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Corticotronpin-releasing hormone inhibits maternal behavior and induces pup-killing
Life Sciences, Vol. 48, pp. 1537-1546 Printed in the U.S.A.
C O R T I C O T R O P I N - R E L E A S I N G HORMONE INHIBITS M A T E R N A L INDUCES P U P - K I L L I N G
Pergamon Press
BEHAVIOR
AND
Cort A. Pedersen, Jack D. Caldwell, M a r s d e n M c G u i r e and Dwight L. Evans Dept. Psychiatry, Brain and D e v e l o p m e n t R e s e a r c h Center, and the N e u r o b i o l o g y Curriculum, Univ. of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7160 (Received in final form February ii, 1991) Summary Behavioral responses to stressors and the effects of stressors on maternal behavior change with m o t h e r i n g experience. C o r t i c o t r o p i n - r e l e a s i n g h o r m o n e (CRH) is r e l e a s e d by stressors and p r o d u c e s stresslike b e h a v i o r a l effects. We tested the effects of ICV infusion of ovine CRH (0.5-4 ug) on p u p - d i r e c t e d b e h a v i o r s in ovariectomized, ovarian s t e r o i d - t r e a t e d v i r g i n rats that were either naive to pups or that had three days of m o t h e r i n g experience. CRH inhibited m a t e r n a l b e h a v i o r in naive and e x p e r i e n c e d rats in a d o s e - r e l a t e d manner. The m a g n i t u d e and d u r a t i o n of inhibition, e s p e c i a l l y at the 1 ug dose, were less in rats with m o t h e r i n g experience. H i g h e r doses of CRH (i - 4 ug) s i g n i f i c a n t l y increased p u p - k i l l i n g in rats that were naive to pups. In contrast, CRH p r o d u c e d no p u p - k i l l i n g in rats with m o t h e r i n g experience. The p r i m a r y c o r t i c o t r o p i n - r e l e a s i n g h o r m o n e (CRH) is a 41 amino acid p e p t i d e o r i g i n a l l y sequenced by Vale et al (i). In response to stressors (2) CRH is secreted into the h y p o p h y s i a l portal v a s c u l a t u r e and stimulates ACTH and, indirectly, adrenal g l u c o c o r t i c o i d release. Pathways c o n t a i n i n g CRH are w i d e l y d i s t r i b u t e d w i t h i n the brain (3,4) as are CRH b i n d i n g sites (5). Intracerebral infusion of CRH produces behavioral changes similar to those seen when animals are stressed (6-14). Additionally, CRH blocks other behaviors, such as c o p u l a t i o n and eating, that are inhibited by stress (15-17). Central administration of a CRH antagonist blocks stress-induced b e h a v i o r a l changes in mice and rats (18-20) s u g g e s t i n g that CRH e n d o g e n o u s to the brain plays a critical role in b e h a v i o r a l r e s p o n s e s to stress. N e w b o r n pups, like other novel stimuli, are a v e r s i v e for v i r g i n female rats (21,22). Behavioral responses t o w a r d young pups change rapidly at the end of p r e g n a n c y from active avoidance or even infanticide to avid mothering (23). Behavioral responses to stressors also shift with the onset of maternal behavior and nursing. These include decreased "emotionality" in a novel open field, d e c r e a s e d startle response and i n c r e a s e d a g g r e s s i o n against intruders (22,24). The effect of a v e r s i v e stimuli on the d i s p l a y of maternal b e h a v i o r m a y also change w i t h m o t h e r i n g experience. The stress of a novel cage 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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s i g n i f i c a n t l y delays the initial onset of maternal b e h a v i o r in rats naive to pups but does not affect the rapid r e e m e r g e n c e of m a t e r n a l b e h a v i o r e x h i b i t e d by e x p e r i e n c e d rat m o t h e r s (25). This o b s e r v a t i o n suggests that n e u r o e n d o c r i n e c o m p o n e n t s of the stress r e s p o n s e may be more d i s r u p t i v e of the initial onset c o m p a r e d to the m a i n t e n a n c e of e s t a b l i s h e d maternal behavior. We have, therefore, tested the effects of i n t r a c e r e b r o v e n t r i c u l a r (ICV) a d m i n i s t r a t i o n of CRH on the onset of maternal b e h a v i o r in naive female rats and on the r e e m e r g e n c e of m o t h e r i n g b e h a v i o r in m a t e r n a l l y e x p e r i e n c e d rats. Methods 17 B - e s t r a d i o l (E2) and p r o g e s t e r o n e (P) (Steraloids) were a d m i n i s t e r e d in capsules made from silastic medical grade tubing (Dow C o r n i n g #602-235). Capsules were made by filling the a p p r o p r i a t e length of silastic tubing with the d e s i r e d amount of E 2 or P, p l u g g i n g the ends of the tubing with pieces of w o o d e n d o w l i n g (5mm) and sealing the ends with silastic 382 medical grade e l a s t o m e r (Dow C o m i n g ) . Capsules were first immersed in pure ethanol for one hr and then in p h o s p h a t e b u f f e r e d saline for 48 hrs p r i o r to SC implantation. A n i m a l s were a n e s t h e t i z e d with ether during i m p l a n t a t i o n and removal of silastic capsules. N u l l i p a r o u s Sprague Dawley rats (zivic M i l l e r Laboratories, 200-250 gm) were o v a r i e c t o m i z e d (OVXed) under ether anesthesia. A f t e r OVX, animals were housed in individual cages on a 12 hr l i g h t / d a r k cycle with free access to food and water. Eight days after OVX one silastic capsule, 1 cm in length and c o n t a i n i n g 8.8 mg of E2, was implanted SC on the right flank of each animal. Ten days after OVX, an additional silastic capsule, 90 mm in length containg 120 mg of P, was implanted SC b e t w e e n the scapulae of each animal. Ovine CRH was obtained from P e n n i n s u l a Laboratories, Inc. (Lot # 007290). CRH was d i s s o l v e d in normal saline (NS) v e h i c l e (pH 5.0) at various c o n c e n t r a t i o n s so that each ICV dose was a d m i n i s t e r e d in a i0 ul volume. F o r t y - e i g h t hrs prior to behavioral testing each animal was p l a c e d in a clear p l e x i g l a s o b s e r v a t i o n cage (44 cm x 21 cm x 20 cm) c o v e r e d by a metal wire top with recesses to hold food p e l l e t s and a w a t e r bottle. Wood chips to a depth of 2-3 cms covered the floor of each o b s e r v a t i o n cage. Twenty pieces of paper t o w e l i n g (4 cm x 6 cm) were placed in each cage to serve as n e s t i n g material. Food and w a t e r were available ad libitum. The l i g h t / d a r k cycle remained the same. Twenty-four hrs prior to behavioral testing silastic implants c o n t a i n i n g P were removed from each animal. One hr prior to b e h a v i o r a l testing, each animal received ICV either one of four doses of CRH (0.5, 1.0, 2.0, or 4.0 ug) or NS v e h i c l e alone; other animals received no ICV treatment. Infusion into the right lateral cerebral v e n t r i c l e was a c h i e v e d by the m e t h o d of Popick (26) while animals were under light ether anesthesia. This m e t h o d results in greater than ninety percent accurate ICV a d m i n i s t r a t i o n as d e t e r m i n e d by e x a m i n a t i o n of the d i s t r i b u t i o n of dye w i t h i n the brain after injection of a c o n c e n t r a t e d s o l u t i o n of Evan's blue dye. were
One hr after ICV infusion three rat pups (1-5 placed 20 cms apart at the apices of an
days old) imaginary
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e q u i l a t e r a l t r i a n g l e c e n t e r e d in the m i d d l e of the o b s e r v a t i o n cage of e a c h animal (the s t a r t i n g position). D u r i n g the initial six hrs of pup c o n t a c t an o b s e r v e r ignorant of ICV t r e a t m e n t s looked for and r e c o r d e d the following c a t e g o r i e s of m a t e r n a l b e h a v i o r d i s p l a y e d by the test animals; initial g r o u p i n g of pups, l i c k i n g of pups, crouching in a n u r s i n g p o s i t i o n over g r o u p e d pups and retrieval of s e p a r a t e d pups. The o b s e r v e r r e t u r n e d the pups to the s t a r t i n g p o s i t i o n at the b e g i n n i n g of the third, fourth, fifth and sixth hr of pup c o n t a c t and b e g a n a new p e r i o d of b e h a v i o r a l o b s e r v a t i o n for each of t h o s e hrs. For each of the o b s e r v a t i o n p e r i o d s (the first two hrs, as well as the third, fourth, fifth and sixth hrs of pup contact) each animal was a s s i g n e d a m a t e r n a l b e h a v i o r a l score b a s e d on one point for each m a t e r n a l b e h a v i o r c a t e g o r y in w h i c h the animal met or exceeded quantitative screening criteria for that behavior. A n i m a l s w e r e c o n s i d e r e d to be fully m a t e r n a l as of the p e r i o d d u r i n g w h i c h they first met or e x c e e d e d s c r e e n i n g c r i t e r i a for all c a t e g o r i e s of m a t e r n a l behavior. Each animal was required to meet the following criteria during an o b s e r v a t i o n p e r i o d to be a s s e s s e d as h a v i n g d i s p l a y e d each of these behaviors. Grouping: The animal carries in its m o u t h all three pups from t h e i r initial s c a t t e r e d p o s i t i o n and clumps t h e m t o g e t h e r in a c o r n e r of the o b s e r v a t i o n cage. It also d i s p l a y s r e g r o u p i n g of pups, i n v o l v i n g r e a r r a n g e m e n t of at least two pups, w i t h i n this area. Licking: The animal licks one or m o r e pups of ten sec or d i s p l a y s at least three b r i e f
for at least a total licks.
Crouching: The animal must adopt a c r o u c h i n g p o s i t i o n for at least a total of 60 sec. All pups m u s t be g r o u p e d b e f o r e c r o u c h i n g can be recorded. The animal m u s t have its h i n d legs spread, its b a c k arched, and its v e n t r u m high e n o u g h off the cage floor to a c c o m m o d a t e pups b e n e a t h it. T h e r e m u s t be at least one pup b e n e a t h it during a crouch. Bouts of two sec or less are not added to the c u m u l a t i v e time. Retrieval: A n y time w i t h i n the p e r i o d a f t e r g r o u p i n g has taken place, all three pups are separated by the observer to a d i s t a n c e of 15-20 cm from the female. Retrieval constitutes c a r r y i n g all pups b a c k to the nest w i t h i n 15 min of separation. R e t r i e v a l m u s t be observed, rather than inferred, since pups may r e t u r n by t h e i r own efforts. Animals that had not met criteria for full maternal b e h a v i o r (FMB) d u r i n g any of the o b s e r v a t i o n p e r i o d s d u r i n g the initial six hr of pup contact were left w i t h pups overnight. Every 24 hr after the initiation of pup contact, t h r e e fresh pups (1-5 days of age) were s u b s t i t u t e d for the pups g i v e n the day before and placed in the starting position in each o b s e r v a t i o n cage. M a t e r n a l b e h a v i o r was o b s e r v e d and r e c o r d e d d u r i n g a one hr p e r i o d after each of these pup exchanges. Each animal was t e s t e d until it met or e x c e e d e d c r i t e r i a for FMB in a one hr o b s e r v a t i o n p e r i o d or until it k i l l e d one of the pups at w h i c h time the r e m a i n i n g pups w e r e immediately removed and b e h a v i o r a l o b s e r v a t i o n s stopped. A n i m a l s that k i l l e d pups w e r e g i v e n a m a t e r n a l b e h a v i o r a l score of "kill".
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F o r t h e p u r p o s e of t e s t i n g l e v e l s of r e e m e r g e n t m a t e r n a l behavior, rats that received no I C V t r e a t m e n t and that met criteria f o r F M B in t h e f i r s t s i x h r s of p u p c o n t a c t were a l l o w e d to s t a y w i t h y o u n g pups. F r e s h 1-5 d a y o l d r a t p u p s w e r e e x c h a n g e d for u s e d p u p s e v e r y 24 hr. A f t e r 72 h r of c o n t i n u o u s e x p o s u r e to p u p s all p u p s w e r e r e m o v e d a n d e a c h a n i m a l r e c e i v e d an I C V i n f u s i o n of C R H (i or 2 ug) or NS v e h i c l e alone. One hr l a t e r t h r e e f r e s h 1-5 d a y o l d rat p u p s w e r e i n t r o d u c e d . An observer ignorant of ICV treatments then recorded maternal b e h a v i o r a l r e s p o n s e s o v e r t h e f i r s t s i x a n d t h e 2 5 t h h r of ree s t a b l i s h e d p u p c o n t a c t as d e s c r i b e d above.
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CRH ()ug ICV) FIG. 1 The cumulative i n c i d e n c e of full m a t e r n a l b e h a v i o r (FMB) in o v a r i a n s t e r o i d - t r e a t e d v i r g i n f e m a l e r a t s at 2, 6 a n d 25 h r s a f t e r I C V i n f u s i o n of C R H (0.5, I, 2 or 4 ug) or n o r m a l saline (NS) v e h i c l e alone. * = p<.05, ** = p<.01, *** <.001, C R H v e r s u s NS ( F i s h e r ' s e x a c t p r o b a b i l i t y test).
Results T h e e f f e c t s of I C V i n f u s i o n of C R H on t h e o n s e t of m a t e r n a l behavior are summarized in F i g u r e i. The ovarian steroid treatment used produced a high incidence of F M B (27/32) in c o n t r o l r a t s r e c e i v i n g NS. In t h e f i r s t t w o h r of p u p contact, t h e i n c i d e n c e of F M B w a s s i g n i f i c a n t l y l o w e r in r a t s r e c e i v i n g I, 2 or 4 u g C R H c o m p a r e d to r a t s r e c e i v i n g NS. T h e s e r e s u l t s s u g g e s t t h a t t h e r e m a y be a l i n e a r r e l a t i o n s h i p b e t w e e n d o s e of C R H a n d i n c i d e n c e of F M B o v e r t h i s t i m e period. A t t h e e n d of
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CRH and Maternal Behavior
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t h e s i x t h h r of p u p c o n t a c t , a n i m a l s r e c e i v i n g I, 2 or 4 u g of CRH still had significantly lower cumulative i n c i d e n c e s of F M B t h a n a n i m a l s r e c e i v i n g NS. By t h e 2 5 t h h r of p u p c o n t a c t , t h e r e were no s i g n i f i c a n t differences among treatment groups. The distribution of m a x i m u m m a t e r n a l b e h a v i o r a l s c o r e s (kill, 0, i, 2, 3, 4) a c h i e v e d b y i n d i v i d u a l a n i m a l s w i t h i n e a c h t i m e p e r i o d was compared between treatment groups using the extended F i s h e r ' s e x a c t p r o b a b i l i t y test. Significant comparisons using this method of statistical analysis precisely paralleled significant comparisons of c u m u l a t i v e i n c i d e n c e of F M B b e t w e e n a n i m a l s r e c e i v i n g 0.5, I, 2, or 4 u g C R H or NS. Animals i n f u s e d w i t h i, 2, or 4 u g of C R H p r i o r to t h e first introduction of p u p s displayed significantly more pup k i l l i n g in t h e f i r s t t w o h r of p u p c o n t a c t t h a n a n i m a l s i n f u s e d w i t h NS. T h e s e r e s u l t s are s u m m a r i z e d in F i g u r e 2. A l l a n i m a l s t h a t k i l l e d p u p s b e g a n to do so in the f i r s t t w o h r of p u p contact although CRH c o n t i n u e d to s i g n i f i c a n t l y inhibit the o n s e t of F M B t h r o u g h at l e a s t the s i x t h h r of p u p c o n t a c t . Most a n i m a l s t h a t k i l l e d p u p s d i d so b y l u n g i n g a n d b i t i n g as t h o u g h t h e y w e r e a t t a c k i n g the pups.
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FIG 2 The incidence of pup-killing in ovarian steroidt r e a t e d v i r g i n r a t s in t w o h r s a f t e r I C V i n f u s i o n of CRH (0.5, I, 2, or 4 ug) or n o r m a l saline (NS) vehicle. * = p<.05, ** = p < . 0 1 C R H vs NS; + = p<.05, 4.0 u g v s 0.5 u g CRH; (Fisher's exact probability test). T h e e f f e c t s of I C V i n f u s i o n of C R H on t h e r e e m e r g e n c e of maternal behavior in a n i m a l s t h a t w e r e a l l o w e d t h r e e d a y s of m o t h e r i n g e x p e r i e n c e a r e s u m m a r i z e d in F i g u r e 3. O v e r t h e f i r s t t w o h r s of r e - e s t a b l i s h e d p u p c o n t a c t t h e i n c i d e n c e of F M B w a s significantly l o w e r in r a t s r e c e i v i n g 1 or 2 u g C R H c o m p a r e d to r a t s r e c e i v i n g NS. By t h e e n d of t h e s i x t h h r of r e - e s t a b l i s h e d
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pup c o n t a c t the c u m m u l a t i v e incidence of m a t e r n a l b e h a v i o r of rats t r e a t e d w i t h 1 ug of CRH was no longer d i f f e r e n t from rats t r e a t e d w i t h NS w h i l e rats t r e a t e d w i t h 2 ug of CRH still had a s i g n i f i c a n t l y lower c u m u l a t i v e incidence of FMB. By the end of the 25th hr of pup contact all but one animal were fully maternal. The d i s t r i b u t i o n of maternal b e h a v i o r a l scores for each treatment group over each period of pup contact was c o m p a r e d u s i n g the e x t e n d e d Fisher's exact p r o b a b i l i t y test. S i g n i f i c a n t c o m p a r i s o n s u s i n g this test p a r a l l e l e d s i g n i f i c a n t c o m p a r i s o n s of c u m u l a t i v e incidences of FMB b e t w e e n t r e a t m e n t groups. None of these animals killed pups. Thus 1 or 2 ug of CRH was s i g n i f i c a n t l y less e f f e c t i v e in s t i m u l a t i n g p u p - k i l l i n g in rats w i t h three days of m o t h e r i n g e x p e r i e n c e c o m p a r e d to rats with no m o t h e r i n g e x p e r i e n c e (0/28 vs 6/30, p <.02; Fisher's exact p r o b a b i l i t y test).
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C R H (,,ug I C Y ) FIG 3 The c u m u l a t i v e incidence of full m a t e r n a l behavior (FMB) in o v a r i a n s t e r o i d - t r e a t e d v i r g i n female rats w i t h three days of p r i o r m o t h e r i n g e x p e r i e n c e at 2, 6 and 25 hrs after ICV infusion of CRH (i or 2 ug) or normal saline (NS) vehicle. * = p<.05, ** = p<.01, CRH vs NS (Fisher's exact p r o b a b i l i t y test).
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In a d d i t i o n to p u p - d i r e c t e d behaviors, C R H - t r e a t e d rats d i s p l a y e d bouts of rooting or b u r r o w i n g with the snout t h r o u g h wood chips, e s p e c i a l l y in the 1-2 hrs after ICV administration. Unfortunately, we did not quantify rooting b e h a v i o r so dose response r e l a t i o n s h i p s were not determined. Because rooting was e x h i b i t e d only i n t e r m i t t e n t l y and ceased by 2 hrs after CRH administration, i.e. well before m a t e r n a l b e h a v i o r r e a p p e a r e d in m o s t animals, the o b s e r v e d inhibition of m a t e r n a l b e h a v i o r by CRH could not be fully a c c o u n t e d for by p r e o c c u p a t i o n w i t h r o o t i n g behavior. Discussion Our results d e m o n s t r a t e that central a d m i n i s t r a t i o n of CRH p o t e n t l y blocks both the initial onset of ovarian steroidinduced m a t e r n a l b e h a v i o r in naive rats and the d i s p l a y of m a t e r n a l b e h a v i o r in rats with m o t h e r i n g experience. However, in the later, the dose response curve of CRH i n h i b i t i o n m a y be shifted to the right as the d u r a t i o n of inhibition at the 1 ug dose is shorter. In addition, CRH s t i m u l a t e d p u p - k i l l i n g in a s i g n i f i c a n t number of rats introduced to pups for the first time but had no such effect in rats with m o t h e r i n g experience. Thus central administration of CRH is quantitatively and q u a l i t a t i v e l y more d i s r u p t i v e of the initial onset than the m a i n t e n a n c e phase of m a t e r n a l behavior. B e h a v i o r a l responses to stressors and stress effects on m a t e r n a l behavior, like CRH effects on p u p - d i r e c t e d behavior, change w i t h m o t h e r i n g experience. Fleming and R o s e n b l a t t (21) r e p o r t e d that "emotionality" in a novel open field d e c l i n e d s i g n i f i c a n t l y in p o s t p a r t u m rats. H a n s e n and F e r r e i r a (24) observed additional behavioral changes in lactating rats including d e c r e a s e d startle response, and increased a g g r e s s i o n against u n f a m i l i a r intruders. In OVXed n u l l i p a r o u s rats the stress of a novel cage s i g n i f i c a n t l y d e l a y e d the initial onset of ovarian s t e r o i d - i n d u c e d maternal b e h a v i o r but had no effect on the r e e m e r g e n c e of maternal b e h a v i o r in e x p e r i e n c e d animals (25). Perhaps stressor released CRH inhibits the initial onset of m a t e r n a l behavior. However, prior a c t i v a t i o n of maternal b e h a v i o r may decrease sensitivity to CRH r e s u l t i n g in the d i m i n i s h e d stress responses observed in actively m o t h e r i n g rats. It will be of interest to d e t e r m i n e if other effects of CRH, e.g. EEG changes, a u t o n o m i c arousal, ACTH and c o r t i c o s t e r o n e release (2,27,28) are also altered by m o t h e r i n g experience. CRH may inhibit maternal b e h a v i o r by r e l e a s i n g opioids within the CNS. Bridges and G r i m m (29) demonstrated that m o r p h i n e p o t e n t l y blocks both the onset and m a i n t e n a n c e of m a t e r n a l behavior. This effect has been localized to the medial p r e o p t i c area (30). CRH inhibits lordosis b e h a v i o r in sexually receptive female rats by releasing B-endorphin in the m e s e n c e p h a l i c central gray (16). Neurones c o n t a i n i n g CRH and their p r o c e s s e s are located in a n u m b e r of brain sites k n o w n to be involved in m a t e r n a l b e h a v i o r including the M P O A (3,4). Perhaps CRH releases opioids in the M P O A thus b l o c k i n g m a t e r n a l behavior. It will be of interest to d e t e r m i n e if t r e a t m e n t with opiate receptor antagonists such as naloxone reverses the i n h i b i t o r y effect of CRH on maternal behavior.
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CRH may inhibit maternal b e h a v i o r by other n e u r o e n d o c r i n e mechanisms as well. CRH increases central noradrenergic n e u r o t r a n s m i s s i o n by a c t i v a t i n g the locus c o e r u l e u s (31). Drug t r e a t m e n t s that release norepinephine, e.g. amphetamine, disrupt maternal behavior (32). CRH p o t e n t l y releases p i t u i t a r y ACTH and, indirectly, c o r t i c o s t e r o n e (2). CRH has been reported to release ACTH in some brain sites (16). However, in our laboratory, ICV a d m i n i s t r a t i o n of ACTH at a wide range of doses had no i n h i b i t o r y effect on maternal behavior (unpublished observations). Studies of the effects of adrenal secretions on maternal behavior are few in number and have produced c o n f l i c t i n g results. A d r e n a l e c t o m y has been reported to shorten the latency of onset of maternal b e h a v i o r in OVXed v i r g i n rats (33), inhibit retrieval b e h a v i o r in p o s t p a r t u m rats (34) and to have no effect on the latency of onset of m a t e r n a l b e h a v i o r in pregnancy-terminated, primigestational rat (35). Both d e x a m e t h a s o n e and a d r e n a l e c t o m y have been o b s e r v e d to reverse the i n h i b i t o r y effect of c a e s a r e a n section or IP injection of saline on the onset of maternal b e h a v i o r (36). The effects of c o r t i c o s t e r o n e on maternal b e h a v i o r have not been tested. CRH s t i m u l a t i o n of the adrenal may also increase p r o g e s t e r o n e levels w h i c h w o u l d inhibit the onset of maternal b e h a v i o r in naive but not m a t e r n a l l y e x p e r i e n c e d rats (23). F a m i l i a r i t y with and active n u r t u r i n g of pups appears to alter CRH effects on pup-directed behavior. Our results conflict, to s o m e extent, with the c o n c l u s i o n s of Sherman and Kalin (37) that the s p e c t r u m of behaviors a f f e c t e d by CRH and the m a g n i t u d e of effects are the same under familiar and novel test cage conditions. How might quantitative as well as q u a l i t a t i v e shifts in CRH effects on p u p - d i r e c t e d b e h a v i o r come about in e x p e r i e n c e d rats mothers? Hormones such as estrogen, o x y t o c i n and, perhaps, p r o l a c t i n play critical roles in the initiation but not the m a i n t e n a n c e of maternal b e h a v i o r (23). Because the m e c h a n i s m s of onset and m a i n t e n a n c e are distinct, it is not s u r p r i s i n g that CRH would d i f f e r e n t i a l l y affect separate phases of maternal responsiveness. Hansen (38) p o s t u l a t e d that p o s t p a r t u m changes in b e h a v i o r (decreased freezing in response to u n e x p e c t e d a u d i t o r y stimuli, increased a g g r e s s i o n against u n f a m i l a r intruders, increased food intake and enhanced p u n i s h e d drinking) are suggestive of increased GABAergic neurotransmission. B e n z o d i a z e p i n e t r e a t m e n t reverses s t r e s s - l i k e b e h a v i o r a l effects of CRH (8,9,11). Perhaps increased G A B A e r g i c n e u r o t r a n s m i s s i o n w i t h i n the CNS of e x p e r i e n c e d rat mothers a n t a g o n i z e s CRH inhibition of maternal b e h a v i o r and prevents pup-killing. The onset and m a i n t e n a n c e phases of the maternal b e h a v i o r a l response, a s s o c i a t e d as they are with changes in b e h a v i o r a l responses to n o v e l t y and other stressors, p r o v i d e an intriquing p a r a d i g m in w h i c h to investigate the s t r e s s - l i k e effects of CRH. Such i n v e s t i g a t i o n s may be of clinical significance. E x c e s s i v e release of CRH has been implicated in mood d i s o r d e r s (39,40). Women are at g r e a t e r risk for affective d i s t u r b a n c e s in the p o s t p a r t u m p e r i o d (41,42). Perhaps changes in s e n s i t i v i t y to CRH in the p u e r p e r i u m contribute to the e m e r g e n c e of m o o d symptoms. Unfortunately, puerperal p s y c h o s i s sometimes includes delusional h o m i c i d a l intent and actions against newborns (41). Perhaps i n f a n t - d i r e c t e d a g g r e s s i o n in puerperal p s y c h o s i s results from
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e x c e s s i v e r e l e a s e of e n d o g e n o u s C R H e v e n in n a i v e r a t s s t i m u l a t e s p u p - k i l l i n g .
as
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Acknowledaements We help. grant
t h a n k Ms. B e t s y S h a m b l e y for h e r e x c e l l e n t s e c r e t a r i a l T h i s w o r k w a s s u p p o r t e d by N I M H g r a n t M H 3 3 1 2 7 a n d N I C H D HD16159. References
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(1985).
C O R T I C O T R O P I N - R E L E A S I N G HORMONE INHIBITS M A T E R N A L INDUCES P U P - K I L L I N G
Pergamon Press
BEHAVIOR
AND
Cort A. Pedersen, Jack D. Caldwell, M a r s d e n M c G u i r e and Dwight L. Evans Dept. Psychiatry, Brain and D e v e l o p m e n t R e s e a r c h Center, and the N e u r o b i o l o g y Curriculum, Univ. of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7160 (Received in final form February ii, 1991) Summary Behavioral responses to stressors and the effects of stressors on maternal behavior change with m o t h e r i n g experience. C o r t i c o t r o p i n - r e l e a s i n g h o r m o n e (CRH) is r e l e a s e d by stressors and p r o d u c e s stresslike b e h a v i o r a l effects. We tested the effects of ICV infusion of ovine CRH (0.5-4 ug) on p u p - d i r e c t e d b e h a v i o r s in ovariectomized, ovarian s t e r o i d - t r e a t e d v i r g i n rats that were either naive to pups or that had three days of m o t h e r i n g experience. CRH inhibited m a t e r n a l b e h a v i o r in naive and e x p e r i e n c e d rats in a d o s e - r e l a t e d manner. The m a g n i t u d e and d u r a t i o n of inhibition, e s p e c i a l l y at the 1 ug dose, were less in rats with m o t h e r i n g experience. H i g h e r doses of CRH (i - 4 ug) s i g n i f i c a n t l y increased p u p - k i l l i n g in rats that were naive to pups. In contrast, CRH p r o d u c e d no p u p - k i l l i n g in rats with m o t h e r i n g experience. The p r i m a r y c o r t i c o t r o p i n - r e l e a s i n g h o r m o n e (CRH) is a 41 amino acid p e p t i d e o r i g i n a l l y sequenced by Vale et al (i). In response to stressors (2) CRH is secreted into the h y p o p h y s i a l portal v a s c u l a t u r e and stimulates ACTH and, indirectly, adrenal g l u c o c o r t i c o i d release. Pathways c o n t a i n i n g CRH are w i d e l y d i s t r i b u t e d w i t h i n the brain (3,4) as are CRH b i n d i n g sites (5). Intracerebral infusion of CRH produces behavioral changes similar to those seen when animals are stressed (6-14). Additionally, CRH blocks other behaviors, such as c o p u l a t i o n and eating, that are inhibited by stress (15-17). Central administration of a CRH antagonist blocks stress-induced b e h a v i o r a l changes in mice and rats (18-20) s u g g e s t i n g that CRH e n d o g e n o u s to the brain plays a critical role in b e h a v i o r a l r e s p o n s e s to stress. N e w b o r n pups, like other novel stimuli, are a v e r s i v e for v i r g i n female rats (21,22). Behavioral responses t o w a r d young pups change rapidly at the end of p r e g n a n c y from active avoidance or even infanticide to avid mothering (23). Behavioral responses to stressors also shift with the onset of maternal behavior and nursing. These include decreased "emotionality" in a novel open field, d e c r e a s e d startle response and i n c r e a s e d a g g r e s s i o n against intruders (22,24). The effect of a v e r s i v e stimuli on the d i s p l a y of maternal b e h a v i o r m a y also change w i t h m o t h e r i n g experience. The stress of a novel cage 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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s i g n i f i c a n t l y delays the initial onset of maternal b e h a v i o r in rats naive to pups but does not affect the rapid r e e m e r g e n c e of m a t e r n a l b e h a v i o r e x h i b i t e d by e x p e r i e n c e d rat m o t h e r s (25). This o b s e r v a t i o n suggests that n e u r o e n d o c r i n e c o m p o n e n t s of the stress r e s p o n s e may be more d i s r u p t i v e of the initial onset c o m p a r e d to the m a i n t e n a n c e of e s t a b l i s h e d maternal behavior. We have, therefore, tested the effects of i n t r a c e r e b r o v e n t r i c u l a r (ICV) a d m i n i s t r a t i o n of CRH on the onset of maternal b e h a v i o r in naive female rats and on the r e e m e r g e n c e of m o t h e r i n g b e h a v i o r in m a t e r n a l l y e x p e r i e n c e d rats. Methods 17 B - e s t r a d i o l (E2) and p r o g e s t e r o n e (P) (Steraloids) were a d m i n i s t e r e d in capsules made from silastic medical grade tubing (Dow C o r n i n g #602-235). Capsules were made by filling the a p p r o p r i a t e length of silastic tubing with the d e s i r e d amount of E 2 or P, p l u g g i n g the ends of the tubing with pieces of w o o d e n d o w l i n g (5mm) and sealing the ends with silastic 382 medical grade e l a s t o m e r (Dow C o m i n g ) . Capsules were first immersed in pure ethanol for one hr and then in p h o s p h a t e b u f f e r e d saline for 48 hrs p r i o r to SC implantation. A n i m a l s were a n e s t h e t i z e d with ether during i m p l a n t a t i o n and removal of silastic capsules. N u l l i p a r o u s Sprague Dawley rats (zivic M i l l e r Laboratories, 200-250 gm) were o v a r i e c t o m i z e d (OVXed) under ether anesthesia. A f t e r OVX, animals were housed in individual cages on a 12 hr l i g h t / d a r k cycle with free access to food and water. Eight days after OVX one silastic capsule, 1 cm in length and c o n t a i n i n g 8.8 mg of E2, was implanted SC on the right flank of each animal. Ten days after OVX, an additional silastic capsule, 90 mm in length containg 120 mg of P, was implanted SC b e t w e e n the scapulae of each animal. Ovine CRH was obtained from P e n n i n s u l a Laboratories, Inc. (Lot # 007290). CRH was d i s s o l v e d in normal saline (NS) v e h i c l e (pH 5.0) at various c o n c e n t r a t i o n s so that each ICV dose was a d m i n i s t e r e d in a i0 ul volume. F o r t y - e i g h t hrs prior to behavioral testing each animal was p l a c e d in a clear p l e x i g l a s o b s e r v a t i o n cage (44 cm x 21 cm x 20 cm) c o v e r e d by a metal wire top with recesses to hold food p e l l e t s and a w a t e r bottle. Wood chips to a depth of 2-3 cms covered the floor of each o b s e r v a t i o n cage. Twenty pieces of paper t o w e l i n g (4 cm x 6 cm) were placed in each cage to serve as n e s t i n g material. Food and w a t e r were available ad libitum. The l i g h t / d a r k cycle remained the same. Twenty-four hrs prior to behavioral testing silastic implants c o n t a i n i n g P were removed from each animal. One hr prior to b e h a v i o r a l testing, each animal received ICV either one of four doses of CRH (0.5, 1.0, 2.0, or 4.0 ug) or NS v e h i c l e alone; other animals received no ICV treatment. Infusion into the right lateral cerebral v e n t r i c l e was a c h i e v e d by the m e t h o d of Popick (26) while animals were under light ether anesthesia. This m e t h o d results in greater than ninety percent accurate ICV a d m i n i s t r a t i o n as d e t e r m i n e d by e x a m i n a t i o n of the d i s t r i b u t i o n of dye w i t h i n the brain after injection of a c o n c e n t r a t e d s o l u t i o n of Evan's blue dye. were
One hr after ICV infusion three rat pups (1-5 placed 20 cms apart at the apices of an
days old) imaginary
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e q u i l a t e r a l t r i a n g l e c e n t e r e d in the m i d d l e of the o b s e r v a t i o n cage of e a c h animal (the s t a r t i n g position). D u r i n g the initial six hrs of pup c o n t a c t an o b s e r v e r ignorant of ICV t r e a t m e n t s looked for and r e c o r d e d the following c a t e g o r i e s of m a t e r n a l b e h a v i o r d i s p l a y e d by the test animals; initial g r o u p i n g of pups, l i c k i n g of pups, crouching in a n u r s i n g p o s i t i o n over g r o u p e d pups and retrieval of s e p a r a t e d pups. The o b s e r v e r r e t u r n e d the pups to the s t a r t i n g p o s i t i o n at the b e g i n n i n g of the third, fourth, fifth and sixth hr of pup c o n t a c t and b e g a n a new p e r i o d of b e h a v i o r a l o b s e r v a t i o n for each of t h o s e hrs. For each of the o b s e r v a t i o n p e r i o d s (the first two hrs, as well as the third, fourth, fifth and sixth hrs of pup contact) each animal was a s s i g n e d a m a t e r n a l b e h a v i o r a l score b a s e d on one point for each m a t e r n a l b e h a v i o r c a t e g o r y in w h i c h the animal met or exceeded quantitative screening criteria for that behavior. A n i m a l s w e r e c o n s i d e r e d to be fully m a t e r n a l as of the p e r i o d d u r i n g w h i c h they first met or e x c e e d e d s c r e e n i n g c r i t e r i a for all c a t e g o r i e s of m a t e r n a l behavior. Each animal was required to meet the following criteria during an o b s e r v a t i o n p e r i o d to be a s s e s s e d as h a v i n g d i s p l a y e d each of these behaviors. Grouping: The animal carries in its m o u t h all three pups from t h e i r initial s c a t t e r e d p o s i t i o n and clumps t h e m t o g e t h e r in a c o r n e r of the o b s e r v a t i o n cage. It also d i s p l a y s r e g r o u p i n g of pups, i n v o l v i n g r e a r r a n g e m e n t of at least two pups, w i t h i n this area. Licking: The animal licks one or m o r e pups of ten sec or d i s p l a y s at least three b r i e f
for at least a total licks.
Crouching: The animal must adopt a c r o u c h i n g p o s i t i o n for at least a total of 60 sec. All pups m u s t be g r o u p e d b e f o r e c r o u c h i n g can be recorded. The animal m u s t have its h i n d legs spread, its b a c k arched, and its v e n t r u m high e n o u g h off the cage floor to a c c o m m o d a t e pups b e n e a t h it. T h e r e m u s t be at least one pup b e n e a t h it during a crouch. Bouts of two sec or less are not added to the c u m u l a t i v e time. Retrieval: A n y time w i t h i n the p e r i o d a f t e r g r o u p i n g has taken place, all three pups are separated by the observer to a d i s t a n c e of 15-20 cm from the female. Retrieval constitutes c a r r y i n g all pups b a c k to the nest w i t h i n 15 min of separation. R e t r i e v a l m u s t be observed, rather than inferred, since pups may r e t u r n by t h e i r own efforts. Animals that had not met criteria for full maternal b e h a v i o r (FMB) d u r i n g any of the o b s e r v a t i o n p e r i o d s d u r i n g the initial six hr of pup contact were left w i t h pups overnight. Every 24 hr after the initiation of pup contact, t h r e e fresh pups (1-5 days of age) were s u b s t i t u t e d for the pups g i v e n the day before and placed in the starting position in each o b s e r v a t i o n cage. M a t e r n a l b e h a v i o r was o b s e r v e d and r e c o r d e d d u r i n g a one hr p e r i o d after each of these pup exchanges. Each animal was t e s t e d until it met or e x c e e d e d c r i t e r i a for FMB in a one hr o b s e r v a t i o n p e r i o d or until it k i l l e d one of the pups at w h i c h time the r e m a i n i n g pups w e r e immediately removed and b e h a v i o r a l o b s e r v a t i o n s stopped. A n i m a l s that k i l l e d pups w e r e g i v e n a m a t e r n a l b e h a v i o r a l score of "kill".
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F o r t h e p u r p o s e of t e s t i n g l e v e l s of r e e m e r g e n t m a t e r n a l behavior, rats that received no I C V t r e a t m e n t and that met criteria f o r F M B in t h e f i r s t s i x h r s of p u p c o n t a c t were a l l o w e d to s t a y w i t h y o u n g pups. F r e s h 1-5 d a y o l d r a t p u p s w e r e e x c h a n g e d for u s e d p u p s e v e r y 24 hr. A f t e r 72 h r of c o n t i n u o u s e x p o s u r e to p u p s all p u p s w e r e r e m o v e d a n d e a c h a n i m a l r e c e i v e d an I C V i n f u s i o n of C R H (i or 2 ug) or NS v e h i c l e alone. One hr l a t e r t h r e e f r e s h 1-5 d a y o l d rat p u p s w e r e i n t r o d u c e d . An observer ignorant of ICV treatments then recorded maternal b e h a v i o r a l r e s p o n s e s o v e r t h e f i r s t s i x a n d t h e 2 5 t h h r of ree s t a b l i s h e d p u p c o n t a c t as d e s c r i b e d above.
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CRH ()ug ICV) FIG. 1 The cumulative i n c i d e n c e of full m a t e r n a l b e h a v i o r (FMB) in o v a r i a n s t e r o i d - t r e a t e d v i r g i n f e m a l e r a t s at 2, 6 a n d 25 h r s a f t e r I C V i n f u s i o n of C R H (0.5, I, 2 or 4 ug) or n o r m a l saline (NS) v e h i c l e alone. * = p<.05, ** = p<.01, *** <.001, C R H v e r s u s NS ( F i s h e r ' s e x a c t p r o b a b i l i t y test).
Results T h e e f f e c t s of I C V i n f u s i o n of C R H on t h e o n s e t of m a t e r n a l behavior are summarized in F i g u r e i. The ovarian steroid treatment used produced a high incidence of F M B (27/32) in c o n t r o l r a t s r e c e i v i n g NS. In t h e f i r s t t w o h r of p u p contact, t h e i n c i d e n c e of F M B w a s s i g n i f i c a n t l y l o w e r in r a t s r e c e i v i n g I, 2 or 4 u g C R H c o m p a r e d to r a t s r e c e i v i n g NS. T h e s e r e s u l t s s u g g e s t t h a t t h e r e m a y be a l i n e a r r e l a t i o n s h i p b e t w e e n d o s e of C R H a n d i n c i d e n c e of F M B o v e r t h i s t i m e period. A t t h e e n d of
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CRH and Maternal Behavior
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t h e s i x t h h r of p u p c o n t a c t , a n i m a l s r e c e i v i n g I, 2 or 4 u g of CRH still had significantly lower cumulative i n c i d e n c e s of F M B t h a n a n i m a l s r e c e i v i n g NS. By t h e 2 5 t h h r of p u p c o n t a c t , t h e r e were no s i g n i f i c a n t differences among treatment groups. The distribution of m a x i m u m m a t e r n a l b e h a v i o r a l s c o r e s (kill, 0, i, 2, 3, 4) a c h i e v e d b y i n d i v i d u a l a n i m a l s w i t h i n e a c h t i m e p e r i o d was compared between treatment groups using the extended F i s h e r ' s e x a c t p r o b a b i l i t y test. Significant comparisons using this method of statistical analysis precisely paralleled significant comparisons of c u m u l a t i v e i n c i d e n c e of F M B b e t w e e n a n i m a l s r e c e i v i n g 0.5, I, 2, or 4 u g C R H or NS. Animals i n f u s e d w i t h i, 2, or 4 u g of C R H p r i o r to t h e first introduction of p u p s displayed significantly more pup k i l l i n g in t h e f i r s t t w o h r of p u p c o n t a c t t h a n a n i m a l s i n f u s e d w i t h NS. T h e s e r e s u l t s are s u m m a r i z e d in F i g u r e 2. A l l a n i m a l s t h a t k i l l e d p u p s b e g a n to do so in the f i r s t t w o h r of p u p contact although CRH c o n t i n u e d to s i g n i f i c a n t l y inhibit the o n s e t of F M B t h r o u g h at l e a s t the s i x t h h r of p u p c o n t a c t . Most a n i m a l s t h a t k i l l e d p u p s d i d so b y l u n g i n g a n d b i t i n g as t h o u g h t h e y w e r e a t t a c k i n g the pups.
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FIG 2 The incidence of pup-killing in ovarian steroidt r e a t e d v i r g i n r a t s in t w o h r s a f t e r I C V i n f u s i o n of CRH (0.5, I, 2, or 4 ug) or n o r m a l saline (NS) vehicle. * = p<.05, ** = p < . 0 1 C R H vs NS; + = p<.05, 4.0 u g v s 0.5 u g CRH; (Fisher's exact probability test). T h e e f f e c t s of I C V i n f u s i o n of C R H on t h e r e e m e r g e n c e of maternal behavior in a n i m a l s t h a t w e r e a l l o w e d t h r e e d a y s of m o t h e r i n g e x p e r i e n c e a r e s u m m a r i z e d in F i g u r e 3. O v e r t h e f i r s t t w o h r s of r e - e s t a b l i s h e d p u p c o n t a c t t h e i n c i d e n c e of F M B w a s significantly l o w e r in r a t s r e c e i v i n g 1 or 2 u g C R H c o m p a r e d to r a t s r e c e i v i n g NS. By t h e e n d of t h e s i x t h h r of r e - e s t a b l i s h e d
1542
CRH and Maternal Behavior
Vol. 48, No. 16, 1991
pup c o n t a c t the c u m m u l a t i v e incidence of m a t e r n a l b e h a v i o r of rats t r e a t e d w i t h 1 ug of CRH was no longer d i f f e r e n t from rats t r e a t e d w i t h NS w h i l e rats t r e a t e d w i t h 2 ug of CRH still had a s i g n i f i c a n t l y lower c u m u l a t i v e incidence of FMB. By the end of the 25th hr of pup contact all but one animal were fully maternal. The d i s t r i b u t i o n of maternal b e h a v i o r a l scores for each treatment group over each period of pup contact was c o m p a r e d u s i n g the e x t e n d e d Fisher's exact p r o b a b i l i t y test. S i g n i f i c a n t c o m p a r i s o n s u s i n g this test p a r a l l e l e d s i g n i f i c a n t c o m p a r i s o n s of c u m u l a t i v e incidences of FMB b e t w e e n t r e a t m e n t groups. None of these animals killed pups. Thus 1 or 2 ug of CRH was s i g n i f i c a n t l y less e f f e c t i v e in s t i m u l a t i n g p u p - k i l l i n g in rats w i t h three days of m o t h e r i n g e x p e r i e n c e c o m p a r e d to rats with no m o t h e r i n g e x p e r i e n c e (0/28 vs 6/30, p <.02; Fisher's exact p r o b a b i l i t y test).
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C R H (,,ug I C Y ) FIG 3 The c u m u l a t i v e incidence of full m a t e r n a l behavior (FMB) in o v a r i a n s t e r o i d - t r e a t e d v i r g i n female rats w i t h three days of p r i o r m o t h e r i n g e x p e r i e n c e at 2, 6 and 25 hrs after ICV infusion of CRH (i or 2 ug) or normal saline (NS) vehicle. * = p<.05, ** = p<.01, CRH vs NS (Fisher's exact p r o b a b i l i t y test).
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CRH and Maternal Behavior
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In a d d i t i o n to p u p - d i r e c t e d behaviors, C R H - t r e a t e d rats d i s p l a y e d bouts of rooting or b u r r o w i n g with the snout t h r o u g h wood chips, e s p e c i a l l y in the 1-2 hrs after ICV administration. Unfortunately, we did not quantify rooting b e h a v i o r so dose response r e l a t i o n s h i p s were not determined. Because rooting was e x h i b i t e d only i n t e r m i t t e n t l y and ceased by 2 hrs after CRH administration, i.e. well before m a t e r n a l b e h a v i o r r e a p p e a r e d in m o s t animals, the o b s e r v e d inhibition of m a t e r n a l b e h a v i o r by CRH could not be fully a c c o u n t e d for by p r e o c c u p a t i o n w i t h r o o t i n g behavior. Discussion Our results d e m o n s t r a t e that central a d m i n i s t r a t i o n of CRH p o t e n t l y blocks both the initial onset of ovarian steroidinduced m a t e r n a l b e h a v i o r in naive rats and the d i s p l a y of m a t e r n a l b e h a v i o r in rats with m o t h e r i n g experience. However, in the later, the dose response curve of CRH i n h i b i t i o n m a y be shifted to the right as the d u r a t i o n of inhibition at the 1 ug dose is shorter. In addition, CRH s t i m u l a t e d p u p - k i l l i n g in a s i g n i f i c a n t number of rats introduced to pups for the first time but had no such effect in rats with m o t h e r i n g experience. Thus central administration of CRH is quantitatively and q u a l i t a t i v e l y more d i s r u p t i v e of the initial onset than the m a i n t e n a n c e phase of m a t e r n a l behavior. B e h a v i o r a l responses to stressors and stress effects on m a t e r n a l behavior, like CRH effects on p u p - d i r e c t e d behavior, change w i t h m o t h e r i n g experience. Fleming and R o s e n b l a t t (21) r e p o r t e d that "emotionality" in a novel open field d e c l i n e d s i g n i f i c a n t l y in p o s t p a r t u m rats. H a n s e n and F e r r e i r a (24) observed additional behavioral changes in lactating rats including d e c r e a s e d startle response, and increased a g g r e s s i o n against u n f a m i l i a r intruders. In OVXed n u l l i p a r o u s rats the stress of a novel cage s i g n i f i c a n t l y d e l a y e d the initial onset of ovarian s t e r o i d - i n d u c e d maternal b e h a v i o r but had no effect on the r e e m e r g e n c e of maternal b e h a v i o r in e x p e r i e n c e d animals (25). Perhaps stressor released CRH inhibits the initial onset of m a t e r n a l behavior. However, prior a c t i v a t i o n of maternal b e h a v i o r may decrease sensitivity to CRH r e s u l t i n g in the d i m i n i s h e d stress responses observed in actively m o t h e r i n g rats. It will be of interest to d e t e r m i n e if other effects of CRH, e.g. EEG changes, a u t o n o m i c arousal, ACTH and c o r t i c o s t e r o n e release (2,27,28) are also altered by m o t h e r i n g experience. CRH may inhibit maternal b e h a v i o r by r e l e a s i n g opioids within the CNS. Bridges and G r i m m (29) demonstrated that m o r p h i n e p o t e n t l y blocks both the onset and m a i n t e n a n c e of m a t e r n a l behavior. This effect has been localized to the medial p r e o p t i c area (30). CRH inhibits lordosis b e h a v i o r in sexually receptive female rats by releasing B-endorphin in the m e s e n c e p h a l i c central gray (16). Neurones c o n t a i n i n g CRH and their p r o c e s s e s are located in a n u m b e r of brain sites k n o w n to be involved in m a t e r n a l b e h a v i o r including the M P O A (3,4). Perhaps CRH releases opioids in the M P O A thus b l o c k i n g m a t e r n a l behavior. It will be of interest to d e t e r m i n e if t r e a t m e n t with opiate receptor antagonists such as naloxone reverses the i n h i b i t o r y effect of CRH on maternal behavior.
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CRH may inhibit maternal b e h a v i o r by other n e u r o e n d o c r i n e mechanisms as well. CRH increases central noradrenergic n e u r o t r a n s m i s s i o n by a c t i v a t i n g the locus c o e r u l e u s (31). Drug t r e a t m e n t s that release norepinephine, e.g. amphetamine, disrupt maternal behavior (32). CRH p o t e n t l y releases p i t u i t a r y ACTH and, indirectly, c o r t i c o s t e r o n e (2). CRH has been reported to release ACTH in some brain sites (16). However, in our laboratory, ICV a d m i n i s t r a t i o n of ACTH at a wide range of doses had no i n h i b i t o r y effect on maternal behavior (unpublished observations). Studies of the effects of adrenal secretions on maternal behavior are few in number and have produced c o n f l i c t i n g results. A d r e n a l e c t o m y has been reported to shorten the latency of onset of maternal b e h a v i o r in OVXed v i r g i n rats (33), inhibit retrieval b e h a v i o r in p o s t p a r t u m rats (34) and to have no effect on the latency of onset of m a t e r n a l b e h a v i o r in pregnancy-terminated, primigestational rat (35). Both d e x a m e t h a s o n e and a d r e n a l e c t o m y have been o b s e r v e d to reverse the i n h i b i t o r y effect of c a e s a r e a n section or IP injection of saline on the onset of maternal b e h a v i o r (36). The effects of c o r t i c o s t e r o n e on maternal b e h a v i o r have not been tested. CRH s t i m u l a t i o n of the adrenal may also increase p r o g e s t e r o n e levels w h i c h w o u l d inhibit the onset of maternal b e h a v i o r in naive but not m a t e r n a l l y e x p e r i e n c e d rats (23). F a m i l i a r i t y with and active n u r t u r i n g of pups appears to alter CRH effects on pup-directed behavior. Our results conflict, to s o m e extent, with the c o n c l u s i o n s of Sherman and Kalin (37) that the s p e c t r u m of behaviors a f f e c t e d by CRH and the m a g n i t u d e of effects are the same under familiar and novel test cage conditions. How might quantitative as well as q u a l i t a t i v e shifts in CRH effects on p u p - d i r e c t e d b e h a v i o r come about in e x p e r i e n c e d rats mothers? Hormones such as estrogen, o x y t o c i n and, perhaps, p r o l a c t i n play critical roles in the initiation but not the m a i n t e n a n c e of maternal b e h a v i o r (23). Because the m e c h a n i s m s of onset and m a i n t e n a n c e are distinct, it is not s u r p r i s i n g that CRH would d i f f e r e n t i a l l y affect separate phases of maternal responsiveness. Hansen (38) p o s t u l a t e d that p o s t p a r t u m changes in b e h a v i o r (decreased freezing in response to u n e x p e c t e d a u d i t o r y stimuli, increased a g g r e s s i o n against u n f a m i l a r intruders, increased food intake and enhanced p u n i s h e d drinking) are suggestive of increased GABAergic neurotransmission. B e n z o d i a z e p i n e t r e a t m e n t reverses s t r e s s - l i k e b e h a v i o r a l effects of CRH (8,9,11). Perhaps increased G A B A e r g i c n e u r o t r a n s m i s s i o n w i t h i n the CNS of e x p e r i e n c e d rat mothers a n t a g o n i z e s CRH inhibition of maternal b e h a v i o r and prevents pup-killing. The onset and m a i n t e n a n c e phases of the maternal b e h a v i o r a l response, a s s o c i a t e d as they are with changes in b e h a v i o r a l responses to n o v e l t y and other stressors, p r o v i d e an intriquing p a r a d i g m in w h i c h to investigate the s t r e s s - l i k e effects of CRH. Such i n v e s t i g a t i o n s may be of clinical significance. E x c e s s i v e release of CRH has been implicated in mood d i s o r d e r s (39,40). Women are at g r e a t e r risk for affective d i s t u r b a n c e s in the p o s t p a r t u m p e r i o d (41,42). Perhaps changes in s e n s i t i v i t y to CRH in the p u e r p e r i u m contribute to the e m e r g e n c e of m o o d symptoms. Unfortunately, puerperal p s y c h o s i s sometimes includes delusional h o m i c i d a l intent and actions against newborns (41). Perhaps i n f a n t - d i r e c t e d a g g r e s s i o n in puerperal p s y c h o s i s results from
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CRH and Maternal Behavior
e x c e s s i v e r e l e a s e of e n d o g e n o u s C R H e v e n in n a i v e r a t s s t i m u l a t e s p u p - k i l l i n g .
as
CRH
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administration
Acknowledaements We help. grant
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