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Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom
Address all correspondence and requests for reprints to: Dr. Astrid C. E. Linthorst, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Clinical Science South Bristol, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, United Kingdom. E-mail: astrid.linthorst{at}bristol.ac.uk.
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Introduction |
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Because there is no routine way to monitor tissue concentrations of glucocorticoids, glucocorticoid responses are normally monitored in the blood. However, an important question is whether blood glucocorticoid concentrations are a true reflection of tissue hormone concentrations because of the presence of tissue barriers such as the blood-brain barrier and specific transporters that can remove glucocorticoids such as the ATP-binding cassette transporter P-glycoprotein (Pgp) (16, 17, 18). Furthermore, the question has arisen whether target tissues such as the brain may only see the peak levels of the blood corticosterone pulses (15). The regulation of tissue glucocorticoid concentrations is of great clinical importance because the availability of glucocorticoids to glucocorticoid receptors in the tissues is critical to their role in health and disease. However, despite at least 5 decades of research on the biology of glucocorticoids, there is a remarkable lack of knowledge regarding the kinetics and concentration of glucocorticoid exposure of the brain and other tissues. We were the first to report that using in vivo microdialysis, it is possible to measure corticosterone concentrations in the extracellular fluid in the brain of conscious, freely behaving rats (19). The extracellular fluid is devoid of corticosterone-binding globulin and other plasma proteins, and therefore, corticosterone measured in the extracellular space reflects the free, biologically active hormone fraction (19, 20). Thus, direct measurement of free corticosterone levels in the brain provides a much better reflection of corticosterone available for binding to the corticosteroid receptors in the brain than the measurement of plasma hormone levels (3, 20).
Using in vivo microdialysis in freely behaving rats, we have now investigated whether in the hippocampus, a major glucocorticoid target in the brain, there is a similar circadian and ultradian pattern of glucocorticoid as to that seen in the plasma. Identification of an ultradian pattern of corticosterone levels within the brain itself will have enormous implications for the development of our understanding of hormone-receptor-gene interactions in this target tissue because ligand availability to the corticosteroid receptors would be pulsatile rather than continuous as often assumed. Furthermore, we addressed whether stressors known to have a differential impact on plasma glucocorticoid concentrations (i.e. exposure to a novel environment and forced swim stress) would also have a differential impact on hippocampal hormone concentrations. Importantly, we also investigated, using a combined microdialysis and blood sampling approach in the same freely behaving rats, whether the stress-induced corticosterone response measured in the brain parallels the response measured in the circulation.
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Materials and Methods |
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Rats were handled once per day (5 min per rat) starting 1 wk before surgery. At the time of surgery, the body weight of the rats was approximately 220 g. All procedures were in accordance with the Animals (Scientific Procedures) Act 1986. The experimental protocols were designed to minimize animal suffering and number of animals used and were approved by the Ethical Review Group of the University of Bristol and the Home Office.
Microdialysis surgical procedures
Eight days before the start of the experiment, under isoflurane anesthesia, a guide cannula (Microbiotech AB, Stockholm, Sweden; MAB 6.14.IC) was implanted just entering the hippocampus or caudate putamen at the dorsal site. Carprofen (4 mg/kg, sc) was given for postoperative pain relief. Coordinates based on the atlas of Paxinos and Watson (21) with the tooth bar set at 3.3 mm and bregma as overall zero were as follows: hippocampal guide cannula, lateral 5.0 mm, posterior 5.2 mm, and ventral 4.0 mm; caudate putamen guide cannula, lateral 1.2 mm, posterior 0.5 mm, and ventral 4.0 mm. Dental cement and three small anchor screws were used to fix the guide cannula and a small metal peg (for later connection to a liquid swivel) to the skull. After surgery, rats were housed individually in Plexiglas cages (length x width x height = 27 x 27 x 35 cm) with food and water ad libitum.
Microdialysis experimental procedures
After 6 d of recovery, a microdialysis probe (polyethersulfone membrane, 15-kDa cutoff, length 4 mm, outer diameter 0.6 mm, MAB 6.14.4; Microbiotech) was inserted into the hippocampus or caudate putamen under light isoflurane anesthesia, and the rats were connected to a liquid swivel and counterbalance arm system (Microbiotech) via the peg on their head. This system allows the animal to move freely in all three dimensions, including full rearing. Microdialysis probes were perfused with sterile, pyrogen-free Ringer solution (Delta Pharma, Pfullingen, Germany; 147 mM NaCl, 4 mM KCl, 2.25 mM CaCl2) at a flow rate of 2 µl/min using a microinfusion pump. Fluorethylenepolymer tubing with a dead volume of 1.2 µl per 100 mm length (Microbiotech) was used for all connections. Microdialysis samples were collected in cooled vials using automated refrigerated sample collectors (Microsampler 820; Univentor, Malta). The samples were stored at –80 C for later determination of the concentrations of corticosterone. Experiments were performed on six animals simultaneously.
Microdialysis experimental design
In the first set of experiments, we investigated the circadian and ultradian rhythm and the stress responsiveness of hippocampal corticosterone. Two days after insertion of the microdialysis probe in the hippocampus, sampling started at 0500 h (i.e. the beginning of the light period) and continued for 48 h. Microdialysis samples were collected in intervals of 10 min (between 0800 and 2200 h) or 30 min (between 2200 and 0800 h). On the first day of sampling, animals were left undisturbed in their home cage (i.e. baseline day). On the second day of sampling (i.e. stress day) at 1100 h, animals were exposed to a novel environment (30 min in a novel cage), submitted to a forced swim stress procedure [15 min, 25 C water, as described in detail previously (22, 23)], or left undisturbed in their home cage. After the stress exposure, rats were returned to their home cage and left undisturbed for the remainder of the experiment while sampling continued. To increase the time resolution of the measurement of the corticosterone response to stress, a sampling interval of 5 min was used between 1100 h (start of stressor) and 1300 h in the stressed animals.
Next, we wanted to study whether the ultradian and stress effects observed in the hippocampus were specific for that brain structure. Therefore, a similar experiment was performed in rats with a microdialysis probe inserted into the caudate putamen, a brain region not intimately involved in the stress response. Thus, using the same experimental protocol as described above, baseline samples were collected for 24 h after which the rats were subjected to a forced swim stress paradigm on the next day.
Subsequently the profile of access of exogenous peripheral corticosterone to the brain was further investigated in a separate experiment. Two days after insertion of the probe into the hippocampus, rats were sc injected at 1100 h with exogenous corticosterone (1 mg/kg body weight, dissolved in 5% ethanol in saline, 1 ml/kg body weight; Sigma-Aldrich Co. Ltd., Gillingham, UK) or with vehicle (1 ml/kg body weight). During the injection the rat was gently held by the experimenter but remained in its home cage. Ten-minute samples were collected between 0900 and 1000 h to establish baseline levels. Between 1100 and 1300 h, the sampling time was 5 min, followed by further 10-min sampling between 1300 and 1400 h.
Simultaneous brain microdialysis and blood sampling: surgical procedures and experimental design
To investigate how concentrations of corticosterone in the blood relate to brain levels of this glucocorticoid, we used a combined brain microdialysis/peripheral blood sampling approach. To achieve this, the jugular vein was cannulated as described previously (10) in addition to implantation of a guide cannula for microdialysis in the hippocampus (see above) during the same surgery. The free end of the jugular vein cannula was exteriorized through a skin incision on the back of the animal and sealed. The jugular vein cannula was flushed daily with heparinized saline (10 IU/ml) to maintain patency.
Four days after surgery, the microdialysis probe was inserted as described above, and the jugular vein cannula was extended with silicon tubing (outer diameter 1.5 mm, inner diameter 0.5 mm). This jugular vein cannula extension was attached to the wire connecting the peg on the rat’s head with the liquid swivel to prevent twisting of the tubing. Two days after the insertion of the microdialysis probe, baseline hormone concentrations in plasma and brain were assessed by collecting blood [50 µl; replaced with same volume of saline; no changes in hematocrit found at the end of the experiment (data not shown)] and microdialysis samples between 1600 and 1800 h in 10-min intervals. On the following day, microdialysis samples were collected between 1000 and 1400 h (10-min samples, except for 1100–1130 h, 5-min samples). The simultaneous collection of blood started with two 10-min baseline samples between 1040 and 1100 h. Next, at 1100 h, the animals were forced to swim for 15 min (25 C). During the first 30 min after the start of forced swimming, six 5-min blood samples were collected, followed by collection of blood samples in 10-min intervals until 1300 h. Blood samples were taken at the midpoint of the dialysate sampling intervals: thus, in case of a 5-min dialysate sample at 2.5 min and in case of a 10-min dialysate sample at 5 min. The dead volume of the microdialysis system was taken into account when synchronizing blood and dialysate sampling. The blood samples were collected, in heparinized tubes, using blunt needles attached to a syringe. Plasma (obtained after centrifugation at 2500 rpm) and dialysate samples were stored at –80 C for measurement of corticosterone.
Measurement of corticosterone
Plasma and dialysate corticosterone concentrations were measured using a commercially available RIA (MP Biomedicals, Irvine, CA) as published previously (19, 24). The detection limits were 0.25 µg/dl for plasma samples (inter- and intraassay variation 7 and 4%) and 0.00125 µg/dl for microdialysis samples (inter- and intraassay variation 16 and 14%). Dialysate levels were not corrected for probe recovery.
Histology
After completion of the experiment, animals were killed using an overdose of pentobarbital (Euthatal, 200 mg/kg body weight, ip). The brains were collected and stored in a 4% buffered paraformaldehyde solution. Brains were cut into 50-µm sections using a cryostat and stained with Cresyl Violet to verify the localization of the microdialysis probe as described previously (19). Only data from rats with correctly placed microdialysis probes were included in the analyses.
Analysis of the ultradian rhythm using the PULSAR algorithm
The PULSAR algorithm (25) was applied on dialysate corticosterone concentrations from the hippocampus (n = 23) and the caudate putamen (n = 5) measured during the baseline day. We performed PULSAR analysis over a 12-h period (0900–2100 h) because microdialysis samples had been collected with a high time resolution (10-min intervals) during this period, thus providing a sufficient number of data points for the analyses of ultradian rhythmicity. Parameters calculated were as follows: 1) pulse frequency (i.e. number of pulses per hour); 2) mean pulse amplitude (i.e. mean height of pulses with respect to a circadian rhythm baseline as calculated by PULSAR, micrograms per deciliter); 3) mean pulse height (i.e. mean height of pulses above zero, micrograms per deciliter); 4) mean corticosterone concentration micrograms per deciliter); and 5) area under the curve (AUC; arbitrary units). Next, we analyzed whether there were any differences between corticosterone pulse characteristics during the morning/early afternoon vs. the late afternoon/early night phase by splitting the 12-h period into two 6-h periods (0900–1500 and 1500–2100 h, respectively). Putative differences in parameters between these two time periods were statistically assessed by paired Student’s t test (level of significance <0.05).
PULSAR analysis was also performed on the corticosterone data collected in the stress control group as described above. These animals were left undisturbed on the baseline day as well as on the second experimental day, and thus, corticosterone data were obtained under baseline conditions on two consecutive days. The pulse characteristics calculated for the first day were statistically compared with those obtained for the second day by paired Student’s t test (level of significance < 0.05).
Similarly, the hippocampal corticosterone data collected after sc injection of vehicle and exogenous corticosterone were analyzed using the PULSAR algorithm. No pulsatile pattern was found. Next, to compare the effect of vehicle and corticosterone injections on hippocampal extracellular levels of corticosterone, the AUC between 1100 and 1300 h was calculated for each group and statistically analyzed using unpaired Student’s t test (level of significance < 0.05).
Further calculations and statistical analyses
To allow direct comparison of the swim stress-induced changes in plasma and brain levels of corticosterone, all values were calculated as percent of the maximal response for the respective compartment of measurement for each rat individually. The time point of the half-maximal response during the rising phase (T
rising phase), the time point of the half maximal decrease during the declining phase (T declining phase) and the AUC were determined for plasma and brain corticosterone in each rat. The T values and the AUCs for plasma and brain were statistically compared by paired Student’s t test (level of significance < 0.05).
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, A and B, depicts two representative profiles of individual rats and Fig. 1C shows the mean values (±SEM) of 23 animals. Importantly, apart from a circadian rhythm, all rats showed a distinct pulsatile ultradian pattern of hippocampal corticosterone levels (Fig. 1, A and B). This ultradian pattern is most clear between 0800 and 2200 h, the period during which 10-min samples were collected. Using the PULSAR algorithm (25), we calculated the parameters characterizing the ultradian corticosterone rhythm in the hippocampus using the 10-min interval data collected between 0900 and 2100 h (Table 1). The mean pulse height was 0.23 ± 0.03 µg/dl, and the mean pulse frequency 1.16 ± 0.04 pulses/h (n = 23), which corresponds with the pulse frequency reported for corticosterone in blood (10) and the adrenal gland (9). Thus, it seems that the ultradian rhythm of corticosterone observed in the circulation is maintained despite the blood-brain barrier. From the visual inspection of all individual profiles, it appeared that the circadian variation in hippocampal corticosterone levels may result from alterations in both pulse height and pulse frequency (Fig. 1, A and B). This notion was therefore further investigated by statistically comparing the pulse characteristics of two 6-h periods, i.e. an early period between 0900 and 1500 h and a late period between 1500 and 2100 h. As shown in Table 1, the pulse frequency, mean pulse amplitude and mean pulse height were all indeed significantly higher between 1500 and 2100 h, compared with the early period between 0900 and 1500 h, resulting in a circadian variation in hippocampal hormone concentrations.
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shows a representative individual 24-h profile, whereas Fig. 2B presents average levels (mean ± SEM) of seven animals over a 4-h time period. All animals showed a pronounced response to forced swimming reaching a maximum corticosterone level of about 0.8 µg/dl in the dialysate at 50 min after the start of the stressor. Hippocampal glucocorticoid concentrations returned to baseline about 110 min after the start of the stressor (Fig. 2B) after which, importantly, the normal circadian and ultradian rhythm was resumed (Fig. 2A).
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displays an individual profile and Fig. 2D the average (mean ± SEM) of four animals. As expected, the novelty stress-induced rise in hippocampal glucocorticoid concentrations was less pronounced than that after forced swim stress (Fig. 2, C and D), reaching maximal levels of about 0.25 µg/dl between approximately 30 and 40 min after the start of the novelty challenge (Fig. 2, C and D). After the stress challenge, hippocampal glucocorticoid concentrations returned rapidly to baseline, resuming their normal ultradian (and circadian) rhythm.
Corticosterone levels in the caudate putamen
To exclude the possibility that the ultradian corticosterone rhythm we found in the hippocampus is specific for this brain structure, we decided to perform the same experimental design (i.e. microdialysis during a baseline day followed by forced swimming on the next day) in animals with a microdialysis probe implanted in the caudate putamen. The caudate putamen was chosen for two reasons: 1) this brain region has no prominent role in the regulation of the hypothalamus-pituitary-adrenal axis activity, and 2) microdialysis can be performed in the caudate putamen using similar probes (with respect to length, diameter, and membrane) as used for the hippocampus experiments, therefore making the data directly comparable.
Figure 3A depicts corticosterone levels in the caudate putamen of a representative rat demonstrating the presence of a clear circadian and ultradian rhythm of corticosterone in this brain structure. Using the profiles of five animals, we calculated the pulse characteristics between 0900 and 2100 h, as described for the hippocampus above, as follows: pulse frequency 1.13 ± 0.10 pulse/h, pulse amplitude 0.07 ± 0.02 µg/dl, pulse height 0.18 ± 0.02 µg/dl, mean corticosterone concentration 0.10 ± 0.01 µg/dl, AUC 1.39 ± 0.10 arbitrary units. These results compared very well with those calculated for the circadian and ultradian rhythm of corticosterone in the hippocampus (Table 1). Furthermore, as can be taken from Fig. 3B, the forced swim stress-induced corticosterone responses in the hippocampus and the caudate putamen are strikingly similar both with respect to maximum effect and time course.
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). Subcutaneous injection of corticosterone caused a rapid and profound increase in hippocampal corticosterone, reaching maximal levels of approximately 1.6 µg/dl between 35 and 40 min later. Subcutaneous injection of vehicle, causing short-lasting injection stress, resulted in a small increase in hippocampal corticosterone. The AUC calculated for the period between 1100 and 1300 h was significantly different between the vehicle and the corticosterone group (3.29 ± 0.8 and 17.68 ± 4.8, respectively, P < 0.05). Importantly, as is shown in Fig. 4, a pulsatile pattern is absent after administration of exogenous corticosterone. PULSAR algorithm analysis showed that there was no discernible pulsatile pattern in hippocampal corticosterone levels after exogenous corticosterone administration, indicating that the observed ultradian pattern of endogenous corticosterone is the result of an endogenous pulsatile release pattern from the adrenal gland to the blood and from the blood to the brain.
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). However, as depicted in Fig. 5B, after forced swimming, the rise in hippocampal glucocorticoid concentrations peaked approximately 20 min later than the rise in plasma hormone concentrations [T rising phase, plasma: 18.7 ± 2.6 min (n = 8), T rising phase, hippocampus: 38.4 ± 3.2 min (n = 8), P < 0.0005; Table 3]. In contrast, glucocorticoid concentrations in hippocampus and plasma fell simultaneously during the declining phase of the stress response [T declining phase, plasma: 73.1 ± 2.9 min (n = 8), T declining phase, hippocampus: 77.0 ± 2.1 min (n = 8), P = 0.108; Fig. 5B and Table 3]. As a consequence, the exposure of the hippocampus to stress-induced glucocorticoid concentrations was significantly (20%) less than would have been expected on the basis of the plasma glucocorticoid response [relative AUCplasma: 5394 ± 216 (arbitrary units, n = 8), relative AUChippocampus: 4297 ± 129 (n = 8), P = 0.002; Table 3].
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Discussion |
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A similar observation was made for the levels of circulating corticosterone after ip injection of the glucocorticoid hormone (35). This led us to conclude that the pulses of corticosterone observed in the hippocampus result from (and thus reflect) the pulsatile release of this glucocorticoid from the adrenal gland. Importantly, we also demonstrated that this is not an observation that is unique to the hippocampus because we could reveal a very similar ultradian rhythm in the caudate putamen, a brain region with no major role in the regulation of the hypothalamus-pituitary-adrenal axis. This is the first demonstration that a glucocorticoid target tissue, here the brain, is, during normal ongoing conditions, not exposed to a continuous level of corticosterone, which gradually increases over the light/dark cycle but to pulses that last about 1 h. These pulses show a circadian rhythm as they increase in frequency as well as in height toward the active (dark period) of the light-dark cycle, which is in agreement with observations in the adrenal gland and the circulation (10, 11).
Importantly, our findings imply that the corticosteroid receptors in the brain are also exposed to a pulsatile pattern of free corticosterone. Although it has not been extensively investigated yet, such pulsatile exposure could well have substantial implications for mineralocorticoid and glucocorticoid receptor-regulated processes in the brain because of the differential affinity of corticosterone for binding to the two receptor types (1). In this respect, it is important to note that Conway-Campbell et al. (35) recently demonstrated that iv pulsatile administration of corticosterone in adrenalectomized rats precipitates a differential nuclear translocation pattern of mineralocorticoid and glucocorticoid receptors in the hippocampus. The most salient finding of their study is that nuclear translocation of the glucocorticoid receptor peaks coincidentally with the peak of a corticosterone pulse and is cleared at the end of such pulse (by a proteosome dependent mechanism), whereas the nuclear retention of the mineralocorticoid receptor is high during the entire duration of the pulse.
We estimated that the extracellular concentration of free corticosterone in the brain at the peak of the pulse is approximately 10–15 nM, depending on the time of the day, which is in good agreement with the affinity constant value for glucocorticoid receptors at 37 C. Therefore, pulsatility could represent, besides the putative role of the enzyme 11β-hydroxysteroid dehydrogenase (36), an additional level of regulation of access of glucocorticoids to their receptors. These observations clearly demonstrate potential profound implications for pulsatile exposure of nuclear receptors to their ligand and could therefore have important implications for glucocorticoid physiology; a subject that will need follow-up investigations.
It is well known that stressors have a different impact on the various aspects of the stress response, depending on their exact nature (e.g. physical vs. psychological aspects and escapable vs. inescapable) and severity. We therefore investigated whether free corticosterone concentrations in the hippocampus also respond to stress in a stressor-dependent way. We found that 15 min forced swim stress, which is a combined physical (body temperature changes, motor activity) and psychological (anxiety, panic) stressor (22), causes a profound increase in hippocampal free corticosterone levels, reaching maximal levels at 50 min after the start. The swim stress-induced increase in hippocampal corticosterone is quite prolonged because levels return to baseline values only 110 min after the beginning of the stressor. In contrast, exposure to a novel environment, a mild psychological form of stress, caused only a moderate and short-lasting (return to baseline after about 60 min) increase in hippocampal corticosterone. Thus, brain corticosterone concentrations respond to stress in a stressor-dependent manner, which is in good agreement with findings on corticosterone in the blood circulation (24, 37). Importantly, although stress induces an extended nonpulsatile elevation of brain corticosterone, glucocorticoid levels resume their ultradian pattern rapidly once prestress levels have been reached again.
Comparison of the time course of the hippocampal corticosterone response to forced swim stress with our earlier work on forced swim stress-induced changes in plasma corticosterone (24) suggested that the brain response may be delayed, compared with the plasma response. Therefore, we performed a series of experiments in which hippocampal dialysis was combined with the simultaneous collection of blood samples. We found that whereas dialysate and plasma levels of corticosterone seemed to vary largely synchronously under baseline conditions, there was a marked delay of up to 20 min in the hippocampal corticosterone response to forced swim stress, compared with the response in the circulation. Intriguingly, the rate of clearance of corticosterone was identical in both compartments of measurement, resulting in an overall significantly shorter exposure of the brain to corticosterone than would have been predicted based on the response found in the plasma. The mechanisms underlying the delayed entrance but simultaneous clearance of corticosterone at the level of the brain are presently unknown. It is possible that the diffusion process of corticosterone from the blood through the blood-brain barrier into the brain parenchyma delays the appearance of corticosterone in the brain. Alternatively, it cannot be excluded that the binding of corticosterone to binding globulin and other plasma proteins plays a role. Another possible mechanism underlying the delayed entrance or accelerated clearance of corticosterone could be the presence of Pgp at the blood-brain barrier. Although there is consensus that Pgp is responsible for transporting cortisol and the synthetic glucocorticoid dexamethasone out of the brain (challenging the long term notion that steroids distribute freely over the whole body), the situation regarding corticosterone is less clear. Thus, after administration of radiolabeled steroids to mice, brain 3H-cortisol and 3H-dexamethasone, but not 3H-corticosterone, levels are significantly higher in abcb1a knockout mice than wild-type animals (17, 38). However, in vitro work has shown that Pgp in multidrug-resistant cells can cause efflux of corticosterone (39). Furthermore, if abcb1a/b double-knockout mice were used, their brains contained higher 3H-corticosterone levels than the wild-type animals (18), suggesting that corticosterone may be a ligand specifically for the abcb1b transporter.
We conclude that in vivo microdialysis is a powerful method to study free corticosterone in target tissues such as the brain (see also Ref. 40). The importance of our study is 2-fold. First, we have provided the first direct evidence that neurons in the brain are exposed to a pulsatile pattern of free (i.e. biologically active) corticosterone. Exposure of brain cells to relatively rapidly varying glucocorticoid concentrations may have functional implications for hormone action in these cells in view of the highly dynamic interaction of glucocorticoids with their cognate receptors (41). Second, we have demonstrated that the measurement of stress-induced changes in circulating plasma corticosterone concentrations may not provide an accurate reflection of the profile of corticosterone concentrations in a target tissue such as the brain. These findings have important implications for research on corticosteroid-regulated pathways and processes in the brain, not only with respect to the issue of continuous vs. pulsatile exposure but also with respect to the exact timing of the brain responses under study.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online March 20, 2008
Abbreviations: AUC, Area under the curve; Pgp, P-glycoprotein; PVN, paraventricular nucleus of the hypothalamus; T, time point of the half-maximal response.
Received January 23, 2008.
Accepted for publication March 10, 2008.
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-melanocyte-stimulating hormone by the ovine anterior and intermediate pituitary. Neuroendocrinology 49:349–360[CrossRef][Medline]This article has been cited by other articles:
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  K. L. Schmidt, E. H. Chin, A. H. Shah, and K. K. Soma Cortisol and corticosterone in immune organs and brain of European starlings: developmental changes, effects of restraint stress, comparison with zebra finches Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2009; 297(1): R42 - R51. [Abstract] [Full Text] [PDF]
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 E. R. de Kloet and R. A. Sarabdjitsingh Everything Has Rhythm: Focus on Glucocorticoid Pulsatility Endocrinology, July 1, 2008; 149(7): 3241 - 3243. [Full Text] [PDF]
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