Adaptation of the glucocorticosterone response to novelty

Physiology & Behavior, Vol. 17, pp. 43--46. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.

Adaptation of the Glucocorticosterone Response to Novelty' H. P E T E R P F I S T E R A N D M A U R I C E G. KING The University o f Newcastle, N e w S o u t h Wales, Australia (Received 4 A u g u s t 1975)

PFISTER, H. P. AND M. G. KING. Adaptation of the glucocorticosterone response to novelty. PHYSIOL. BEHAV. 17(1 ) 4 3 - 4 6 , 1976. - The 11-hydroxycorticosterone (11-OHCS) response in male rats to novelty as a stressor was examined. Since the emphasis to date has been on short durations of exposure to novel stimuli, up to 45 rain only, the present study aimed at investigating the elevation and adaptation of the 11-OHCS response to prolonged exposure to novel environments. It was found that the l 1-OHCS elevation due to a novel environment takes about 4 hr to return to resting levels. Net elevations of 11-OHCS induced by novelty in the rat appear to be affected by the underlying circadian rhythm. In addition such elevations due to novelty are not constant over time but are subject to a ceiling effect. 11-Hydroxycorticosterone

Circadian rhythm

Novelty

A D E R and F r i e d m a n [ 1 ] first r e p o r t e d t h a t b r i e f e x p o s u r e of t h e rat to a novel e n v i r o n m e n t resulted in a rapid rise of 11-hydroxycorticosterone (11-OHCS) output from the adrenal cortex. The effect o f longer periods o f e x p o s u r e ( u p to 45 rain) to novel e n v i r o n m e n t s o n elevation of I 1-OHCS were investigated b y Bassett, Cairncross and King [ 3 ] . One c o n s e q u e n c e i n t r o d u c e d by e x t e n d e d e x p o s u r e is t h e i n t e r a c t i o n b e t w e e n (1) the response o f 11-OHCS due to novelty, and (2) t h e variations in l l - O H C S due t o t h e u n d e r l y i n g d i u r n a l variations. M e a s u r e m e n t s of the resting levels o f free plasma l l-OHCS in rats revealed diurnal variations [6] and such circadian r h y t h m s in n o n s t r e s s e d rats are n o w well established [1, 15, 18]. Plasma I I - O H C S reaches a peak just prior to o n s e t of t h e dark period w h i c h is t h e active time for rats [4, 5, 13]. The fall of free plasma l l - O H C S is gradual to a t r o u g h w h i c h occurs prior to o n s e t of the light period. The a d r e n o c o r t i c a l r e s p o n s e to stress in the rat results in a m a r k e d e l e v a t i o n o f c o r t i c o s t e r o i d s s u p e r i m p o s e d on the circadian r h y t h m [ 1 , 5 , 10]. Ader, F r i e d m a n and G r o t a [2] r e p o r t e d a rise o f 11-OHCS due to o p e n field testing in rats. T h e elevation was a b o u t the same w h e t h e r testing o c c u r r e d at the peak or t h e t r o u g h of the circadian r h y t h m . F r o m this it was suggested t h a t the stress r e s p o n s e as m e a s u r e d b y A d e r et al. [2] simply adds to the value resulting f r o m the u n d e r l y i n g circadian r h y t h m . D u n n et al. [5] d e m o n s t r a t e d a m a r k e d increase of c o r t i c o s t e r o n e level on the circadian r h y t h m at t h e trough, using n o v e l t y as a stressor. When testing was carried o u t prior to offset of the light p e r i o d (i.e. at t h e peak of t h e circadian r h y t h m ) n o significant elevation of t h e 11-OHCS level a b o v e t h e n o n s t r e s s peak resulted. However, testing (using n o v e l t y as the stressor) at d i f f e r e n t t i m e s d u r i n g a 24 hr p e r i o d suggested t h a t the 1 I-OHCS response in male rats reaches a ceiling level

irrespective o f t h e u n d e r l y i n g circadian r h y t h m . D u n n et al. [5] c o n c l u d e d t h e r e f o r e t h a t the stress-induced net elevat i o n of free plasma l l-OHCS varied w i t h the phase of t h e circadian r h y t h m . With r e f e r e n c e to t h e l a t e n c y of a d a p t a t i o n of 11-OHCS levels, S t o c k h a m [ 1 6 ] , a n d J o n e s and S t o c k h a m [7] r e p o r t e d a p r o l o n g e d elevation of l l-OHCS of 4 hr d u r a t i o n a f t e r a second stressful s t i m u l u s was i n t r o d u c e d in close t e m p o r a l p r o x i m i t y to the initial one. F u r t h e r evidence for a b o u t a 4 h r delay of l l - O H C S to r e t u r n to base level was r e p o r t e d by Underhill et al. [ 1 7 ] . T h u s it could be e x p e c t e d t h a t rats exposed for 4 h r to a novel testing a p p a r a t u s should have a d a p t e d to it and t h a t n o significant elevation above the t r o u g h n o n s t r e s s level of 11-OHCS s h o u l d be f o u n d . It would also be e x p e c t e d t h a t a 1 hr d u r a t i o n of e x p o s u r e to a novel testing a p p a r a t u s would be i n s u f f i c i e n t t i m e for the 1 I-OHCS level to r e t u r n to base level [ 3 ] . Hence the 11-OHCS r e s p o n s e in the rat appears to a d a p t to a novel testing a p p a r a t u s b e t w e e n 1 and 4 hr o f exposure. One aim of the p r e s e n t e x p e r i m e n t was to d e t e r m i n e the t i m e course of the n o v e l t y - i n d u c e d elevation of the 11-OHCS and its r e t u r n to resting level. The e x p e r i m e n t was also designed to clarify the degree of elevation of novelty i n d u c e d 11-OHCS response. The main q u e s t i o n u n d e r investigation was w h e t h e r the n o v e l t y i n d u c e d elevation of 1 I-OHCS is of a c o n s t a n t additive n a t u r e as p o s t u l a t e d by A d e r et al. [1] or, w h e t h e r the elevation is subject to a ceiling level i n d e p e n d e n t of the u n d e r l y i n g circadian r h y t h m as p o s t u l a t e d by D u n n et al. [51. METHOI)

Animals Eighty male Wistar rats 90

100 days old at the start of

1This research was supported in part by a Grant from the Australian Research Grants Committee A65/15506 to M. G. King. 43

44 testing were used. T h e rats were raised in a fully airc o n d i t i o n e d (22 -+ I ° C ) a n i m a l h o u s e w i t h all a n i m a l s h a v i n g access t o n a t u r a l d a y light c o n d i t i o n s . T h r e e weeks p r i o r to t e s t i n g all rats were t h e n h o u s e d singly in b o x e s ( 3 0 x 4 0 x 12) in a fully a i r - c o n d i t i o n e d h o l d i n g r o o m at 22 -+ I°C. Artificial light was o n f r o m 0 4 0 0 - 1 8 0 0 h r daily, o t h e r w i s e ( 1 8 0 0 - 0 4 0 0 h r ) a n i m a l s were k e p t in c o m p l e t e darkness. A t n o t i m e was any s o u r c e o f n a t u r a l light available. F o o d a n d w a t e r were p r o v i d e d ad lib.

Glucocorticoid Assay T h e stress r e s p o n s e m e a s u r e d was t h e p l a s m a level of free c o r t i c o s t e r o n e (11-OHCS), t h e p r e d o m i n a n t glucoc o r t i c o i d secreted in t h e rat [ 1 1 , 1 4 ] . A t t h e t i m e s i n d i c a t e d in t h e p r o c e d u r e a n i m a l s were sacrificed b y d e c a p i t a t i o n . T h e b l o o d was c o l l e c t e d in h e p a r i n i z e d t u b e s a n d c e n t r i f u g e d to o b t a i n cell-free p l a s m a w h i c h was t h e n frozen. C o r t i c o s t e r o n e levels in p l a s m a were o b t a i n e d subsequently by the fluorimetric method of Mattingley [ 12] w h i c h is specific for free p l a s m a 11-OHCS.

Apparatus T h e novel a p p a r a t u s c o n s i s t e d of a 2 c o m p a r t m e n t s h u t t l e b o x of clear Plexiglas, w i t h e x t e r n a l d i m e n s i o n s of 60 × 12.5 x 12.5 cm. T h e grid floor was of stainless steel bars w i t h a dia. o f 0.5 c m a n d spaced 1.2 cm apart. T h e guillotine d o o r s e p a r a t i n g t h e 2 c o m p a r t m e n t s was o p e n throughout the experiment.

Procedure F o r t y - e i g h t c o n t r o l (C) a n i m a l s were r a n d o m l y allocated to 6 g r o u p s o f 8 each. W i t h i n e a c h of t h e 6 g r o u p s a n i m a l s were r a n d o m l y allocated n u m b e r s 1 - 8 . On each day for 8 s u b s e q u e n t days, a rat f r o m e a c h of t h e 6 g r o u p s was r e m o v e d f r o m t h e h o t d i n g r o o m in its h o m e cage. W i t h i n 9 0 sec o f r e m o v a l f r o m t h e h o l d i n g r o o m rats were sacrificed as described earlier. A n i m a l s were r e m o v e d f r o m t h e h o l d i n g r o o m at 1400, 1700, 2 0 0 0 , 2 3 0 0 , 0 2 0 0 or 0 5 0 0 hr. T h u s o n t h e first day, at 1400 h r t h e first a n i m a l f r o m C o n t r o l G r o u p 1 was sacrificed, f o l l o w e d b y A n i m a l 1 f r o m C o n t r o l G r o u p 2 at 1700 hr. A t 2 0 0 0 h r A n i m a l 1 f r o m C o n t r o l G r o u p 3 was sacrificed, f o l l o w e d b y t h e first a n i m a l f r o m C o n t r o l G r o u p s 4 a n d 5 at 2 3 0 0 and 0 2 0 0 h r respectively. T h e last a n i m a l sacrificed o n Day 1 was R a t N u m b e r 1 f r o m C o n t r o l G r o u p 6 at 0 5 0 0 hr. T h u s at t h e e n d of Day 1, all n u m b e r 1 a n i m a l s f r o m G r o u p s 1 t o 6 h a d b e e n sacrificed. T h e n , c o m m e n c i n g Day 2, R a t 2 f r o m C o n t r o l G r o u p 1 was sacrificed at 1400 hr. R a t 2 f r o m C o n t r o l G r o u p 2 f o l l o w e d at 1700 hr. H e n c e all t h e r e m a i n i n g n u m b e r 2 a n i m a l s f r o m C o n t r o l G r o u p s 3, 4, 5 a n d 6 were sacrificed at 2 0 0 0 , 2 3 0 0 , 0 2 0 0 and 0 5 0 0 h r respectively. A t t h e e n d o f Day 2, all n u m b e r 2 rats f r o m C o n t r o l G r o u p s 1 t o 6 h a d b e e n sacrificed. F o l l o w i n g t h e p r e c e d i n g p a t t e r n , o n t h e t h i r d day all n u m b e r 3 animals, a n d o n t h e f o u r t h day all n u m b e r 4 a n i m a l s were sacrificed. This was f o l l o w e d b y all a n i m a l s w i t h t h e n u m b e r s 5, 6, 7 a n d 8 w h i c h were sacrificed o n t h e fifth, sixth, s e v e n t h and e i g h t h days respectively. T h e 32 e x p e r i m e n t a l a n i m a l s were r a n d o m l y allocated t o 4 g r o u p s o f 8, w i t h i n e a c h of t h e 4 g r o u p s rats were randomly allocated the numbers 1-8: (1) A 4 h r e x p o s u r e g r o u p ( 4 H E ) was tested at the

PHSTER

A N D KING

t r o u g h ( 0 2 0 0 h r ) o f t h e circadian r h y t h m ( 4 H E t ) . At 2 2 0 0 h r t h e first o f t h e 8 rats of t h e 4 H E I g r o u p was r e m o v e d f r o m tts h o m e cage and placed in t h e test b o x for 4 hr. At 0 2 0 0 h r the rat was r e m o v e d f r o m t h e s h u t t t e b o x and t a k e n o u t of t h e h o l d i n g r o o m . Within 90 ~ec of r e m o v a l the ral was sacrificed as described previously. The f o l l o w i n g day Rat 2 o f t h e 4 H E t g r o u p was t a k e n from its h o m e cage and placed in the test box for 4 hr. At 0 2 0 0 hr t h e rat was r e m o v e d f r o m t h e s h u t t t e b o x and t a k e n out of the h o l d i n g r o o m . Within 90 sec of removal the rat was sacrificed as described previously. Similarly on the nexl day Rat 3 was tested. This p r o c e d u r e was repeated daily u n t i l all 8 rats ot t h e 4 H E t g r o u p were tested. t21 A second 4 hr e x p o s u r e g r o u p was tested at the peak t 1 7 0 0 hr) o f the circadian r h y t h m 44HEp) g r o u p was r e m o v e d f r o m its h o m e cage and placed in the test b o x t o l 4 hr. At 1700 h r the rat was r e m o v e d f r o m the test box and t a k e n o u t of t h e h o l d i n g r o o m . Within 90 sec ot removal the rat was d e c a p i t a t e d . At 1300 hr the following day, Rat 2 o f t h e 4 H E p g r o u p was r e m o v e d f r o m its h o m e cage and placed in the test b o x for a hr. At 1700 hr the rat was r e m o v e d f r o m the test b o x and t a k e n out of the h o l d i n g room. Within 90 sec o f removal the ra~ was d e c a p i t a t e d . Similarly o n the next day Rat 3 of ~he 4 H E p g r o u p was sacrificed. T h e same p r o c e d u r e was r e p e a t e d daily until all 8 rats of t h e 4 H E p g r o u p had b e e n tested (3J A 2 h r e x p o s u r e g r o u p was rested a~ the Irough ( 0 2 0 0 hr) of t h e circadian r h y t h m ( 2 l t [ tL A t m i d n i g h t the first ot t h e 8 rats o f the 2HE t g r o u p was r e m o v e d from its h o m e cage and placed in the test box for 2 hr. At 0 2 0 0 h ~ the rat was r e m o v e d f r o m t h e test box and t a k e n o u t of the h o l d i n g r o o m . D e c a p i t a t i o n followed within 90 sec o f r e m o v a l f r o m t h e test box. This p r o c e d u r e was r e p e a t e d on t h e following day, testing Rat 2 of the 2HE t group. T h e n e x t day Rat 3 of the 2 H E t g r o u p was tested. Hence. this p r o c e d u r e was r e p e a t e d daily until ~11 8 rats ot ~he 2HE 1 g r o u p h a d been tested. (4J A second 2 h r e x p o s u r e g r o u p was tested at the peak q1700 hr) of t h e circadian r h y t h m ( 2 H E p ) At 1 5 0 0 h r t h e first of t h e 8 rats of the 2 H E p g r o u p was r e m o v e d f r o m its h o m e cage a n d placed in t h e test box f~r 2 hr. At 1700 hr t h e rat was r e m o v e d f r o m t h e test box and t a k e n o u t of the h o l d i n g room. Within 90 se¢ of removal the rat was d e c a p i t a t e d as described previously. The same p r o c e d u r e was repeated on the following d a ~ testing Rat 2 of ~he 2HEp group. The n e x t day Rat 3 (,t ihe 2HEp group was tested. T h e same p r o c e d u r e was repeated u n t i l all 8 rats of t h e 2HEp g r o u p had been tested RESUI~TS

Figure 1 shows the s u m m a r t z e d m e a n values and s t a n d a r d e r r o r scores of 1 I-OHCS for the 6 g r o u p s in the baseline study. T h e m e a n values and s t a n u a r d e r r o r scores ot the 2 c o n t r o l groups: t r o u g h at 0 2 0 0 hr (Ct) a n d p e a k at 1700 h r (Cp) are s u m m a r i z e d in T a b l e 1~ Mean values and s t a n d a r d e r r o r scores for t h e 4 e x p e r i m e n t a l g r o u p s are also given in Table I. T h e 11-OHCS m e a n s of the 2 c o n t r o l G r o u p s (Ct a n a Cp) a n d t h e 4 e x p e r i m e n t a l g r o u p s ( 4 H E t, 4 H E p , 2 H E t a n d 21~IEp) were s u b j e c t e d to a p l a n n e d c o n t r a s t s analysis [ 9 ] . The c o m p a r i s o n s tested are s u m m a r i z e d and t h e results p r e s e n t e d in T a b l e 2. T h e 5 c o m p a r i s o n s resulted in 3 i n s t a n c e s of a c c e p t a n c e of t h e null h y p o t h e s i s and 2

GLUCOCORTICOSTERONE AND NOVELTY

20

T

175

/±

a_ 15 ~6 E O

TROUGH TEST

PEAK TEST

<~

45

\,

125

{- ...... 4.

±\,

10 75

r-

L) t Oi

5 25 LIGHT OFF I

I

1700

1400

TrME

I

2000

2300

IN

HOURS

I

0200

I

050O

FIG. 1. Diurnal variation in the resting ll-hydroxycorticosterone levels in blood plasma of male Wistar rats.

given only 2 hr in the novel e n v i r o n m e n t and tested at the trough of the circadian r h y t h m did not reach the base level. However rats which were given 2 hr in the novel environment and tested at the peak of the circadian r h y t h m did not differ significantly in their 11-OHCS levels from the control group which were also tested at the peak of the circadian r h y t h m (4Ho). Thus, the net elevations of 11-OHCS of rats which were exposed for 2 hr to the novel e n v i r o n m e n t are significantly different (5Ho). F r o m this it follows the elevations of the 11-OHCS response due to novelty differ with the circadian rhythm. Rats which were given 4 hr in the novel e n v i r o n m e n t and killed either at the trough (1Ho) or the peak (2Ho) o f the circadian r h y t h m did not exhibit in blood plasma any significant elevation of the 11-OHCS level. The 2 missing points of the circadian r h y t h m (0800 hr and 1100 hr) were not included, since in the present study only the peak (1700 hr) and the trough (0200 hr) were of interest. In order to locate the peak and the trough only one additional p o i n t on either side, i.e. 1400 and 2000 hr for the peak and 2300 and 0500 hr for the trough need be included. DISCUSSION

TABLE 1 MEAN VALUES OF I I-OHCS (IN /~g/100 ML OF PLASMA) FOR CONTROL AND EXPERIMENTAL GROUPS

Group

Hr of Exposure Novel Environment

Free Plasma Mean

Ct Cp 4HEr 4HEp 2HEt 2HEp

0 0 4 4 2 2

7.66 17.85 10.26 17.39 16.03 23.00

11-OHCS Levels S.E. 1.26 2.23 1.09 1.46 3.12 0.92

8 8 8 8 8 8

TABLE 2 COMPARISONS TESTED AND STATISTICAL DECISIONS Mathematical Statement of Null Hypothesis lHo:/xCt - #4HEt 2Ho:~ Cp - u 4HEp 3Ho: ~Ct - u 2HEr 4Ho: u Cp - u 2HEp 5Ho: (¢ 2HEt - ~ Ct) ( ~ 2 H E p - uCp)

Statistical

Decision Accept Accept Reject* Accept Reject*

*p<0.01. rejections of the null hypothesis at the level of p < 0 . 0 1 . Rats exposed for 2 hr to a novel e n v i r o n m e n t and killed at the trough of the circadian r h y t h m show a significant elevation of 11-OHCS level with respect to control rats measured at the trough of the circadian r h y t h m (3Ho). Thus, the elevation of 11-OHCS levels in rats which were

The circadian r h y t h m for 1 !-OHCS of male rats reported by Dunn e t al. [5] was substantiated in the present study (Fig. 1). With respect to adaptation of the response to the novel environment, the present findings suggest that more than 2 hr and up to 4 hr is required for the 1 1-OHCS response to return to normal (Table 2). This is in agreement with studies reported by S t o c k h a m [ 1 6 ] , Jones and S t o c k h a m [7] and Underhill e t al. [17]. Male rats exposed to the novel e n v i r o n m e n t for a period of 2 hr showed a significant rise of 1 1-OHCS only when tested at the trough; if tested at the peak, the elevation failed to reach significance (Table 2). With regard to the degree of elevation of the 1 1-OHCS response to novelty, it was found that the net elevation at the trough is significantly larger than the corresponding one at the peak of the circadian r h y t h m (Table 2). This supports Dunn e t al. [5] who proposed a ceiling level effect for 1 1-OHCS elevations. The present findings with respect to the 2 hr groups are consistent with the rate sensitive negative feedback control of A C T H proposed by Jones, Brush and Neame [8]. They reported that the rate of secretion of plasma steroids is negatively related to the level of the response. Thus in the 2 hr groups the a m o u n t over time and therefore the rate of secretion at the peak due to novelty was relatively less than for the same exposure at the trough. Thus the rate of secretion due to novelty would appear to be governed by the higher basal rate of secretion at the peak rather than at the trough of the circadian rhythm. In summary, the elevation of 1 1-OHCS due to novelty is affected by the underlying circadian rhythm. A d a p t a t i o n of 1 1-OHCS of rats to the novel e n v i r o n m e n t is slow, and 4 hr should be allowed for the 1 1-OHCS elevation to return to base level. It would appear also that this effect is not of a simple additive nature as proposed by Ader e t al. [2] but rather that of a ceiling level as proposed by Dunn e t al. [5]. E x p e r i m e n t s e m p l o y i n g the l l - O H C S response to novelty would best be carried out during the trough phase

46

P F I S T E R AND KINC,

o f t h e circadian r h y t h m since resulting changes o f 1 1-OHCS o u t p u t can t h e n vary over a larger range. If rats are tested at

t h e peak o f t h e circadian r h y t h m , p o t e n t i a l changes in ~:he a d r e n o c o r t i c a l o u t p u t are r e d u c e d .

REFERENCES 1. Ader, R., and S. B. Friedman. Plasma corticosterone response to environmental stimulation: Effects of duration of stimulation and the 24-hour adrenocortical rhythm. Neuroendocrinology 3: 378-386, 1968. 2. Ader, R., S. B. Friedman and L. J. Grota. Emotionality anti adrenal cortical function: Effects of strain, test and the 24-hour corticosterone rhythm. Anim. Behav. 1 5 : 3 7 44, 1967. 3. Bassett, J. R., K. D. Cairncross and M. G. King~ Parameters of novelty, shock predictability and response contingency in corticosterone release in the rat. PhysioL Behav. 10: 901-907, 1973. 4. Chiefetz, P. N. The daily rhythm of secretion of corticotrophin and corticosterone in rats and mice. Proceedings of the Society for Endocrinology, J. Endocr. 49:11 - 12, 1971. 5. Dunn, J., L. Scheving and P. Millet. Circadian variation in stress-evoked increases in plasma corticosterone. Am. .l. Physiol. 223: 402-406, 1972. 6. Guillemin, R., W. E. Dear and R. Liebelt. Nychthermal variation in plasma free corticosteroid levels of the rat. Proc. Soc. exp. Biol. Med. 101: 394-395, 1959. 7. Jones, M. T., and M. A. Stockham. The effects of previous stimulation of the adrenal cortex by adrenocorticotrophin on the function of the pituitary-adrenocortical axis in response to stress. J. Physiol. 184: 741-750, 1966. 8. Jones, M. T., F. R. Brush and R. L. B. Neame. Characteristics of fast feedback control of corticotrophin release by corticosteroids. J. Endocr. 55: 489-497, 1972. 9. Keppel, G. Design and Analysis, New Jersey: Prentice-Hall, 1973.

10. Knigge, K. M., C. It. Penrod and W. J. Schindler. h7 LT#ro and in vivo adrenal eorticosteroid secretion following stress. A m . . 1 Physiol. 196:579 582. 1959. 11. LaPlanta, C., C. J. Geroud and .i. Stachenko. Lack ol appreciable 17-hydroxylase activity in the normal and regenerated rat adrenal tissues. Endocrinotog_y 75: 825-827. 1964. 12. Mattingley, D. A. A simple fluorimetrie method for estimation of free l l-hydroxycorticosteroids in human plasma..L ctin. Path. 15:374 379. 1962. 13. McCarthy, J. L., R. C. Corely and M. X. Zarro~-. Diurnal rhythm in plasma corticosterone and lack of diurnal rhythm in plasma component Fdike material in the rat. Proc ,%c erp Biol. Meal. 104: 787-789, 1960. 14. Peron, F. G. The isolation and idemification of some aarent,corticosteroids released by rat adrenal tissue incubated 07 vttre) Fndocrin. 66: 458-469, 1960. 15. Scheving, L. E. and J. E. Pauly. Effects of lighl on corucosterone levels in plasma of rat~ Am. J. Physiol. 210: 1112 1117.1966. 16. Stockham, M. A. Changes of plasma anti adrenal corlicosterone levels in lhe rat after repeated slimuli. ,Z Physiol. 173: 149 159. 1964. 17. Underhill, W. R.. W. B. Rueker. C. (~ McDiarmid and S. B Sparber. Time factors in behavioral control c~f brain n~repinephrine and plasma corticosterone levels. Proc. 81st a Cony. Am. P~ychol. Ass. 1021--1022, 1973. 18. Zimmerman, E., and V. Critchlow. Effects ot diurnal variation m plasm~ corticosterone levels on adrenocortical response to stress. Pro('. Soc, exp. Biol. Med. 125:658 - 663. 1967