A pulsatile mode of signalling underlies the activity of the HPA axis, a system crucial for maintaining basal and stress-related homeostasis by regulating the circulating levels of vital glucocorticoid hormones (cortisol in man, corticosterone in rodents). Glucocorticoids govern a broad range of physiological functions, including the regulation of cardiovascular, metabolic, cognitive and immunological activity (7–10). The regulation of HPA activity depends on multiple inputs (Fig. 1). In the hypothalamus, the paraventricular nucleus (PVN) receives an indirect input from the SCN which regulates circadian variation in HPA activity, as well as afferent information from brainstem nuclei responding to physical stressors such as hypotension and inflammation, and from limbic areas of the central nervous system that respond to cognitive and emotional stressors (11). The PVN regulates corticotroph activity in the anterior pituitary via two neuropeptides [corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP)], which are released into the pituitary portal circulation and hence the anterior pituitary. Upon stimulation, corticotrophs secrete adrenocorticotrophic hormone (ACTH) into the general circulation, through which it accesses cells of the adrenal cortex initiating the synthesis and secretion of glucocorticoids.
Figure 1. Glucocorticoid release is regulated by the hypothalamic-pituitary adrenal (HPA) axis. Neurones of the paraventricular nucleus (PVN) receive circadian signals from the suprachiasmatic nucleus (SCN), as well as information from brainstem nuclei responding to physical stressors, and from limbic areas of the central nervous system (CNS) that respond to cognitive and emotional stressors. These neurones project to the median eminence where they release corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) into the portal blood. These peptides act on corticotroph cells in the anterior pituitary to secrete adrenocorticotrophic hormone (ACTH) into the general circulation, which in turn stimulates the synthesis and release of glucocorticoid hormones (CORT) from cells of the adrenal cortex. Glucocorticoids feed back at the pituitary and hypothalamus to inhibit ACTH and CRH/AVP secretion, respectively.
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Glucocorticoid hormones act at target sites via their two cognate receptors [the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR)], the expression of which is widespread in areas regulating HPA activity (12–14). The classical effect of glucocorticoids is via activation of their receptors that translocate to the nucleus acting as ligand dependent transcription factors binding to glucocorticoid response elements at promoter regions of glucocorticoid sensitive genes (15–18). In addition to these classic genomic effects, there has been considerable recent interest in rapid nongenomic mechanisms through which glucocorticoids can also act (19,20).
In the basal state, glucocorticoids are released in an ultradian pulsatile fashion from the adrenal cortex, which results in rapidly changing levels of hormone concentration observable in blood plasma (21–24) (Fig. 2), as well as within target tissues such as the brain (25,26). The classic circadian glucocorticoid rhythm, which peaks in the morning in man (27) and evening in the rodent (28), is the result of amplitude (and to a lesser extent frequency) modulation of the underlying ultradian rhythm (22,23,29–33). A particularly striking feature of glucocorticoid pulsatility is the appearance of a more distinct ultradian rhythm during the peak of the circadian cycle, which operates at a frequency of approximately one pulse per hour in rats (see shaded region 2 of Fig. 3). Variations in amplitude and frequency of the ultradian rhythm not only compose the circadian rhythm, but also characterise changes in HPA activity that occur during early life programming (34), chronic stress (35), lactation and ageing (36), and a number of other physiological and pathological conditions (37).
Figure 3. Different ways in which the hypothalamic-pituitary adrenal axis encodes glucocorticoid pulsatility. In region 1, where mean corticotrophin-releasing hormone (CRH) levels are low, glucocorticoid pulsatility may well reflect the irregular high-frequency fluctuations in CRH and other corticotrophin-releasing factors acting on the anterior pituitary. In region 2, higher mean levels of CRH drive on the anterior pituitary are sufficient to excite the intrinsic rhythmicity of the pituitary-adrenal loop, which gives rise to the more distinct approximately hourly rhythm in glucocorticoid secretion. CRH data are adapted from a previous study (51); glucocorticoid data adapted from a previous study (21). All data were obtained from male Sprague–Dawley rats.
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The importance of glucocorticoid pulsatility resides in its ability to provide a digital signalling system that can respond rapidly to stress or changes in environmental conditions. A recent study by Stavreva et al. (38) clearly shows that the pattern of glucocorticoid presented to a tissue is critical for its transcriptional response, and this effect is seen both in vivo and in vitro. The details of the mechanisms underlying both genomic and nongenomic signalling of glucocorticoid hormones are beyond the scope of this review, but they not only act directly on multiple genes throughout the body, but also have major effects on the ‘clock genes’ and through them on circadian physiology (39,40).
Despite the significance of glucocorticoid pulsatility (36), surprisingly little is known about the mechanisms that encode this rhythm. Although the role of the SCN is well founded in regulating circadian HPA activity (41,42), evidence suggests it is not required for the generation of ultradian rhythmicity. Indeed, lesioning of the SCN completely abolishes the circadian glucocorticoid rhythm in both the adrenals and blood plasma (43–45), although it has little effect on the ultradian rhythm, which persists at amplitudes and frequencies comparable to those observed at the peak of the native circadian rhythm (E. Waite, personal communication). Furthermore, pulsatility in the HPA axis appears to be regulated independently of the ultradian rhythmicity found in other neuroendocrine systems. Glucocorticoids and luteinising hormone (LH) are both secreted in pulses with similar frequencies, for example, although their release is not concurrent (37).
Given the wealth of evidence showing that the circadian pacemaker resides within the hypothalamus (46), it is perhaps not surprising that it has also been assumed that the ultradian rhythm was the result of a hypothalamic neural pulse generator. We review this hypothesis, covering what we feel to be the most important experimental studies and go on to discuss recent developments from theoretical studies, which have led us to propose a new model for the encoding mechanisms of glucocorticoid pulsatility.
The ‘hypothalamic pulse generator’ hypothesis
Neural signalling to the anterior pituitary is encoded in the dynamic patterns of hypothalamic neuropeptides released into the portal circulation (47). Both in man and the rat, the major ACTH secretagogues are CRH and to a lesser extent AVP, although oxytocin, norepinephrine and epinephrine may modulate corticotroph activity (48). Moreover, CRH is the only corticotrophin-releasing factor known to regulate pro-opiomelanocortin gene expression in the rat (48). Whilst, CRH is also an important ACTH secretagogue in ovine species there is evidence to suggest that the predominant corticotroph secretagogue is AVP (49).
Push–pull perfusion studies of the median eminence in freely moving rats show that CRH follows an irregular pulsatile mode of secretion, with a frequency of approximately three pulses per hour (50). Interestingly, no significant difference is seen in CRH pulse frequency between the morning and evening, indicating a relatively steady frequency over the diurnal period (51). On the other hand, mean CRH concentration levels do vary significantly over the 24-h period, with evening concentration levels being almost two-fold greater than those of the morning (51). These increased levels result from the fact that mean pulse nadir and peak levels, as well as pulse amplitude, and to a lesser extent pulse duration, are all significantly higher in the evening (51).
An episodic mode of secretion for CRH and AVP has also been demonstrated in vivo in a number of other species under basal conditions (48). In the portal blood of unrestrained conscious rams, a pulsatile pattern of CRH and AVP secretion is evident (52), and measurement of AVP in the pituitary venous effluent of the unanesthetised horse also reveals an episodic secretory pattern (53). In conscious sheep also, both CRH and AVP display a pulsatile secretory pattern in the portal blood (54).
The regulatory oscillator(s) underlying the pulsatile mode of CRH secretion remains to be defined. It is certainly interesting to compare the pulses of CRH with the much more organised pulsatile secretion of gonadotrophin-releasing hormone (GnRH) into portal blood. GnRH and LH release has a relatively consistent frequency, which correlates well with organised increases in electrical activity in networks of GnRH-producing neurones (55). By contrast, the irregularity of episodic CRH secretion not only suggests a different underlying mechanism, but may also reflect an inability of CRH-producing neurones to effectively synchronise with one another in a coherent way (51).
ACTH pulsatility and its relationship to CRH
Given this episodic release of CRH, it was not surprising that pulsatile patterns of ACTH measured in blood plasma were also found in many species (4). What has been more surprising however, is that in most cases, the relationship between pulsatile neuropeptides and ACTH rhythmicity is far from straightforward.
In the rat, two ultradian ACTH rhythms are found simultaneously (56). Concentrations of ACTH in blood plasma display fast episodic bursts of variable amplitude that occur at a frequency of approximately three pulses per hour (so-called ‘micropulses’), as well as larger and more prolonged episodes of secretion that appear approximately every 1–2 h (we shall refer to this as ‘ultradian’). The two rhythms in ACTH are related in the sense that it is in fact the rhythmic variation in micropulse amplitude that makes up the slower ultradian rhythm (56).
Blockade of endogenous CRH by passive immunoneutralisation in the rat results in a significant reduction in micropulse amplitude without any effect on micropulse frequency (57), which could be attributed to the pulsatile release of other corticotrophin-releasing factors, an intrinsic rhythmicity of the corticotroph, or a combination of these factors (58). Moreover, blockade of endogenous CRH practically completely destroys the slower ultradian rhythm (57), which has prompted speculation that the slower ultradian rhythm in ACTH is driven by rhythmic secretion of CRH (57). However, there are currently no solid data that support this hypothesis. In fact, this hypothesis is actually contradictory to data on the frequency of pulsatile CRH measured in rats of the same strain (male Sprague-Dawley), which was three pulses per hour across the circadian cycle (50,51,59), and which clearly correlates well with the ACTH micropulse frequency.
In larger species, which allow for the simultaneous measurements of portal blood and blood plasma, a straightforward connection between pulsatile CRH/AVP and pulses of ACTH is also absent. In rams, for example, a clear relationship between portal levels of AVP/CRH and temporal patterns of ACTH is not seen (52). In sheep, a large proportion of CRH/AVP pulses are not followed by a significant rise in ACTH, and a significant number of ACTH pulses are not preceded by a pulse in CRH or AVP (54). Moreover, in sheep that have undergone surgical disconnection of the hypothalamus from the pituitary, pulsatility in ACTH and cortisol is maintained (60).
Taken together, of these diverse experimental studies highlight the lack of any clear causal relationship between changes in portal CRH concentrations and the ultradian rhythmicity of ACTH and glucocorticoid hormones. This suggests that the generation of HPA ultradian activity must involve other factors, presumably at a sub-hypothalamic level.
Network encoding of pulsatility
Some biological systems are endogenously rhythmic and oscillate under the influence of constant stimulation, or even in the absence of stimulation; such systems are often referred to as ‘pacemakers’ (61). In hepatocytes (liver cells), for example, continuous stimulation with physiological levels of AVP induces intracellular Ca2+ oscillations, the frequency of which increases with increasing concentrations of agonist (62). The endogenous rhythmicity of these systems finds its roots in the underlying regulatory mechanisms that govern the dynamics of that system (3), and these often involve some form of feedback, which may be positive and/or negative, nonlinear in nature, and is often time-delayed (3,61,63). Indeed, circadian time-keeping in neurones of the SCN is achieved through an intricate molecular circuitry involving ‘clock genes’ that take part in a complex regulatory network consisting of transcriptional and translational feedback loops (64). Feedback also underlies the generation of ultradian rhythmicity at the cellular level. For example, serum treatment of cultured cells induces oscillatory expression of the transcription factor Hes1 at ultradian frequencies (period approximately 2 h), which is the result of a negative feedback loop whereby Hes1 binds directly to regulatory sequences in the Hes1 promoter, thereby repressing transcription of its own gene (65). The network comprising the HPA axis also features some of the properties that are common to pacemaker circuitry. In particular, the anterior pituitary, PVN and higher centres are all targets for negative feedback by circulating glucocorticoids (66–69). In addition to feedback, delays are inherent in the HPA network, which arise from transmission times through the blood, as well as delayed response times.
The existence of pacemaker traits in the HPA network has prompted speculation that the generation of pulsatile glucocorticoid secretion is not necessarily a result of the neural pulse generator in the hypothalamus, but may actually arise from the complex network of excitatory and inhibitory interactions between CRH, ACTH and the glucocorticoids. This view is supported by a number of studies that have developed mathematical models characterising the dynamic interactions between HPA hormones (70–72). Most of these studies assume that feedback at the level of both the pituitary and the hypothalamus gives rise to the ultradian rhythm. If this were the case, then CRH, ACTH and glucocorticoids would all oscillate over the same time frame. However, there is no good evidence for this. Specifically, in the rat, the approximately hourly rhythm observed in ACTH (56) and corticosterone (22) is not observable in CRH during either the nadir or peak of the circadian rhythm (51) and, if glucocorticoid feedback at the hypothalamic level was important in generating ultradian rhythmicity, then removal of this feedback should ablate CRH pulsatility. However, in cultured explants of the macaque hypothalamus, the pulsatile release of CRH persists both in the presence and absence of glucocorticoids (73) and, in conscious rats, CRH pulsatility in the median eminence is maintained following adrenalectomy (59).
We have recently taken a mathematical approach to explore the encoding mechanisms underlying glucocorticoid pulsatility. Our mathematical model builds upon one describing dynamic interactions within the HPA axis (74), and focuses on the effects of nonlinear feedback of glucocorticoids mediated by GR at the level of the anterior pituitary. Furthermore, we incorporate the delayed response of glucocorticoid secretion following ACTH stimulation (75), which results from the lack of releasable pools of glucocorticoids and the need to synthesise the hormone before secretion.
Our theoretical results do not discount the possibility of a hypothalamic pulse generator but suggest that ultradian pulsatility in ACTH and glucocorticoids can also occur even in the absence of pulsatile input from the hypothalamus, providing that the mean levels of hypothalamic drive are within a certain range. Thus, for very low or high levels of constant hypothalamic stimulation, the mathematical model predicts a steady-state response in ACTH and glucocorticoid levels, whereas, for intermediate levels of hypothalamic drive, sustained oscillations in ACTH and glucocorticoid levels occur (76). These oscillations are born out of the excitatory–inhibitory loop formed by the interactions between the anterior pituitary and the adrenal gland. Implicit in this idea is that there is a close coupling between ultradian rhythms in ACTH and glucocorticoids, and there is indeed good experimental evidence supporting such a relationship (Fig. 2).
Although yet to be tested in vivo, it follows from our work that the HPA axis may have multiple ways of encoding glucocorticoid pulsatility (Fig. 3). During the nadir of the circadian rhythm, when mean CRH levels are low, our mathematical results suggest that fluctuations in ACTH and glucocorticoids most likely reflect the activity of episodic CRH and other corticotrophin-releasing factors. During the peak of the circadian cycle, however, when mean CRH levels are significantly higher, the theoretical model predicts that these higher mean CRH levels are sufficient to generate the endogenous hourly rhythm in ACTH and glucocorticoids, an intrinsic property of the pituitary-adrenal system (76).
Under both basal and nonbasal conditions, the HPA axis functions as a closed-loop control system, heavily influenced by negative feedback from circulating glucocorticoids. The regulation of pulsatility in the HPA axis likely involves a number of factors. The complex network of excitatory and inhibitory connections coupled with the pulsatile activity of hypothalamic-releasing factors renders the task of unravelling mechanisms regulating glucocorticoid pulsatility extremely difficult. Experimental studies have so far been unsuccessful in identifying either the mechanistic or anatomical origin of the ultradian rhythm. We have been able to demonstrate, using a mathematical approach, that glucocorticoid negative feedback at the level of the anterior pituitary may not just be important for the homeostatic regulation of optimal levels of ACTH and glucocorticoids, but also could actually be involved in generating ultradian HPA activity. The implications of this finding are that the pulsatile patterns of glucocorticoids that we observe in blood plasma may well reflect the integrated activity of pulsatile hypothalamic forcing on an endogenously rhythmic pituitary-adrenal system.