HPA Axis-Rhythms

Francesca Spiga,1 Jamie J. Walker,1,2 John R. Terry,2 and Stafford L. Lightman*1

ABSTRACT
The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating levels of glucocorticoid hor-
mones, and is the major neuroendocrine system in mammals that provides a rapid response and
defense against stress. Under basal (i.e., unstressed) conditions, glucocorticoids are released
with a pronounced circadian rhythm, characterized by peak levels of glucocorticoids during the
active phase, that is daytime in humans and nighttime in nocturnal animals such as mice and
rats. When studied in more detail, it becomes clear that the circadian rhythm of the HPA axis is
characterized by a pulsatile release of glucocorticoids from the adrenal gland that results in rapid
ultradian oscillations of hormone levels both in the blood and within target tissues, including the
brain. In this review, we discuss the regulation of these circadian and ultradian HPA rhythms, how
these rhythms change in health and disease, and how they affect the physiology and behavior of
the organism.  C 2014 American Physiological Society. Compr Physiol 4:1273-1298, 2014.

Introduction circulation, glucocorticoids access target tissues, such as the

The HPA axis
The hypothalamic-pituitary-adrenal (HPA) axis regulates cir-
culating levels of glucocorticoid hormones, and is the major
neuroendocrine system in mammals that provides a rapid
response and defense against stress (Fig. 1A). Glucocorti-
coids (cortisol in humans and corticosterone in rodents) are
vital hormones that regulate many physiological functions,
including glucose, fat, and protein metabolism (32, 121, 138).
In addition, glucocorticoids exert anti-inflammatory and
immunosuppressive actions and can affect mood and cog-
nitive function (35, 49, 128). Under basal (i.e., unstressed)
conditions, the dynamics of the HPA axis is characterized by
both a circadian and an ultradian rhythm of hormone secre-
tion (Fig. 1B). In addition to this, the activity of the HPA
axis increases when the organism is exposed to stress. This
stress response is initiated by the activation of several afferent
neural pathways, including those originating from limbic and
brain-stem structures (196), which in turn induce the release
of corticotropin-releasing hormone (CRH) and arginine vaso-
pressin (AVP) from parvocellular neurons of the hypothala-
mic paraventricular nucleus (PVN) (62, 159, 167, 197). Upon
stimulation, these neuropeptides are released from neuronal
terminals at the level of the median eminence into the por-
tal vessels (6). Via the portal circulation, CRH and AVP
reach the anterior pituitary, where they bind to their spe-
cific receptors, CRH receptor 1 (CRHR1) and AVP 1B recep-
tor (V1bR), respectively (7, 30), to stimulate the release of
adrenocorticotropic hormone (ACTH) from corticotroph cells
into the general circulation. ACTH, in turn, stimulates the
adrenal gland to produce and secrete glucocorticoid hor-
mones (42, 45). In contrast to peptide hormones, which are
presynthesized and stored within the cytoplasm and released
upon activation, glucocorticoids are synthesized de novo upon
stimulation of the adrenal. Once released into the blood
liver, the heart and vascular tissues, to exert metabolic and
cardiovascular effects, respectively, and the brain, where for
example, glucocorticoids promote cognitive processes neces-
sary to cope with a threatening situation (47).
Glucocorticoids also regulate the activity of the HPA axis,
and thus their own production, through feedback mechanisms,
acting at the level of the pituitary gland where they inhibit
ACTH release (91), and at the level of the PVN where they
inhibit the synthesis and release of CRH and AVP (42, 43,
91). In addition, glucocorticoids indirectly regulate HPA axis
activity via modulation of other brain structures, including the
hippocampus, the amygdala, and the prefrontal cortex, which
in turn regulate the activity of the PVN (196).
Adrenal glucocorticoid synthesis
Glucocorticoids are synthesized in the zona fasciculata of
the adrenal cortex. Glucocorticoid steroidogenesis is acti-
vated when ACTH binds to its specific cell surface G-protein-
coupled receptor, the melanocortin type-2 receptor (MC2R)
(137). Upon ACTH binding, MC2R undergoes conforma-
tional changes that activate adenylyl cyclase, leading to an
increase in intracellular levels of cyclic adenosine monophos-
phate (cAMP). This intracellular increase in cAMP activates
downstream signaling pathways, including the protein kinase
A (PKA) pathway. Activation of PKA induces acute syn-
thesis of glucocorticoids via both genomic and nongenomic
* Correspondence to Stafford.Lightman@bristol.ac.uk
1 University of Bristol, Henry Wellcome Laboratories for Integrative
Neuroscience & Endocrinology, Bristol, United Kingdom
2 University of Exeter, College of Engineering, Mathematics and
Physical Sciences, Exeter, United Kingdom
Published online, July 2014 (comprehensivephysiology.com)
DOI: 10.1002/cphy.c140003
Copyright  C American Physiological Society.

Volume 4, July 2014 1273

HPA Axis-Rhythms Comprehensive Physiology

Circadian and stress inputs

(A)
(B)
PVN
Hypothalamus
100
CRH and AVP

Corticosterone (ng/mL)
80

Corticotroph cells 60
Pituitary
gland 40
ACTH
20

CORT 0
0900 1500 2100 0300 0900
Clock time (h)
Adrenal gland Adrenal cortex

Figure 1 The hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid rhythms. (A) The hypothalamic
paraventricular nucleus (PVN) receives circadian input from the SCN and stress inputs from the brainstem and
from regions of the limbic system such as the hippocampus and amygdala. The PVN projects to the median
eminence where it releases corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) into the
hypothalamic-pituitary portal circulation. CRH passes through this vascular route to access corticotroph cells
in the anterior pituitary, which respond with the rapid release of adrenocorticotropic hormone (ACTH) from
preformed vesicles into the general circulation. In turn, ACTH reaches the adrenal cortex where it activates the
synthesis and secretion of glucocorticoid hormones (CORT). Glucocorticoids regulate the activity of the HPA axis,
and thus their own production, through feedback mechanisms acting at the level of the pituitary gland where they
inhibit ACTH release, and at the level of the PVN where they inhibit the release of CRH and AVP. Reproduced with
permission from (116). (B) Under basal (i.e., unstressed) conditions, the dynamics of glucocorticoid secretion is
characterized by both a circadian (gray line) and an approximately hourly ultradian rhythm (black line). In the
rat, peak levels of corticosterone occur during the active evening phase. Shaded region indicates the dark phase.
Reproduced with permission from (200).

mechanisms. The rate-limiting step in glucocorticoid synthe-
sis is the mobilization of cholesterol and its transfer inside the
mitochondria, and this process is mediated by the activity of
the steroidogenic acute regulatory protein (StAR) (118, 185).
Activation of the PKA signaling pathway activates nuclear
transcription factors, such as the cAMP response element
(CRE) binding protein (CREB), which in turn induce tran-
scriptional activation of a number of steroidogenic genes,
including StAR. In parallel to StAR transcription, PKA also
induces posttranslational modification of StAR, resulting in
phosphorylation at a specific site that activates StAR and
increases its ability to transport cholesterol across the mito-
chondrial membrane (9). Within the mitochondria, choles-
terol is subjected to enzymatic modifications by a cascade of
steroidogenic regulators, which ultimately lead to glucocorti-
coid synthesis (21, 36, 80).
Glucocorticoid receptors and genomic
responses
Glucocorticoids exert their effects by binding to specific
receptors expressed in target tissues. Two receptors for glu-
cocorticoids are known: the glucocorticoid receptor (GR)
and the mineralocorticoid receptor (MR). These receptors
belong to the superfamily of nuclear receptors (48, 123).
When inactive, they reside in the cytoplasmic compartment,
stabilized by chaperone molecules (140, 147, 215), including
the 90-kDa heat-shock protein (HSP-90) (149). Upon glu-
cocorticoid binding, the chaperone-receptor complex dissoci-
ates to enable the ligand-receptor complex to translocate to the
nucleus, where it functions as transcription factor to modulate
genomic events via activation or repression of transcription
of glucocorticoid target genes.
GR and MR differ in their affinity for glucocorticoids. The
affinity of the endogenous hormones corticosterone and cor-
tisol for MR is approximately 10-fold higher than the affinity
for GR. Therefore, variations in circulating levels of gluco-
corticoids lead to shifts in the balance between MR and GR
activity (158). The high-affinity MR is activated at very low
concentrations of glucocorticoids, whereas the lower affinity
GR is only activated at high concentrations. In basal (non-
stressed) physiological states, when glucocorticoid concen-
trations are low, MR is activated and localized in the nucleus,
whereas GR remains latent in the cytoplasm. Once gluco-
corticoid concentrations increase above a threshold level, for
example during the circadian peak or following exposure to
stress, GR translocates into the nucleus where it exerts its
genomic effects (39, 50, 106, 156, 157, 177).
Differences between MR and GR are also found in their
distribution throughout the body. While GR is ubiquitously
expressed in the periphery and in the brain, the distribution of
MR is more localized to specific organs such as the kidney and

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Comprehensive Physiology
the heart. In the brain, the distribution of these receptors is also
not uniform (156, 157). GR is almost ubiquitously expressed
in neurons and glial cells, with particularly high density in the
hippocampus and in parvocellular neurons of the PVN. In con-
trast, MR localization is restricted to the hippocampus, pre-
frontal cortex, and amygdala (3, 10, 198), structures essential
for learning, memory, and emotional behavior. Significantly,
both receptors are expressed abundantly in areas regulating
the HPA axis, including a number of limbic structures that
regulate the activity of the PVN, such as the hippocampus
and amygdala, as well as in the anterior pituitary (79).
Nongenomic effects of glucocorticoids
In addition to their genomic effects, glucocorticoids can also
modulate cellular activity much more rapidly (i.e., within sec-
onds to minutes) through nongenomic actions; that is, inde-
pendently of gene expression and de novo synthesis of mRNA
and protein.
Rapid nongenomic effects of glucocorticoids occur
through a mechanism mediated by G-protein coupled
membrane-associated receptors, with a pharmacological pro-
file different from the well known intracellular GR (141,142).
Given the rapidity with which glucocorticoids inhibit the
activity of the HPA axis, this effect is likely to be mediated by
nongenomic mechanisms at both the level of the pituitary
and the brain. For example, corticosterone—administered
peripherally—suppresses rat hippocampal cell firing within
20 min (146), while mEPSC frequency increases within min-
utes in CA1 pyramidal cells from rat hippocampal slices
infused with corticosterone. In the rat, a rapid inhibitory
effect of glucocorticoids has also been observed in the
activity of PVN neurons projecting to the median emi-
nence (102, 115, 164). Furthermore, a fast inhibitory effect
of corticosterone on CRF-induced ACTH secretion has been
observed in the rat within 15 min of corticosterone admin-
istration (75). In addition, a rapid (within 20 min) effect of
the synthetic glucocorticoid methylprednisolone on corticos-
terone secretion has also been observed (178).
Studies from Tasker and colleagues have shown that
nongenomic inhibitory effects of glucocorticoids in the PVN
are mediated by a mechanism involving glucocorticoid-
induced endocanabinoid synthesis. Indeed, Di et al. (51) have
shown that rapid glucocorticoid effects on PVN neuronal
activity are mediated by the activation of postsynaptic recep-
tors and the dendritic synthesis and release of an endogenous
endocanabinoid, which in turn suppresses excitatory synap-
tic inputs by feeding back onto glutamatergic pre-synaptic
terminals.
Evidence for rapid glucocorticoid actions in the brain is
also supported by behavioral studies. For example, corticos-
terone has been shown to inhibit male reproductive behav-
ior as well as medullary neuronal firing in newts (160).
Similarly, corticosterone has been shown to have a rapid
effect on aggressive behavior, by modulating glucocorticoid-
responsive hypothalamic areas (108).
Volume 4, July 2014
HPA Axis-Rhythms
Circadian Rhythms
The suprachiasmatic nucleus and the clock
gene network
From the Latin circa diem, circadian means “around a day,”
and circadian rhythms refer to biological oscillations with a
period of approximately 24 h. Circadian rhythms are highly
conserved among living organisms, from cyanobacteria to
mammals. Circadian rhythms are characteristic of a num-
ber of biochemical, physiological, endocrine, and behavioral
functions, and this allows the organism to anticipate and pre-
pare for predictable environmental changes (i.e., the light/dark
cycle). The result is an optimization of energy expenditure by
timely coordination of physiological processes.
In mammals, circadian rhythms are generated and syn-
chronized by a central clock in the suprachiasmatic nucleus
(SCN) of the ventral hypothalamus (126,135,188). The activ-
ity of the SCN is entrained by the light input received from the
retina via the retino-hypothalamic tract, and this allows our
physiology to be entrained to solar time (72,136). The molecu-
lar mechanism regulating the circadian activity of each single
cell within the SCN consists of an oscillating transcriptional
network, including the “clock genes” circadian locomotor
output cycles kaput (CLOCK), brain and muscle aryl hydro-
carbon receptor nuclear translocator-like (BMAL1), period1-
3 (PER1-3), and cryptochrome1-2 (CRY1-2) (20, 61, 104). A
network of positive and negative transcriptional, translational
and posttranslational feedback loops regulates the rhythmic
transcription of these genes, with subsequent rhythmic expres-
sion of their protein products (155). Signaling between the
SCN and the periphery involves both hormonal and neuronal
mechanisms (112). Rhythmic activity of the SCN is translated
into circadian patterns of gene expression in target tissues,
which in turn determine circadian rhythms in the physiologi-
cal function of that specific organ.
In the SCN, the expression of vasoactive intestinal
polypeptide (VIP) is under regulation of the clock machin-
ery. VIP is released with a 24-h cycle, and activates and syn-
chronizes SCN neuronal activity (5, 120). In rodents, lesion-
ing of the SCN results in a loss of physiological circadian
rhythmicity, including body temperature, locomotor activity,
and hormone secretion (1, 17, 182, 200), even when the 24-h
light-dark cycle is maintained, and the circadian rhythm of
locomotor activity can be reinstated in arrhythmic animals by
transplantation of an intact SCN (153).
In addition to the central master clock in the SCN,
clock genes are expressed rhythmically in peripheral tissues,
including liver, kidney, skeletal muscle, lung, and adrenal
(16, 70, 71, 187). However, although the molecular dynamics
of clock genes are the same in the SCN and the peripheral
clocks, only the SCN functions as a self-sustaining pace-
maker at the whole-tissue level (139, 208, 216); while the
SCN can independently generate and maintain its own circa-
dian rhythms, peripheral clocks require SCN-dependent drive
to maintain synchronicity with the light-dark cycle (139,208).
Nevertheless, the existence of peripheral clocks suggests that
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HPA Axis-Rhythms Comprehensive Physiology

local clock gene networks can generate and regulate the cir- circadian pacemaker that can regulate glucocorticoid synthe-
cadian rhythm of a number of cellular functions, such as daily sis independently from the SCN (144, 175).
variations in metabolic and cardiovascular activity, and hor-
mone secretion. Indeed, disruption of clock gene expression
in the brain and muscle has been associated with a loss of cir-
SCN regulation of HPA axis circadian rhythms
cadian rhythmicity in locomotor activity (127). Furthermore,
ablation of clock genes in the liver leads to a dysfunction Neuro-anatomical tracing studies have identified direct axonal
in glucose and lipid metabolism (109, 110), and disruption projections from the SCN to the PVN, as well as indi-
of clock genes in the pancreas is associated with diabetes rect connections via the dorsomedial hypothalamus (DMH)
mellitus in mice (124). (204, 205). In turn, circadian secretion of CRH regulates cir-
cadian release of ACTH from the anterior pituitary (26, 206).
Interestingly, the expression of StAR in the adrenal cor-
tex, which is dependent on ACTH signaling, is also char-
Circadian Rhythm of Glucocorticoid acterized by a circadian rhythm, as shown by changes in
Secretion both mRNA and protein levels throughout the 24-h cycle
(63, 145, 175, 195), and this in turn regulates circadian syn-
Consistent with other circadian physiological functions, the thesis and release of glucocorticoids (Fig. 2).
circadian rhythm of the HPA axis is primarily regulated by A number of studies have demonstrated a role for vaso-
the central pacemaker located in the SCN (95). The SCN pressin release from the SCN in modulating HPA circadian
regulates the circadian rhythm of glucocorticoids in part by activity. Vasopressin-containing neurons in the dorsal SCN
modulating the release of CRH from neurons in the PVN, and have been shown to be an important component of the SCN
in part via the autonomic nervous system (ANS) through a output (94), and experiments using micro-infusions of vaso-
multisynaptic neural pathway from the SCN to the adrenal pressin and its antagonists in the PVN and the DMH have
gland. Additionally, there is evidence of an intra-adrenal shown that vasopressin released from SCN terminals strongly

(A) (B)
140 Adrenal 400
Plasma
400
Corticosterone (ng/mg)

Corticosterone (ng/mL)
120
300
ACTH (pg/mL)

100 300
200
80 200
100
60
100
40 0
0
5 9 13 17 21 1 5 5 9 13 17 21 1 5
Clock time (h) Clock time (h)

(C) (D)
8 mRNA 1.5
Protein
MC2R mRNA (fold change)
StAR protein (fold change)
StAR mRNA (fold change)

2.0
6 1.25
1.6
4 1

1.2
2 0.75

0.8
0 0.5
5 9 13 17 21 1 5 5 9 13 17 21 1 5
Clock time (h) Clock time (h)

Figure 2 Circadian rhythms in the HPA axis. Rat ACTH (A) and corticosterone (B) profiles
show a clear circadian variation over the 24-h period with peak levels during the active
phase. Steroidogenic acute regulatory protein (StAR), the rate limiting protein in adrenal
glucocorticoid synthesis, is also expressed in the adrenal with a circadian pattern that is
similar to that observed for ACTH and corticosterone (C). In contrast, the expression pattern
of the specific ACTH receptor melanocortin 2 (MC2R) appears to be in antiphase with both
StAR and ACTH (D). Reproduced with permission from (145).

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Comprehensive Physiology
inhibits the release of adrenal corticosterone in the rat (93).
Furthermore, a decrease in vasopressin release from these
SCN terminals in the DMH toward the active dark phase
is necessary for the circadian increase in plasma corticos-
terone (97). The important role of vasopressin in regulating
low corticosterone levels during the circadian nadir of HPA
axis activity has also been confirmed in a series of experi-
ments in vitro using brain slices in which removal of the SCN
from the slice results in a loss of circadian rhythm in the
spontaneous firing rate of PVN neurons (194). However, as
shown in the same study, this rhythm can be reinstated either
by using co-cultures of the slice with SCN tissue, or by a
rhythmic (12 h on, 12 h off) perfusion of vasopressin.
In nocturnal animals, where light entrains the SCN dur-
ing their inactive phase, vasopressin release is important to
ensure low circulating levels of corticosterone during the cir-
cadian nadir (96). In diurnal species (e.g., man), vasopressin
release from the SCN is also induced by light, suggesting that
vasopressin has an excitatory effect on HPA axis activity in
diurnal organisms. Indeed, this is in accordance with the well-
characterized excitatory effect of vasopressin on its neuronal
targets (89, 107). These observations clearly suggest that the
mechanism through which vasopressin exerts an inhibitory
or excitatory effect on CRH release in nocturnal or diurnal
species, respectively, does not involve a direct effect of vaso-
pressin on CRH neurons, but depends on vasopressin acti-
vation of either an inhibitory or excitatory neuronal pathway
regulating CRH release in the PVN. Indeed, although there
are direct projections from the SCN to the PVN (204, 205),
there is currently no evidence for a direct connection between
fibers from SCN vasopressin neurons and CRH neurons in
the PVN (18, 199).
A detailed anatomical scheme of the SCN neuronal con-
nections in both nocturnal and diurnal species has been pro-
posed by Kalsbeek and colleagues by comparing the effect
of vasopressin on CRH release in the nocturnal rat and in
the diurnal Sudanian grass rat (Arvicanthis ansorgei) (95). In
accordance with their model, vasopressin is released from the
SCN during the day in both nocturnal and diurnal species. In
nocturnal species, vasopressin released from the SCN will
activate gamma-aminobutyric acid (GABA)ergic interneu-
rons within the PVN and the DMH, which in turn will directly
or indirectly (via the DMH) inhibit CRH release during the
circadian nadir. In contrast, in the diurnal species, vasopressin
release from the SCN will activate glutamatergic interneu-
rons in the PVN and DMH, which in turn will stimulate
CRH release. Although the importance of SCN-secreted vaso-
pressin for inhibition of PVN activity has been well described
in the rat, a neurotransmitter responsible for SCN stimula-
tory effect on the HPA axis during the circadian peak has not
been clearly identified. A number of studies have also sug-
gested that a stimulatory signal from the SCN via the release
of VIP modulates the evening rise in HPA activity (5, 120).
Furthermore, a role for neuromedin U in regulating the stim-
ulatory effect of the SCN on HPA axis activity has also been
proposed (64).
Volume 4, July 2014
HPA Axis-Rhythms
ANS regulation of the glucocorticoid
circadian rhythm
Although a circadian rhythm of ACTH has been described
in the rat, the circadian variation in corticosterone release
throughout the 24-h cycle is more pronounced than the circa-
dian variation in ACTH release. Indeed, the circadian rhythm
in ACTH release is often undetectable (4, 44, 97, 98, 145, 195,
210). This dissociation between ACTH and corticosterone
during the circadian cycle suggests a diurnal variation in the
adrenal sensitivity to ACTH, with higher responsiveness dur-
ing the peak phase of glucocorticoid secretion. Indeed, it has
been proposed that, in addition to the regulatory effect on
CRH secretion, the SCN can also regulate adrenal activity
independently from hypothalamic-pituitary drive by modulat-
ing adrenal sensitivity to ACTH. The adrenal gland receives
sympathetic innervation via the thoracic splanchnic nerve.
Using transneuronal virus tracing, Buijs and colleagues (19)
have identified direct connections between the adrenal gland
and neurons located in the intermedio-lateral column of the
spinal cord, which receive input from the autonomic division
of the PVN, which in turn is innervated by vasopressin neu-
rons from the SCN. This is consistent with studies from Jasper
and Engeland (87) showing that transection of the splanchnic
nerve in the rat increases corticosterone secretion in the morn-
ing but has no effect in the evening, suggesting an inhibitory
effect of the splanchnic nerve on corticosterone secretion dur-
ing the nadir phase of the circadian rhythm. Further studies
from the same group have also demonstrated a role for the
splanchnic nerve in the regulation of adrenal sensitivity to
ACTH in nonstressed rats (88). The mechanism by which the
splanchnic nerve regulates adrenal sensitivity to ACTH is not
fully understood. A study in which the adrenal responsiveness
to different doses of exogenous ACTH was investigated (195)
has shown that the decrease in corticosterone levels induced
by splanchnic nerve transection during the peak phase of hor-
mone secretion is associated with a decrease in the levels of
adrenal cAMP. Since ACTH induces glucocorticoid synthe-
sis via the cAMP/PKA signaling pathway, these data suggest
that splanchnic nerve activity regulates adrenal sensitivity to
ACTH by affecting intracellular signaling pathways involved
in glucocorticoid synthesis. Via a signaling pathway involv-
ing cAMP, PKA, and CREB, ACTH induces transcriptional
activation of several steroidogenic genes, including StAR.
However, in this study, the circadian rhythm of StAR was not
affected by lesioning the splanchnic nerve, suggesting that the
effects of splanchnic activation at the level of adrenal sensitiv-
ity to ACTH may be modulated by a nongenomic mechanism,
such as phosphorylation of StAR.
Circadian clock genes in the adrenal gland
regulate rhythmic glucocorticoid synthesis
In addition to the SCN, key components of the HPA axis,
including the PVN, pituitary, and adrenal cortex, express
circadian clock genes (63, 81, 95, 143, 144). Indeed, using
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HPA Axis-Rhythms
microarray and qPCR techniques to analyze circadian gene
expression in the mouse adrenal gland, Oster and col-
leagues have recently shown that approximately 5% of the
whole genome—including several canonical clock genes—
demonstrates rhythmic expression (143). Evidence for the
involvement of adrenal clock genes in circadian glucocor-
ticoid synthesis has been shown in vivo using mice defec-
tive in components of the circadian clock system. In fact,
studies in clock gene knockout mice have shown that dis-
ruption of the circadian clock in these animals is associated
with a disruption of the HPA circadian rhythm. Dallmann and
colleagues (46) have shown that, while both Per1 KO mice
and Per2 KO mice have markedly elevated levels of circu-
lating glucocorticoids, only Per1 KO mice lack a detectable
circadian hormone rhythm. In agreement with this study, a
loss of circadian rhythm of corticosterone has been found in
Per2/Cry1−/−double mutant mice, whereas circadian rhyth-
micity of ACTH is maintained in these mice (144). Interest-
ingly, in these animals, a number of clock genes, including
Bmal1, Per1, and Per3, which are all rhythmically expressed
in the wild-type animals, lack transcriptional circadian rhyth-
micity. The same authors have shown that transplantation of
wild-type adrenal glands restores corticosterone rhythmic-
ity, whereas transplanting adrenal glands from arrhythmic
Per2/Cry1 double mutant mice to wild-type adrenalectomized
mice dampens the corticosterone circadian rhythm. Further-
more, the circadian rhythmicity of clock genes in the adrenal
is independent of circadian rhythmicity of ACTH, as has been
shown in a recent study in which circadian rhythms of clock
genes such as Per1, Per2, and Bmal1 are maintained in rats
after hypophysectomy (58). Taken together, these observa-
tions suggest that, although the central pacemaker in the SCN
controls peripheral clock gene rhythmicity, clock genes in the
adrenal may have an important role in regulating circadian
rhythms of glucocorticoid synthesis.
Several rhythmically expressed genes in the adrenal
gland encode proteins involved in steroidogenic processes,
including proteins involved in cholesterol biosynthesis and
transport (e.g., StAR), and proteins implicated in ACTH
receptor signaling [e.g., MC2R and the melanocortin 2 recep-
tor accessory protein (MRAP)] (144, 145, 175). Park and
colleagues have found that the pattern of StAR expression
is consistent with the pattern of ACTH and corticosterone
secretion; that is, StAR mRNA and protein levels are max-
imal during the active phase. Interestingly, the pattern of
expression of MC2R mRNA appears to be in antiphase with
both StAR expression and ACTH and corticosterone secretion
(Fig. 2D). A recent study has shown that, while the ACTH
circadian rhythm is unchanged in Bmal1 deficient mice, cir-
cadian variations of corticosterone are reduced, with lower
corticosterone levels during the peak phase (111). Interest-
ingly, the effect of Bmal1 deficiency on the corticosterone
rhythm in these animals is associated with downregulation in
the expression of several key genes involved in cholesterol
trafficking, including StAR, while the expression of MC2R is
elevated.
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Comprehensive Physiology
The mechanisms underlying the regulation of adrenal
steroidogeneis by adrenal clock genes have recently been elu-
cidated using Bmal1 adrenal knockdown mice (175). This
study has shown that the circadian expression of StAR
appears to be directly regulated by the Clock:Bmal1 com-
plex at the level of the StAR promoter in the adrenal gland.
Bmal1 adrenal knockdown mice display a flattened circadian
rhythm of StAR protein expression, and although the circa-
dian rhythm of corticosterone is maintained in these animals
when they are kept under a normal light-dark cycle, corti-
costerone rhythmicity is lost in mutant mice maintained in
constant darkness. This suggests that, while in normal light
conditions adrenal circadian rhythmicity is primarily under
the control of the SCN via both hypothalamic-pituitary and
splanchnic nerve regulation, the adrenal clock pathway may
become important for regulation of steroidogenic genes when
the central circadian input is disrupted.
A more recent study has shown that in addition to StAR,
several of its transcriptional regulators are also rhythmi-
cally expressed in the adrenal gland (145), and this includes
positive regulators such as the nuclear receptors steroido-
genic factor 1 (SF-1) (189) and nerve Growth factor IB
(NGFIB, also known as Nur77 or NR4A1) (176), as well the
negative regulator nuclear receptor dose-sensitive sex rever-
sal, adrenal hypoplasia congenita determining region on the
X-chromosome-1 (DAX1) (217). Consistent with their role in
regulating the expression of adrenal StAR, SF-1 and Nur77
mRNA and protein levels are high during the hormonal cir-
cadian peak, whereas levels of expression of DAX1 are high
during the circadian nadir (145). Whether circadian variation
in StAR’s transcriptional regulators is important for the circa-
dian corticosterone rhythm, via regulation of the StAR circa-
dian rhythm, is not known. However, Park and colleagues have
shown that disruption of the corticosterone circadian rhythm
in rats kept in constant light is associated with a disrupted
circadian rhythm of StAR and its transcriptional regulators
(145). Whether transcriptional activation of StAR’s regula-
tors is under control of the clock gene machinery, as is the
case for StAR, is also not currently known. Taken together,
these data suggest that, while the SCN is indispensable for
maintaining a circadian rhythm of glucocorticoid secretion,
the adrenal clock provides an additional level of control by
regulating the steroidogenic pathway.
Ultradian Rhythm of Glucocorticoid
Secretion
As shown for other neuroendocrine systems that sig-
nal through the hypophysial portal system, such as the
gonadotropic hormone and growth hormone pathways, the
basal activity of the HPA axis is characterized by pulsatile
activity. The circadian variation in glucocorticoid levels over
the 24-h cycle is not made up of a smooth change in hor-
mone levels, but is in fact characterized by a rapid ultradian,
Volume 4, July 2014
Comprehensive Physiology
pulsatile pattern of hormone secretion, with a periodicity of
approximately one hour (Fig. 1B). The ultradian glucocorti-
coid rhythm has been reported in numerous species, including
rat (86,214), rhesus monkey (76,192), sheep (59), and human
(73, 113, 207).
The ultradian rhythm of corticosterone in the rat was first
described by Jasper and Engeland using intra-adrenal micro-
dialysis in nonstressed freely behaving animals (86). In addi-
tion to the hourly corticosterone pulse frequency, they also
observed that both the ultradian frequency and amplitude of
the pulses are modulated in a circadian manner (87).
The pattern of corticosterone secretion has also been
investigated in blood plasma. An automated blood-sampling
(ABS) system developed by Clark et al. (37) enables the
remote collection of small blood samples at a high frequency
(e.g., every 5-10 min) over extended periods of time (214).
This system provides a powerful tool to study glucocorticoid
levels in “unstressed” rats over the whole 24-h cycle, and
has been used to show that corticosterone levels in the blood
are also pulsatile with an ultradian frequency. Analysis of
pulse characteristics using the PULSAR algorithm (130) has
shown that the pulse amplitude varies in a circadian manner,
with low amplitudes in the morning and higher amplitudes in
the evening (213).
Ultradian rhythms of the HPA axis
There is good evidence that the circadian rhythm of glucocor-
ticoids is regulated by the SCN. However, the mechanisms
generating the ultradian pulsatile dynamics of the system
remain poorly understood. In rats with SCN lesions, the cir-
cadian rhythm of corticosterone is disrupted, but the ultradian
pulsatile pattern of corticosterone is maintained (200), which
suggests that the origin of the ultradian rhythm of corticos-
terone is generated independently of the SCN (Fig. 3).
A number of studies have shown that, in addition to
endogenous glucocorticoids, an ultradian pattern underlies
(A)
100
Corticosterone (ng/mL)
80
60
40
20
0
0900 1500 2100 0300
Clock time (h)
Figure 3 Ultradian corticosterone pulsatility is maintained following lesioning of the SCN. (A)
Plasma corticosterone levels in a control (sham lesion) rat. (B) Plasma corticosterone levels in a
rat with SCN lesion. Corticosterone was measured in blood samples collected at 10 min intervals
from freely behaving male Sprague-Dawley rats using an automated blood sampling system.
Gray curves indicate circadian trend. Shaded regions indicate the dark phase. Reproduced with
permission from (200).
Volume 4, July 2014
HPA Axis-Rhythms
other components of the HPA axis. Indeed, a small number
of studies, both in vitro and in vivo, have reported episodic
release of CRH from macaque hypothalamic explants (131),
rapid fluctuations in CRH levels in the median eminence of
freely behaving rats (83,85), and pulsatile patterns of CRH and
AVP in the hypophysial-portal circulation of conscious sheeps
(24, 55). In addition to this, ultradian pulses of ACTH have
been observed in the rat (25,26,29), sheep (8), and human (73).
Observations of pulsatile CRH have led to a general
acceptance that a ‘hypothalamic “pulse generator”’ drives the
ACTH and glucocorticoid ultradian rhythm. However, a num-
ber of findings are in contrast to this hypothesis. Significantly,
there is a mismatch between the frequency of CRH pulses and
the frequency of ACTH and corticosterone oscillations. In the
rat, CRH pulse frequency is higher (∼3 pulses/h) (83) than
the near-hourly oscillation in ACTH (26) and glucocorticoids
(214) (Fig. 4). Furthermore, studies in the sheep have shown
that pituitary-adrenal ultradian activity is maintained follow-
ing hypothalamic disconnection from the pituitary (56), sug-
gesting that a supra-pituitary pulsatile drive is not required
for pulsatility in ACTH and glucocorticoids.
The pituitary-adrenal system is characterized by both
a positive feed-forward pathway whereby pituitary ACTH
drives glucocorticoid release from the adrenal cortex, and a
negative feedback mechanism through which adrenal glu-
cocorticoids feed back on the anterior pituitary to inhibit
ACTH release. A short time delay in the pituitary-adrenal
feed-forward pathway has been observed both in humans
and in the rat, where each pulse of ACTH is followed by
a delayed response in cortisol or corticosterone, respectively
(27,73) (Fig. 5). Furthermore, a rapid inhibitory effect of glu-
cocorticoids on CRH-induced ACTH secretion from the ante-
rior pituitary has been shown (75, 90, 92, 122, 161-163, 209).
The observation of this fast inhibitory feedback within the
pituitary-adrenal system has led to the hypothesis that this
process could provide a potential mechanism within the sys-
tem for generating oscillatory dynamics.
(B)
0900 0900 1500 2100 0300 0900
Clock time (h)
1279
HPA Axis-Rhythms Comprehensive Physiology

(A) 40 800
CRH-41 (pg/5 min fraction) 20

Plasma ACTH (pg/mL)

Serum cortisol (nmol/l)

ACTH
15 30 600 Cortisol

10 20 400

5
10 200
0
1700 1800 1900 2000
Clock time (h) 0 0
(B) 200 0200 0500 0800 1100 1400 1700
Clock time (h)
Plasma ACTH (pg/mL)

160
Figure 5 Human cortisol and ACTH dynamics. Ultradian ACTH and
cortisol oscillations in a healthy man are tightly correlated with ACTH
120 leading cortisol. ACTH and cortisol were measured in blood samples
collected at 10 min intervals using an automated blood sampling sys-
tem. Light was off between 2300 and 0700. Adapted and reproduced
80
with permission from (73).

40
oscillations with a physiological ultradian frequency, even
0 under conditions of constant CRH drive to the anterior pitu-
1000 1100 1200 1300 1400 1500 itary. Analysis of this model has provided a number of the-
Clock time (h) oretical predictions (Fig. 6). First, the model predicts that
(C) 120
oscillations in ACTH and glucocorticoids can be generated
for “intermediate” constant levels of CRH, and that these
Corticosterone (ng/mL)

100
80
60
40
20
0
1500 1700 1900
Clock time (h)
Figure 4 An ultradian pattern underlies all the components of the
HPA axis. (A) Pulsatile release of CRH in the rat median eminence.
CRH was measured in perfusate from push-pull cannulas implanted in
the rat median eminence; samples were collected at 5 min intervals
from an unanesthetized male rat under basal conditions. Reproduced
with permission from (84). (B) ACTH levels in rat plasma samples col-
lected every 2 min from the jugular vein via an indwelling cannula.
Reproduced with permission from (27). (C) Corticosterone levels in rat
blood samples collected every 5 min using an automated blood sam-
pling system. Reproduced with permission from (201). Note that in the
rat, CRH pulse frequency is higher (∼3 pulses/h) than the near-hourly
oscillation in ACTH and corticosterone.
The origin of the glucocorticoid
ultradian rhythm
The interaction between the pituitary corticotrophs and the
adrenal fasciculata cells has been modeled mathematically
utilizing differential equations that incorporate the rapid glu-
cocorticoid inhibition of ACTH secretion (202). Numerical
analysis of the model suggests that the pituitary-adrenal sys-
tem can support self-sustained ACTH and glucocorticoid
oscillations occur at the same frequency as endogenous cor-
ticosterone oscillations observed in the rat during the peak
phase of the circadian cycle (Fig. 6, A and B). Moreover,
the model also suggests that these glucocorticoid oscillations
become “damped” for higher (or lower) levels of CRH (Fig. 6,
A and C). These predictions are consistent with experimental
observations that ACTH and corticosterone pulse amplitude
increases during the early dark phase are accompanied by
2100 increased mean levels of CRH in the median eminence (84),
and with the loss of pulsatile corticosterone secretion that
occurs both at the circadian nadir of HPA axis activity and
following an acute stress response (214), when the levels of
CRH are respectively lower and higher than during the cir-
cadian peak. The third key prediction of this mathematical
model is that glucocorticoid oscillations induced by constant
CRH stimulation are driven by oscillations in ACTH, with
a phase shift between ACTH and glucocorticoids. This is
again consistent with experimental data showing that ultra-
dian ACTH oscillations have been observed in the rat (26,27)
and that pulsatile ACTH is a critical factor in regulating pul-
satile corticosterone secretion from the adrenal cortex (180).
Consistent with both mathematical and experimental data,
high-frequency blood sampling in humans under basal, non-
stressed, conditions has revealed a close temporal relationship
between pulses of ACTH and cortisol, with a short phase lag
between the two hormones (73) (Fig. 5).
The hypothesis that the sub-hypothalamic pituitary-
adrenal system can function as an ultradian hormone oscil-
lator has been tested in vivo by studying the dynamics of
corticosterone responses to different levels of constant CRH
stimulation in conscious freely behaving male rats (201). In

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Comprehensive Physiology HPA Axis-Rhythms

Oscillation period (min)

(A)
45 60 75

60 (B) ACTH
2.33 2
CORT

CORT (AU)
ACTH (AU)
50 Steady state

40 C 0 0
0 1 2 3 4 5
CRH (AU)

Time (h)
30
Oscillation
B (C) 2.33 2 ACTH

CORT (AU)
ACTH (AU)
20 CORT

10
0 0
0 1 2 3 4 5
0 Time (h)
0 5 10 15 20
Adrenal delay (min)

Figure 6 Mathematical modeling predictions of the pituitary-adrenal response to different levels

of constant CRH drive. (A) Computed two-parameter bifurcation diagram shows that different
combinations of constant CRH drive and adrenal delay result in qualitatively different dynamic
responses from the pituitary-adrenal system. On one side of the transition curve, the pituitary-
adrenal system responds with oscillations in ACTH and glucocorticoids (CORT), despite the fact
that the CRH drive is constant (point B). On the other side of the transition curve, the pituitary-
adrenal system responds with steady state levels in ACTH and CORT (point C). (B) Model predictions
for ACTH (grey) and CORT (black) corresponding to point B in panel (A). (C) Numerical simulations
for ACTH (grey) and CORT (black) corresponding to point C in panel (A). Model predictions are
shown for a 5-h period after transient dynamics have decayed. AU, arbitrary units. Reproduced
with permission from (202).

the male Sprague-Dawley rat, the HPA circadian cycle is char-
acterized by a distinct and prolonged nadir phase (7 am-1 pm),
when endogenous CRH secretion is minimal (84) and there is
no detectable pulsatile secretion of ACTH or corticosterone
(28,214). This experimental model allows manipulation of the
system by controlling the level of CRH and simultaneously
measuring the levels of ACTH and corticosterone, without
interference from endogenous HPA hormone release. Con-
stant infusions of CRH (during the circadian nadir) have been
shown to induce different temporal patterns of corticosterone
secretion that are dependent on the level of CRH. In keep-
ing with the predictions of the mathematical model (202),
a constant infusion of CRH induces sustained ultradian cor-
ticosterone oscillations that persist throughout the infusion
period, with pulse amplitude remaining relatively constant
throughout the infusion (Fig. 7A). Furthermore, there is a
phase shift between ACTH and corticosterone, with ACTH
oscillations preceding corticosterone oscillations, also pre-
dicted by the mathematical model (Fig. 7, B and C). This
time delay between ACTH and corticosterone pulses reflects
the time taken for de novo biosynthesis and release of corticos-
terone from the adrenal cortex upon stimulation with ACTH
(186). Interestingly, this oscillatory response to CRH is dose
dependent, and becomes disrupted for higher levels of CRH.
In summary, the hypothesis that a systems-level dynamic
balance between positive feed-forward and negative feedback
provides a mechanism for generating ultradian oscillations in
ACTH and glucocorticoid hormone secretion has been sup-
ported by both mathematical modeling and experimental data
and these findings provide good evidence for an oscillating
mechanism outside the central nervous system.
Adrenal regulation of glucocorticoid pulsatility
Glucocorticoid hormones are lipophilic and therefore can-
not be simply packaged in readily releasable vesicles within
steroidogenic cells in the adrenal cortex, but need to be
newly synthesized in response to ACTH. Given that pulsatile
secretion of glucocorticoids is generated by pulses of ACTH,
as shown by both mathematical modeling and experimental
work (201, 202), it is clear that within the adrenal there must
be a remarkably responsive steroid synthesis mechanism that
is able to respond rapidly to each pulse of ACTH. Indeed,
a recent study in the rat has shown that pulsatile ACTH
is necessary for optimal release of corticosterone (180); in
rats in which endogenous HPA activity has been suppressed
by the addition of a synthetic glucocorticoid (methylpred-
nisolone, MP) to the drinking water, pulsatile infusion of
ACTH results in a normal pattern of pulsatile corticosterone
secretion (Fig. 8A), whereas an identical dose of ACTH
infused at a constant rate has no effect on the adrenal cor-
ticosterone secretion.

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HPA Axis-Rhythms Comprehensive Physiology

(A) 150 (B) 80 140 (C) 100 160

ACTH ACTH

Corticosterone (ng/mL)

Corticosterone (ng/mL)
120 140
Corticosterone (ng/mL) CORT CORT
80 120

ACTH (pg/mL)

ACTH (pg/mL)
100
100 60
100
80
60 80
60
60
50 40
40 40 40
20 20
0 20 0 20 0
0600 0800 1000 1200 0700 0800 0900 0 10 20
Clock time (h) Clock time (h) Time (min)

Figure 7 Constant infusion of CRH induces pulsatile secretion of ACTH and corticosterone. (A) Individual corticosterone
response to constant CRH infusion. In line with the modeling hypothesis, constant infusion of CRH (0.5 μg/h) induces ultradian
corticosterone oscillations that persist throughout the infusion period. This oscillatory response is characterized by an initial
rapid increase in corticosterone within approximately 15 min following the onset of the CRH infusion, reaching peak levels by
approximately 30 min and returning to low levels by approximately 80 min. Following the initial pulse, the amplitude of the
oscillation is relatively constant throughout the infusion. Samples were collected every 5 min from a freely behaving male rat
using an automated blood sampling system. Grey bar indicates the period of infusion. (B) ACTH and corticosterone responses to
constant CRH infusion. Samples were collected manually at 20 min intervals throughout the infusion via an indwelling cannula
implanted in the jugular vein. In agreement with the modeling predictions, CRH induces ACTH and corticosterone oscillations
that persist throughout the infusion period. (C) Phase-shifted ACTH and corticosterone response to constant CRH infusion
(0.5 μg/h) over the initial activation phase (0-25 min) of the oscillation. This phase shift between ACTH and corticosterone
presumably reflects the time taken for de novo biosynthesis and release of corticosterone in the adrenal cortex. Reproduced with
permission from (201).

To better understand the mechanism underlying the in vivo
generation of pulsatile corticosterone secretion from adrenal
fasciculata cells within the adrenal cortex, the dynamics of
steroidogenic gene transcription in relationship to the dynam-
ics of corticosterone secretion has been investigated over a
detailed time-course following a single ultradian pulse of
ACTH (179). In the rat, pulsatile release of corticosterone,
induced by intravenous (IV) administration of a small ACTH
pulse, is associated with pulsatile transcription of steroido-
genic genes, including StAR (118, 186) (Fig. 8B). This has
been shown by measuring rapid changes in the levels of het-
eronuclear RNA (hnRNA), which is a reliable marker of gene
transcription. Specifically, StAR hnRNA levels peak at 15 min
after ACTH administration, and return to basal levels within
30 min. A similar dynamic pattern of transcription has been
observed for CYP11A, the gene encoding for cholesterol side-
chain cleavage cytochrome P450 (P450scc) (36), which catal-
yses the cleavage of the cholesterol side chain to produce
pregnenolone in the inner mitochondrial membrane. In keep-
ing with these findings, Spiga and colleagues (179) have also
shown that activation of CREB and its transcriptional regu-
lator TORC2 (transducer of regulated CREB activity 2, also
known as CRTC2) also occurs in a highly dynamic pattern
following pulsatile ACTH stimulation: PKA-mediated activa-
tion of CREB and TORC2, by phosphorylation and dephos-
phorylation respectively, occurs within 5 min of exposure to
a pulse of ACTH, supporting an involvement of these pro-
teins in the rapid (within 15 min) transcription of StAR. An
involvement of TORC2 in the CREB-mediated transcriptional
regulation of StAR in adrenalcortical cells has been shown
in vitro (190, 191). A recent study has shown that a pulse of
ACTH also induces pulsatile transcription of the gene encod-
ing for MRAP (119), a protein that is crucial in regulating
the level and activity of MC2R at the cell surface, and thus
the cell’s responsiveness to ACTH (132) (Fig. 8C). Taken
together, these studies clearly suggest that pulsatile secretion
of corticosterone is associated with pulsatile transcription of
steroidogenic genes in the adrenal gland.
Although corticosterone levels peak within 5 min of an
ACTH pulse (given IV), hnRNA StAR levels do not peak until
15 min after the ACTH pulse. The fact that the peak of the
secretory event precedes the transcription peak of the steroido-
genic enzyme is consistent with evidence that nongenomic
posttranscriptional and posttranslational changes in the StAR
protein regulate the rapid steroidogenic response. Indeed,
rapid activation of proteins involved in steroidogenesis by
phosphorylation, such as phosphorylation of StAR on Ser194
mediated by PKA, plays a role in the immediate initiation
of steroidogenesis and glucocorticoid secretion upon stimu-
lation of the adrenal by ACTH (31, 133, 134, 148). Therefore,
it is possible that the pulsatile transcription of StAR following
each pulse of ACTH may be acting as mechanism for replen-
ishing depleted intracellular stores of StAR (that have been
used to generate a pulse of corticosterone) prior to subsequent
ACTH pulses. Furthermore, these observations are consistent
with previous data showing that only newly synthesized StAR
is involved in posttranslational events mediating the rapid
synthesis of corticosterone (11). Whether nongenomic events
regulating rapid steroidogenesis are also characterized by a
pulsatile pattern of activity is currently under investigation.

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Comprehensive Physiology HPA Axis-Rhythms

(A) 100

Corticosterone (ng/mL)
75

50

25

0700 1300 1900 0100 0700

Clock time (h)

(B) 2.5 (C) 10

2.0 8
Fold induction hnRNA

Fold induction hnRNA

(MRAP/GAPDH)
(StAR/GAPDH)

1.5 6

1.0 4

0.5 2

0 0
0 5 15 30 60 0 5 15 30 60
Time (min) Time (min)

Figure 8 Effect of pulsatile ACTH administration on plasma corticosterone levels and StAR and
MRAP primary transcript levels. (A) Pulsatile corticosterone secretion in rats chronically treated with
methylprednisolone (MP) and infused with pulsatile ACTH (4 ng/h, 5 min pulse/h). ACTH infusion
started at 10 am and blood samples were collected every 10 min using an automated blood sampling
system. Data are the mean of 9 different rats. Data reproduced with permission from (180). (B and
C). Administration of a small dose of ACTH (4ng/rat; i.v.) induces a rapid but transient increase in the
primary transcript of StAR (B) and MRAP (C), suggesting that pulsatile expression of steroidogenic genes
is important for the ultradian rhythm of corticosterone secretion. Transcriptional response of StAR and
MRAP was measured by RT-qPCR using specific primers targeting the gene primary transcript. Data in
(B) reproduced with permission from (179). Data in (C) reproduced with permission from (119).

Glucocorticoid Rhythms in Health
and Disease
Over the last 20 years, we have used our ABS system to define
corticosterone pulsatility in freely behaving rats in their home
cages. These studies have revealed that both the circadian
and the ultradian pattern shows marked sexual diergism and
genetic variation. Furthermore, corticosterone rhythms are
remarkably plastic, with changes seen both in physiological
conditions, such as during lactation and ageing, and in patho-
logical conditions, particularly stress-related disorders.
Physiological changes: Gonadal steroids
A marked difference in the levels of basal corticosterone
secretion between male and female rodents is well recognized.
Recently, studies using frequent blood sampling to investigate
corticosterone pulsatility in the rat have shown a profound
difference in the pulsatile profile of corticosterone release
over the 24-h cycle between male and female rats (169-172).
Compared to male rats, basal corticosterone secretion is
enhanced in female rats (169) (Fig. 9, A and B), and this is
due to: (i) an increase in the total number of pulses over the
24-h cycle (28 pulses for females vs. 10 pulses for males);
(ii) an increase in pulse magnitude (approximately 60 ng of
corticosterone/pulse for females vs. 24 ng/pulse for males);
and (iii) a greater number of pulses over the 24h period.
Gonadal steroids have major effects on the pulsatile pat-
tern of corticosterone release in the rat. Following gonadec-
tomy, male rats have increased overall corticosterone secre-
tion similar to that observed in female rats, characterized
by an increased number of pulses, pulse amplitude, and

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HPA Axis-Rhythms Comprehensive Physiology

120

Corticosterone (ng/mL)
(A) (B)
100
80
60
40
20
0
0700 1300 1900 0100 0700 0700 1300 1900 0100 0700
Clock time (h) Clock time (h)
160
Corticosterone (ng/mL)

(C) (D)
120

80

40

0
0500 1100 1700 2300 0500 0500 1100 1700 2300 0500
Clock time (h) Clock time (h)
120 (E)
Corticosterone (ng/mL)

(F)
100
80
60
40
20
0
1800 0000 0600 1200 1800 1800 0000 0600 1200 1800
Clock time (h) Clock time (h)

Figure 9 Changes in ultradian glucocorticoid dynamics in different physiological and

pathological states. (A and B) Ultradian corticosterone oscillations in a female (A) and male
(B) Sprague-Dawley rat. Reproduced with permission from (169) (C and D). Ultradian corti-
costerone oscillations in juvenile (C) and adults (D) rats (unpublished observations). (E and F)
Ultradian corticosterone oscillations in a control male PVG rat (E) and a male PVG rat with
active immune-mediated adjuvant-induced arthritis (F). Reproduced with permission from
(212). In all experiments, corticosterone was measured in blood samples collected at 10 min
intervals from freely behaving rats. Shaded regions indicate the dark phase.

pulse frequency. On the other hand, ovariectomized females
have reduced overall corticosterone levels similar to those
observed in male rats, with a reduction in the number of
pulses, and decreased pulse amplitude and frequency (169).
To better understand whether these changes are due to a
direct effect of gonadal hormone withdrawal, further stud-
ies have been carried out to investigate the effect of androgen
or oestrogen replacement on corticosterone secretion in the
gonadectomized male and female, respectively (170): andro-
gen replacement reverses the increase in corticosterone pulse
frequency and amplitude induced by castration in male rats;
similarly, oestradiol reverses the changes in corticosterone
secretory activity in ovariectomized female rats.
Neonatal masculinization or feminization also affects
basal corticosterone secretion patterns in adult life in female
and male rats, respectively (171,172). Exposure of the female
neonate to a single injection of testosterone within 24 h of birth
results, in adult animals, in a reduction in the number of cor-
ticosterone pulses, as well as pulse amplitude and frequency,
over the 24-h period, compared to untreated adult females, and
these pulse characteristics are comparable to those observed
in normal adult male rats (171). Consistent with this, changes
in the adult pulsatile pattern of corticosterone secretion are
observed in male rats exposed to neonatal feminization (172):
here, rats deprived of pre and postnatal testosterone activity
by exposure to the antiandrogen flutamide and the aromatase
inhibitor 1,4,6-androstatriene-3,17-dione, respectively, show
increased basal corticosterone secretion characterized by an
increase in the number, frequency, and amplitude of corti-
costerone pulses. Interestingly, the amplitude of the corticos-
terone pulses of prenatal testosterone-deprived rats is similar
to those observed in intact females and castrated males (169).
The mechanisms underlying the effects of gonadal
steroids on the ultradian rhythm of glucocorticoids are not
clear. There is evidence suggesting that androgens and estro-
gens can affect HPA axis activity at the level of the adrenal
gland, anterior pituitary and PVN [reviewed in (67)]. This is
supported by an overlap in the expression of gonadal hormone
receptor and glucocorticoid hormone receptors in key areas
regulating the activity of the HPA axis (67).

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Comprehensive Physiology
Differences in female corticosterone levels across the
estrus cycle have been observed. During the diestrous phase,
corticosterone levels in female rats are similar to those
observed in male rats; in contrast, during the proestrous phase,
when the concentrations of progesterone and estradiol are ele-
vated, corticosterone levels are increased (34, 45), and this
is paralleled by an increase in CRH mRNA expression in
the PVN (82). Consistent with this, inhibition of HPA axis
activity by androgens is paralleled by a reduction in CRF-
immunoreactivity in the PVN (15). Gonadal steroid hormones
can also regulate basal glucocorticoid secretion by affecting
GR signaling. Indeed, estrogens regulate the transcriptional
of a number of proteins involved in GR activity, such as the
chaperone protein FKBP51 (78) and the nuclear GR coacti-
vator SRC1 (22). Taken together, these studies suggest that
sex steroids can affect the glucocorticoid ultradian rhythm by
modulating both hypothalamic CRH drive and GR-mediated
glucocorticoid negative feedback.
Physiological changes: Ageing
Studies on ageing rats have revealed marked changes in the
pattern of corticosterone pulsatility across their life cycle
(117) (Fig. 9, C and D). In juvenile and adults rats, marked
changes in pulse amplitude over the 24-h cycle have been
observed, with higher amplitude pulses during the peak phase,
resulting in the well defined circadian rhythm of corticos-
terone. In elderly (12-month-old) rats, however, there is a
loss of circadian rhythmicity predominantly due to a decrease
in pulse amplitude during the peak phase, but also due to
an overall loss of ultradian pulsatility throughout the 24 h.
These observations are important and suggest a potential link
between disrupted corticosterone pulsatility and age-related
disorders.
Physiological changes: Reproduction
Changes in corticosterone rhythms have also been observed in
female rats over the course of their reproductive cycle (211).
Circadian and ultradian rhythms of corticosterone have been
studied in virgin rats, rats on day 9 of lactation, weaned dams
2 days after pups removal, and postlactating dams 13 days
after pups removal. During lactation, although there is a sig-
nificant circadian variation in corticosterone release, this is
associated with a flattening of the rhythm—due to a decrease
in the evening peak levels and an increase in the number of cor-
ticosterone pulses throughout the 24 h. Very marked changes
in corticosterone release are also observed in rats 2 days after
experimental weaning. In this group, corticosterone levels are
significantly suppressed throughout the 24-h period, with the
number of pulses and pulse amplitude significantly lower than
any other group. Interestingly, these effects are reversible, as
no differences in either circadian or ultradian rhythms of cor-
ticosterone are observed between 13 days postlactating dams
and virgin rats.
Volume 4, July 2014
HPA Axis-Rhythms
Physiological changes: Genetic background
Changes in corticosterone pulsatility patterns related to
genetic background have also been observed. Ultradian corti-
costerone rhythms have been investigated in a number of rat
strains, including Wistar, Sprague Dawley, Lewis, and Fisher
344. Although they all exhibit a pulsatile pattern of corti-
costerone release, there are significant differences between
strains (213). Lewis rats have a clear circadian rhythm, as
seen in female and male Sprague Dawley and Wistar rats, due
to changes in pulse amplitude over the 24-h cycle, which is
greater in the evening than in the early morning. In contrast,
female Fischer rats have higher mean circulating corticos-
terone concentrations and lack any discernible circadian vari-
ation, and this is due to increased pulse amplitude in the morn-
ing, which is similar to pulse amplitude in the evening. The
mechanism underlying the different pattern of corticosterone
secretion between the two strains is not known. Given that
female Fisher rats maintain high-pulse amplitudes throughout
the morning, it has been proposed that insensitivity to corti-
costerone feedback, mediated by activation of both GR and
MR, might be responsible for the lack of circadian rhythm.
Furthermore, in light of the role of splanchnic innervation of
the adrenal in the regulation of circadian adrenal responsive-
ness to ACTH (195), the differential dynamics in pulsatile
secretion between the two strains could also be due to differ-
ences in sympathetic regulation of adrenal sensitivity. Inter-
estingly, Lewis rats are known to be susceptible to a range of
inflammatory conditions, including streptococcal cell wall-
induced arthritis (183, 184), whereas the Fisher rat does not
develop any of these conditions. This suggests that differences
in disease susceptibility may be linked to differences in the
dynamics of corticosterone secretion.
Pathological changes: Chronic stress
Chronic inflammation is a model of chronic stress that has
high clinical relevance. In particular, a model of chronic
inflammatory disease that has been extensively studied in the
rat is Mycobacterium-adjuvant-induced arthritis. Increased
circulating corticosterone and ACTH concentrations, and loss
of the normal circadian rhythm of HPA activity, have been
observed in Piebald-Viral-Glaxo (PVG) rats infected with the
Mycobacterium-adjuvant (212) (Fig. 9, E and F). While con-
trol PVG rats demonstrate the expected circadian and ultra-
dian patterns of corticosterone release, significant changes in
the dynamics of corticosterone secretion occur both prior to
the development of arthritis (presymptomatic rats sampled at
6 days after injection of the mycobacterium) and following
the development of hind-joint inflammation (13 days after
injection). In presymptomatic rats, although a diurnal pattern
of corticosterone secretion is still observed, there are changes
in the characteristics of the pulses. While no changes in the
number of pulses or their amplitude have been observed across
the 24-h cycle, in presymptomatic rats the average length
of time during which hormone levels rise from baseline to
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HPA Axis-Rhythms
peak is decreased. Specifically, within each pulse, corticos-
terone levels rise to peak concentrations more quickly than in
control rats. During the symptomatic period, rats show dra-
matic changes in pulsatility, with an almost doubled number
of pulses throughout the 24-h cycle related to continued pul-
satility during the normally quiescent lights-on period. Inter-
estingly, neither the amplitude or the duration of the pulses
are different from control rats, indicating that the increase in
circulating hormone is solely due to this increased number of
pulses. It is important to note that in rats exposed to adjuvant-
induced arthritis, a decrease in CRH synthesis and secretion
has also been observed, together with a very marked increase
in pituitary portal levels of vasopressin (2,34,68). The lack of
changes in corticosterone pulsatility across the 24-h cycle in
these animals suggests a consistent level of CRH (and AVP)
activation of the pituitary across the day within the range that
will generate pulsatile ACTH and corticosterone secretion,
as predicted by the mathematical model described by Walker
and colleagues (201, 202).
Another model of chronic stress that has been found to
affect corticosterone pulsatility in the rat is exposure to con-
stant light. Experimental animals are normally kept under a
12-h light-dark cycle, with light input acting as a zeitgeber
processed through the SCN. Disruption of the SCN signal,
either by physical lesioning of the SCN or by disruption of
the light input, induces a loss of circadian rhythmicity and
this is associated with changes in corticosterone pulsatility.
Rats maintained under conditions of constant light for a pro-
longed period of time (5 weeks) lose their circadian corticos-
terone rhythm, which is due to increased pulsatility during the
nadir phase of the circadian cycle, with no difference between
the nadir and the peak phase pulse amplitude (200). These
same rats have increased levels of CRH mRNA in the morn-
ing, which probably accounts for the increased corticosterone
secretion during the nadir phase. This suggests that removal
of the inhibitory SCN input to the hypothalamic PVN results
in sufficient CRH secretion throughout the 24 h to maintain
ultradian pituitary-adrenal activity.
The SCN also mediates corticosterone secretion by regu-
lating adrenal sensitivity to ACTH through a mechanism that
involves the ANS and splanchnic nerve. It has been shown
that light can activate adrenal secretion of corticosterone in
a way that is independent of pituitary release of ACTH, but
that involves activation of the splanchnic nerve (19, 81). It is
therefore possible that the effect of constant light exposure on
corticosterone pulsatility may also occur due to a direct effect
on the adrenal gland.
Pathological changes: Neonatal programming
Exposure to stress during the neonatal period has profound
effects on physiological functions that can be observed in
adult life. This phenomenon is known as neonatal program-
ming. Early life stress can also program the development of
the HPA axis with changes that persist for the rest of the life
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Comprehensive Physiology
of the organism. The effect of neonatal programming on cor-
ticosterone pulsatility in the rat has been investigated using a
model of early life infection (174). In rats exposed to endo-
toxin (Salmonella enteritidis) in neonatal age (days 3 and 5
postpartum), basal levels of corticosterone are higher both
during the nadir and peak phase of the circadian cycle in
adult life. This is due to an increase in both the number and
amplitude of corticosterone pulses throughout the 24-h cycle.
The Importance of Pulsatility for
Hormonal and Behavioral Response
to Stress
The functional interaction between glucocorticoid pulsatility
and the response to stress has been investigated in a number of
studies where rats are exposed to stress during different phases
of their ultradian corticosterone secretory profile (165, 213).
Windle and colleagues (213) have shown that the timing of
the stressor relative to the phase of the underlying ultradian
rhythm is crucial in determining the magnitude of the cor-
ticosterone response. Specifically, in rats exposed to a mild
stressor such as white noise, the corticosterone response is
considerably greater when the stressor is applied during the
rising phase of a corticosterone pulse than when the stressor
is applied during the falling phase (Fig. 10), which suggests
a facilitated stress response during the rising phase and/or
an inhibitory effect during the falling phase. This relation-
ship between pulse-phase and the ability of the HPA axis
to respond to acute stress has been observed in both male
and female rats. These observations indicate the existence of
a dynamic interaction between basal glucocorticoid pulsatil-
ity and the ability of an animal to mount a hormonal stress
response.
Mathematical modeling and in vivo experimental work
suggest that ultradian glucocorticoid oscillations can be gen-
erated by the feedforward-feedback interaction between the
anterior pituitary and adrenal cortex, independent of pulsatile
hypothalamic stimulation (201, 202). Therefore, exposure to
a stressor, and its associated release of hypothalamic CRH
and AVP, can be considered as a transient perturbation to the
endogenous oscillatory activity of the pituitary-adrenal sys-
tem. This mathematical model has also been used to under-
stand in more detail the interaction between pulsatility and
stress responsiveness (154). In addition to explaining ear-
lier observations that the magnitude of the stress response
depends on the timing of the stress, analysis of this model
has also shown that an external stress can act as a resetting
mechanism to the phase of the endogenous ultradian rhythm.
The mechanism underlying the differential HPA axis
response to stress in relationship to the ultradian corticos-
terone rhythm has been further investigated in adrenalec-
tomized rats in which the endogenous hormone is replaced
with an IV infusion of hourly pulses of corticosterone (165).
Volume 4, July 2014
Comprehensive Physiology
(A)
600
Corticosterone (% of basal)
300
0
–60 –40 –20 0 20
Time (min)
(B)
600
Corticosterone (% of basal)
300
0 is reflected in differences in the corticosterone response to
–60 –40 –20 0 20
Time (min)
Figure 10 Stress responsiveness is dependent on the phase of
the ultradian corticosterone rhythm. Corticosterone response in rats
exposed to a noise stress (10 min, 114 dB); blood samples were col-
lected every 10 min using an automated blood sampling system. Rats
stressed during the rising phase of an endogenous corticosterone pulse
(A) show much greater corticosterone responses than animals stressed
during the falling phase (B). Reproduced with permission from (214).
Consistent with Windle et al. (214), exposure of these rats to
noise stress results in an increase in ACTH, and this response
is more pronounced when the stressor is applied during the
rising phase of the corticosterone ultradian pulse than during
the falling phase. The differential ACTH response to noise
stress, relative to the phase of corticosterone pulse, is also
associated with a different behavioral response to the stressor.
Indeed, the behavioral response of rats exposed to noise stress
is higher in rats that are stressed during the rising phase of
the corticosterone pulse, compared to rats exposed to stress
during the falling phase of the pulse. Interestingly, in addi-
tion to a differential hormonal and behavioral response to
noise relative to the phase of the corticosterone pulse, differ-
ential neuronal activation in the amygdala occurs when the
noise stress is delivered during the rising phase rather than
the falling phase of the ultradian pulse (165). These findings
are consistent with earlier behavioral studies showing that
rats exposed to a male intruder during the rising phase of an
endogenous corticosterone pulse are more aggressive than rats
exposed to the intruder during the falling phase of the pulse
(65, 66).
Volume 4, July 2014
HPA Axis-Rhythms
Differential response to stress is associated with
changes in the ultradian glucocorticoid rhythm
Both experimental data and mathematical modeling have
shown that a facilitated corticosterone response to stress
occurs when the stressor is applied during the rising phase of
an ultradian pulse (i.e., when the HPA axis is already secret-
ing hormone), or during an interpulse period (when the axis
is quiescent). In contrast, the axis is inhibited and unable to
respond when the stress is applied during the falling phase of
a pulse (154, 214). It is clear therefore that the characteristics
40 60 of the corticosterone ultradian rhythm are important not only
for basal corticosterone secretion but also for maintaining
hormonal responsiveness to stress. In light of this, the rela-
tionship between the timing of the exposure to stress relative
to the phase of the corticosterone pulse has been investigated
in a number of experimental models where differences in cor-
ticosterone pulsatility have been observed, such as in rats of
different strain (213), reproductive stage (173, 211), gender
(169-172), and in rats exposed to chronic stress (174, 212).
Differences in the pattern of ultradian rhythmicity have
been observed between female Fisher and Lewis rats, and this
40 60 stress in these animals (213). The corticosterone response to
noise stress is greater in the Fischer than in the Lewis female
rat. In the female Lewis rat, stress-induced corticosterone
secretion does not occur when the noise is applied during the
falling phase of the ultradian pulse. In contrast, female Fisher
rats respond equally to a noise stress regardless of when it
occurs in relation to the phase of the endogenous corticos-
terone rhythm. A pronounced difference in the duration of
stress-induced corticosterone release has also been observed
between the two strains, with Fischer rats showing a more
prolonged response.
Differential responses to stress have been observed fol-
lowing manipulation of the gonadal steroid environment of
both male and female rats, and changes in the ultradian rhythm
are associated with an altered response to both psychological
noise stress and LPS-induced immune challenge (169-172).
Although there is a significant and prolonged increase in
plasma corticosterone in both males and females in response
to noise and LPS, the amplitude of the response is higher
in females compared to males. Furthermore, gonadectomy
reverses this pattern with corticosterone profiles and stress-
induced peak corticosterone release in castrated males that are
similar to those seen in sham females. In contrast, the effect of
ovariectomy results in an ultradian profile and stress-induced
peak response similar to those in sham males (169). As seen
for basal corticosterone pulsatility, the effects of gonadectomy
on the stress response in both male and female are reversed
by androgen and oestradiol replacement in male and female,
respectively (170). Similarly, neonatal masculinization, which
results in reduced pulse amplitude, number, and frequency in
adult female rats, is associated with reduced responsiveness to
both noise and LPS stress, as seen in normal adult males (171).
In contrast, pre and postnatal deprivation of testosterone in
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HPA Axis-Rhythms
male rats, which results in increased pulse number, amplitude
and frequency, is associated with significantly elevated cor-
ticosterone secretion after noise stress or LPS administration
(172).
Differential responses to stress have also been observed in
female rats at different reproductive stages (173, 211). While
in virgin rats noise stress causes a rapid increase in plasma
corticosterone concentration, in the lactating group, which
has an increased number of pulses across the 24-h cycle,
no effects on plasma corticosterone levels are seen follow-
ing noise stress. Furthermore, in the experimentally weaned
group, which show lower basal corticosterone levels and a
lower number of pulses, the response to noise is of similar
amplitude, but more prolonged, compared to the virgin group
(211).
A different response to stress has been observed in rats
exposed to chronic inflammation (adjuvant-induced rheuma-
toid arthritis). In these rats, the relationship between the pulse
phase and the timing of the stress is maintained through-
out the development of the inflammation (212). However, the
overall corticosterone response to noise stress is significantly
lower in symptomatic rats than in controls. Thus, it is possi-
ble that as the number of pulses increases and the interpulse
periods reduce in symptomatic rats, there is a greater propor-
tion of time when the rats are unable to respond to stress.
These data provide evidence for a direct relationship between
increased basal HPA axis activity, associated with chronic
disease, and a decreased response to acute stress, and this
is indeed consistent with previous data showing a decreased
corticosterone response to acute stress in rats with adjuvant-
induced arthritis (2, 69), and also in humans with rheuma-
toid arthritis (33). Interestingly, there is no difference in the
corticosterone response to LPS between rats with adjuvant-
induced arthritis and control rats.
Changes in basal corticosterone pulsatility in rats neona-
tally exposed to an inflammatory stress are also associated
with a differential corticosterone response to stress (174).
Rats that receive endotoxin in early life show an increase in
both the amplitude and number of corticosterone pulses dur-
ing basal conditions in adult life, and are also hyper-reactive
to stress (noise and LPS).
Ultradian Rhythm of Glucocorticoids
in Target Tissues
CBG regulates the level of glucocorticoids at
target tissues
Once they have been released into the general circulation,
approximately 95% of glucocorticoid molecules become
bound to carrier proteins, which prevents the steroid from
diffusing into cells in target tissues. In the rat, approximately
80% of circulating corticosterone is bound to corticosteroid-
binding globulin (CBG), and 10% to 15% is bound to albumin
(113). The remaining fraction of circulating corticosterone is
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Comprehensive Physiology
unbound and free to diffuse across cell membranes and bind to
intracellular GRs and MRs in target tissues. Therefore, steroid
hormones bound to carrier proteins are considered biologi-
cally inactive and provide a reservoir of inactive circulating
hormone, and the levels of CBG regulate the amount of free
hormone available for diffusion into tissue. CBG is secreted
from hepatocytes (103) with a circadian pattern in the plasma
that is in antiphase with the circadian variation in plasma cor-
ticosterone levels (77, 114). This results in an increased level
of free corticosterone during the circadian peak. In addition,
CBG is also released from the liver in response to acute stress
in parallel with the adrenal release of corticosterone, and this
is thought to be important for maintaining low levels of free
corticosterone at target tissues (53, 152).
With respect to the role of CBG in regulating the ultradian
rhythm of active free unbound glucocorticoid, CBG acts to
accentuate pulse profiles at the tissue level. In fact, it is known
that in human, CBG has a relatively low saturation point: 400
to 500 nmol/L cortisol (12). Therefore, when levels of hor-
mone are high, such as during the peak of each ultradian
pulse, CBG is already saturated. This results in pulses of
free cortisol that are superimposed on pulses of total corti-
sol in which only about 10% is in the free state. This type
of augmented ultradian rhythm of corticosterone has been
demonstrated experimentally in the rat (53).
It has also been shown in the rat that free corticosterone
levels in the blood show distinct circadian and ultradian
rhythms, with a pulse frequency of approximately one pulse
per hour (151), consistent with the pulse characteristics of
total glucocorticoid hormone in plasma (214). Given that the
level of free glucocorticoid depends on the availability of CBG
and its affinity for the hormone, a recent study has investigated
whether there are dynamic changes in the affinity of cortisol
for CBG in human (23). This study has shown that the affinity
of CBG for cortisol is remarkably temperature sensitive—
the affinity of cortisol for CBG drops approximately 16-fold
as the temperature increases from 35 to 42◦ C. Interestingly,
there are circadian and ultradian differences in core temper-
ature that can be between 1 and 4◦ C in rodents and around
1◦ C in man. Based on the findings of Cameron et al. (23),
these changes in temperature may be sufficient to cause rapid
changes in the availability of free glucocorticoid over the
course of both the circadian and ultradian rhythms of hormone
secretion.
Temperature dependence of glucocorticoids binding to
CBG is also an important factor for the free/bound hormone
ratio in situations when robust changes in body temperature
occur. For example, an increase in body temperature of as little
as 1◦ C in febrile patients may have profound effects, markedly
increasing the amplitude of free pulsatile bio-available gluco-
corticoid at the tissue and cellular level. Indeed, studies in rats
have shown changes in the ultradian rhythm of corticosterone
secretion in animals exposed to inflammation (174,212), sug-
gesting that changes in the body temperature of rats exposed to
inflammatory stress may have an effect on the corticosterone
Volume 4, July 2014
Comprehensive Physiology HPA Axis-Rhythms

0.4

Hippocampal free corticosterone (μg/dL)

0.3

0.2

0.1

0
0700 1300 1900 0100 0700
Clock time (h)

Figure 11 Ultradian rhythm of hippocampal free corticosterone measured by in vivo micro-

dialysis in a freely behaving male Wistar rat. Hippocampal dialysate samples were collected every
10 min between 0800 and 2200 h and every 30 min between 2200 and 0800 h. Male Wistar rats
show a clear and distinct circadian and ultradian pattern of corticosterone in the hippocampus
with a pulse frequency that is similar to that observed in plasma corticosterone. Reproduced with
permission from (53).

binding to CBG, and thus on the pattern of free corticosterone
pulsatility.
Ultradian rhythm of glucocorticoid is
maintained in target organs
Recent studies have shown that the ultradian rhythm of corti-
costerone secretion is not only observed in the blood but also
in tissue targets such as the brain and subcutaneous tissue
(52-54, 152). Using the technique of microdialysis in the rat,
Droste et al. (53) have shown that the corticosterone ultradian
rhythm can be detected in discrete brain regions such as the
hippocampus and the striatum, mirroring the pulses detected
in the peripheral circulation. In the hippocampus, the fre-
quency of the pulses is approximately one per hour, which
fits well with the pulse frequency of corticosterone observed
in the blood circulation and in the adrenal gland (Fig. 11).
Interestingly, in contrast with plasma corticosterone, the ultra-
dian rhythm of hippocampal corticosterone is not affected by
gender (54). Indeed, while both plasma corticosterone levels
and pulse amplitude are higher in female rats compared to
male rats, there is no difference in either hippocampal cor-
ticosterone levels or pulse characteristics between male and
female. Although this discrepancy cannot be fully explained,
it should be noted that while the plasma corticosterone levels
reported by Windle and colleagues (214) and by Seale and col-
leagues (169) represent both CBG-bound and free hormone,
the corticosterone levels in brain measured by Droste and
colleagues (53, 54) represent only the free biologically active
fraction. Therefore, given that the levels of plasma CBG are
higher (by up to 2.5-fold) in female than in male rats (60,
125), the CBG-bound corticosterone fraction can account for
most of the differences observed between male and female. In
addition to CBG, other factors contribute to the regulation of
corticosterone levels in the brain, including the ATP-binding
cassette transporter P-glycoprotein (Pgp) (99). However, to
date, there are no reported differences in Pgp levels between
male and female rats.
The ultradian rhythm of free corticosterone is highly syn-
chronized between the blood and the target tissues both in the
rat and in man (14,152). Dual-probe microdialysis techniques
have been used to directly compare, within the same ani-
mals, the corticosterone ultradian rhythm between the blood
and the subcutaneous tissue, and between the blood and the
hippocampus. These studies have shown that corticosterone
secretion is highly synchronized between the blood and both
the subcutaneous tissue and the brain. However, although
there are no differences in the pulse amplitude between the
blood, the subcutaneous tissue, and the brain during the nadir
phase of the circadian cycle, free corticosterone levels are
slightly lower in target tissues, compared with the blood, dur-
ing the active phase.
As has been seen for plasma corticosterone, changes in the
ultradian rhythm have also been observed in the hippocam-
pus. For example, there are changes in the ultradian and cir-
cadian rhythms of free corticosterone concentrations in the
hippocampus of rats that have been exposed to long-term
(4 weeks) voluntary exercise (52). Specifically, exercising
rats display increased pulse amplitudes and mean free corti-
costerone levels during the peak phase of the circadian cycle,
compared with control animals during this period, but show
no differences in pulse frequency. In contrast, there are no dif-
ferences in ultradian rhythm characteristics between the two
groups during the nadir phase of the circadian cycle.

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HPA Axis-Rhythms
In man, studies have also been performed showing ultra-
dian and circadian rhythmicity in subcutaneous tissue (14).
The same authors have now shown a close correlation between
total cortisol levels in the blood and free cortisol levels in the
blood and subcutaneous tissue (13).
The Importance of Glucocorticoid
Pulsatility for GR Signaling
Glucocorticoids act via two distinct intracellular receptors,
MR and GR, which upon activation by ligands, translocate
into the nucleus where they induce or repress gene transcrip-
tion. Given that GR is widely expressed throughout the body,
the effects of glucocorticoids at target organs are mediated
primarily by activation of GR. When glucocorticoid levels
are low, GR is retained in the cytoplasm, where it is bound
to chaperone molecules, including HSP90 and p23 (149).
These molecular chaperones hold GR in a conformation that
exposes the ligand binding cleft, and therefore allow GR to
be activated by the ligand (150). Once activated, GR under-
goes conformational changes that induce dissociation from
the chaperone complex, leading to exposure of nuclear local-
ization sequences (NLS) in the GR hinge domain and ligand-
binding domain (140). These sequences are important so that
importin molecules can recognize and bind at these NLS
sequences, and facilitate active transport of GR through the
nuclear pore. Once in the nucleus, GR can dimerize and bind
to specific regulatory sequences in the DNA, termed glu-
cocorticoid response elements (GREs) (168). This event is
followed by the recruitment of cofactors, including members
of the p160 family (129) and the histone acetyl transferase
(HAT) proteins P300 and CBP (105). Importantly, P300 and
CBP are involved in the reorganization of the local chro-
matin structure to allow access of RNA polymerase and the
basal transcriptional machinery at the transcriptional start site
(203), ultimately leading to transcription of target genes. It is
important to understand how the dynamic ultradian oscilla-
tions in glucocorticoid levels we have described above affect
the activity of these receptors and their downstream influences
on gene transcription.
Effect of ultradian glucocorticoid rhythm on GR
activity: in vitro studies
The importance of glucocorticoid pulsatility for the physio-
logical regulation of GR activity has been investigated in vitro
in a number of cell lines, including mouse corticotroph AtT-
20, human HeLa, rat liver HTC, and mouse MCF7(3617) cell
lines (38,40,181). Exposure of cells to pulses of ligand (corti-
sol for human HeLa cells and corticosterone for rat HTC and
mouse AtT-20 cells) results in cyclical GR activation. In all
three cell lines, each pulse of glucocorticoid initiates a “wave”
of GR activation and DNA association. This GR-GRE asso-
ciation decreases rapidly after hormone washout, returning to
baseline levels before addition of the next pulse 45 min later.
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Comprehensive Physiology
The pattern of DNA binding has been investigated in more
detail in the MCF7(3617) mouse cell line (181). This cell line
stably expresses GR protein fused to a green fluorescent pro-
tein (GFP) tag, allowing visualization of receptor movement
in the living cell. Additionally, these cells also contain an
expanded MMTV long terminal repeat giving an array of
approximately 1200 GRE sequences. Using live cell imaging
and photobleaching techniques, Stavreva et al. have investi-
gated the real-time effect of pulses of corticosterone applied to
these cells and visualized the intracellular effect of the treat-
ment on GR activity (Fig. 12A). They have found that each
corticosterone pulse is paralleled by cyclic GFP-GR bind-
ing to the GREs and that dissociation of GFP-GR from GRE
occurs within 10 min from the beginning of the hormone
washout period. Further studies in both AtT20 and MCF7
cells (40) have shown that this transient activation, typical
of normal ultradian rhythmicity, only occurs in response to
the endogenous glucocorticoids corticosterone and hydrocor-
tisone, whereas a single pulse of the synthetic glucocorticoid
(A) Uninduced Pulse 1 Washout 1
Pulse 2 Washout 2 Pulse 3
(B) 30
Pulsatile
Constant
Fold change
20
10
0
0 20 40 60 80 100 120
Time (min)
Figure 12 Ultradian corticosterone pulses induce pulsatile GR-GRE
interactions and pulsatile gene transcription. (A) Real time single cell
imaging of GFP-labeled GR loading at an engineered array of GREs
in the MCF-7 (3617) mouse cell line. Each pulse of corticosterone
causes a “wave” of GR-GRE binding (fluorescent green points) that is
rapidly reversed (GR-GRE dissociation) following hormone withdrawal
(washout). (B) Dependence of transcriptional dynamics on the pattern
of hormone stimulation. Ultradian corticosterone treatment results in
pulsatile transcription of the Period 1 gene (Per1) (black), whereas
constant corticosterone induces sustained transcription (grey). Levels of
Per1 primary transcript were measured in the 3134 cell line by real-
time quantitative PCR. Data reproduced with permission from (181).
Volume 4, July 2014
Comprehensive Physiology
analogue dexamethasone results in a prolonged GR activation
profile. The dynamics of GR dissociation from, and reasso-
ciation with, the ligand, and the mechanism underlying these
events, has also been investigated in MCF7 cells (181). When
these cells are exposed to pulses of corticosterone, GR rapidly
dissociates from the ligand following washout. However, for
GR to be able to rebind the ligand when the next pulse of
corticosterone arrives, and therefore initiate a second cycle
of transcriptional activity, it requires the formation of a com-
plex with chaperone proteins in the nucleus. Indeed, when
the chaperone complex formation is blocked using the Hsp90
inhibitor geldanamycin, GR cannot bind corticosterone, and
this results in GR failure to associate with GRE, and an accel-
erated proteasome-mediated degradation of GR.
In other studies, it has been shown that pulsatile exposure
to corticosterone also results in dynamic recruitment of Poly-
merase II (Pol II) at GRE sites, with periods of reduced Pol
II exchange with DNA that coincide with maximal GR-GRE
binding. Using chromatin immunoprecipitation (ChIP), it has
been shown that in both MCF7 (181) and AtT20 (38) cells,
consistent with the pattern of GR binding to DNA, there is
also a pulsatile pattern of GR binding to GREs in the promoter
sequences of naturally occurring glucocorticoid-responsive
genes, including genes encoding metallothionein1 and Period
1 (Per1), and the negatively regulated pro-opiomelanocortin
(38). This is also reflected in the pattern of transcription
of a number of GR-responsive genes, as measured by real
time quantitative PCR (RTqPCR) using primers designed to
recognize nascent RNA transcripts (or heteronuclear RNA,
hnRNA). Pulsatile corticosterone exposure results in a rapid
and transient increase in hnRNA from several GR-regulated
genes (38, 181), whereas constant exposure to corticosterone
is associated with a continuous increase in hnRNA (181)
(Fig. 12B). In addition to this, constant treatment with corti-
costerone results in accumulation of higher levels of mature
RNA (mRNA) and protein, when compared with pulsatile cor-
ticosterone (181). Interestingly, when the cells are treated with
a synthetic glucocorticoid, such as Dexamethasone, which is
known to have a higher affinity for GR—and therefore pro-
longed GR association with GRE—the duration of the tran-
scriptional response is longer (181).
The mechanism underlying the dynamics of pulsatile gene
transcription has been further investigated in AtT20 cells. In
this case, GR-mediated cyclical transcription of the Per1 gene,
induced by a pulse of corticosterone, is associated with rapid,
but transient, generalized protein acetylation at the GRE site
within the Per1 promoter, and ChIP assays have revealed the
nucleosome core protein H4 as an acetylation target (38).
Furthermore, the HAT complex members CBP and p300 have
also been found to undergo cyclical recruitment to the GRE
region in the Per1 promoter. The pattern of both CBP and
p300 recruitment at the Per1 promoter is consistent with the
pattern of both GR and RNA Pol II recruitment at the Per1
promoter region. In turn, the pattern of Pol II recruitment is
consistent with the pattern of Per1 transcription in this cell
line (181). Furthermore, it has been shown that GR-mediated
Volume 4, July 2014
HPA Axis-Rhythms
cyclical transcriptional activity at the level of the Per1 pro-
moter involves cyclical intranuclear chaperone protein activ-
ity. Indeed, consistent with its effect on corticosterone binding
to GR, inhibition of HSP90 with geldanamycin results in com-
plete ablation of ultradian cyclical transcriptional activity at
the Per1 gene, as shown by lack of GR, CBP, or p300 recruit-
ment at the GRE and reduced acetylation and transcriptional
rate (38).
Effects of glucocorticoid pulsatility on
GR activity: in vivo studies
Given the importance of pulsatile glucocorticoid pulsatility
for gene regulation in vitro, the question arises as to the
importance of pulsatility for GR-mediated gene regulation
in the living animal. Therefore, the effect of pulsatile corti-
costerone in target tissue has been explored in detail using
adrenalectomized rats, in which corticosterone is replaced by
hourly pulses delivered by IV injection (39, 41, 181).
The dynamics of GR and MR activation over each ultra-
dian pulse can be measured by determining nuclear translo-
cation of the receptors (39), and this has been achieved by
using subcellular fractionation and Western blotting to detect
GR and MR in cytoplasmic and nuclear compartments. Hip-
pocampal GR and MR have been found to be rapidly acti-
vated following each pulse. Following nuclear translocation
and binding to DNA, GR is rapidly cleared from the nucleus,
with nuclear levels dropping significantly within 30 min and
returning to baseline levels by 60 min after each pulse. In
contrast, MR is not rapidly cleared from the nucleus, with
levels remaining high for up to 60 min after each pulse. As
this timing is coincident with the duration of the physio-
logical inter-pulse interval, these findings demonstrate that
in the hippocampus, pulsatile exposure to corticosterone is
a sufficient stimulus to retain the high affinity MR in the
nucleus, while causing the lower affinity GR to translocate
in and out of the nucleus, in parallel with fluctuating steroid
levels.
The dynamics of nuclear localization of GR has been
investigated using immunofluorescent confocal imaging of
GR in hippocampal CA1 neurons (Fig. 13A) (41). This study
has shown that a rapid but transient increase in nuclear GR
levels occurs in response to an IV corticosterone pulse. Prior
to the pulse, GR immunoreactivity is predominantly cyto-
plasmic, whereas a significant nuclear translocation of GR is
detectable at 15 min after the pulse. By 60 min after the pulse,
nuclear GR immunofluorescence has returned to baseline lev-
els.
The mechanism underlying the rapid nuclear clearance of
GR in the hippocampus, and therefore, its rapid inactivation
mediated by proteosomal activity, has also been investigated
(39). Using the specific 26S proteasome inhibitor MG132,
injected intra-cerebraventricularly, Conway-Campbell et al.
have shown that irreversible proteasomal inhibition abolishes
the rapid clearance of GR from the nucleus, and thus restores
elevated levels of nuclear activated GR.
1291
HPA Axis-Rhythms Comprehensive Physiology

(A)
0 min 15 min 60 min 120 min

(B) (C)

Per1 hnRNA (fold change)

800
Corticosterone (ng/mL)

2.0
600
1.5
400
1.0
200

0 0.5
0 1 60 61 120 121 180 181 0 30 60 90 120 150 180 210
Time (min) Time (min)

Figure 13 Corticosterone ultradian rhythm induces pulsatile GR activation and GR-mediated gene tran-
scription in the rat hippocampus. To determine the effect of pulsatile corticosterone treatment on GR nuclear
translocation and transcription of the Period 1 (Per1) gene, multiple corticosterone pulses (100ng/pulse;
i.v.) were given at hourly intervals. (A) GR immunofluorescence in the CA1 region of the hippocampus
throughout the time course of one corticosterone pulse in adrenalectomized rats. At time 0 min, GR
immunoreactivity is predominantly cytoplasmic. A clear transient increase of approximately twofold can
be seen in nuclear GR within 15 min of the corticosterone injection, which returns to baseline levels by
60 min. (B) Circulating corticosterone levels induced by four corticosterone pulses in adrenalectomized
rats. Corticosterone is maximally increased in plasma at 1 min after each injection and then subsequently
cleared according to the characterized half-life of corticosterone in the blood. (C) Each corticosterone
pulse induces a pulse of Per1 gene transcription. Per1 primary transcript levels increase rapidly, reaching
a maximum at 30 min after each injection and, consistent with the pattern of GR dissociation from the
DNA as corticosterone is cleared from the circulation, return to basal levels at 60 min after each pulse.
Reproduced with permission from (41).

Consistent with the effect of pulses of corticosterone on
cyclic GR nuclear translocation, pulsatile corticosterone infu-
sions induce “waves” of GR activation and DNA binding in
the hippocampus of rats (41). Using ChIP, this has been con-
firmed by showing that the pulses of GR activation result in
cyclical recruitment of GR to a GRE in the promoter of the
Per1 gene. Furthermore, each pulse of corticosterone induces
a pulse of transcription of Per1, as shown by measurements of
Per1 hnRNA (Fig 13B). Interestingly, despite a pulsatile pat-
tern of hnRNA, pulsatile corticosterone administration results
in an increase in Per1 mRNA that reaches constant levels, sug-
gesting that ultradian activity with pulses at hourly intervals
is optimized to maintain Per1 protein level at a steady state.
In addition to the hippocampus, the effect of glucocor-
ticoid pulsatility on GR-induced gene transcription has also
been investigated in the liver (181). In a similar manner, pul-
satile corticosterone induces cyclic GR-GRE binding, and
this is associated with pulsatile binding of GR to regulatory
regions in the target clock gene Period1 (Per1). This in turn
is followed by cyclic changes in Per1 hnRNA levels over the
duration of the corticosterone pulse. As seen for GR dynamic
activity in the hippocampus, these results indicate that in
the liver, glucocorticoid signaling is highly dynamic and
intrinsically dependent on the secretion pattern of corticos-
terone released from the adrenal gland. This is very important
given that glucocorticoids in the liver regulate numerous vital
functions, including glucose metabolism.
Nongenomic effects of glucocorticoid pulsatility
A number of studies have shown that the ultradian rhythm
of glucocorticoids is important for the transcription of GR-
regulated genes in glucocorticoid responsive target organs.
In addition to their genomic effects, glucocorticoids also
have rapid nongenomic effects on target tissues, such as
rapid effects on neuronal activity in the brain (57, 74, 193).
Indeed, rapid effects of corticosterone on excitatory and
inhibitory inputs to the hippocampus have been described
(101). Recently, rapid nongenomic effects of glucocorticoid
pulsatility on neuronal activity have been studied using elec-
trophysiology techniques (100). Using a paired-pulse stimula-
tion paradigm, Karst and colleagues have shown that exposure
to two pulses of corticosterone increases mEPSC frequency
in CA1 hippocampal pyramidal cells, and that this effect is
comparable for both pulses. In contrast, when two corticos-
terone pulses are applied in the basolateral amygdala nucleus,

1292 Volume 4, July 2014

Comprehensive Physiology
neurons in this region respond to the first pulse with increased
mEPSC frequency—but display a reduced mEPSC frequency
in response to the second pulse (100).
Effect of Disrupted Ultradian Rhythm
on Hormonal and Behavioral
Response to Stress
The existence of an interaction between hormonal and behav-
ioral responsiveness to stress and the ultradian rhythm of cor-
ticosterone has been shown in the rat (65, 66, 165, 214). The
mechanism underlying the differential HPA axis response to
stress in relationship to the ultradian corticosterone rhythm
has been further investigated in adrenalectomized rats in
which corticosterone is replaced either as a pulsatile or con-
stant infusion (165). In rats infused with constant levels of
corticosterone, the ACTH response to stress is suppressed,
when compared to rats infused with either vehicle or pul-
satile corticosterone. Consistent with these data, disruption
of the ultradian pattern of corticosterone secretion results in
changes in GR receptor sensitivity with profound effects on
the glucocorticoid responsiveness of target tissues (166). In
rats implanted with a corticosterone pellet (to abolish corti-
costerone pulsatility), GR in the hippocampus is downregu-
lated. This downregulation results in decreased GR nuclear
translocation and transcription of the GR target gene GILZ
in hippocampal CA1 cells in response to a corticosterone
injection.
Clinical relevance
It is clear that for optimal transcriptional and nongenomic
responses tissues need to be exposed to oscillating concentra-
tions of cortisol. Current hormone replacement regimes fail
in two respects. First, they do not provide an anticipatory
burst of cortisol release prior to awakening, which is impor-
tant to ensure that cognitive and metabolic functions are ready
for the activities needed first thing in the morning. Second,
oral replacement therapy results in smooth levels of steroid
throughout the day, which do not provide ultradian regulation
for GR signaling mechanisms. It is perhaps not surprising
that patients on glucocorticoid replacement have double the
age related mortality and, often, severe morbidity in terms
of lassitude, tiredness and poor cognitive function. Similarly,
little appreciation has gone into the timing of glucocorticoid
therapy for inflammatory or malignant disease to maximize
effect and minimize side effects.
Conclusions
The HPA axis is a highly dynamic system. Ultradian rhyth-
micity emerges due to the interaction between the dynamic
release of hypothalamic hormones and an intrinsically
Volume 4, July 2014
HPA Axis-Rhythms
oscillating pituitary-adrenal network. On a slower time scale,
circadian rhythmicity entrained by the SCN modifies these
rapidly oscillating levels of glucocorticoid to create a pattern
of pulses that vary in amplitude throughout the day. These
pulsatile patterns of hormone are vital for maintaining home-
ostasis, stress responsiveness, and optimal metabolic and cog-
nitive function.
Acknowledgements
The work from Stafford Lightman and colleagues described
in this review has been supported by grants from the Welcome
Trust, the Medical Research Council (MRC), the Biotechnol-
ogy and Biological Sciences Research Council (BBSRC), the
Neuroendocrinology Charitable Trust, and the Engineering
and Physical Sciences Research Council (EPSRC).
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