CRF receptor regulation and sensitization of ACTH responses to acute ether stress during chronic intermittent immobilization stress

Brain Research, 532 (1990) 34-40 Elsevier

34

BRES 15986

CRF receptor regulation and sensitization of ACTH responses to acute ether stress during chronic intermittent immobilization stress*'** Richard L. Hauger 1, Marge Lorang 1, Michael Irwin I and Greti Aguilera 2 1Veterans Administration Medical Center and Department of Psychiatry, University of California, San Diego, CA (U.S.A.) and eEndocrinology and Reproduction Research Branch, National Institutes of Health, Bethesda, MD (U.S.A.) (Accepted 8 May 1990)

Key words: Corticotropin releasing factor; Adrenocorticotropic hormone; Corticosteroid; Chronic stress

The relationship between corticotropin releasing factor (CRF) receptors and pituitary-adrenal responses was determined after chronic intermittent immobilization (2.5 h restraint/day) to examine the hypothesis that CRF receptor regulation is involved in the sensitization of the pituitary-adrenocortical axis to novel stimuli during repeated stress. Following the ll-fold stimulation of ACTH secretion on the first day of restraint stress, a desensitization of the pituitary ACTH response to immobilization was observed over the next 9 days of chronic intermittent stress. In contrast, the magnitude of the restraint-stimulated release of corticosterone on the 2nd and 4th day of stress was similar to the day 1 adrenocortical response. Furthermore, the significant stimulation of corticosterone secretion by restraint stress persisted to the 16th day of immobilization (P < 0.001), even though significant increases in plasma ACTH were absent. The concentration of anterior pituitary CRF receptors was unchanged after a single period of restraint; however, a down-regulation of anterior pituitary CRF receptors was observed following 4 days (P < 0.001) and 10 days (P < 0.005) of repeated immobilization stress. CRF receptors in the olfactory bulb were unchanged following acute or chronic restraint stress, consistent with previous observations that brain CRF receptors are neither changed by adrenalectomy, glucocorticoid administration, nor 18-48 h of continuous restraint stress. The concentration of CRF receptors in the intermediate lobe of the pituitary also was not influenced by immobilization stress. Despite the desensitization of the ACTH response during repeated immobilization, a highly significant potentiation (P < 0.001) of ACTH release following 5 min of ether vapor was observed 24 h following 9 or 15 days of repeated restraint stress. Consequently, the loss of anterior pituitary CRF receptors cannot account for the sensitization of the ACTH response to acute ether exposure following chronic immobilization stress. The data support the hypothesis that the increased pituitary corticotroph responses to novel stressful stimuli during chronic stress involves the 'facilitation' of stimulatory inputs to CRF-releasing neurons in the hypothalamus and the integrative actions of CRF and other ACTH regulators at the post-CRF receptor level.

INTRODUCTION Biologic stress elicits a biphasic response of the hypothalamic-pituitary-adrenal ( H P A ) axis 2"9"21-23'28-31. Acute stress increases the secretion of CRF, A C T H and other P O M C - d e r i v e d peptides, and adrenal corticosteroids. This large stimulation of H P A axis h o r m o n e release during the initial period of stress is usually transient followed by a decline in the concentration of circulating stress hormones to near basal levels during the adaptation phase of the response to continuous chronic stress. A similar desensitization of H P A axis responses is observed during intermittent exposure to chronic stress. The regulation of H P A axis h o r m o n e secretion during chronic stress could occur at the following levels of the brain-pituitary-adrenal system: (1) extrahypothalamic centers modulating PVN C R F neurons, (2) hypothalamic sites releasing C R F and other A C T H secretagogues, (3)

ACTH-secreting cells in the anterior pituitary, and (4) the adrenal cortex. It is well-known, however, that the magnitude of the acute stress-stimulated increases in H P A axis hormonal secretion and the temporal pattern of A C T H , fl-endorphin, and corticosteroid responses are influenced by the type of stressful stimulus and its intensity1'zl-24'31. For example, the acute hypersecretion of A C T H provoked by a single exposure of laboratory animals to ether, leg fracture, or tourniquet stress persists during continuous or repeated exposure to these stimuli in contrast to the loss of A C T H responses to chronic footshock or immobilization stress 5"14'17'21-23'28-31. H P A axis responses to novel stimuli can also vary with the type of prior chronic stress exposure. 'Systemic' forms of stress such as hypoglycemia can inhibit the release of A C T H provoked by a novel, superimposed stressor, while 'neurogenic' stress (e.g., skin incision, laparotomy, immobilization, forced swim)

* The work described in 'CRF receptor regulation and sensitization of ACTH responses to acute ether stress during chronic intermittent immobilization stress' was done as part of our employment with the federal government and is therefore in the public domain. ** Portions of these data were presented in abstract form at the American Federation for Clinical Research, San Diego, CA, April, 1987. Correspondence: R.L. Hauger, (present address:) Oregon Health Sciences University, 3181 S.W. Sam Talleron Park Road, Portland, OR 97201-3098, U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

35 can facilitate the A C T H r e s p o n s e to a s u b s e q u e n t stress 14"21-23. I n a d d i t i o n , n e u r o g e n i c stress causes a large s t i m u l a t i o n of A C T H s e c r e t i o n f r o m the n e u r o i n t e r m e diate l o b e w h i l e h u m o r a l stressors m a i n l y A C T H - r e l e a s i n g cells in the a n t e r i o r l o b e z2'23.

activate

W e h a v e p r e v i o u s l y d e m o n s t r a t e d that a large r e d u c tion in a n t e r i o r pituitary C R F r e c e p t o r s occurs in rats e x p o s e d to the s e v e r e and c o n t i n u o u s stress of 18-48 h of immobilization

TM.

In the p r e s e n t study, we h a v e e x a m -

ined the relationship between CRF receptor regulation and t h e h o r m o n a l r e s p o n s e s of the p i t u i t a r y - a d r e n a l axis d u r i n g r e p e a t e d restraint (2.5 h/day) which r e p r e s e n t s a stressor of m o d e r a t e intensity and b r i e f duration. T h e c h r o n i c i n t e r m i t t e n t i m m o b i l i z a t i o n stress p a r a d i g m allows for a d a p t a t i o n a l r e s p o n s e s of the H P A axis during the

period

each

day

when

the

animals

are

not

i m m o b i l i z e d 23. W e n o w r e p o r t that a d o w n - r e g u l a t i o n of C R F r e c e p t o r s in the a n t e r i o r pituitary, but not in the i n t e r m e d i a t e l o b e o r central n e r v o u s system, also occurs f o l l o w i n g 4 - 1 0 days of chronic i n t e r m i t t e n t i m m o b i l i z a tion stress and that the d i r e c t i o n of this C R F r e c e p t o r c h a n g e is consistent with the loss of the pituitary A C T H response

to restraint.

However,

a highly significant

p o t e n t i a t i o n o f t h e A C T H r e s p o n s e to acute e t h e r stress occurs f o l l o w i n g c h r o n i c i m m o b i l i z a t i o n stress, despite the d e c r e m e n t in C R F r e c e p t o r c o n c e n t r a t i o n , suggesting that the sensitization of the pituitary c o r t i c o t r o p h by c h r o n i c stress results f r o m a c o m p l e x i n t e r a c t i o n b e t w e e n post-CRF afferent

receptor pathways

mechanisms in the

brain

and

a facilitation of

which

stimulate

the

s e c r e t i o n of h y p o t h a l a m i c c o r t i c o t r o p i n releasing factor.

MATERIALS AND METHODS

Animals In all experiments adult male Sprague-Dawley rats weighing 300-350 g (Charles River, Boston, MA) were housed in groups of 3 animals per cage and maintained in a controlled temperature (24 °C) and light environment (12L;12D; lights on 06.00). The animals had free access to food pellets and water before and after being subjected to stress or while being housed in their home cage for 14 days before starting the stress experiments. Stress procedure After the initial period of environmental acclimation, groups of rats (n = 6-16/group) were randomly selected for the immobilization stress paradigms or served as non-restrained controls (e.g., left undisturbed in their home cages throughout the experiment). The body weight of animals was determined at the beginning of the experiment, before the first stress exposure, and 24 h prior to decapitation. Daily restraint stress consisted of exposing rats to 2.5 h/day of immobilization (from 08.00 to 10.30) for periods ranging from 1 to 16 days using a modification of the restraint method of Stone et ai. 32. Following each stress session, rats were returned to their home cage and were able to eat and drink ad libitum for the remainder of the day. CRF receptors and plasma hormone patterns were measured in chronically stressed rats immediately prior-to or following the last immobilization period on days 4, 10 or 16, and compared to animals acutely restrained for 2.5 h or controls. In

addition, the hormonal responses were measured in a subgroup of rats exposed to repeated immobilization for 3, 9, or 15 days and to 5 min of acute ether stress on the last stress day (days 4, 10, or 16). In experiments involving CRF binding in the intermediate lobe and brain, CRF receptors were also measured after 18-48 h of continuous immobilization as previously described TM.

Hormone analysis Immediately following the final stress period, all animals underwent decapitation in a separate room within 5 s of being removed from their home cage, released from 2.5 h of immobilization, or exposed to 5 min of ether vapors. Approximately 5 ml of truncal blood was collected in plastic conical centrifuge tubes containing 200 /~1 of a solution of 50 mg/ml EDTA and 500 klU of aprotinin (Sigma Co., St. Louis, MO). Plasma ACTH was measured by radioimmunoassay (RIA) in lyophilized eluates after extraction onto C18 Sep-Pak cartridges (Waters Associates, Inc., Milford, MA) and elution with 60% acetonitrile in triethylamine formate buffer, pH 3.2 (refs. 13, 14). Plasma corticosterone was measured by RIA as previously described 13A4'34. CRF receptor assay Pituitary glands were rapidly removed and separated into anterior and neural-intermediate lobes. Brains were promptly dissected on ice and brain regions collected in ice-cold PBS for measurement of CRF binding. CRF receptors were measured in brain and pituitary membrane-rich particles prepared as previously described 13"14'36. The CRF receptor assay consisted of incubating in 1.5-ml polystyrene microfuge tubes 100-/A aliquots of the membrane suspension with 100000 cpm (0.1 nM) of [125I]Tyr-ovine CRF (oCRF) (Dupont-NEN, Boston, MA) in a total volume of 300/~1 of buffer 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCI2, 2 mM EGTA, 0.1% BSA, 100 kIU/ml of aprotinin, and 1 mM DTT. Non-specific binding was measured as CRF binding in the presence o f 10 -6 M oCRE After incubation for 60 rain at 22 °C, tubes were centrifuged twice at 10,000 g for 3 min following the addition of 1 ml of 7.5% polyethylene glycol in 50 mM Tris-HCl, pH 7.4. The resultant pellets were analyzed for bound radioactivity in a gamma-spectrometer. Protein concentrations were measured by the Pierce BCA assay. Statistical analysis Calculation of receptor affinities and concentrations were performed by computer analysis of the equilibrium binding data using the non-linear least squares curve-fitting program LIGAND 25. Hormonal and binding data are presented as arithmetic mean _+ S.E.M. Statistical evaluations were made with paired and unpaired Student's t-test and with one-way analyses of variance (ANOVA) using Student-Newman-Keuls multiple-range tests to compare individual groups.

RESULTS

Pituitary-adrenocortical responses to chronic intermittent immobilization stress Fig. 1 depicts H P A axis h o r m o n a l r e s p o n s e s to r e p e a t e d r e s t r a i n t and a c u t e e t h e r stress. I m m o b i l i z a t i o n stress for 2.5 h on day 1 r e s u l t e d in l l - f o l d increases in the p l a s m a c o n c e n t r a t i o n s o f A C T H and c o r t i c o s t e r o n e which w e r e highly significant ( P < 0.001) c o m p a r e d to basal h o r m o n e levels in n o n - s t r e s s e d controls. H o w e v e r , t h e r e was a dissociation b e t w e e n the s e c r e t o r y r e s p o n s e s of the p i t u i t a r y c o r t i c o t r o p h and t h e a d r e n a l c o r t e x during c h r o n i c i n t e r m i t t e n t r e s t r a i n t stress. T h e i n c r e a s e in p l a s m a A C T H levels after i m m o b i l i z a t i o n o n day 4 was

36 significantly smaller than the A C T H response on day 1 (P < 0.001). Significant decreases in the A C T H responses to restraint occurred on days 10 ( P < 0.001) and 16 ( P < 0.05) c o m p a r e d to the day 4 A C T H response. Plasma A C T H levels on the 10th and 16th days of immobilization stress were not significantly different ( P > 0.05 by N e w m a n - K e u l s a posteriori test) from A C T H concentrations in non-stressed controls due to this progressive reduction in restraint-stimulated A C T H secretion. These d a t a suggest that a desensitization occurs in the plasma A C T H responses to restraint during chronic intermittent immobilization stress. In contrast to the p a t t e r n for A C T H responses, there were no statistically significant changes in the plasma

A.

800 •

[] []

Basalipre-Rastraint) RestraintStress(2,5h/day) AcuteEtherStress(5 rain)

a,b ,,

600

N ~

.~

400

a ~200

Day 1

B.

30

Day 4

Day 10

Day 16

e e

== 20 2m

•

Basal ~re-Restralnt)

i

RestraintStress(2.5 h/day) AcuteEtherStress(5 rain)

e, b

8

~ ~

"1~

~-

Day 1

Day 4

,

Day 10

,

Day 16

Fig. 1. Plasma concentrations of ACTH and corticosterone following chronic intermittent immobilization stress and/or acute ether stress. Values are the mean + S.E.M. of data obtained in 5-60 rats per time point. In a previous publication, a small subset of the plasma ACTH values after repeated restraint (but not pre-restraint or ether stress ACTH levels) have been correlated to splenic natural killer (NK) cytotoxicity in the same immobilized animals 17. The adaptation of pituitary ACTH responses to chronic restraint stress occurs earlier than the adaptational changes in splenic NK activity 17. A: ACTH values across the treatment groups were significantly different (F = 80.1, df 11,221, P < 0.001 by one-way ANOVA); B: corticosterone values across the treatment groups were significantly different (F = 41.0, df 11,228, P < 0.001 by one-way ANOVA). When the unpaired Student t-test or Newman-Keuls a posteriori test were used to compare individual groups, statistically significant differences were as follows: ap < 0.001 vs. non-stressed control; bp < 0.001 vs. day 1 restraint; cp < 0.05 vs. non-stressed control.

corticosterone responses to restraint on day 2 (21.1 + 2.3 /~g/dl) and day 4 (Fig. 1). M o r e o v e r , p l a s m a corticosterone responses on day 10 and d a y 16 continued to be significantly greater than basal levels in non-stressed cont-rols ( P < 0.001), although the magnitudes of the restraint-stimulated corticosterone responses were statistically lower than the responses on day 4 ( P < 0.001). This persistence of adrenocortical responses to chronic intermittent immobilization was associated with significant increases ( P < 0.05) in a d r e n a l weight on days 4, 10, and 16 of r e p e a t e d restraint stress (Table I).

Effect of chronic intermittent immobilization stress on pituitary-adrenocortical responses to acute ether stress The secretory responses of the H P A axis to 5 min of ether vapor after various periods of chronic intermittent immobilization is depicted in Fig. 1. A l t h o u g h the magnitude of the plasma A C T H response to acute ether stress was similar to that for acute restraint on day 1, a progressive sensitization of the e t h e r response was observed during chronic intermittent immobilization. A highly significant potentiation ( P < 0.001) of the A C T H response to e t h e r was m e a s u r e d on day 10 and day 16 of r e p e a t e d restraint stress c o m p a r e d to the day 1 A C T H response to e t h e r in non-restrained controls (Fig. 1). In contrast, a decline in the ether-stimulated increases in plasma corticosterone levels resulted after exposure to chronic immobilization, which was not statistically significant. T h e r e f o r e , no sensitization of adrenocortical responses to acute ether stress was o b s e r v e d following r e p e a t e d restraint stress (Fig. 1). The ratio of plasma A C T H : c o r t i c o s t e r o n e responses to ether in rats immobilized for 9 or 15 days is significantly increased ( P < 0.05). Since these changes in the A C T H : c o r t i c o s t e r o n e ratio indicate that the secretion of adrenal corticosteroids in response to the very large ether-induced increases in plasma A C T H levels is r e d u c e d on days 10 and 16, the adrenal cortex appears to be desensitized to A C T H (Table I). H o w e v e r , the significant decreases in the ratio of A C T H : c o r t i c o s t e r o n e responses to r e p e a t e d restraint stress alone on those days provides evidence that very low levels of A C T H can stimulate substantial amounts of corticosterone release (Table I). Therefore, adrenocortical responsivity to A C T H r e l e a s e d by immobilization vs. e t h e r is differentially regulated.

CRF receptor regulation during chronic immobilization stress H a b i t u a t i o n of rats to handling has been r e p o r t e d to lower p l a s m a corticosterone concentrations in truncal blood samples 35. H o w e v e r , in two e x p e r i m e n t s the C R F receptor content in the anterior pituitary (440 + 53 fmol/mg) of rats h a b i t u a t e d to 2 min of daily handling

37 TABLE I The effect o f chronic intermittent immobilization stress on body weight and adrenal weight, and A CTH: corticosterone response ratios

Values are the mean + S.E.M. Days o f immobilization stress 0

ANOVA

1

4

10

16

Final body weight (g)

317 + 9

305 + 5

279 + 4*

271 + 3*

254 + 3*

F = 1 9 . 7 ; d f = 4 , 5 2 ; P
Adrenal weight (mg/100 g b.wt.)

15.3 + 0.6

16.6 + 0.5

18.4 + 0.6*

18.9 + 0.7*

19.6 + 0.6*

F = 7.1;df=4,54;P
Plasma ACTH: corticosterone response to restraint ~

-

9.33 + 1.76

5.4 + 0.6*

3.74 + 0.48*

5.09 + 1.09

F = 6.8; d f = 3,82; P < 0.001

Plasma corticosterone response to ether a

-

12.37 + 1.38

14.48 + 5.31

76.7 + 14.6"

105 + 32*

F = 6.5; d f = 3,19; P < O.O05

* P < 0.05 vs. control (no restraint stress) or day I immobilization group using the Newman-Keuls a posteriori test. a The ACTH:corticosterone response ratios were calculated by dividing the plasma ACTH level (pg/ml) by the plasma corticosterone value ~g/dl) in the individual animal immediately following the last restraint exposure (n = 8-33 rats/group) or after acute ether challenge (n = 6 rats/group).

o u t s i d e of their h o m e cage for 10 days was similar to Bma x v a l u e s in n o n - h a n d l e d controls (Table II). Basal levels of

+ 5 . 3 % on day 10 ( P < 0.005) c o m p a r e d to Bma x v a l u e s in n o n - s t r e s s e d c o n t r o l s (Table II and Fig. 2). T h e r e w e r e

p l a s m a A C T H (23.2 + 4.6 pg/ml) and c o r t i c o s t e r o n e (2.2

no statistically significant c h a n g e s in t h e b i n d i n g affinity

_+ 0.7/~g/dl) in h a n d l e d rats (n = 13) w e r e also similar to

(Ko) for a n t e r i o r p i t u i t a r y C R F r e c e p t o r s f o l l o w i n g single

A C T H (19.9 ___ 2.5 pg/ml) and c o r t i c o s t e r o n e values (3.1

o r r e p e a t e d i m m o b i l i z a t i o n stress c o m p a r e d to Ko v a l u e s

_+ 0.9/~g/dl) in a n i m a l s (n = 11) n o t e x p o s e d to handling.

in controls.

Since daily h a n d l i n g of n o n - s t r e s s e d animals p r i o r to

o b s e r v a t i o n s ) , we h a v e also o b s e r v e d a 21.6 + 4 . 0 %

killing did not consistently alter the basal state of the

d e c r e a s e in t h e Bma x for C R F r e c e p t o r s in t h e a n t e r i o r

pituitary-adrenal

pituitary, w i t h o u t any K d c h a n g e s , d u r i n g t h e a d a p t a t i o -

axis,

handling

habituation

was

not

p e r f o r m e d in o u r e x p e r i m e n t s . In contrast corticosterone

there

experiments

(unpublished

nal r e d u c t i o n in p i t u i t a r y - a d r e n a l h o r m o n e r e s p o n s e s to

to the acute activation o f A C T H secretion,

In p r e l i m i n a r y

were

no

and

c h r o n i c a u d i o g e n i c stress (1 h o f 108 d B n o i s e e x p o s u r e

significant

daily for 4 o r 10 days18). T h i s finding p r o v i d e s f u r t h e r

c h a n g e s in C R F b i n d i n g to the a n t e r i o r pituitary after 2.5 h of a c u t e r e s t r a i n t stress on day 1 (ref. 14). H o w e v e r , r e p e a t e d i m m o b i l i z a t i o n significantly d e c r e a s e d t h e concentration

(Bmax) of C R F

receptors

in the

o.15

anterior

p i t u i t a r y by 34.0 + 5 . 9 % on day 4 ( P < 0.001) and 26.0 0.10 TABLE II The effect o f chronic intermittent immobilization stress on anterior pituitary CRF receptors

z

O 005

Values are the mean + S.E.M. Days o f immobilization stress

0 4 10

K,~ (nM)

1.64 + 0.15 1.30 + 0.21 1.75+0.17

CRF receptor concentration (fmol/mg)

490 + 16 (n = 11) 334 + 25** (n = 6) 364+26" ( n = 7 )

A significant difference in the CRF receptor concentration (Bmax) in the anterior pituitary was determined to be present across the treatment groups using one-way ANOVA IF = 9.8224; d f = 3,24; P < 0.001]. When the unpaired Student t-test were used to compare individual group differences, statistically significant differences were as follows: *P < 0.005 vs. control, **P < 0.001 vs. control.

I

~

I00

200

~00

400

500

BOUND 1::'51-TYR-oCRF(fmol/mg) Fig. 2. Scatchard analysis of the binding of [laSI]Tyr-oCRF to the anterior pituitary from control (0) and rats exposed to 10 days of daily immobilization stress (O). Data points are the mean of duplicate binding determinations for the pool of 8 pituitaries from each group. The Bmax decreased from the control concentration of 535 to 375 fmol/mg following chronic restraint stress without any changes in the binding affinity (K d values: 1.91 nM control vs. 1.60 nM stress).

38 receptor content of the olfactory bulb was unchanged after 18 h of continuous immobilization14. C R F binding

~m~ 500~ i~ ii

~,_~

was also unchanged following 18 h of continuous restraint stress in the amygdala (96.1 _ 3.8% of control, n = 5) and after 18 or 48 h of immobilization in the frontal cortex

400 I 3°°

(103.0 --- 12.8 and 106.5 + 7.7% of control, n = 5).

--' ~ 2°0

DISCUSSION

tOO E m 0

Anterior Lobe Pituitary

Intermediate Lobe Pituitary

Olfactory Bulb

Fig. 3. Effect of chronic immobilization stress on the concentration of CRF receptors. The B.... values are the mean + S.E.M. of duplicate binding determinations in pooled tissue for the anterior pituitary (n = 6-8), intermediate pituitary (n = 12), and brain (n = 3-4) of restrained or non-stressed rats obtained in separate experiments (n = 2-6) by Scatchard analysis. The concentration of anterior pituitary CRF receptors was found to be significantly different using one-way ANOVA across the treatment groups IF = 22.7; d f = 2,16; P < 0.001]. The anterior pituitary Bmax for CRF receptors after 48 h restraint was significantly lower than the decrease in receptors on day 10 (P < 0.05 by the Newman-Keuls a posteriori test). The B m a x values for the anterior pituitary following 48 h of continuous restraint have been previously reported and are included here for comparision to CRF binding values in intermediate lobe tissue measured in the same animals~4. No changes in K a values for CRF binding to the anterior pituitary were observed (controls: 2.02 + 0.17 nM, 10 days restraint: 1.91 + 0.22 nM, 48 h restraint: 2.59 + 0.51 nM). Chronic restraint did not cause significant differences (P > 0.05) in B m a x and binding affinity (Ko) values for CRF receptors in the intermediate lobe [Ka values (nM): control = 3.45 + 0.58, 10 days restraint = 3.06 + 0.02, 48 h restraint = 2.93 + 0.72] and olfactory bulb [Ka (nM) values: control = 4.21 + 0.35, 4 days restraint = 4.68 + 1.04, 10 days restraint = 4.25 + 0.32]. *P < 0.01 vs. control, **P < 0.001 vs. control (by unpaired Student's t-test).

evidence that chronic stress of moderate intensity can cause C R F receptor down-regulation. The effect of continuous immobilization (18-48 h) or repeated, intermittent restraint stress (2.5 h/day) on C R F receptors in the intermediate lobe of the pituitary gland is depicted in Fig. 3. In contrast to the down-regulation of anterior pituitary C R F receptors by 48 h of continuous immobilization (48 + 2% decrease, n = 5) or 10 days of repeated restraint (19 + 3% decrease, n = 5), no changes were detected in the C R F receptor content of the intermediate lobe in the same animals subjected to chronic stress. Acute restraint stress (2.5 h) or 18 h of immobilization also did not alter C R F binding to the intermediate lobe data not shown. C R F binding in brain tissue was measured during intermittent or continuous restraint stress (Fig. 3). The concentrations of C R F receptors in the olfactory bulb of rats exposed to 1 (data not shown), 4, or 10 days of intermittent restraint stress were not statistically different from Bmax values in non-restrained controls (Fig. 3), consistent with our previous finding that the C R F

We have demonstrated that 4-10 days of repeated restraint results in a significant loss of C R F receptor sites in the anterior pituitary. The C R F receptor loss in the anterior pituitary following chronic stress was not due to occupancy by endogenous C R F as shown by the lack of effect of acute stress on C R F receptors when endogenous C R F release from the hypothalamus is high. Also, only a slight decrease in pituitary C R F receptors is observed in Brattleboro rats after adrenalectomy, despite the presence of C R F hypersecretion suggesting that occupancy does not interfere with our receptor measurements 15. Although no changes were observed in C R F receptors in the intermediate lobe of the pituitary during chronic continuous or i n t e r m i t t e n t immobilization stress, we have observed that the exogenous administration of high corticosterone doses or chronic cold stress increases C R F binding to intermediate pituitary tissue 11-13. Similar to previous findings after adrenalectomy, glucocorticoid administration, or continuous immobilization stress, C R F receptors in the brain were unchanged by 4 or 10 days of daily restraint stress suggesting that the cerebral responses to stress are not associated with the regulation of extrahypothalamic C R F receptor sites 8' 11,13.14.36. In dissociated fetal brain cell cultures, 3 days of exposure to very high C R F concentrations (10 -6 M oCRF) in vitro results in a 36% decrease in the C R F receptor concentration of extrahypothalamic cells2°. Significant reductions in C R F receptors in the brain have also been observed in vivo after extreme conditions such as the intracisternal administration of very high doses of C R F or 3-14 days of severe footshock stress 12'19. U n d e r similarly severe chronic stress conditions, the content of immunoreactive C R F in the locus coeruleus increases and C R F m R N A levels in the olfactory bulb decrease, which suggests that extreme stress can change local C R F release in extrahypothalamic sites of the brain 4'16. However, other stressors such as acute insulin-induced hypoglycemia do not change C R F m R N A levels in the cerebral cortex, while increasing hypothalamic C R F m R N A levels to 186% of control values 33. At present, there are no data available on extrahypothalamic C R F synthesis, release, and turnover responses during chronic exposure to mild stress conditions. Therefore, it is not clear whether the inability of chronic intermittent restraint stress to alter

39 brain C R F receptors in the present experiments is due to little change in r e c e p t o r exposure to C R F at these CNS sites. Alternatively, the ligand/receptor mechanisms in the brain may be different from those in the pituitary whereby r e c e p t o r occupancy results in m a r k e d regulatory changes in C R F r e c e p t o r content. Consistent with previous observations in different stress models, there was a transient hypersecretion of A C T H followed by a loss of the A C T H response during chronic immobilization stress. This desensitization of the A C T H response to stress occurs over 3 - 6 h if the stress exposure is continuous or over several days when the stress exposure is intermittent and involves a long stress-free p e r i o d each day as in the present study. A l t h o u g h the reduction in anterior pituitary C R F receptors was t e m p o r a l l y related to the desensitization of the A C T H response to chronic restraint stress, a hyperresponsiveness of A C T H release to acute ether exposure was observed in rats subjected to 9-15 days of immobilization. Therefore, C R F r e c e p t o r down-regulation and the loss of A C T H responsiveness to a chronic stressor do not represent secretory exhaustion of the pituitary corticotroph. F u r t h e r m o r e , since chronic restraint stress augments acute ether-stimulated ACTI-I secretion, stress adaptation must involve the enhancement of CNS inputs to the h y p o t h a l a m u s which stimulate the secretion of corticotropin releasing factors. The present findings with r e p e a t e d restraint and our recent study of 18-48 h of continuous immobilization indicate that C R F r e c e p t o r regulation is not the p r i m a r y site for the control of corticotroph function during stress. H o w e v e r , we have observed that vasopressin m a r k e d l y potentiates in vitro cyclic A M P and A C T H responses to C R F in cultured pituitary cells from rats immobilized for 48 h despite the loss of C R F receptors 14. This finding and the recent observation that 6 h of daily immobilization for 5 weeks m a r k e d l y enhances the A C T H responses to an arginine vasopressin injection suggest that vasopressin m a y be an i m p o r t a n t regulator of pituitary responsiveness to novel stimuli during chronic stress ~°'15. Finally, stress adaptation at the adrenal level is characterized by persistent increases in plasma concenREFERENCES 1 Armario, A., Hidalgo, J. and Giratt, M., Evidence that the pituitary-adrenal axis does not cross-adopt to stressors: comparison to other physiological variables, Neuroendocrinology, 47 (1988) 263-267. 2 Axelrod, J. and Reisine, T.D., Stress hormones: their interaction and regulation, Science, 224 (1984) 452. 3 Burchfield, S.R., Woods, S.C. and Elich, M.S., Pituitary adrenocortical response to chronic intermittent stress, Physiol. Behav., 24 (1980) 297-302. 4 Chappel, P.B., Smith, M.A., Kilts, C.D., Bissette, G., Ritchie, J., Anderson, C. and Nemeroff, C.B., Alterations in cortico-

trations of corticosterone despite the decrease in A C T H release on days 10 and 16 of r e p e a t e d immobilization. A l t h o u g h these sustained high levels of circulating glucocorticoids could inhibit via negative f e e d b a c k the A C T H response to r e p e a t e d restraint, A C T H release becomes desensitized during chronic stress in adrenalectomized rats 6"21'29. The progressive reduction in the A C T H : c o r t i c o s t e r o n e response ratio to daily immobilization indicates that the corticosterone response to stress-stimulated A C T H release has increased consistent with the o b s e r v e d adrenal h y p e r t r o p h y . H o w e v e r , during acute ether challenge an increased adrenocortical sensitivity to A C T H was not o b s e r v e d in rats e x p o s e d to r e p e a t e d immobilization. This finding is consistent with other studies where a significant reduction in the corticosterone response to e t h e r stress was m e a s u r e d after several weeks of chronic daily restraint or crowding stress, despite the ether-stimulated secretion of A C T H being similar in stressed rats and controls 7"22. Consequently, the sensitivity of the adrenal cortex to A C T H during chronic stress m a y be m o d u l a t e d by h u m o r a l factors differentially released in immobilization vs. ether stress. Since no significant increases in the pre-restraint secretion of corticosterone were o b s e r v e d before the next exposure to immobilization stress on days 4, 10, and 16, we did not find any evidence that a d a p t a t i o n a l responses of the H P A axis to chronic intermittent stress involve 'a conditioned endocrine r e s p o n s e ' (ref. 3). In conclusion, the down-regulation of C R F receptors does not a p p e a r to be a generalized mechanism for the adaptational sensitization of the H P A axis in chronically stressed rats. T h e r e f o r e , the m o d u l a t o r y effects of vasopressin and o t h e r regulators of C R F action at p o s t - C R F receptor sites m a y contribute to the hyper-responsiveness of A C T H responses to acute e t h e r stress after chronic restraint stress. Acknowledgements. The authors wish to thank Maria Bongiovanni for her excellent typing of the manuscript. This work was supported by VA Merit Review and VA Research Associate grants to R. Hauger who is also a recipient of Pfizer Scholars and VA Clinical Investigator Awards; and NIMH Grants MH44275-01, MH30914 and VA Merit Review to M. Irwin.

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