European Journal of Pharmaeolog?', 171 (1989) 9-15
Elsevier LIP 51030
The action of corticosterone on schedule-induced wheeirunning W e n j u a n Lin, G e o r g e Singer a n d J o h n F u n d e r 1 Brain Behaeiour Research Institute, Department of Ps),cholog)', lxztrobe Unwersity. Bundoora 3083, and / Medical Research Centre. Prince tleno"s Hospital, Melbourne 3004. Austraha
Received 3 August 1989,accepted 15 August 1989
Previous studies have shown that schedule-induced wheelrunning is dependent on an intact pituitary-adrenal axis, and thus the presence of circulating corticosterone. In the present study, the mechanism of action of corticosterone on schedule-induced wheelrunning was explored in two ways. In the first series of studies, the effect of different levels of corticosterone on schedule-induced wheelrunning in adrenalectomized rats was investigated; the results of this study show a dose-response relationship between levels of corticosterone and schedule-induced wheelrunning. In the second study, the glucocorticoid receptor subtype involved was determined by examining the effect of dexamethasone, a synthetic glucocorticoid, on schedule-induced wheelrunning in adrenalectomized rats. A low dose of dexamethasone effectively reversed the suppressant effect of adrenalectomy, suggesting that the behavioural action of glucocorticoids is mediated through classical (Type Ii) glucocorticoid receptors, and not through Type I, corticosterone-preferring receptors. Schedule-induced wheelrunning: Adrenalectomy: Corticosterone; Dexamethasone: (Rat)
1. Introduction We have recently observed that adrenalectomy abolishes schedule-induced wheelrunning and that the suppressant effect of adrenalectomy can be reversed by corticosterone replacement; in contrast, these manipulations do not significantly influence non-scheduled wheelrunning ( L i n e t al., 1988). These findings suggest that corticosterone plays an important role in the development and occurrence ofschedule-inducedwheelrunning. The present study was undertaken to further characterize the mechanism of corticosterone action in schedule-induced wheelrunning, In Experiment 1, a dose-response study was carried out to determine whether variation of corticosterone levels within the physiological range
Correspondence to: J. Funder, Medical Research (?enter, Prince tlenry's Hospital, Melbourne3004, Australia.
leads to variation in the level of schedule-induced wheelrunning, and thus to establish whether the action of corticosterone is permissive or regulatory. In Experiment 2, the mechanism of the behavioural action of corticosterone was further explored. There are two types of glucocorticoid receptors in the brain and pituitary. The first are classical dexamethasone-binding glucocorticoid receptors, identified in the hypothalamus, the midbrain, and especially in the pituitary (De Kloet et al.. 1975). The second are high affinity corticosterone-preferring glucocorticoid receptors, found in abundance in the hippocampus (McEwcn et al., 1976) and which are clearly identical to mineralocorticoid receptors in the kidney and other aldosterone target tissues (Funder, 1986; Evans and Arriza, 1989). It has been suggested that glucocorticoids may have the same or different effects on behaviour, depending on whether they activate one or both of these receptor systems. Since the hippocampal
0014-2999/89/$03.50 .,~.1989 Elsevier Science Publishers B.V. (Biomedical l)ivision)
Type I sites have a much higher affinity for corticosterone than for dexamethasone (De Kloet et al., 1975; McEwen el al., 1976), determination of the effect of dexamethasone on schedule-induced wheelrunning in adrenalectomized rats should indicate whether Type I and Type It glucocorticoid receptors are involved in schedule-induced wheelrunning,
2. Materials and methods
2.1. Animals Sixty-six experimentally naive, male hooded Long-Evans rats with an initial body weight of about 300 g and aged between 90-120 days were used. Rats were housed individually in one of two laboratories under temperature controlled conditions (22 + 1 o C) and 12 h light/12 h dark cycles, with lights on an 08:00 h in the first laboratory and 13:00 h in the second. Following a period of acclimatization to the laboratory, the animals were body weight reduced over a 14 day period of restricted food intake to 80% of their free-feeding body weight, and were maintained at this weight throughout the experiment, unless otherwise stated. During the whole experimental period, animals were weighed daily and the availability of food was manipulated to maintain each animal at the desired weight. On a population basis, animals in all groups required the same amount of food to remain at 80% free-feeding weight, except those with 100% corticosterone or 10% dexamethasone pellets, in which groups weight was maintained at 70-80% of control on the same food intake,
2.2. Preparation of pellets Corticosterone pellets were produced by a method modified from Meyer et al. (1979). Corticosterone (100 mg; Sigma, St. Louis, MO) and 100 mg cholesterol (Sigma) were placed in a 10 ml beaker, melted and mixed carefully in a controlled laboratory heater/stirrer. The mixture was pelleted in a common canning mold, and pellets were individually weighed and trimmed to 100 nag with a scalpel blade. Pure cholesterol, pure corticosterone and other pellets with different ratios of
cholesterol to corticosterone or dexamethasone were prepared similarly.
2.3. Surgery Bilateral adrenalectomy was performed via the dorsal approach with animals under 60 m g / k g pentobarbitone sodium anaesthesia. The adrenal glands, together with surrounding fatty tissue, were removed: sham adrenalectomy animals underwent the sarne surgical procedure, but the adrenal glands were left intact. Adrenalectomized rats with glucocorticoid replacement had a glucocorticoid pellet implanted s.c. in the nape of the neck at the completion of surgery: adrenalectomized rats with a 100% cholesterol pellet served as vehicle-iraplanted controls. All adrenalectomized rats, with or without pellets, were maintained on 0.9% NaCI solution to drink. Animals were allowed a 7 day recovery period prior to testing. Absence of adrenal tissue and the bioavailability of administered corticosterone were confirmed by plasma corticosterone levels.
2.4. Apparatus The test chamber was made of clear Perspex with a stainless steel barred floor, and measured 33.5 x 28 × 42 cm. A food cup was located on one end wall of the chamber approximately 3.5 cm above the floor. Delivery of food pellets was automaritally controlled by standard relay circuitry, and Noyes standard formula 45 nag food pellets were used. The chamber was illuminated by a 40 W globe and ventilation fans provided masking against external sounds. A 26 cm diameter running wheel was at the rear of the chamber and turned in either a clockwise or counter-clockwise direction: the number of wheel revolutions ,*'as recorded on a five-digit electromechanical counter. Experimental sessions were conducted in eight chambers simultaneously, and all experiments were conducted at the same time each day to eliminate possible circadian effects.
2.5. Procedure: first study Forty-four rats were used, of which 40 were randomly assigned to one of five equal-sized
groups (eight per group). Four groups of rats (ADX, ADX (10% B), A D X (25% B), A D X (50% B)) were adrenalectomized and then implanted with a pellet weighing 100 mg and containing 0, l(), 25 or 50% of cortieosterone (B) with the residuum made up of cholesterol. The doses used in this experiment were chosen in the knowledge that a pellet consisting of 50 mg corticosterone and 50 mg cholesterol is fully effective in reinstating schedule-induced wheelrunning in adrenalectomized rats (Lin et al., 1988). One remaining group served as a sham-adrenalectomized control group, Animals were allowed a 7 day post-operative recovery period prior to testing. Five squads of eight rats were tested daily for 1.5 h at the same time of day for 10 consecutive days in a FT 120 s non-reinforcement contingent schedule. Each squad consisted of animals from the three of the five groups, thereby enabling experimental rats from each of three different groups to run simultaneously. In a subsequent small-scale study, four adrenalectomized rats were implanted with 100 mg of pure corticosterone as a pellet, and their schedule-induced wheelrunning performance evaluated as described above, 2.6. Procedure." second study Twenty-two rats were randomly assigned to one of three groups. Fourteen rats were adrenalectomized. Eight of these received a pellet consisting of 10 mg dexamethasone and 90 mg cholesterol pellets (ADX, 10% DEX), while the other six received cholesterol pellets as vehicle (ADX). The remaining eight rats were sham-adrenalectomized (sham). The dose chosen in this experiment was based on the results of the first study, and the knowledge that the half-life of dexamethasone is 5-6 times that of corticosterone (De Kloet et al., 1974). Thus, if dexamethasone has the same behavioural action in schedule-induced wheelrunning as corticosterone, 10% dexamethasone pellets would be expected to show a considerable effect. Other experimental procedures were the same as for the first study, 2. 7. Assam's Animals were killed by decapitation between 3 and 7 p.m. immediately after completing the tenth
test session. Animals were exsanguinated within 1 min; trunk blood was collected into heparinized tubes, centrifuged and the plasma stored at - 8 0 ° C . Plasma corticosterone levels were measured by a modification of the competitive protein binding assay of Murphy (1967), with horse serum as the binding protein source, [3H]corticosterone as tracer and Dextran-charcoal to adsorb unbound steroid. The limit of sensitivity was approximately 0.5 ng corticosterone, and the intraand inter-assay coefficients of variation were 10 and 13% respectively. 2.8. Statistics Data were analysed by' a two-way analysis of variance (ANOVA), followed where appropriate by simple main effect tests and post hoc Newman-Keuls tests.
3. Results The mean numbers of wheel revolutions over the 10 day period for ADX rats, corticosteronetreated A D X rats and sham A D X rats are shown in fig. 1. A treatments (5) x d a y s (10) two-way A N O V A with one repeated measure showed a significant treatment effect (F(4,35)= 7.10; P < 0.0001), day effect (F(9,315)= 32.20: P < 0.0001) and treatment × day interaction (F(9,315)= 2.36: P<0.0001). Simple main effect tests indicated that schedule-induced wheelrunning was totally absent only in the adrenalectomized group with vehicle implantation. However, although all doses of corticosterone facilitated the response over the 10 day testing period, inspection of fig. 1 shows that the levels of schedule-induced wheelrunning increased as the corticosterone doses increased. Further post hoc Newman-Keuls tests on group means of day data showed that significant (P < 0.05) increases were seen in sham-operated rats and A D X rats treated with 50% corticosterone, compared with vehicle-treated ADX rats, from the 4th day of testing. For the A D X rats treated with the 25 and 10% corticosterone pellets, differences from A D X were found on the 6th and the 7th day of testing respectively. These results show that
]2 MEAN 300 "
NO. OF WHEEL
ADX ( 10=,, B
ADX ( 25% B )
ADX ( 50% B ]
0 -" 0
" • ~4
. . . . . . . . . . . . . . . . 2 4 6
ADX * 10% B
ADX * 25% B
ADX • 50% B
AOX • 100% B
DAYS t"ig. l. Mean number of wheel revolutions over 1.5 h of scheduled food exposure in adrenalectomized (ADX) rats, in ADX rats implanted with 100 mg pellets containing 0, 10, 25 or 50q~ corticosterone (B), or in sham-adrenalectomized rats. For each group, n = 8.
Fig. 3. l)lasma corticostcron¢ Ic,.'cls ( m e a i ' ~ _ + S [:..,%1 ) i n adrcnalectonli/ed [AI.)X) rat>,, in AI')X rats imphmted ,aith 1(~'~ rag, pellets containing 10, 25. 50 anti 100% corticosterone (B). and in sharn-adrenalectomJzed rats. All plasma samples were from b l t ~ d taken within I min of finishing the final SIW exposure.
different doses of corticosterone administration not only produce different levels of scheduleinduced wheelrunning over 10 days, but also influence the rate of acquisition of the response, To examine further the corticosterone dose-response relationship, an additional four adrenalectomized rats implanted with a pellet consisting of 100 nag corticosterone were tested• These rats (,fig. 2) did more wheelrunning than sham-operated
evidence that the response develops in a dose-dependent manner, and in addition demtmstratmg that in the intact, sham-adrenalectomized rat schedule-induced wheelrunning does not reach maximum despite stress levels of endogenous glucocorticoids. The plasma corticosterone levels (mean ~_ S•t-., /,g/100 ml) for the various groups are given in fig. 3. As shown in this figure, an increase in corti-
rats (F(I,10) -- 7.09; P < 0•025), providing further MEAN MEAN
NO. OF WHEEL
NO. OF WHEEL
4 0 0 ~" ~
ADX (100% B)
3 0 0 '-
A D X ', 10% D E X
. . . . .
. . . . . .
DAYS Fig. 2. Mean number of wheel revolutions over 1.5 h of scheduled f ~ d exposure in adrenalectomi7ed (AI)X) rats iraplanted 'xith 100% 100 mg corticosterone (B) pellet,', (n ~ 4). and Of sham-operated rats (n :-g). Shov,'n are mean a-_S.E.M, values for each point,
/ / ~
DAYS Fig. 4. Mean number of ~heel revolutions over 1.5 h of
~,cheduled food exposure in adrenalcctonfized ( A I ) X ) r a t s iraplanted with 100 mg pellets containing (I or 10q: dexamethasone, or in ~,ham-adrenalectomized rails. Value>, ~hown are mean +_S.|'.M.: for AI)X alone n : 6. and for the other two group~, n
13 costerone composition of the implants is followed by a dose-related increase in plasma corticosterone. In the four adrenalectomized rats with 100% corticosterone implants, the plasma corticosterone level was not significantly different from that in sham-ADX rats. The mean numbers of wheel revolutions over the 10 day period for the three groups of animals in the second study are shown in fig. 4. A treatments (3) × d a y s (10) two-way A N O V A with one repeated measure analysis showed a significant treatment effect (F(2,20) = 6.84: P < 0.005) and day effect (F(18,180)=33.89: P<0.0001). Further main effect tests indicated that both sham-opcrated and dexanethasone-treated animals exhibited schedule-induced wheelrunning, while it was absent in ADX rats treated with vehicle, Inspection of fig. 4 shows that a 10% dexamethasone pellet is effective in reinstating scheduleinduced wheelrunning in adrenalectomized rats to levels at least as high as those of sham-ADX controls from day 4 onwards,
4. Discussion Although there is clearly a dose-response relationship between corticosterone and scheduleinduced wheelrunning, the behavioural effect of exogenous corticosterone cannot simply be cornpared with that of the same measured level of endogenous corticosterone. Endogenous corticosterone levels at any time are the net result of circadian patterns of secretion, environmental inputs including stress, and the effects of pituitaryadrenal feedback control; in contrast, the level of exogenous corticosterone from a pellet is relatively stable. Thus. although the plasma corticosterone levels in ADX rats treated with 50% of corticosterorle pellets were considerably lower than those in sham-operated controls, the schedule-induced wheelrunning levels in the former group were at least as high as those in the latter group, presumably' reflecting the maintained rather than episodic nature of the corticosterone, The results of the first study clearly' show that circulating corticosterone concentration determined the level of schedule-induced wheelrun-
ning: the higher the level of corticosterone, the higher the level of response. These results suggest that the action of corticosterone is not permissive or 'all or none', but dose-dependent. Several lines of evidence from the literature lend support to this finding. First. Tazi et al. (1986) found a trend (P < 0.10) in the correlation of plasma corticosterone and schedule-induced wheelrunning in intact rats. In pigs there is also a positive relationship between the amount of schedule-induced chain nibbling and pre-session plasma cortisol levels: animals which are more active have higher plasma cortisol levels than those which nibble their chain less intensively (Dantzer and Morm6de, 1981). Secondly, both food deprivation and intermittent scheduling of food delivery, which are the main factors determining the occurrence and development of schedule-induced behaviour (Falk, 1971). are involved in activating adrenal cortical function. It has been shown that prolonged food deprivation resulting in body' weight reduction activates the hypothalamo-pituitary-adrenal axis and increases the secretion of corticosterone from the adrenals (Krieger, 1974). It is also clear that animals exposed to schedule conditions show higher levels of corticosterone than those exposed to non-schedule conditions (Wallace et al.. 1983: Tang et al., 1984). Third. rats tested during the dark phase of the light/dark cycle exhibit both higher levels of schedule induced drinking and higher levels of plasma corticosterone compared with animals tested during the light phase. Animals tested during the dark phase even develop and maintain moderate levels of schedule-induced drinking without the need for the traditional requirement of body weight reduction (Gibson, 1987). Thus, given the evidence reported in this study as well as the related evidence in the literature, it is clear that an increase in plasma corticosterone, whether experimentally induced or endogenous, leads to an increase in schedule-induced activity levels. It should be noted, however, that although levels of schedule-induced behaviour may be determined by' corticosterone levels, the behaviour itself does not necessarib, feed back to further increase the corticosterone response. In contrast, from reports
14 in the literature, the behaviour may relieve the stimulus for increased hormonal response in a stressful situation. It has, for example, been repeatedly found that animals which have developed schedule-induced drinking during schedule conditions exhibit a significant reduction in plasma corticosterone levels, pre-session to post-session (Brett and Levine, 1979: 1981: Dantzer and Mormede, 1981', Tazi et al., 1986). Thus, the appatently contradictory relationships between schedule-induced behaviour and corticosterone (Brett and Levine, 1979: 1981; Dantzer and Mormede, 1981: Finlay and Wallace, 1981; Wallace et al., 1983: Tang et al., 1984: Tazi et al., 1986) may actually reflect different aspects of hormone-behaviour interaction, with different baselines taken for comparison of control and experimental groups, The results of the second study show that dexanaethasone completely reverses the suppressant effect of adrenalectomy, an effect essentially identical to that of corticosterone. This suggests that the behavioural action of corticosterone is not through the Type I. corticosterone-preferring glucocorticoid receptor system which is predominantly found in the hippocampus and septal areas, Similarly. the dose-response relationship seen in the first study is consistent with the behavioural action of corticosterone being mediated through moderate affinity receptors (such as Type I1 classical glucocorticoid receptors) rather than a high affinity receptor system, Certainly, the metabolic effects of the glucocorticoids are exerted via the Type 2 receptor. In view of the energy-expending characteristics of
eating that the metabolic effects on body weight do not run parallel with the dose-dependent response of corticosterone on schedule-induced wheclrunning. Furthermore, although both 100c/c, corticosterone and 10% dexamethasone implants resulted in an almost identical reduction in body weight, the behavioural effect of these two steroids is clearly different. It is thus unlikely that the action of corticosterone is uniquely through a peripheral metabolic mechanism: conclusive answers, however, must await studies in which glucocorticoids are applied locally into the brain. Taken together, these data provide limited information on the possible location, hippocampal or otherwise, of the neural areas subserving the development of schedule-induced wheelrunning. In previous studies, Devenport (1978) has suggested that corticosterone may affect the development of schedule-induced behaviour via the hippocampus. Tang et al. (1984) however, have shown that neither clectrol\,tic nor 6-hydroxydopamine lesions of the hippocampus influence scheduleinduced drinking, suggesting that hippocampal neurons may not be involved in this type of behaviour. The studies detailed in the present paper show that type 11 receptors, which are widely distributed in the brain including the hippocampus, mediate the effects of glucocorticoids on schedule-induced wheelrunning. The question of whether the hippocampus has a role in the regulation of any or all forms of schedule-induced behaviour thus remains unresolved.
wheelrunning, the possibility that the effect of glucocorticoids on schedule-induced wheelrunning could be due to the peripheral metabolic effects of glucocorticoids cannot be excluded, ltowever. since all animals received almost the same rations
Brett. L.P. and S. l.evine, 1979, Schedule induced polydipsia suppresses pituitar?.-adrenal activit?, in rats..1. Comp. Ph',siol. Psychol. 93, 941% Bren. I..P. and S. I,evme, 1981, The pituitary-adrenal response t,, immunized schedule-induced drinking. Physiol. Behax. 26. 153. Dantzer.R. and P. Mormdde, 1981, Pituitary-adrenal consequences of adjunctive activities in pigs, tiorm. Beha~. 15. 3~,. Devenport.I,.D., 1978, Schedule-induced pol.',dipsia in rats: Adrenocortical and hippt~_'ampal modul,ation, J. ('omp. Phvsiol. Psvchol. 92. 651. l)e Kloel. Ii.R.. J. Van tier Vies lind D. l)e Wied, 1q~74,"the site of suppressive action of dexamethasone on pmiitar5adrenal acti,,ity, Endocrinology94. 61.
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