Eleanor Waite

The origin of glucocorticoid hormone oscillations.

Characterization of a peripheral hormonal system identifies the origin and mechanisms of regulation of glucocorticoid hormone oscillations in rats.

Stress responsiveness varies over the ultradian glucocorticoid cycle in a brain-region-specific manner

Glucocorticoid hormones are released in rapid hourly hormone bursts by the adrenal gland. These ultradian oscillations are fundamental to hypothalamic-pituitary-adrenal activity and transcriptional regulation of glucocorticoid responsive genes. The physiological relevance of glucocorticoid pulsatility is however unknown. Using a novel automated infusion system, we artificially created different patterns (modulating pulse amplitude) of corticosterone (cort). Identical amounts of cort either in constant or in hourly pulses were infused into adrenalectomized rats. At the end of the infusion period, either during rising or falling concentrations of a cort pulse, animals were exposed to 99 dB noise stress (10 min). Pulsatile cort infusion led to a differential stress response, dependent on the phase of the pulse during which the stress was applied. Although constant administration of cort resulted in a blunted ACTH response to the stressor, a brisker response occurred during the rising phase of plasma cort than during the falling phase. This phase-dependent effect was also seen in the behavioral response to the stressor, which was again greater during the rising phase of each cort pulse. Within the brain itself, we found differential C-fos activation responses to noise stress in the pituitary, paraventricular nucleus, amygdala, and hippocampus. This effect was both glucocorticoid pulse amplitude and phase dependent, suggesting that different stress circuits are differentially responsive to the pattern of glucocorticoid exposure. Our data suggest that the oscillatory changes in plasma glucocorticoid levels are critical for the maintenance of normal physiological reactivity to a stressor and in addition modulate emotionality and exploratory behavior.

ACTH-dependent ultradian rhythm of corticosterone secretion.

The activity of the hypothalamic-pituitary-adrenal axis is characterized by an ultradian pulsatile pattern of glucocorticoid secretion. Despite increasing evidence for the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, little is known about the mechanism underlying the pulsatility of glucocorticoid synthesis and release. We tested the hypothesis that pulsatile ACTH release is critical for optimal adrenocortical function. Hypothalamic-pituitary-adrenal activity was suppressed by oral methylprednisolone, and ACTH (4 ng/h) was infused for 24h either as a constant infusion or in 5-min pulses at hourly intervals. Control methylprednisolone-treated rats had very low plasma corticosterone (CORT) levels with undetectable pulses and also had steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage (P450scc) heteronuclear RNA levels reduced to approximately 50% of that seen in untreated animals. Pulsatile but not constant ACTH infusion restored pulsatile CORT secretion, and this was accompanied by parallel rises in StAR and P450scc heteronuclear RNA levels during the rising phase of the CORT pulse, which then fell during the falling phase. The pulsatile pattern of StAR and P450scc was paralleled by pulsatile transcription of the melanocortin 2 receptor accessory protein. Pulsatile ACTH activation of the adrenal cortex not only is critical for the secretion of CORT but also induces episodic transcription of the rate-limiting enzymes necessary for physiological steroidogenic responses. Because constant infusion of identical amounts of ACTH did not activate CORT secretion, pulsatility of ACTH provides a more effective signaling system for the activation of adrenocortical activity.

Ultradian corticosterone secretion is maintained in the absence of circadian cues

Plasma levels of corticosterone exhibit both circadian and ultradian rhythms. The circadian component of these rhythms is regulated by the suprachiasmatic nucleus (SCN). Our studies investigate the importance of the SCN in regulating ultradian rhythmicity. Two approaches were used to dissociate the hypothalamic‐pituitary‐adrenal (HPA) axis from normal circadian input in rats: (i) exposure to a constant light (LL) environment and (ii) electrolytic lesioning of the SCN. Blood was sampled using an automated sampling system. As expected, both treatments resulted in a loss of the circadian pattern of corticosterone secretion. Ultradian pulsatile secretion of corticosterone however, was maintained across the 24 h in all animals. Furthermore, the loss of SCN input revealed an underlying relationship between locomotor and HPA activity. In control (LD) rats there was no clear correlation between ultradian locomotor activity and hormone secretion, whereas, in LL rats, episodes of ultradian activity were consistently followed by periods of increased pulsatile hormone secretion. These data clearly demonstrate that the ultradian rhythm of corticosterone secretion is generated through a mechanism independent of the SCN input, supporting recent evidence for a sub‐hypothalamic pulse generator.

Acute stress causes rapid synaptic insertion of Ca2+-permeable AMPA receptors to facilitate long-term potentiation in the hippocampus

The neuroendocrine response to episodes of acute stress is crucial for survival whereas the prolonged response to chronic stress can be detrimental. Learning and memory are particularly susceptible to stress with cognitive deficits being well characterized consequences of chronic stress. Although there is good evidence that acute stress can enhance cognitive performance, the mechanism(s) for this are unclear. We find that hippocampal slices, either prepared from rats following 30 min restraint stress or directly exposed to glucocorticoids, exhibit an N-methyl-d-aspartic acid receptor-independent form of long-term potentiation. We demonstrate that the mechanism involves an NMDA receptor and PKA-dependent insertion of Ca2+-permeable AMPA receptors into synapses. These then trigger the additional NMDA receptor-independent form of LTP during high frequency stimulation.

Organizational role for pubertal androgens on adult hypothalamic‐pituitary‐adrenal sensitivity to testosterone in the male rat

The inhibitory effect of androgens on the hypothalamic‐pituitary‐adrenal (HPA) axis in basal and stress conditions in adult male rats is well documented. Major sex‐related neuroendocrine changes take place during puberty. There is a robust rise in production and secretion of gonadal steroids, which is thought to underlie numerous neural and behavioural changes brought on after puberty. The present study investigated the effect of the pubertal rise in gonadal steroid levels on the subsequent adult corticosterone profile, particularly the sensitivity of the adult HPA axis to testosterone. Animals were castrated either prepubertally (28 days) or in adulthood (11 weeks) and adult animals were subsequently treated with subcutaneous implants containing either testosterone or cholesterol. Using an automated blood sampling system, blood was collected from each freely moving, conscious rat every 10 min (i) over a 24 h period; (ii) in response to 10 min of noise stress, and (iii) following an immunological challenge with lipopolysaccharide (LPS). Analysis revealed that testosterone treatment did not significantly affect overall corticosterone release over the 24 h period in adult animals castrated before puberty in contrast to animals castrated in adulthood in which testosterone significantly suppressed corticosterone secretion. Following either a noise stress or LPS injection, testosterone treatment did not affect the hypothalamic or adrenal stress response in animals castrated prepubertally. Testosterone significantly suppressed the corticotrophin‐releasing hormone and arginine vasopressin mRNA as well as the corticosterone response to LPS in castrated animals that had had their testes intact over puberty. These data provide evidence that puberty is a critical organizational period during which rising levels of gonadal steroids programme the sensitivity of the adult HPA axis to gonadal steroids in adulthood.

Different degrees of somatotroph ablation compromise pituitary growth hormone cell network structure and other pituitary endocrine cell types.

We have generated transgenic mice with somatotroph-specific expression of a modified influenza virus ion channel, (H37A)M2, leading to ablation of GH cells with three levels of severity, dependent on transgene copy number. GH-M2(low) mice grow normally and have normal-size pituitaries but 40-50% reduction in pituitary GH content in adult animals. GH-M2(med) mice have male-specific transient growth retardation and a reduction in pituitary GH content by 75% at 42 d and 97% by 100 d. GH-M2(high) mice are severely dwarfed with undetectable pituitary GH. The GH secretory response of GH-M2(low) and GH-M2(med) mice to GH-releasing peptide-6 and GHRH was markedly attenuated. The content of other pituitary hormones was affected depending on transgene copy number: no effect in GH-M2(low) mice, prolactin and TSH reduced in GH-M2(med) mice, and all hormones reduced in GH-M2(high) mice. The effect on non-GH hormone content was associated with increased macrophage invasion of the pituitary. Somatotroph ablation affected GH cell network organization with limited disruption in GH-M2(low) mice but more severe disruption in GH-M2(med) mice. The remaining somatotrophs formed tight clusters after puberty, which contrasts with GHRH-M2 mice with a secondary reduction in somatotrophs that do not form clusters. A reduction in pituitary beta-catenin staining was correlated with GH-M2 transgene copy number, suggesting M2 expression has an effect on cell-cell communication in somatotrophs and other pituitary cell types. GH-M2 transgenic mice demonstrate that differing degrees of somatotroph ablation lead to correlated secondary effects on cell populations and cellular network organization.

A glucocorticoid sensitive biphasic rhythm of testosterone secretion.

Studies of the hypothalamic‐pituitary‐adrenal (HPA) axis and the hypothalamic‐pituitary‐testicular (HPT) axis have revealed a reciprocal relationship between these two endocrine pathways. In rats, for example, disruption of the HPT axis alters the circadian secretion of corticosterone. Stress, on the other hand, can have varying effects on testosterone secretion in both rats and humans. Furthermore, in contrast to humans, where several pulses of testosterone secretion can be detected across the 24‐h period with the largest in the morning, rats appear to exhibit a diurnal rhythm of testosterone secretion. In the present study, we used an automated blood sampling system to investigate the true circadian pattern of testosterone secretion under basal conditions and investigated how this responds to changes in levels of circulating corticosteroids. Analysis of plasma testosterone revealed the expected bimodal pattern of basal testosterone secretion. The two secretory episodes were 12.59 h ± 41 min apart and 4.04 h ± 16 min long, with one in the light phase and the other in the dark phase of the cycle. Interestingly, when both testosterone and corticosterone diurnal profile were compared, we found that the circadian rise in plasma corticosterone levels falls neatly between the two testosterone secretory episodes. Treatment of rats with the synthetic glucocorticoid methylprednisolone in their drinking water abolished the normal bimodal profile of testosterone secretion. These rats show transient pulses of testosterone throughout the 24 h, but no circadian pattern. By contrast, adrenalectomised rats maintain their bimodal circadian pattern, suggesting that an intact HPA axis is not necessary for generation of the endogenous HPT rhythm. Thus, although the circadian rhythm of testosterone does not depend on normal HPA function, increased levels of glucocorticoids can abolish normal HPT rhythmicity.

GHRH Receptor-Targeted Botulinum Neurotoxin Selectively Inhibits Pulsatile GH Secretion in Male Rats

Botulinum neurotoxin is a potent inhibitor of acetylcholine secretion and acts by cleaving members of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor family, which are critical to exocytotic vesicular secretion. However, the potential of botulinum neurotoxin for treating secretory disease is limited both by its neural selectivity and the necessity for direct injection into the relevant target tissue. To circumvent these limitations, a technology platform called targeted secretion inhibitors (TSIs) is being developed. TSIs are derived from botulinum neurotoxin but are retargeted to specific cell types to inhibit aberrant secretion. A TSI called qGHRH-LHN/D, with a GHRH receptor targeting domain and designed to specifically inhibit pituitary somatotroph GH release through cleavage of the N-ethylmaleimide-sensitive factor-attachment protein receptor protein, vesicle-associated membrane protein (VAMP), has recently been described. Here we show this TSI activates GHRH receptors in primary cultured rat pituicytes is internalized into these cells, depletes VAMP-3, and inhibits phorbol-12-myristate-13-acetate-induced GH secretion. In vivo studies show that this TSI, but not one with an inactive catalytic unit, produces a dose-dependent inhibition of pulsatile GH secretion, thus confirming its mechanism of action through VAMP cleavage. Selectivity of action has been shown by the lack of effect of this TSI in vivo on secretion from thyrotrophs, corticotrophs, and gonadotrophs. In the absence of suitable in vivo models, these data provide proof of concept for the use of somatotroph-targeted TSIs in the treatment of acromegaly and moreover raise the potential that TSIs could be used to target other diseases characterized by hypersecretion.