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European Journal of Pharmacology 583 (2008) 255–262www.elsevier.com/locate/ejphar

Review

The significance of glucocorticoid pulsatility

Stafford L. Lightman ⁎, Crispin C. Wiles, Helen C. Atkinson, David E. Henley, Georgina M. Russell,Jack A. Leendertz, Mervyn A. McKenna, Francesca Spiga, Susan A. Wood,

Becky L. Conway-Campbell

Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY

Accepted 7 November 2007Available online 9 February 2008

Abstract

Glucocorticoids are secreted in discrete pulses resulting in an ultradian rhythm in all species that have been studied. In the rat there is anapproximately hourly rhythm of corticosterone secretion, which appears to be regulated by alternating activation and inhibition of the HPA axis.At the level of signal transduction, the response to these pulses of corticosterone is determined by its dynamic interaction with the twotranscription factors — the glucocorticoid and mineralocorticoid receptors. While the mineralocorticoid receptor remains activated throughout theultradian cycle, the glucocorticoid receptor shows a phasic response to each individual pulse of corticosterone. This phasic response is regulatedby an intranuclear proteasome-dependent rapid downregulation of the activated glucocorticoid receptor.© 2008 Elsevier B.V. All rights reserved.

Keywords: Glucocorticoid receptor; Mineralocorticoid receptor; Proteasome; Ultradian rhythm

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2552. Basal glucocorticoid pulsatility and stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2563. Glucocorticoid feedback and pulsatility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2574. Cellular responsiveness to glucocorticoid pulsatility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2575. Molecular mechanisms of glucocorticoid response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2596. Pulsatility and glucocorticoid replacement paradigms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2607. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

1. Introduction

Timing is everything in endocrinology. Frequency encodingmediated by the ultradian pattern of circulating hormones as ameans of signalling within mammalian systems is well es-tablished. Pulsatile release of gonadotropin-releasing hormone

⁎ Corresponding author. Tel: +44 117 3313167; fax: +44 117 3313169.E-mail address: Stafford.Lightman@bristol.ac.uk (S.L. Lightman).

0014-2999/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.11.073

(GnRH) is essential for luteinising hormone (LH) and folliclestimulating hormone (FSH) secretion (Belchetz et al., 1978), andsexually dimorphic differences in the pattern of pulsatile growthhormone (GH) secretion results in differential expression of liverenzymes and JAK kinase dependent STAT 5 responses (Clarket al., 1987; Waxman et al., 1995). Indeed, ultradian pulsatility iscritical for normal physiological function of a variety of hormonesincluding LH (Knobil et al., 1980), and insulin (Matthews et al.,1983). This knowledge has led to the introduction of a novel typeof clinical therapy where manipulation of the temporal pattern of

Fig. 1. Ultradian cortisol rhythm in a healthy human female volunteer. A: Serumcortisol concentrations obtained utilising a human automated blood-sampling systemat 10-minute intervals reveals the ultradian pattern underlying a circadian profile(unpublished results). B: Deconvolution analysis technique (Johnson et al., 2004)estimates 24 secretory events with mean secretion pulse height 15.3 nmol/L/min,pulse mass 115.1 nmol/L/min and interpulse interval 59.2 min (Henley et al.,unpublished data).

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ligand exposure to their cognate receptors results in therapeuticbenefits (Amato et al., 2000; Kesrouani et al., 2001).

Similarly, the hypothalamic–pituitary–adrenal (HPA) axisexhibits a pulsatile pattern of secretion of corticotrophinreleasing hormone (CRH), adrenocorticotrophic hormone(ACTH) and glucocorticoids (cortisol in humans, corticosteronein rats and mice). This pulsatility has now been reported innumerous species including rat (Atkinson et al., 2006b; Jasper

Fig. 2. Examples of circulating corticosterone profiles from a male and a female Spragdiurnal profile using an automated blood-sampling system. Note that the profile from tthat of the male profile. Horizontal bars indicate lights off. Data from Seale et al. (2

and Engeland, 1991, 1994;Windle et al., 1998a), rhesus monkey(Sarnyai et al., 1995; Tapp et al., 1984), Syrian hamster (Loudonet al., 1994; Lucas et al., 1999), horse (Cudd et al., 1995), sheep(Engler et al., 1989a,b), and goat (Carnes et al., 1992, 1990). Inman, an ultradian pattern of cortisol release is also evident andforms the basis of the typical diurnal rhythm (Hellman et al.,1970; Veldhuis et al., 1990; Weitzman et al., 1971; Young et al.,2004). It is likely that the pulsatile nature – the relativedifferences between peak and trough for each individual cortisolpulse – of free cortisol in human plasmawill be even greater thanthat for total cortisol, as corticosteroid binding globulin (CBG)will re-equilibrate with free cortisol in the minutes after eachsecretory episode. A typical human 24-hour cortisol profile froma healthy adult female is shown in Fig. 1.

Over the last decade we have used our automated blood-sampling system to define corticosterone pulsatility in freerunning rats in their home cages. These studies have revealed acomplex ultradian rhythm of endogenous levels of corticosterone,with discrete peaks occurring at approximately hourly intervals.Modulation of the amplitude of these ultradian peaks generatesthe well-characterised circadian profile of corticosterone. Inter-estingly, the ultradian pattern shows marked sexual diergism(Seale et al., 2004a,b) (Fig. 2), and is remarkably plastic, withchanges seen during lactation and ageing (Lightman et al., 2000),the presence of or susceptibility to disease (Windle et al., 2001,1998a), and early life programming (Shanks and Lightman,2001). Despite the apparent universality of glucocorticoidpulsatility and the longstanding associations between changesin HPA axis activity and disease (Gibbons, 1964; Young et al.,2004), there has been relatively little research into the significanceof the glucocorticoid ultradian rhythm.

2. Basal glucocorticoid pulsatility and stress

There are several clues indicating that basal glucocorticoidpulsatility may be significant for normal HPA axis function. Ithas been demonstrated (Windle et al., 1998b) that the time ofonset of a stressor in relation to the phase of an endogenousbasal pulse can determine the physiological response. FemaleSprague–Dawley rats undergoing automated blood microsam-pling were exposed to the mild stressor of white noise at 114 dB

ue–Dawley rat. Blood samples were collected at 10-minute intervals across a fullhe female rat displays both increased pulse frequency and amplitude compared to004a,b).

Fig. 4. Rapid feedback of endogenous corticosterone by methylprednisolone.Blood samples were collected around the diurnal peak at 5-minute intervalsusing an automated blood-sampling system. Within half an hour of anintravenous bolus of methylprednisolone (250 μg; vertical line), endogenouscorticosterone concentrations are low and pulsatility is shut off (Atkinson et al.,unpublished data).

Fig. 3. Mean profiles from “responder” and “non-responder” groups indicate thedifferential response to noise stress depending on the relative timing of noise-stress onset with endogenous basal pulsatility. The dotted rectangle indicates thetime of onset and offset of the noise stressor (Windle et al., 1998b).

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for 10 min. Subsequent examination of the hormone profilesindicated two separate groups of animals; those whichresponded to the stressor, and those which did not. Thoseanimals which had rising endogenous basal corticosteronelevels immediately prior to the onset of the stressor respondedwith an additional release of corticosterone. Those in whichbasal corticosterone levels were falling at stressor onset showedlittle or no corticosterone response (Fig. 3).

This indicates that basal glucocorticoid pulsatility has adynamic interaction with the ability of an animal to mount astress response. Additionally, the inability of animals to mount astress response when the stressor onset occurred during thedown-phase of an endogenous pulse suggests that during thisperiod the HPA axis is quiescent or even inhibited. This fits withthe observation that the rate of corticosterone clearance duringthe down-phase of an endogenous corticosterone pulse is almostidentical to the clearance rate of an intravenous bolus ofexogenous corticosterone (Windle et al., 1998b), indicating thatno secretion occurs during this phase. A subsequent experiment(Windle et al., 1998a) showed that pituitary responsiveness toexogenous CRH is not affected by pulse phase. Thus theregulation of glucocorticoid pulsatility seems to be a result ofrapidly alternating periods of activity and inhibition within theHPA axis, controlled at a supra-pituitary site.

3. Glucocorticoid feedback and pulsatility

Glucocorticoid feedback operates at multiple time domainsand involves both rapid non-genomic responses as well as theclassical genomic responses (Hinz and Hirschelmann, 2000;Keller-Wood and Dallman, 1984). Although the mechanisms ofnon-genomic glucocorticoid feedback have proved elusive andnovel (Di et al., 2003; Karst et al., 2005) the existence of fast rate-

sensitive feedback has long been recognised (Dallman and Yates,1969).When a rat is exposed to rapidly increasing concentrationof exogenous steroids, it has limited capacity to mount a HPAresponse to stress (Dallman, 2005). Using our automated blood-sampling system, we have been able to show that administrationof an intravenous bolus of steroid (which results in rapidlyincreasing steroid concentrations) can rapidly inhibit basal HPAactivity in conscious animals. Circulating concentrations ofendogenous corticosterone are significantly lower within30min (see Fig. 4) of exogenous steroid injection and consideringthat the half life for corticosterone clearance is approximately10 min (Glenister and Yates, 1961; Woodward et al., 1991), thefast feedback signal generated by the steroid bolus is presumablynon-genomic.We propose that because this effect is rapid enoughto function within the timespan of ultradian pulsatility it mayexplain why pulses are composed of alternating phases ofactivation and inhibition (see Fig. 5). Thus, during the secretoryphase of a pulse the rapid rise in corticosterone causes a fastfeedback signal that results in the inhibitory phase of the pulsewhere corticosterone is cleared according to its half life. Thisinhibitory phase is short in duration thus allowing a new pulse tobe initiated within a short time-frame. We also have evidence toshow that this fast rate-sensitive feedback of basal HPA activitycan be blocked by a selective mineralocorticoid receptorantagonist but not by glucocorticoid receptor antagonists(Atkinson et al., 2006a; Spiga et al., 2007).

4. Cellular responsiveness to glucocorticoid pulsatility

For glucocorticoid pulsatility to be significant there must bea cellular signalling system capable of dynamic responses torapidly fluctuating hormone levels. The genomic effects ofglucocorticoids are mediated via two cognate receptors, the typeI high affinity mineralocorticoid receptor and the type II lowaffinity glucocorticoid receptor (de Kloet et al., 1990). Bothmineralocorticoid receptor and glucocorticoid receptor aremembers of the nuclear receptor superfamily, one of the mostabundant classes of transcriptional regulators in vertebrates(Mangelsdorf et al., 1995). The primary function of thesereceptors is to act as ligand activated transcription factors, able

Fig. 6. Schematic representation of corticosterone replacement via differentforms of delivery (i.p. versus i.v.) into adrenalectomised rats.

Fig. 7. Schematic representation of glucocorticoid receptor and mineralocorticoidreceptor nuclear translocation and retention kinetics after two bolus i.v. injections attimes 0 and 120 min. The peripheral corticosterone levels measured by automatedsampling at 5-minute intervals show a rapid spike of corticosterone into thecirculation after each i.v. bolus injection. This is followed by a rapid clearanceconsistent with the approximate 10-minute half life of corticosterone in blood. Theconsequent activation and nuclear translocation kinetics of glucocorticoid receptorsand mineralocorticoid receptors are traced over the profile. Glucocorticoidreceptors exhibit rapid nuclear translocation, then clearance after each bolus i.v.injection. In contrast, nuclear mineralocorticoid receptor levels remain high for60 min after each bolus i.v. injection.

Fig. 5. Amodel of corticosteroid pulsatility. The rapid rise in corticosteroids duringthe secretory phase provides a fast feedback signal that shuts off endogenouscorticosteroid secretion thus providing an inhibitory phase during whichcorticosteroids are cleared according to their half lives. The duration of inhibitionby fast feedback is brief and a new secretory event is soon initiated.

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to detect circulating steroid levels and transmit that informationinto intranuclear transcriptional responses in target cells. Todate, there has been relatively little information on howphysiologically relevant pulsatile glucocorticoid presentationaffects target organs in vivo.

A particularly interesting glucocorticoid target tissue asso-ciated with stress-related memory and learning is the hippo-campus. Glucocorticoids can readily enter the brain, and can bemeasured at significant levels in hippocampal extracellular fluid(ECF) by microdialysis (Linthorst et al., 1994). Furthermore, arecent study has shown that corticosterone exhibits an ultradianrhythmicity within discrete brain structures including thehippocampus as measured by microdialysis in the brains offreely behaving rats (Droste et al., 2006). Extracellularglucocorticoids are able to diffuse freely into the cell, to bindand activate cytoplasmic glucocorticoid receptors and miner-alocorticoid receptors, initiating their translocation into thenucleus and subsequent genomic regulatory effects. We havebeen able to show that glucocorticoid receptors and miner-alocorticoid receptors, which are both abundantly co-expressedin the hippocampus, respond in a defined and distinct mannerdepending on the pattern of pulsatile presentation (Conway-Campbell et al., 2007). Receptor activation was measured bydetermining nuclear translocation of receptors using subcellularfractionation and Western blotting of glucocorticoid receptorsand mineralocorticoid receptors. Utilising different corticoster-one administration protocols in adrenalectomised rats (Fig. 6),we have compared a corticosterone profile similar to that seenfollowing an acute stress with a profile more similar to anendogenous corticosterone pulse. Hippocampal glucocorticoidreceptors and mineralocorticoid receptors were both rapidlyactivated following both the acute pulse and the more prolonged“stress” increase in corticosterone. Following the longerduration increase in corticosterone during a “stress” mimickingpulse, both mineralocorticoid receptors and glucocorticoidreceptors remained elevated in the nucleus for the duration ofthe increased plasma corticosteroid levels (Fig. 7). What wasextraordinary however, was the response to the rapid pulse ofcorticosterone. After activation, glucocorticoid receptors werecleared from the nucleus extremely swiftly with nuclear levelsdropping significantly within 30 min and returning to baseline

levels by 60 min after each pulse. Interestingly, mineralocorti-coid receptors were not cleared so rapidly with levels remaininghigh up to 60 min after each pulse. This timing is coincidentwith the duration of the physiological interpulse interval. Wewere therefore able to demonstrate in the hippocampus that thepulsatile exposure to corticosterone is a sufficient stimulus toretain the high affinity mineralocorticoid receptor in thenucleus, while the lower affinity glucocorticoid receptortranslocates in parallel with changing steroid levels. Thisdifferential nuclear expression of both receptors is likely toresult in differential effects on the transactivating andtransrepressing activities of circulating glucocorticoids andmay be an important factor in the dual action of corticosteronewith mineralocorticoid receptors preventing disturbance ofhomeostasis and glucocorticoid receptors promoting recoveryfollowing stress (de Kloet et al., 1998).

We were further able to establish a mechanism for the rapidinactivation of glucocorticoid receptors as targeted proteindegradation dependent on the cellular ubiquitin–proteasomesystem. Using the specific irreversible proteasome inhibitorMG132 given intracerebroventricularly, we were able to abolish

Fig. 8. Proposed model of pulsatile corticosterone regulation of glucocorticoidreceptors in the hippocampus. Schematic depiction of circulating corticosteronepulsatility, showing how the peak and trough of each pulse differentially affectglucocorticoid receptors and mineralocorticoid receptors. During the peak of eachpulse, both glucocorticoid receptors andmineralocorticoid receptors aremaximallyactivated and localised to the nuclear compartment where they are known to bind toGREs in the promoters of glucocorticoid target genes in the genome. Then duringthe trough of each pulse, nuclear glucocorticoid receptors are specifically down-regulated by a proteasome-dependent mechanism while mineralocorticoidreceptors are retained in the nucleus. This mechanism allows nuclear glucocorti-coid receptors to be constantly and rapidly cleared ensuring dynamic interactionwith fluctuating glucocorticoid levels (Conway-Campbell et al., 2007).

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the rapid clearance of glucocorticoid receptor from the nucleusthereby restoring elevated levels of nuclear activated gluco-corticoid receptor throughout the timecourse. Finally andimportantly, we found that in the hippocampus there is a largereservoir of cytoplasmic glucocorticoid receptors available, sothat each wave of glucocorticoid receptor translocation to thenucleus has only minimal effects on the levels of total cyto-plasmic glucocorticoid receptor. This in turn means that thisreservoir of unliganded glucocorticoid receptor will still beavailable for subsequent pulses of corticosterone.

5. Molecular mechanisms of glucocorticoid response

At the molecular level, differential clearance rates forhippocampal glucocorticoid receptor and mineralocorticoidreceptor may simply reflect their different affinities for the ligand(Reul and de Kloet, 1985). While glucocorticoid receptorsdissociate from glucocorticoid quite rapidly (t1/2=5 min forhuman glucocorticoid receptor binding to cortisol), mineralocorti-coid receptors remain bound for significantly longer (t1/2=45 minfor human mineralocorticoid receptor binding to cortisol). Thisraises the possibility that increased ligand affinity may also conferincreased stability of mineralocorticoid receptors compared toglucocorticoid receptors, effectively protecting mineralocorticoidreceptors from rapid proteasome-dependent downregulation.Evidence both in support of, and against this theory exists in theliterature. Stavreva et al. have shown in a series of elegant cellculture experiments that the stability of glucocorticoid receptor ona target promoter was dependent on glucocorticoid receptorsaffinity for ligand (Stavreva et al., 2004). Although notinvestigating mineralocorticoid receptor, glucocorticoid receptordynamics were examined with either corticosterone or the highaffinity synthetic agonist dexamethasone using live cell imagingwith a green fluorescent protein tagged glucocorticoid receptor(GFP-glucocorticoid receptor). This study was performed in a cellline engineered to stably integratemultiple arrays of glucocorticoidresponse elements in themurine mammary tumour virus (MMTV)promoter, so that fluorescence in situ hybridisation (FISH) wasable to detect this array of GREs as two bright spots within thenucleus. The recruitment of GFP-glucocorticoid receptor to thearray was observed after 5 min of treatment with eithercorticosterone or dexamethasone. Interestingly, after a stringentwash over a 5-minute period to remove all the ligand from theculture medium, transcription from the MMTV promoter wascompletely abolished in the case of corticosterone, but continued at50% ofmaximal levels in the case of dexamethasone. On the otherhand, Meijsing et al. concluded that ligand dissociation was notrequired for glucocorticoid receptor dissociation from GREs,based onmobility after photobleaching (FRAP) experiments usingdexamethasone modified to bind covalently to glucocorticoidreceptor (Meijsing et al., 2007). They concluded however, that theligand binding domain (LBD) was important, as LBD deletionconstructs of glucocorticoid receptor dissociated faster from theGRE. It remains to be seen how relevant these covalently boundligands and glucocorticoid receptor mutants are to endogenouscorticosterone-induced transcriptional activity, but certainly theglucocorticoid receptor LBD appears to play a critical role.

Another possible mechanism for the removal of glucocorti-coid receptor from the nucleus is nuclear export to thecytoplasm for degradation or recycling. It is clear that gluco-corticoid receptors do not utilise the classical CRM1 mediatednuclear export pathway, although some groups have identified acalreticulin mediated nuclear export mechanism for glucocorti-coid receptors (Holaska et al., 2001, 2002; Walther et al., 2003).In fact, when a classical nuclear export sequence (NES) isengineered into the glucocorticoid receptor coding sequence,nuclear export is significantly increased. There is also evidencefor a nuclear retention signal in the sequence of the gluco-corticoid receptor DNA binding domain (DBD) which appearsto be required for transcriptional activity (Carrigan et al., 2007).Our results also argue against the export of glucocorticoidreceptor as the mechanism for its rapid clearance from thenuclear fraction as we can detect significant changes in nuclearlevels without significant changes in cytoplasmic levels aftertreatment with MG132. It is well established that ligand ac-tivated glucocorticoid receptor is both a substrate for ubiqui-tination and a target for degradation by the proteasome (Derooet al., 2002; Garside et al., 2006; Kinyamu and Archer, 2003;Wallace and Cidlowski, 2001; Wang et al., 2002). Theimportance of the rate limiting component of the cellularubiquitin–proteasome system termed CHIP E3 ligase (carboxyterminus of heat shock protein 70-interacting protein E3 ligase)in glucocorticoid receptor regulation has been clearly shown ina series of experiments where cells naturally deficient in CHIPE3 ligase were unable to downregulate glucocorticoid receptorafter chronic dexamethasone treatment, yet glucocorticoidreceptor downregulation was reinstated with CHIP overexpres-sion (Wang and DeFranco, 2005). Furthermore, it has been

Fig. 9. Seven-hour corticosterone profile of a single adrenalectomised ratundergoing infused pulsatile corticosterone replacement during the automatedblood-sampling procedure. Both the frequency and amplitude of these pulsesmimic those seen in an adrenal-intact animal (Wiles et al., unpublished data).

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shown that the 19S subunit of the proteasome complex iscolocalised with GFP-glucocorticoid receptor on GREs of theMMTV promoter and in surrounding regions within the nucleus(Stavreva et al., 2004). Increasing amounts of glucocorticoidreceptor at the promoter were correlated with increasedproteasome immunofluorescence, suggesting glucocorticoidreceptor recruits proteasome machinery to the target site.

We should therefore like to put forward the following putativemodel (Fig. 8) describing our hypothesis that dissociation ofligand from glucocorticoid receptors but not mineralocorticoidreceptors (or alternatively a non-ligand LBD related mechanism)is the first step in destabilising the active complex from itsassociation with the GRE on target promoters prior to gluco-corticoid receptor being recruited to the proteasome for clearance.The functional significance of proteasome-dependent rapid turn-over of activated glucocorticoid receptors in the nucleusmay be toallow dynamic and continuous responses to the highly fluctuatinghormone levels observed in pulsatile glucocorticoid secretion,while mineralocorticoid receptors maintain a continuous basallevel of homeostasis. This mechanism may underlie the dualaction of corticosterone via its two receptors.

6. Pulsatility and glucocorticoid replacement paradigms

All current corticosteroid replacement paradigms aim eitherto clamp the blood corticosterone at a fixed level over time, or tomimic the diurnal rhythm with a corticosterone peak during thedark phase and a subsequent nadir during the light phase.Similarly in human Addisonian patients, cortisol replacement isadministered in the morning to approximately recapitulate thenormal endogenous diurnal pattern with peak levels in themorning, and nadir levels between midnight and 3 am.

The most commonly used method of replacing corticoster-one levels in the adrenalectomised rat is by clamping plasmalevels with a subcutaneous pellet of corticosterone fused intocholesterol. The resultant constant level of corticosterone how-ever, does not reflect the normal endogenous circadian patternseen in adrenal-intact animals. This lack of diurnal variation canitself attenuate serotonin (5-HT) receptor (5-HT1a) function(Leitch et al., 2003), and reduce the effectiveness of selectiveserotonin reuptake inhibitor (SSRI) treatment for elevatingforebrain 5-HT levels (Gartside et al., 2003). Furthermore, thediurnal rhythm of corticosterone appears to be necessary forneurogenesis in the hippocampus (Huang and Herbert, 2006).These studies are of particular importance, as several studiessuggest that there is a flattening of the diurnal rhythm of cortisolsecretion in patients with depression (Deuschle et al., 1997;Gibbons, 1964; Wong et al., 2000; Yehuda et al., 1996).

The recognition of the importance of the diurnal variation ofcorticosterone has led to new replacement paradigms, mostcommonly the addition of corticosterone to drinking water. Sincethe rodent has a diurnal pattern of drinking activity, with little orno drinking occurring until the onset of the dark phase, this simplereplacement regime results in diurnal variation of the bloodcorticosterone level, which is able to normalise key physiologicalparameters such as thymusweight andACTH levels (Akana et al.,1985). Furthermore, corticosterone replacement in the drinking

waterwasmore effective than subcutaneous pellets in normalisingstress induced ACTH secretion, indicating the importance of thepattern rather than simply the amount of corticosterone replace-ment. An alternative method of maintaining the diurnal rhythminvolves the implantation of a low concentration corticosteronepellet followed by a subcutaneous injection of corticosterone atthe onset of the dark phase (Huang and Herbert, 2006).

The evidence that specific patterns of corticosterone replace-ment can have physiological consequences, combined with thestriking endogenous pulsatility of glucocorticoids, and thefindings in related fields that ultradian pulsatility is critical fornormal physiological function, all highlight the need for aphysiologically realistic method of corticosterone replacement.The potential importance of the ability to respond to rapid andshort-lived changes in glucocorticoid levels has already beenestablished by Mikics et al. (2004) who have demonstrated howaggressive behaviour is promoted by rapid non-genomicmechanisms. As no extant corticosterone replacement paradigmsmimic the physiological ultradian rhythm of corticosteronesecretionwe have therefore addressed this problem by developingan infusion protocol with which we are able to selectively replacecorticosteronewith pulsatile characteristics of our own choice intoadrenalectomised animals. In order to achieve this, we usecorticosterone solubilised in a cyclodextrin vehicle, which isinfused into the femoral vein and is controlled by a computer-driven infusion pump. This allows us to reproduce physiologicalcorticosterone pulses at a rate and amplitude similar to thosefound in adrenal-intact animals. Combining our automatedinfusion system with automated blood sampling directly fromthe jugular vein every 10 min, we have been able to verify theinfusion pattern (Fig. 9). This infusion system gives us a novelway to directly compare the effects of different patterns ofcorticosterone administration on a slew of physiological andbehavioural variables, and should be able to provide us withcritical insights into the true physiological significance of thestriking endogenous glucocorticoid pulsatility.

7. Summary

It is now well established that glucocorticoids are secretedepisodically in an ultradian pattern and regulation of this pulsatility

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appears to be due to alternate activation and inhibition within theHPA axis. Termination of the active phase of the corticosteronepulse by rapid feedback inhibition may be important in thegeneration of such pulses. The cellular response to glucocorticoidsis determined by a dynamic interaction between the fluctuatinglevels of corticosterone and differential activation kinetics of thetwo glucocorticoid receptors, glucocorticoid receptor and miner-alocorticoid receptor. Glucocorticoid receptors display phasicresponse patterns closely regulated by the pattern of glucocorticoidpresentation, with specific proteasome-dependent rapid down-regulation of activated glucocorticoid receptors determining thedynamic response to pulsatile glucocorticoid secretion. Thedevelopment of an automated corticosterone infusion system incombination with automated blood microsampling has produced apowerful tool to further investigate the physiological relevance ofthis phenomenon.

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