels. In these studies rat pups are handled from the day following birth until weaning. The animals are then housed normally in groups and tested as fully mature adults. With the exception of some subtle differences (see Ref. 7) the handling effect on HPA function is the same for both males and females. Briefly, postnatal handling reduces adult HPA responses to stress and this is apparent in plasma levels of either ACI’H or corticosterone.g-12 Interestingly, there is no difference (as best as we can detect) between handled (H) and nonhandled (NH) animals in basal levels of ACTH or corticosterone.lOJ1 At the level of the paraventricular n. of the hypothalamus, H animals how lower levels of the nRNAs for corticotropin releasing-hormone (CRH) and arginine vaso-

The interaction between prenatal stress and neonatal handling on nociceptive response latencies in male and female rats

Neonatal handling produces physiological and behavioral changes that persist into adulthood. These effects are opposite to those resulting from prenatal stress (PS). We examined the interaction between PS and handling on nociception in adult male and female rats. Randomly selected pregnant rats were subjected to restraint stress on days 13-17 of gestation for 25 min each day, or left undisturbed. At birth, selected stressed/nonstressed litters were assigned to be handled. handling consisted of 15 min of separation from the dam, once per day, from postnatal days 1-14. At 4 months of age, rats were placed on a 50 degrees C hot plate, and their latencies to paw lick were recorded. Prenatal stress and handling interacted to affect latencies in male rats. Handled (H)/PS rats had significantly lower paw lick latencies than nonhandled (NH)/PS rats (p < 0.05). However, handling had no effect on the male offspring of control dams. Handling elevated paw lick latencies in the female offspring of control dams, an effect that was most pronounced in diestrous vs. estrous rats. The NH/PS rats showed significantly elevated latencies compared to NH/NS rats (p < 0.05). These results suggest that handling effects on nociception are most apparent in rats subjected to PS; in males at least, these effects would otherwise not be present.

Muscarinic antagonists are anxiogenic in rats tested in the black-white box

Central cholinergic (ACh) projections have been shown to modulate stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis and are integral to the expression of electrophysiological correlates of arousal, namely hippocampal theta rhythm. The degree to which these actions of ACh are behaviorally relevant has received comparatively less attention, and we sought to investigate if manipulations of ACh systems might also affect behaviors related to stress and arousal. We chose to examine indices of anxiety as revealed by changes in behavior elicited by the black-white box test, a relatively novel and recently validated model of rodent anxiety. Groups of rats were injected with either scopolamine hydrobromide (SCOP; 0, 0.05, and 0.10 mg/kg i.p.) or the peripherally acting scopolamine methyl bromide (methyl-SCOP; 0, 0.05, and 0.10 mg/kg i.p.) to compare and contrast the effects of central and peripheral ACh blockade on measures of anxiety. SCOP pretreatment significantly lowered latencies for rats to escape from the white to black compartment, while methyl-SCOP elevated latencies to reenter the white chamber from the black. Both drugs increased the amount of time rats spent in the black compartment and also suppressed exploration as revealed by decreased episodes of intercompartmental locomotion. Neither drug deleteriously affected locomotor activity, however; in fact, SCOP significantly increased locomotion in the white chamber. In the absence of motor disturbances to account for any group differences, we contend that both central and peripheral ACh blockade may affect measures of anxiety, perhaps by directly or indirectly affecting HPA activity. Central ACh systems may underlie sensory filtering whereby irrelevant stimuli are excluded from sensory processing. Antagonism of ACh transmission may render an animal incapable of correctly processing sensory information leading to hyperresponsiveness, which can manifest itself as enhanced anxiety and fear.

The effects of intrahippocampal scopolamine infusions on anxiety in rats as measured by the black-white box test.

Hippocampal cholinergic projections mediate attention to arousing stimuli as demonstrated by behavioral, electrophysiological, and endocrine studies. We recently reported that peripheral injections of the cholinergic antagonist scopolamine (SCOP) increased anxiety-like behaviour (ALB) in rats and we sought to investigate if this response might be hippocampally mediated. Adult male, Lister Hooded rats were implanted bilaterally with hippocampal cannulae 3 weeks prior to testing. On the test day, rats were injected with vehicle (VEH; artificial CSF at 3 microl), 15 or 30 microg SCOP, 20 min prior to being placed into the white chamber of the black-white box (n = 10/group). Rats were scored for latencies to exit and reenter the white chamber, total time spent in the white chamber, intercompartmental crossings, and activity. SCOP at 30 microg significantly reduced time to exit the white arena, while both doses of SCOP elevated latencies to reenter the white chamber. There were no effects of SCOP on intercompartmental crossing, time spent in the white chamber, or on activity levels. Loss of hippocampal cholinergic function impairs processing of threatening stimuli that manifests itself as increased ALB.

Hippocampal cholinergic blockade enhances hypothalamic-pituitary-adrenal responses to stress

We examined the role of the hippocampal cholinergic system, which is known to mediate processes related to fear and anxiety, in the regulation of stress-induced hypothalamic-pituitary-adrenal (HPA) activity. Bilateral intra-hippocampal injections (30 microg per side) of the muscarinic antagonist Scopolamine augmented adrenocorticotropin and corticosterone responses to restraint without altering basal HPA activity compared to vehicle-treated animals. These results suggest that the hippocampal cholinergic system regulates stress-induced HPA activity and may serve to coordinate behavioral and neuroendocrine responses to stress.