Animal husbandry and cannulation
All experiments were performed on virgin female Sprague Dawley rats (250–350 g) obtained from Bantin and Kingman (Hull, UK) and housed in the local animal facility at least 7 d before experimentation, initially in groups of four to six per cage. Female rats were used because the higher basal levels of corticosterone compared with males (36
) enabled better resolution of the temporal profile of glucocorticoid inhibition. Animals were housed under standard environmental conditions: 14-h light, 10-h dark cycle (lights on at 0500 h) and ad libitum
access to water and rat chow. The iv cannulation of the right jugular vein was performed as previously described (37
). Surgery was performed at least 3 d before blood sampling to allow for postoperative recovery and adaptation to the sampling environment. Animals were housed singly after surgery, and a swivel allowed complete freedom of movement within the home cage. Cannulae were flushed with a small volume of heparinized saline (10 U/ml) daily to maintain patency. Procedures were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 and the European Community Council Directive (86/609/EEC). Each animal underwent only one period of sampling.
Blood sampling and drug administration
Blood sampling was undertaken in a sound-isolated room using an automated sampling system (38
). Animals were connected to the system through a liquid swivel and samples (37 μl whole blood diluted 1:5 in heparinized saline) automatically withdrawn every 5 or 10 min and replaced by heparinized saline. This procedure for multiple blood sampling does not cause any adverse effects (8
). The initial 1 h of samples was discarded to avoid any nonspecific release and measurements commenced either at 1600 h (late light phase) or 0700 h (early light phase). Injections of steroids, CRH, or vehicle were performed manually by briefly disconnecting the iv cannula from the swivel and injecting through the sampling cannula. This procedure was completed between two samples and caused minimal disturbance. Drug effects were controlled by comparison with injections of saline.
Study 1: measures of clearance rate of corticosterone and steroid cross-reaction
Because studies involved the determination of plasma corticosterone levels during injection of glucocorticoid agonists, a study was performed to examine whether measurements were affected by cross-reaction of the agonists. Seven animals underwent bilateral adrenalectomy at the same time as cannulation. In the postoperative period, they were allowed access to drinking water containing 0.9% NaCl and 0.25 mg/ml corticosterone to maintain hydromineral balance. On the fourth day at 1200 h, this solution was replaced by drinking water containing 0.9% NaCl alone to allow the corticosterone to clear from the circulation. At 1600 h, blood samples were withdrawn every 5 min for a period of 6 h. At 25, 125, and 225 min of sampling, each animal received injections of 400 μg corticosterone (Sigma-Aldrich, St. Louis, MO), 2000 μg methylprednisolone (sodium succinate, Solu-Medrone; Pharmacia & Upjohn Ltd., Milton Keynes, UK), and 500 μg dexamethasone (dexamethasone-21-phosphate; Sigma-Aldrich), respectively. These doses represented the maximum doses administered during the later pharmacological studies. Samples were assayed for corticosterone-like immunoreactivity and measured values used to calculate cross-reaction. Exponential regression was used to calculate the clearance rate (y = y0e−kt) and half-life [t½ = (ln 2)/k], where y0 is the peak corticosterone value (nanograms per milliliter), k is the rate constant, and t is time.
Study 2: effect of glucocorticoid agonists and antagonist on basal HPA activity
To examine the acute effects of glucocorticoid agonists on basal HPA activity, animals were administered injections of methylprednisolone (5 μg, n = 8; 50 μg, n = 10; 500 μg, n = 12; 2000 μg, n = 10), dexamethasone (5 μg, n = 8; 50 μg, n = 6; 500 μg, n = 7), or saline vehicle (n = 11), each in a volume of 0.1 ml. Injections were made during the late light period to coincide with the phase of the diurnal cycle when basal corticosterone secretion is near maximum (23
). The dynamics of the response to agonist injection were examined by rapid blood sampling every 5 min for 3 h and then every 10 min for 2 h. In all cases, samples were collected from 1600 h (3 h before lights off), and agonist injections were given at 1645 h. To determine the effect of glucocorticoid antagonism on the response to methylprednisolone, additional groups of animals were injected with either 50 μg/rat (n = 6) or 500 μg/rat (n = 6) of the antagonist RU38486 (mifepristone; gift from Dr. J. K. Belanoff, Corcept Therapeutics Inc., Menlo Park, CA) 15 min before injection of 50 μg methylprednisolone.
Study 3: CRH challenge during glucocorticoid-induced suppression
To determine whether glucocorticoid-induced suppression of HPA activity was due to suppression of pituitary-adrenal responsiveness, animals were tested for responses to CRH injection. Samples were collected every 5 min starting at 1500 h, although measurements did not start until 1600 h. Acute injection of methylprednisolone (500 μg) or saline was given at 1700 h, and CRH (1 μg/rat, iv) was injected at either 1800 or 2000 h. CRH (Bachem, Torrance, CA) was diluted in heparinized saline and stored at 4 C until use. The two time points for CRH injection (60 or 180 min after methylprednisolone) were selected to coincide with either 1) the period of maximal rate of change of corticosterone levels or 2) the period of maximal suppression of the axis. Blood sampling continued every 5 min for a minimum of 2 h after the CRH injection before changing to every 10 min.
Study 4: rapid glucocorticoid suppression of stress-induced HPA activity
To determine whether glucocorticoid feedback could rapidly suppress HPA activity induced by acute psychological stress, methylprednisolone (500 μg) or saline was injected iv 15 min before the onset of a 30-min noise (104 dB). Sampling was undertaken in the early light phase starting at 0600 h when basal corticosterone levels were low, and the noise stress commenced at 0800 h. Blood samples were collected every 5 min until 1200 h. At 15 min after the end of the noise stress, all animals received an iv injection of 1 μg CRH to determine pituitary-adrenal axis reactivity during the period of maximal poststress decline in corticosterone levels.
Finally, to demonstrate both the dose dependency and central site of action for the glucocorticoid-mediated suppression of stress-induced HPA activity, groups of animals were injected iv 15 min before the onset of a noise stress (30 min at 104 dB) with different doses of methylprednisolone (5 μg, n = 6; 50 μg, n = 5; or 500 μg, n = 5) or saline (n = 6). No automated sampling was conducted, but animals were decapitated immediately after the end of the noise, and trunk blood (~4 ml) was collected. Six other animals injected with saline were not exposed to noise but were euthanized at the equivalent time after injection. Trunk blood samples were centrifuged at 5000 rpm for 5 min, and plasma was collected and stored at −20 C. Brains were removed and immediately stored at −80 C for later determination of c-fos mRNA expression.
Measurement of corticosterone
Total plasma corticosterone levels were measured by RIA in diluted whole blood samples (automated sampling) or in plasma (trunk blood) using a citrate buffer (pH 3.0) to denature the binding globulin, [125
I]corticosterone (0.37 MBq/ml activity; ICN, High Wycombe, UK), and a specific rabbit antirat corticosterone primary antibody (gift courtesy of Professor G. Makara, Institute of Experimental Medicine, Budapest, Hungary) (see Ref. 8
for additional details). All samples from an individual animal were assayed together.
In situ hybridization histochemistry for c-fos mRNA
The hypothalamic PVN was cryosectioned at 12 μm and sections mounted on gelatin-coated slides, with the presence of the PVN confirmed by staining adjacent sections with toluidine blue. Before hybridization, sections were fixed in 4% paraformaldehyde, washed in saline/triethanolamine containing 0.25% acetic anhydride, dehydrated in ethanol and chloroform, and air dried. The c-fos
mRNA levels were measured by in situ
hybridization using a cDNA riboprobe as described elsewhere (39
). Hybridized sections were apposed to autoradiographic film for 14 d. Relative levels of c-fos
mRNA expression were calculated as OD measurements using computerized image analysis software (Image; National Institutes of Health, Bethesda, MD).
Hormone data are presented as the mean and sem of the absolute measures of plasma corticosterone. However, to determine the time course of the effect of acute agonist injection on plasma corticosterone levels, a normalization procedure was also applied. This normalization involved first calculating the average corticosterone levels in the preinjection period (0–45 min.) for each animal and then dividing all data for that animal by this average value. Second, to overcome the nonstationary nature of the data, the rescaled value at each time point was divided by the respective mean value for that time point in the saline control group. Thus, the mean normalized value at all time points for the saline controls was fixed at 1, and treatment groups are displayed relative to this to show the drug effect. Both raw and normalized data are presented. To determine the period of significant steroid-induced effect, t tests were performed on these normalized values at each time point to determine the first and last points that differed significantly (P < 0.05) from controls. Other comparisons were performed using t test or ANOVA, as appropriate.