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Brain norepinephrine and 5-hydroxytryptamine as a function of time after avoidance training and footshock
Pharmacology Biochemistry and Behavior, Vol. l, pp. 615-618. ANKHO International Inc., 1973. Printed in the U.S.A.
Brain Norepinephrine and 5-Hydroxytryptamine as a Function of Time after Avoidance Training and Footshock R I C H A R D H. B A U E R 2
Department o f Psychiatry, School o f Medicine. University o f California, Los Angeles, California 90024
( R e c e i v e d 19 F e b r u a r y 1973)
BAUER, R. H. Brain norepinephrme and 5-hydroxytryptamine as a function of time after avoidance training and footshock. PHARMAC. BIOCHEM. BEHAV. 1 (6) 6 1 5 - 6 1 8 , 1973.-Male Sprague-Dawley rats acquired a one-way avoidance task more rapidly 10 min and 24 hr after partial avoidance training or inescapable shock in the start box than 3.5 hr after these treatments. Endogenous levels of brain 5-hydroxytryptamine (5-HT) were not altered 10 min, 3.5 hr, or 24 hr after avoidance training or inescapable shock in the start box. Norepinephrine (NE) was lower 10 rain and 3.5 hr after training and inescapable shock but not 24 hr later. Since neither 5-HT nor NE exhibited a U or inverted U function following avoidance training or footshock, these results indicate that endogenous levels of 5-HT and NE are not related to poorer avoidance at intermediate retest intervals. Avoidance
Norepinephrine
5-Hydroxyt ryptamine
[ 10]. However, m o r e r e c e n t evidence has n o t s u p p o r t e d t h e h y p o t h e s i s t h a t a l t e r a t i o n o f the p i t u i t a r y - a d r e n a l s y s t e m is related to a v o i d a n c e deficits I - 8 hr after training [7, 13, 1 6 ] . T h e r e f o r e , e v i d e n c e relating t h e p i t u i t a r y adrenal s y s t e m to this p h e n o m e n o n is at best controversial. A l t h o u g h the p i t u i t a r y - a d r e n a l s y s t e m m a y n o t a c c o u n t for p o o r e r a v o i d a n c e 1- 8 hr a f t e r training, e v i d e n c e suggests t h a t a l t e r a t i o n of brain n o r e p i n e p h r i n e (NE) m a y be related to this p h e n o m e n o n . A n u m b e r of studies have r e p o r t e d r e d u c e d levels o f e n d o g e n o u s b r a i n NE s h o r t l y a f t e r a variety of stressors [4,111 and a f u r t h e r decrease 2 - 4 hr f o l l o w i n g stress [ 2 , 1 5 ] . Weiss, S t o n e and Harrell [19] f o u n d t h a t rats w h i c h c o u l d escape and avoid s h o c k have increased NE w h e n sacrificed 2 0 - 4 0 min a f t e r t r a i n i n g b u t animals t h a t could n e i t h e r escape n o r avoid s h o w e d n o change. Drugs w h i c h release NE also increase activity and a v o i d a n c e [8, 15, 17]. These results suggest t h a t poststress r e d u c t i o n of brain NE can p e r h a p s a c c o u n t for a v o i d a n c e deficits 1 - 8 hr a f t e r training. T h e m a j o r p u r p o s e o f the present e x p e r i m e n t is to determ i n e if rats sacrificed 3.5 h r a f t e r a v o i d a n c e t r a i n i n g have lower levels o f e n d o g e n o u s brain NE a n d / o r 5 - h y d r o x y t r y p t a m i n e (5-HT) t h a n t h o s e sacrificed 10 min or 24 hr a f t e r training. In a d d i t i o n , rats given inescapable and una v o i d a b l e s h o c k in the start b o x of a one-way a v o i d a n c e a p p a r a t u s 5 rain and 24 hr prior to a v o i d a n c e training
A T T H E p r e s e n t t i m e t h e r e is c o n s i d e r a b l e c o n t r o v e r s y regarding the t h e o r e t i c a l i n t e r p r e t a t i o n of p o o r e r a v o i d a n c e at i n t e r m e d i a t e retest intervals ( I - 8 h r ) as c o m p a r e d to t h o s e e i t h e r earlier or later. A n u m b e r of papers suggested t h a t p o o r a v o i d a n c e at i n t e r m e d i a t e intervals is d u e to an initial increase and s u b s e q u e n t decrease o f fear [ 6 ] . decreased l o c o m o t i o n and an increase in i n c o m p a t i b l e responses, w h i c h i n t e r f e r e with a v o i d a n c e , are p r e s u m e d to b e related to fear. More r e c e n t l y , Spear, Klein, and Riley [ 14] have suggested t h a t physiological changes w h i c h follow a stressful e v e n t m a y be r e s p o n s i b l e for a m e m o r y retrieval deficit 1 - 8 h r a f t e r aversive c o n d i t i o n i n g . I n t e r n a l c o n d i tions p r e s e n t d u r i n g learning are a s s u m e d t o b e altered at i n t e r m e d i a t e r e t e n t i o n intervals and c o n s e q u e n t l y m e m o r y o f originial learning is m o r e difficult to retrieve. Thus, p o o r e r a v o i d a n c e I -.-8 hr a f t e r t r a i n i n g m a y b e m e d i a t e d b y a m e c h a n i s m similar to s t a t e d e p e n d e n t learning f o u n d w i t h various drugs [ 1 2 ] . At the behavioral level, a n u m b e r of workers have e x a m i n e d this U-shaped r e t e n t i o n f u n c t i o n b u t relatively litzle research has b e e n d e v o t e d to investigating physiological changes w h i c h m i g h t a c c o u n t for this p h e n o m e n o n . Earlier papers suggested t h a t a decrease in c o r t i c o s t e r o n e , w h i c h parallels the initial p e r f o r m a n c e d e c r e m e n t b u t n o t t h e s u b s e q u e n t i m p r o v e m e n t in a v o i d a n c e , was r e s p o n s i b l e for the a n i m a l ' s i n a b i l i t y to cope b e h a v i o r a l l y with stress
This research was supported in part by Public Health Service Grant NIMH-06960-11 to Daniel Sheer. The author gratefully acknowledges the assistance of Edward Butler, William Jones, and Peggy Hall. 2 Requests for reprints should be sent to Richard H. Bauer, Department of Psychiatry, School of Medicine, University of California, Los Angeles, California, 90024. 615
616
BAUER
acquired the one-way avoidance task m o r e rapidly than rats trained 4 hr after this t r e a t m e n t [ I ] . Inescapable and unavoidable f o o t s h o c k was, t h e r e f o r e , a d m i n i s t e r e d in the start box of a one-way avoidance apparatus in an a t t e m p t to replicate these results and d e t e r m i n e if NE or 5-liT o f trained and stressed animals is d i f f e r e n t l y a f f e c t e d . Thus, the present e x p e r i m e n t e x a m i n e d the possibility that altered brain NE a n d / o r 5-1IT are related to p o o r e r avoidance at i n t e r m e d i a t e retest intervals and a test of the generality o f the Weiss, Stone, and Harrell [1~)] results. MET1 I()1)
Animals and Experimental Design The animals were 156 e x p e r i m e n t a l l y naive 1 2 0 - 1 8 0 d a y o l d m a l e albino Sprague-Dawley rats weighing 3 0 0 - - 4 5 0 g. These animals were m a i n t a i n e d on ad lib food (Pt, rina rat c h o w ) and water, and housed in the l a b o r a t o r y in individual cages for at least two weeks b e f o r e experimental t r e a t m e n t . Light onset and offset in the c o l o n y r o o m were at 6:00 a,m. and t):00 p.m. respectively. All e x p e r i m e n t a l t r e a t m e n t s were given b e t w e e n 11:00 a.m, and 4 : 0 0 p.m. These rats were run by t w o e x p e r i m e n t e r s w h o were not a c q u a i n t e d with previous research on this phenomenon. S e v e n t y - t w o animals received one-way avoidance training ( A I ) with a t o n e CS. A tone and inescapable and unavoidable f o o t s h o c k were p r e s e n t e d in a s e m i r a n d o m order in the start box o f the one-way avoidance apparatus for 72 additional animals ¢RTS, i.e., r a n d o m t o n e and shock). One-half o f these two groups was given one-way avoidance training l0 rain, 3.5 hr, or 24 hr later ( h e r e a f t e r referred to as the retest) while lhe remaining animals were sacrificed at one o f these time intervals and their brains later analyzed for NE and 5-HT. There were 12 animals in each o f these 12 i n d e p e n d e n t groups. Twelve u n t r e a t e d animals were also sacrificed and their brains analyzed for NE and 5-HT.
Apparatus The walls of the one-way avoidance apparatus were Plexiglas and the floor was c o n s t r u c t e d o f 2 m m in dia. stainless steel bars placed 14 mm apart ( c e n t e r - c e n t e r ) . The start box and goal box were each 30 cm long, 12 cm wide, and 16 cm high. A 4.0 cm high hurdle and hand o p e r a t e d d o o r separated the t w o c o m p a r t m e n t s . The side o f the d o o r facing the goal box was black. Raising and lowering the d o o r b e t w e e n the two c o m p a r t m e n t s activated and terminated the CS, US, and a timer. The CS was a 75-dh 4,5000 (TPS tone generated by a Mallory Sonalert (Model SC62gH) placed 3 0 c m above the apparatus. The US was a 1.5-ma f o o t s h o c k generated by a Foringer s h o c k g e n e r a t o r and scrambler. Pro {'~!t.lurt' During avoidance training and the retest the start box walls were covered on the outside with m e d i u m gray paper and the goal b o x walls were covered with black paper. The animals were a d a p t e d to the start b o x for 3 0 - 6 0 sec and a trial was begun only when the animal was facing the door. Avoidance training required the rat to cross from the start box to the goal box before the 5 sec ('S-US interval elapsed. When the rat crossed into the goal b o x the d o o r was lowered and the animal was c o n f i n e d there for 5 sec. The
animals spent tile 30 sec intertrial interval in a 40 cnl high holding box. All animals were t r a n s p o r t e d m their h o m e cage and h a n d l e d by the base of the tail. The AT animals were trained to one avoidance following at least one escape and r e t u r n e d to the c o l o n y r o o m until sacrificed or receiving a retest consisting of 15 additional avoidance training trials 10 rain. 3.5 hr or 24 br later. A total o f 36 AT animals (n = 6 per g r o u p ) received avoidance training and the retest before any of the RTS animals were run. This p r o c e d u r e was followed because the CS and tJS were p r e s e n t e d in the start box o f the avoidance a p p a r a t u s for the RTS animals and it was. t h e r e f o r e , impossible to use a y o k e d c o n t r o l design. l h u s . the n u m b e r o f ('Ss and l.!Ss and d u r a t i o n of the USs received by the RTS animals was d e t e r m i n e d on the basis of the avoidance training results o f the first 36 AT animals. These AT animals received a mean o f 5.2 shocks with a mean d u r a t i o n of 1 . 1 , 2 . 7 , 1.0, 0.7 and 0.5 sec respectively before an avoidance. The RTS animals received five inescapable and unavoidable shocks of these d u r a t i o n s . Duration o f the five tones was always 5 sec and tone and shock were separated by 5 15 sec. T h e g r a y paper was r e m o v e d from the start box walls and a white card was placed over the start box d o o r when animals received the ('S and US at r a n d o m in the start box. The 30 sec intertrial interval was s p e n t in the same holding box used during training and the retest. The RTS animals were r e t u r n e d to the c o l o n y r o o m until sacrificed or receiving 15 retest trials, as described above. 10 rain, 3.5 hr, or 24 hr later. Sacrificed animals were t r a n s p o r t e d from the colony r o o m to a r o o m adjoining the e x p e r i m e n t a l r o o m and decapitated with large shears. Their brains were removed and i m m e d i a t e l y frozen at 17"(' before d e t e r m i n a t i o n of e n d o g e n o u s NE and 5-lIT by the m e t h o d of I,averty and Taylor [ 9 ] . The brains were coded and NE and 5-11"1 analysis was d o n e blind. I'~,l'iNU l ,'i ,%
Behavioral The median n u m b e r o f trials to the first avoidance during one-way avoidance training was not significantly different for the first (6.1) and last (5.0) 36 AT animals. These results indicate that the n u m b e r and d u r a t i o n o f shocks received by RTS animals c o n s t i t u t e d an a d q u a t e c o n t r o l . Median n u m b e r o f trials to the first avoidance during training for animals given the retest ( 5 . 2 ) o r sacrificed ( 4 . 7 ) a l s o did not differ significantly. T h e r e f o r e , retested and sacrificed animals received a similar n u m b e r o f f o o t s h o c k s . Since n u m b e r o f avoidances during the retest and trials to the first avoidance yielded a similar p a t t e r n o f results only trials to the first avoidance are r e p o r t e d . Figure 1 presents the median n u m b e r of trials to the first avoidance during the retest for the two t r e a t m e n t groups at each of the three retest intervals. The heavy black dol represents the median n u m b e r of trials to the first avoidance during avoidance training for sacrificed and retested animals. Inspection o f Fig. 1 suggests that animals retested I0 min and 2 4 h r a f t e r avoidance training or f o o t s h o c k required fewer trials to the first avoidance than animals retested 3.5 hr later. Mann Whitney I.~ tests revealed that animals retested 10rain and 24 hr after avoidance training made the first avoidance in fewer Irials than those retested 3.5 hr later (p-~0.01). Rats retested 10 rain and 24 hr after fools h o c k also avoided in significantly fewer trials than animals retested 3 . 5 h r a f t e r f o o l s h o c k l / ) , 0 . 0 1 ) . C o m p a r i s o n s
BRAIN NOREPINEPHRINE
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the p r e s e n t m e t h o d of analysis 117]. A 2 × 3 factorial analysis of variance of the NE data revealed n o significant effects of prior t r e a t m e n t or the t r e a t m e n t - s a c r i f i c e interval ( p < 0 . 1 0 ) . C o m p a r i s o n s h e t w e e n n o n s h o c k e d c o n t r o l s and each t r e a t m e n t g r o u p i n d i c a t e d t h a t AT-10 min, AT-3.5 hr, and RTS-3.5 animals had signficantly lower NE levels ( p < 0 . 0 1 ; one-tailed). T h e RTS-10 min animals also had l o w e r NE levels t h a n c o n t r o l s b u t this d i f f e r e n c e was of lesser m a g n i t u d e ( p < 0 . 0 5 ) .
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RETEST INTERVAL FIG. I. Median number of trials to the first avoidance for AT and R-T5 animals tested at one of three retest intervals. The heavy black dot is the median number of trials to the first avoidance of AT animals during avoidance training. b e t w e e n AT and RTS animals at each retest interval were n o n s i g n i f i c a n t . T h u s a U shaped f u n c t i o n of a p p r o x i m a t e l y the same m a g n i t u d e was f o u n d for rats receiving a v o i d a n c e t r a i n i n g a n d the CS and US in the start box. As would be e x p e c t e d , Fig. 1 also indicates t h a t A T animals required fewer trials to the first a v o i d a n c e w h e n r e t e s t e d 10 min and 24 hr a f t e r t r a i n i n g t h a n d u r i n g avoidance training, l l o w e v e r , RTS animals also a p p e a r to require fev, er trials to the first a v o i d a n c e w h e n retested 10 min and 24 hr a f t e r f o o t s h o c k t h a n A T animals d u r i n g a v o i d a n c e training. M a n n - W h i t n e y U tests revealed t h a t A T and RTS animals r e t e s t e d 10 min and 24 hr a f t e r t r e a t m e n t required fev~er trials to the first a v o i d a n c e t h a n sacrificed animals d u r i n g a v o i d a n c e t r a i n i n g ( p < 0 . 0 5 ; in these c o m p a r i s o n s a v o i d a n c e t r a i n i n g data of 12 r a n d o m l y selected sacrificed animals were i n c l u d e d to avoid m i x i n g c o r r e l a t e d and u n c o r r e l a t e d data and to form samples of equal size). Trained and foot s h o c k e d animals r e t e s t e d 3.5 hr after t r e a t m e n t did not differ significantly from these sacrificed animals. These results i n d i c a t e t h a t w h e n retested 1 0 r a i n and 2 4 h r b u t n o t 3.5 hr later b o t h A T and RTS animals r e q u i r e d fewer trials to the first awfidance t h a n naive animals d u r i n g a v o i d a n c e training.
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T R E A T M E N T - SACRIFICE INTERVAL HG. 2. Mean 5-HT and NE in #g/g of brain tissue for nonshocked controls (black dot). AT, and RTS animals sacrificed at one of the three intervals after treatment. Verticle lines _*SEM.
I)ISCUSSION
('heroical T h e u p p e r panel of Fig. 2 p r e s e n t s m e a n 5-HT levels in ug/g o f brain tissue for t h e c o n t r o l g r o u p and six experim e n t a l groups. As can be seen, 5-1tT was n o t altered b y a v o i d a n c e t r a i n i n g or f o o t s h o c k . A 2 × 3 factorial analysis of variance of the 5-1qT data revealed n o significant differences. T-tests b e t w e e n n o n s h o c k e d c o n t r o l s and each of t h e six e x p e r i m e n t a l g r o u p s were also n o n s i g n i f i c a n t . "['he l o w e r panel of Fig. 2 shows m e a n NE levels in of brain tissue for the c o n t r o l group and six e x p e r i m e n t a l groups. I n s p e c t i o n of Fig. 2 suggests t h a t trained and stressed animals have lower NE levels 1 0 r a i n and 3.5 hr a f t e r t r e a t m e n t b u t n o t 24 hr later. N o r e p i n e p h r i n e values for c o n t r o l s are similar to t h o s e r e p o r t e d previously with
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T h e p r e s e n t behavioral results i n d i c a t e t h a t a U-shaped a v o i d a n c e f u n c t i o n of a p p r o x i m a t e l y the same m a g n i t u d e o c c u r r e d following a v o i d a n c e t r a i n i n g and f o o t s h o c k in the start b o x of the a v o i d a n c e a p p a r a t u s . F u r t h e r m o r e . animals retested 10 min and 24 hr b u t n o t 3.5 hr a f t e r a v o i d a n c e t r a i n i n g or f o o t s h o c k were s u p e r i o r to naive animals. These results i n d i c a t e t h a t p o o r aw)idance at i n t e r m e d i a t e retest intervals can o c c u r in the a b s e n c e of escape and a v o i d a n c e training. P o o r e r a v o i d a n c e has previously b e e n f o u n d with rats given unsignaled, u n a v o i d a b l e , and inescapable s h o c k in the start b o x of a one-way a v o i d a n c e a p p a r a t u s 4 hr prior to a v o i d a n c e t r a i n i n g [ 1 ] . In c o n t r a s t to these results, Brush [5] f o u n d no U shaped f u n c t i o n w h e n unsignaled shock
BAUER
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was given in a h o m o g e n e o u s shuttle b o x prior to avoidance training. Since results in the one-way avoidance task suggest that avoidance deficits at i n t e r m e d i a t e intervals are due to high levels o f fear c o n d i t i o n e d to specific apparatus cues failure to find this effect in a h o m o g e n e o u s s h u t t l e b o x following unsignaled shock may be due to the animal's inability to discriminate the s h o c k e d from the n o n s h o c k e d side. The b i o c h e m i c a l results indicate that rats receiving avoidance training and unavoidable and inescapable shock in the start b o x have lower whole brain NE levels than n o n s h o c k e d controls 10 min and 3.5 hr after these treatm e n t s but not 24 hr later. These results are c o n s i s t e n t with a n u m b e r o f o t h e r r e p o r t s indicating that f o o t s h o c k lowers NE [4,11] Failure to find any change in 5-HT following avoidance training or inescapable shock is also c o n s i s t e n t with previous reports [4, 11, 18]. Since n e i t h e r NE nor 5-HT e x h i b i t e d a U or inverted U f u n c t i o n following avoidance training or f o o t s h o c k , alteration o f these amines is a p p r e n t l y not related to inferior avoidance at interm e d i a t e retest intervals. These results suggest that changes in whole brain NE or 5-HT are not the m e c h a n i s m responsible for either a deficient coping response or state d e p e n d e d m e m o r y retrieval. It also appears unlikely that regional changes in brain NE can a c c o u n t for p o o r e r avoidance at i n t e r m e d i a t e intervals because f o o t s h o c k reduces NE u n i f o r m l y t h r o u g h o u t the brain 13,4]. l t o w e v e r , these results do not c o m p l e t e l y eliminate NE or 5-HT from c o n s i d e r a t i o n since t u r n o v e r rates o f NE and 5-HT are
increased a f t e r f o o t s h o c k [ 4 , 1 8 ] . Increased t u r n o v e r o f NE a n d / o r 5-lIT 1 - 8 hr following f o o t s h o c k may a c c o u n t for p o o r e r avoidance at these i n t e r m e d i a t e retest intervals. The present NE results are at variance with the r e p o r t e d NE increase following avoidance training but no change in y o k e d c o n t r o l s receiving unavoidable and inescapable shock [ l t ) ] . A n u m b e r o f procedural d i f f e r e n c e s could a c c o u n t for these discrepant findings. First, in the Weiss, S t o n e and Harrell s t u d y shock was a d m i n i s t e r e d to the tail and increased s y s t e m a t i c a l l y across trials whereas in the present e x p e r i m e n t f o o t s h o c k was used and i n t e n s i t y remained c o n s t a n t . A wide variely o f stress situations are r e p o r t e d to reduce brain NE and it is, t h e r e f o r e , unlikely that these d i f f e r e n c e s can a c c o u n t for the conflicting results. As in the present e x p e r i m e n t , rats in E x p e r i m e n t 1 o f the Weiss, S t o n e and ltarrell s t u d y received a p p r o x i m a t e l y five shocks but were given a total o f 70 trials requiring 2 . 5 - 3 hr. T h e r e f o r e , time s p e n t in the avoidance apparatus and level o f avoidance training differed considerably in the two studies. As few as five f o o t s h o c k s a m i n u t e for 5 m i n [ 11 ] and six per m i n u t e for 15 min [4] are r e p o r t e d to decrease e n d o g e n o u s NE but six shocks per m i n u t e for 1 0 m i n alternated with 20 min o f rest for 3 hr p r o d u c e no change [ 1 8 ] . T h e r e f o r e , there is a possibility that c o n f i n e m e n t in the avoidance apparatus for periods with no shock can a c c o u n t for these discrepant findings. However. level o f avoidance training d i f f e r e d considerably and this factor remains a likely possibility.
REFERENCES 1. Anisman, H and G. T. Waller. Effects of conflicting response requirements and shock-compartment confinement on the Kamin effect in rats. J. comp. physiol. Psychol. 77:240 244, 1971. 2. Barchas, J. 1). and D. X. Freedman. Brain amines: Response to physiological stress. Biochem. Pharmac. 12:1232-1235, 1963. 3. Bliss, E. I. and J. Zwanziger. Brain amines and emotional stress. J. psychiat. Res. 4: 189-198, 1966. 4. Bliss, E. L, J. Ailion and J. Zwanziger. Metabolism of norepinephrine, serotonin, and dopamine in rat brain with stress. J. Pharmae. exp. Ther. 164:122 134, 1968. 5. Brush, F. R. Retention of aversively motivated behavior. In: Aversive Conditioning and Learning, edited by I.. R. Brush. New York: Academic Press, 1971, pp. 401 465. 6. Denny, M. R. and R. E. Ditchman. ]'he locus of maximal "Kamin effect" in rats. J. comp physiol. Psychol. 55: 1069-1070, 1962. 7. Kasper-Pandi, P., R. ltansing and I). R. Usher. The effect of dexamethasone blockage of ACTH release on avoidance learning. Physiol. Behav. 5:361--363, 1970. 8. Kriekhaus, E. E., N. E. Miller and P. Zimmerman. Reduction of freezing behavior and improvement of shock avoidance by d-amphetamine. J. comp. physiol. Psychol. 60: 36-40, 1965. 9. Laverty, R. and K. M. Taylor. The flurometric assay of catecholamines and related compounds: Improvements and extension to the hydroxyindole technique. Analyt. Bioehem. 22: 269 2 7 9 , 1968. 10. l,evine, S. and 1.. R. Brush. Adrenocortical activity and avoidance learning as a function of time after avoidance training. Physiol. Behav. 2: 385- 388, 1967. 11. Maynert. E. W. and R. Levi. Stress-induced release of brain norepinephrine and its inhibiton by drugs. J. Pharmac exp. l'her. 143:90 -95, 1964.
12. Overton, D. A. Dissociated learning in drug states (state dependent learning). In: Psychopharmacology: A review o f progress. 1957 1967, edited by D. H. Efron and E. Usdin. (Public Ilealth Service Publication No. 18361 Washington, D. C.: United State Government Printing Office, 1968. 13. Snider, N., H. A. Marquis. M. Black and M. 11. Sul)oski. Adrenal corticosteroids and the Kamin effect. Psychon. Sci. 22: 3119-310. 1971. 14. Spear, N. t:., S. B. Klein and E. P. Riley. The Kamin effect as "state-dependent learning": Memory-retrieval failure in the rat. J. comp. physiol. Psychol. 55:416 -425, 1971. 15. Stone, E. A. Swim-stress-induced inactivity: Relation to body temperature and brain norepinephrine, and effect of d-amphetamine. Psychosom. Med. 32: 51--59, 1970. 16. Suboski, M. D., H. A. Marquis, M. Black and P. Platenius. Adrenal and amygdala function in the incubation of aversively conditioned responses. Physiol. Behav. 5:283 289, 1970. 17. Taylor, K. M. and S. H. Snyder. Differential effects of D-and l,-amphetamine on behavior and on catecholamine disposition in dopamine and norepinephrine containing neurons of rat brain. Brain Res. 28:295 -309, 1971. 18. Thierry, A., I.. Javoy, J. Glowinski and S. S. Kety. Effects of stress on the metabolism of norepinephrine, dopamine and serotonin in the central nerw~us system of the rat. 1. Modifications of norepinephrine turnover. J. Pharmac. exp. l'her. 163: 163-171, 1968. 19. Weiss, J. M., E. A. Stone and N. Ilarrell. Coping behavior and brain norepinephrine level in rats. J. comp physiol. Psychol. 72:153 160, 1970.
Brain Norepinephrine and 5-Hydroxytryptamine as a Function of Time after Avoidance Training and Footshock R I C H A R D H. B A U E R 2
Department o f Psychiatry, School o f Medicine. University o f California, Los Angeles, California 90024
( R e c e i v e d 19 F e b r u a r y 1973)
BAUER, R. H. Brain norepinephrme and 5-hydroxytryptamine as a function of time after avoidance training and footshock. PHARMAC. BIOCHEM. BEHAV. 1 (6) 6 1 5 - 6 1 8 , 1973.-Male Sprague-Dawley rats acquired a one-way avoidance task more rapidly 10 min and 24 hr after partial avoidance training or inescapable shock in the start box than 3.5 hr after these treatments. Endogenous levels of brain 5-hydroxytryptamine (5-HT) were not altered 10 min, 3.5 hr, or 24 hr after avoidance training or inescapable shock in the start box. Norepinephrine (NE) was lower 10 rain and 3.5 hr after training and inescapable shock but not 24 hr later. Since neither 5-HT nor NE exhibited a U or inverted U function following avoidance training or footshock, these results indicate that endogenous levels of 5-HT and NE are not related to poorer avoidance at intermediate retest intervals. Avoidance
Norepinephrine
5-Hydroxyt ryptamine
[ 10]. However, m o r e r e c e n t evidence has n o t s u p p o r t e d t h e h y p o t h e s i s t h a t a l t e r a t i o n o f the p i t u i t a r y - a d r e n a l s y s t e m is related to a v o i d a n c e deficits I - 8 hr after training [7, 13, 1 6 ] . T h e r e f o r e , e v i d e n c e relating t h e p i t u i t a r y adrenal s y s t e m to this p h e n o m e n o n is at best controversial. A l t h o u g h the p i t u i t a r y - a d r e n a l s y s t e m m a y n o t a c c o u n t for p o o r e r a v o i d a n c e 1- 8 hr a f t e r training, e v i d e n c e suggests t h a t a l t e r a t i o n of brain n o r e p i n e p h r i n e (NE) m a y be related to this p h e n o m e n o n . A n u m b e r of studies have r e p o r t e d r e d u c e d levels o f e n d o g e n o u s b r a i n NE s h o r t l y a f t e r a variety of stressors [4,111 and a f u r t h e r decrease 2 - 4 hr f o l l o w i n g stress [ 2 , 1 5 ] . Weiss, S t o n e and Harrell [19] f o u n d t h a t rats w h i c h c o u l d escape and avoid s h o c k have increased NE w h e n sacrificed 2 0 - 4 0 min a f t e r t r a i n i n g b u t animals t h a t could n e i t h e r escape n o r avoid s h o w e d n o change. Drugs w h i c h release NE also increase activity and a v o i d a n c e [8, 15, 17]. These results suggest t h a t poststress r e d u c t i o n of brain NE can p e r h a p s a c c o u n t for a v o i d a n c e deficits 1 - 8 hr a f t e r training. T h e m a j o r p u r p o s e o f the present e x p e r i m e n t is to determ i n e if rats sacrificed 3.5 h r a f t e r a v o i d a n c e t r a i n i n g have lower levels o f e n d o g e n o u s brain NE a n d / o r 5 - h y d r o x y t r y p t a m i n e (5-HT) t h a n t h o s e sacrificed 10 min or 24 hr a f t e r training. In a d d i t i o n , rats given inescapable and una v o i d a b l e s h o c k in the start b o x of a one-way a v o i d a n c e a p p a r a t u s 5 rain and 24 hr prior to a v o i d a n c e training
A T T H E p r e s e n t t i m e t h e r e is c o n s i d e r a b l e c o n t r o v e r s y regarding the t h e o r e t i c a l i n t e r p r e t a t i o n of p o o r e r a v o i d a n c e at i n t e r m e d i a t e retest intervals ( I - 8 h r ) as c o m p a r e d to t h o s e e i t h e r earlier or later. A n u m b e r of papers suggested t h a t p o o r a v o i d a n c e at i n t e r m e d i a t e intervals is d u e to an initial increase and s u b s e q u e n t decrease o f fear [ 6 ] . decreased l o c o m o t i o n and an increase in i n c o m p a t i b l e responses, w h i c h i n t e r f e r e with a v o i d a n c e , are p r e s u m e d to b e related to fear. More r e c e n t l y , Spear, Klein, and Riley [ 14] have suggested t h a t physiological changes w h i c h follow a stressful e v e n t m a y be r e s p o n s i b l e for a m e m o r y retrieval deficit 1 - 8 h r a f t e r aversive c o n d i t i o n i n g . I n t e r n a l c o n d i tions p r e s e n t d u r i n g learning are a s s u m e d t o b e altered at i n t e r m e d i a t e r e t e n t i o n intervals and c o n s e q u e n t l y m e m o r y o f originial learning is m o r e difficult to retrieve. Thus, p o o r e r a v o i d a n c e I -.-8 hr a f t e r t r a i n i n g m a y b e m e d i a t e d b y a m e c h a n i s m similar to s t a t e d e p e n d e n t learning f o u n d w i t h various drugs [ 1 2 ] . At the behavioral level, a n u m b e r of workers have e x a m i n e d this U-shaped r e t e n t i o n f u n c t i o n b u t relatively litzle research has b e e n d e v o t e d to investigating physiological changes w h i c h m i g h t a c c o u n t for this p h e n o m e n o n . Earlier papers suggested t h a t a decrease in c o r t i c o s t e r o n e , w h i c h parallels the initial p e r f o r m a n c e d e c r e m e n t b u t n o t t h e s u b s e q u e n t i m p r o v e m e n t in a v o i d a n c e , was r e s p o n s i b l e for the a n i m a l ' s i n a b i l i t y to cope b e h a v i o r a l l y with stress
This research was supported in part by Public Health Service Grant NIMH-06960-11 to Daniel Sheer. The author gratefully acknowledges the assistance of Edward Butler, William Jones, and Peggy Hall. 2 Requests for reprints should be sent to Richard H. Bauer, Department of Psychiatry, School of Medicine, University of California, Los Angeles, California, 90024. 615
616
BAUER
acquired the one-way avoidance task m o r e rapidly than rats trained 4 hr after this t r e a t m e n t [ I ] . Inescapable and unavoidable f o o t s h o c k was, t h e r e f o r e , a d m i n i s t e r e d in the start box of a one-way avoidance apparatus in an a t t e m p t to replicate these results and d e t e r m i n e if NE or 5-liT o f trained and stressed animals is d i f f e r e n t l y a f f e c t e d . Thus, the present e x p e r i m e n t e x a m i n e d the possibility that altered brain NE a n d / o r 5-1IT are related to p o o r e r avoidance at i n t e r m e d i a t e retest intervals and a test of the generality o f the Weiss, Stone, and Harrell [1~)] results. MET1 I()1)
Animals and Experimental Design The animals were 156 e x p e r i m e n t a l l y naive 1 2 0 - 1 8 0 d a y o l d m a l e albino Sprague-Dawley rats weighing 3 0 0 - - 4 5 0 g. These animals were m a i n t a i n e d on ad lib food (Pt, rina rat c h o w ) and water, and housed in the l a b o r a t o r y in individual cages for at least two weeks b e f o r e experimental t r e a t m e n t . Light onset and offset in the c o l o n y r o o m were at 6:00 a,m. and t):00 p.m. respectively. All e x p e r i m e n t a l t r e a t m e n t s were given b e t w e e n 11:00 a.m, and 4 : 0 0 p.m. These rats were run by t w o e x p e r i m e n t e r s w h o were not a c q u a i n t e d with previous research on this phenomenon. S e v e n t y - t w o animals received one-way avoidance training ( A I ) with a t o n e CS. A tone and inescapable and unavoidable f o o t s h o c k were p r e s e n t e d in a s e m i r a n d o m order in the start box o f the one-way avoidance apparatus for 72 additional animals ¢RTS, i.e., r a n d o m t o n e and shock). One-half o f these two groups was given one-way avoidance training l0 rain, 3.5 hr, or 24 hr later ( h e r e a f t e r referred to as the retest) while lhe remaining animals were sacrificed at one o f these time intervals and their brains later analyzed for NE and 5-HT. There were 12 animals in each o f these 12 i n d e p e n d e n t groups. Twelve u n t r e a t e d animals were also sacrificed and their brains analyzed for NE and 5-HT.
Apparatus The walls of the one-way avoidance apparatus were Plexiglas and the floor was c o n s t r u c t e d o f 2 m m in dia. stainless steel bars placed 14 mm apart ( c e n t e r - c e n t e r ) . The start box and goal box were each 30 cm long, 12 cm wide, and 16 cm high. A 4.0 cm high hurdle and hand o p e r a t e d d o o r separated the t w o c o m p a r t m e n t s . The side o f the d o o r facing the goal box was black. Raising and lowering the d o o r b e t w e e n the two c o m p a r t m e n t s activated and terminated the CS, US, and a timer. The CS was a 75-dh 4,5000 (TPS tone generated by a Mallory Sonalert (Model SC62gH) placed 3 0 c m above the apparatus. The US was a 1.5-ma f o o t s h o c k generated by a Foringer s h o c k g e n e r a t o r and scrambler. Pro {'~!t.lurt' During avoidance training and the retest the start box walls were covered on the outside with m e d i u m gray paper and the goal b o x walls were covered with black paper. The animals were a d a p t e d to the start b o x for 3 0 - 6 0 sec and a trial was begun only when the animal was facing the door. Avoidance training required the rat to cross from the start box to the goal box before the 5 sec ('S-US interval elapsed. When the rat crossed into the goal b o x the d o o r was lowered and the animal was c o n f i n e d there for 5 sec. The
animals spent tile 30 sec intertrial interval in a 40 cnl high holding box. All animals were t r a n s p o r t e d m their h o m e cage and h a n d l e d by the base of the tail. The AT animals were trained to one avoidance following at least one escape and r e t u r n e d to the c o l o n y r o o m until sacrificed or receiving a retest consisting of 15 additional avoidance training trials 10 rain. 3.5 hr or 24 br later. A total o f 36 AT animals (n = 6 per g r o u p ) received avoidance training and the retest before any of the RTS animals were run. This p r o c e d u r e was followed because the CS and tJS were p r e s e n t e d in the start box o f the avoidance a p p a r a t u s for the RTS animals and it was. t h e r e f o r e , impossible to use a y o k e d c o n t r o l design. l h u s . the n u m b e r o f ('Ss and l.!Ss and d u r a t i o n of the USs received by the RTS animals was d e t e r m i n e d on the basis of the avoidance training results o f the first 36 AT animals. These AT animals received a mean o f 5.2 shocks with a mean d u r a t i o n of 1 . 1 , 2 . 7 , 1.0, 0.7 and 0.5 sec respectively before an avoidance. The RTS animals received five inescapable and unavoidable shocks of these d u r a t i o n s . Duration o f the five tones was always 5 sec and tone and shock were separated by 5 15 sec. T h e g r a y paper was r e m o v e d from the start box walls and a white card was placed over the start box d o o r when animals received the ('S and US at r a n d o m in the start box. The 30 sec intertrial interval was s p e n t in the same holding box used during training and the retest. The RTS animals were r e t u r n e d to the c o l o n y r o o m until sacrificed or receiving 15 retest trials, as described above. 10 rain, 3.5 hr, or 24 hr later. Sacrificed animals were t r a n s p o r t e d from the colony r o o m to a r o o m adjoining the e x p e r i m e n t a l r o o m and decapitated with large shears. Their brains were removed and i m m e d i a t e l y frozen at 17"(' before d e t e r m i n a t i o n of e n d o g e n o u s NE and 5-lIT by the m e t h o d of I,averty and Taylor [ 9 ] . The brains were coded and NE and 5-11"1 analysis was d o n e blind. I'~,l'iNU l ,'i ,%
Behavioral The median n u m b e r o f trials to the first avoidance during one-way avoidance training was not significantly different for the first (6.1) and last (5.0) 36 AT animals. These results indicate that the n u m b e r and d u r a t i o n o f shocks received by RTS animals c o n s t i t u t e d an a d q u a t e c o n t r o l . Median n u m b e r o f trials to the first avoidance during training for animals given the retest ( 5 . 2 ) o r sacrificed ( 4 . 7 ) a l s o did not differ significantly. T h e r e f o r e , retested and sacrificed animals received a similar n u m b e r o f f o o t s h o c k s . Since n u m b e r o f avoidances during the retest and trials to the first avoidance yielded a similar p a t t e r n o f results only trials to the first avoidance are r e p o r t e d . Figure 1 presents the median n u m b e r of trials to the first avoidance during the retest for the two t r e a t m e n t groups at each of the three retest intervals. The heavy black dol represents the median n u m b e r of trials to the first avoidance during avoidance training for sacrificed and retested animals. Inspection o f Fig. 1 suggests that animals retested I0 min and 2 4 h r a f t e r avoidance training or f o o t s h o c k required fewer trials to the first avoidance than animals retested 3.5 hr later. Mann Whitney I.~ tests revealed that animals retested 10rain and 24 hr after avoidance training made the first avoidance in fewer Irials than those retested 3.5 hr later (p-~0.01). Rats retested 10 rain and 24 hr after fools h o c k also avoided in significantly fewer trials than animals retested 3 . 5 h r a f t e r f o o l s h o c k l / ) , 0 . 0 1 ) . C o m p a r i s o n s
BRAIN NOREPINEPHRINE
AFTER AVOIDANCE TRAINING
the p r e s e n t m e t h o d of analysis 117]. A 2 × 3 factorial analysis of variance of the NE data revealed n o significant effects of prior t r e a t m e n t or the t r e a t m e n t - s a c r i f i c e interval ( p < 0 . 1 0 ) . C o m p a r i s o n s h e t w e e n n o n s h o c k e d c o n t r o l s and each t r e a t m e n t g r o u p i n d i c a t e d t h a t AT-10 min, AT-3.5 hr, and RTS-3.5 animals had signficantly lower NE levels ( p < 0 . 0 1 ; one-tailed). T h e RTS-10 min animals also had l o w e r NE levels t h a n c o n t r o l s b u t this d i f f e r e n c e was of lesser m a g n i t u d e ( p < 0 . 0 5 ) .
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RETEST INTERVAL FIG. I. Median number of trials to the first avoidance for AT and R-T5 animals tested at one of three retest intervals. The heavy black dot is the median number of trials to the first avoidance of AT animals during avoidance training. b e t w e e n AT and RTS animals at each retest interval were n o n s i g n i f i c a n t . T h u s a U shaped f u n c t i o n of a p p r o x i m a t e l y the same m a g n i t u d e was f o u n d for rats receiving a v o i d a n c e t r a i n i n g a n d the CS and US in the start box. As would be e x p e c t e d , Fig. 1 also indicates t h a t A T animals required fewer trials to the first a v o i d a n c e w h e n r e t e s t e d 10 min and 24 hr a f t e r t r a i n i n g t h a n d u r i n g avoidance training, l l o w e v e r , RTS animals also a p p e a r to require fev, er trials to the first a v o i d a n c e w h e n retested 10 min and 24 hr a f t e r f o o t s h o c k t h a n A T animals d u r i n g a v o i d a n c e training. M a n n - W h i t n e y U tests revealed t h a t A T and RTS animals r e t e s t e d 10 min and 24 hr a f t e r t r e a t m e n t required fev~er trials to the first a v o i d a n c e t h a n sacrificed animals d u r i n g a v o i d a n c e t r a i n i n g ( p < 0 . 0 5 ; in these c o m p a r i s o n s a v o i d a n c e t r a i n i n g data of 12 r a n d o m l y selected sacrificed animals were i n c l u d e d to avoid m i x i n g c o r r e l a t e d and u n c o r r e l a t e d data and to form samples of equal size). Trained and foot s h o c k e d animals r e t e s t e d 3.5 hr after t r e a t m e n t did not differ significantly from these sacrificed animals. These results i n d i c a t e t h a t w h e n retested 1 0 r a i n and 2 4 h r b u t n o t 3.5 hr later b o t h A T and RTS animals r e q u i r e d fewer trials to the first awfidance t h a n naive animals d u r i n g a v o i d a n c e training.
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I)ISCUSSION
('heroical T h e u p p e r panel of Fig. 2 p r e s e n t s m e a n 5-HT levels in ug/g o f brain tissue for t h e c o n t r o l g r o u p and six experim e n t a l groups. As can be seen, 5-1tT was n o t altered b y a v o i d a n c e t r a i n i n g or f o o t s h o c k . A 2 × 3 factorial analysis of variance of the 5-1qT data revealed n o significant differences. T-tests b e t w e e n n o n s h o c k e d c o n t r o l s and each of t h e six e x p e r i m e n t a l g r o u p s were also n o n s i g n i f i c a n t . "['he l o w e r panel of Fig. 2 shows m e a n NE levels in of brain tissue for the c o n t r o l group and six e x p e r i m e n t a l groups. I n s p e c t i o n of Fig. 2 suggests t h a t trained and stressed animals have lower NE levels 1 0 r a i n and 3.5 hr a f t e r t r e a t m e n t b u t n o t 24 hr later. N o r e p i n e p h r i n e values for c o n t r o l s are similar to t h o s e r e p o r t e d previously with
ug/g
T h e p r e s e n t behavioral results i n d i c a t e t h a t a U-shaped a v o i d a n c e f u n c t i o n of a p p r o x i m a t e l y the same m a g n i t u d e o c c u r r e d following a v o i d a n c e t r a i n i n g and f o o t s h o c k in the start b o x of the a v o i d a n c e a p p a r a t u s . F u r t h e r m o r e . animals retested 10 min and 24 hr b u t n o t 3.5 hr a f t e r a v o i d a n c e t r a i n i n g or f o o t s h o c k were s u p e r i o r to naive animals. These results i n d i c a t e t h a t p o o r aw)idance at i n t e r m e d i a t e retest intervals can o c c u r in the a b s e n c e of escape and a v o i d a n c e training. P o o r e r a v o i d a n c e has previously b e e n f o u n d with rats given unsignaled, u n a v o i d a b l e , and inescapable s h o c k in the start b o x of a one-way a v o i d a n c e a p p a r a t u s 4 hr prior to a v o i d a n c e t r a i n i n g [ 1 ] . In c o n t r a s t to these results, Brush [5] f o u n d no U shaped f u n c t i o n w h e n unsignaled shock
BAUER
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was given in a h o m o g e n e o u s shuttle b o x prior to avoidance training. Since results in the one-way avoidance task suggest that avoidance deficits at i n t e r m e d i a t e intervals are due to high levels o f fear c o n d i t i o n e d to specific apparatus cues failure to find this effect in a h o m o g e n e o u s s h u t t l e b o x following unsignaled shock may be due to the animal's inability to discriminate the s h o c k e d from the n o n s h o c k e d side. The b i o c h e m i c a l results indicate that rats receiving avoidance training and unavoidable and inescapable shock in the start b o x have lower whole brain NE levels than n o n s h o c k e d controls 10 min and 3.5 hr after these treatm e n t s but not 24 hr later. These results are c o n s i s t e n t with a n u m b e r o f o t h e r r e p o r t s indicating that f o o t s h o c k lowers NE [4,11] Failure to find any change in 5-HT following avoidance training or inescapable shock is also c o n s i s t e n t with previous reports [4, 11, 18]. Since n e i t h e r NE nor 5-HT e x h i b i t e d a U or inverted U f u n c t i o n following avoidance training or f o o t s h o c k , alteration o f these amines is a p p r e n t l y not related to inferior avoidance at interm e d i a t e retest intervals. These results suggest that changes in whole brain NE or 5-HT are not the m e c h a n i s m responsible for either a deficient coping response or state d e p e n d e d m e m o r y retrieval. It also appears unlikely that regional changes in brain NE can a c c o u n t for p o o r e r avoidance at i n t e r m e d i a t e intervals because f o o t s h o c k reduces NE u n i f o r m l y t h r o u g h o u t the brain 13,4]. l t o w e v e r , these results do not c o m p l e t e l y eliminate NE or 5-HT from c o n s i d e r a t i o n since t u r n o v e r rates o f NE and 5-HT are
increased a f t e r f o o t s h o c k [ 4 , 1 8 ] . Increased t u r n o v e r o f NE a n d / o r 5-lIT 1 - 8 hr following f o o t s h o c k may a c c o u n t for p o o r e r avoidance at these i n t e r m e d i a t e retest intervals. The present NE results are at variance with the r e p o r t e d NE increase following avoidance training but no change in y o k e d c o n t r o l s receiving unavoidable and inescapable shock [ l t ) ] . A n u m b e r o f procedural d i f f e r e n c e s could a c c o u n t for these discrepant findings. First, in the Weiss, S t o n e and Harrell s t u d y shock was a d m i n i s t e r e d to the tail and increased s y s t e m a t i c a l l y across trials whereas in the present e x p e r i m e n t f o o t s h o c k was used and i n t e n s i t y remained c o n s t a n t . A wide variely o f stress situations are r e p o r t e d to reduce brain NE and it is, t h e r e f o r e , unlikely that these d i f f e r e n c e s can a c c o u n t for the conflicting results. As in the present e x p e r i m e n t , rats in E x p e r i m e n t 1 o f the Weiss, S t o n e and ltarrell s t u d y received a p p r o x i m a t e l y five shocks but were given a total o f 70 trials requiring 2 . 5 - 3 hr. T h e r e f o r e , time s p e n t in the avoidance apparatus and level o f avoidance training differed considerably in the two studies. As few as five f o o t s h o c k s a m i n u t e for 5 m i n [ 11 ] and six per m i n u t e for 15 min [4] are r e p o r t e d to decrease e n d o g e n o u s NE but six shocks per m i n u t e for 1 0 m i n alternated with 20 min o f rest for 3 hr p r o d u c e no change [ 1 8 ] . T h e r e f o r e , there is a possibility that c o n f i n e m e n t in the avoidance apparatus for periods with no shock can a c c o u n t for these discrepant findings. However. level o f avoidance training d i f f e r e d considerably and this factor remains a likely possibility.
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