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5,7-Dihydroxytryptamine lesions of the ascending 5-hydroxytryptamine pathways: Habituation, motor activity and agonistic behavior
Pharmacology Biochemistry & Behavior, Vol. 7, pp. 205--210. Copyright © 1977 by AN KHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
5,7-Dihydroxytryptamine Lesions of the Ascending 5-Hydroxytryptamine Pathways: Habituation, Motor Activity and Agonistic Behavior I KJELL
HOLE, GEORG E S P O L I N J O H N S O N A N D O D D - G E I R
BERGE
Institute o f Physiology, University o f Bergen, Norway ( R e c e i v e d 19 J a n u a r y 1 9 7 7 ) HOLE, K., G. E. JOHNSON AND O . - G . BERGE. 5,7-Dihydroxytryptamine lesions of the ascending 5-hydroxymotor activity and agonistic behavior. PHARMAC. BIOCHEM. BEHAV. 7(3) 2 0 5 - 2 1 0 , 1 9 7 7 . - Rats were injected stereotactically in mesencephalon with 5,7-dihydroxytryptamine (5,7-DHT) in the medial 5-hydroxytryptamine (5-HT) pathway (n = 8) and in the medial plus the lateral 5 - H T pathways (n = 7) or injected with vehicle (n = 8), or sham-operated (n = 8). The 5,7-DHT lesions reduced the in vitro 3H-5-HT uptake in the hypothalamus and the cortex cerebri to 2 7 - 5 1 % of control values, 3H-noradrenaline uptake was not significantly changed. 5,7-DHT lesions of the medial, and of the medial plus the lateral, 5 - H T pathways induced mouse killing behavior and increased number of boxing positions in the shock elicited fighting test. Both lesions also reduced the rate of habituation to touch, but only the lesion of the medial plus the lateral 5 - H T pathway significantly reduced the rate of habituation to acoustic stimulation. Activity in the home cage was not significantly changed by the lesions. It was concluded that selective chemical lesions of the ascending 5 - H T pathways result in prolonged habituation of the orienting response and increase in particular components of agonistic behavior. The increase in locomotor activity observed after electrolytic lesions of nucleus raphe medianus seems not to be due only to lesion of the 5 - H T neurons ascending from this nucleus.
tryptamine pathways: habituation,
5-Hydroxytryptamine Motor activity
5,7 ~Dihydroxytryptamine
Habituation of orienting
Agonistic behavior
5 - H T p a t h w a y s are due to d a m a g e o f o t h e r n e u r o n s t h a n the 5 - H T n e u r o n s . The b e h a v i o r a l effects of selective c h e m i c a l lesions o f the 5 - H T p a t h w a y s t h e r e f o r e s h o u l d be f u r t h e r investigated. In t h e p r e s e n t s t u d y the e f f e c t o f 5 , 7 - D H T lesions o f the a s c e n d i n g 5 - H T p a t h w a y s o n h a b i t u a t i o n o f t h e o r i e n t i n g response, agonistic b e h a v i o r , a n d m o t o r activity in t w o d i f f e r e n t s t i m u l u s s i t u a t i o n s in the h o m e cage, were studied.
IT HAS b e e n suggested t h a t the 5 - h y d r o x y t r y p t a m i n e ( 5 - H T ) n e u r o n s o r i g i n a t i n g in the m i d b r a i n r a p h e n u c l e i are i m p o r t a n t in c e n t r a l c o n t r o l of pain sensitivity [ 1 2 ] , m o t o r activity [9, 15, 18, 2 4 ] , h a b i t u a t i o n or sensitization to s e n s o r y s t i m u l a t i o n [3, 4, 5 ] , a n d aggressive b e h a v i o r [6, 17, 2 2 ] . In the m a j o r i t y of p r e v i o u s studies the f u n c t i o n o f the 5 - H T n e u r o n s was altered b y electrolytic lesions, p a r a c h l o r o p h e n y l a l a n i n e (PCPA) t r e a t m e n t , or o t h e r p h a r m a c o l o g i c a l m a n i p u l a t i o n s . These m e t h o d s affect o t h e r t y p e s o f n e u r o n s as well as t h e 5 - H T n e u r o n s . R e c e n t l y it was o b s e r v e d t h a t w h e n the 5 - H T n e u r o n s are l e s i o n e d selectively with, 5 , 7 - d i h y d r o x y t r y p t a mine ( 5 , 7 - D H T ) i n j e c t e d s t e r e o t a c t i c a l l y i n t o t h e 5 - H T p a t h w a y s , o p e n field l o c o m o t o r activity is r e d u c e d a n d pain sensitivity is u n c h a n g e d [ 1 5 , 1 6 ] , in c o n t r a s t to t h e increase in l o c o m o t o r activity [24] a n d increase in pain sensitivity [12] o b s e r v e d a f t e r e l e c t r o l y t i c lesions. Possibly some o f the b e h a v i o r a l changes o b s e r v e d a f t e r electrolytic lesions o f the m i d b r a i n r a p h e nuclei or the a s c e n d i n g
METHOD
Animals T h i r t y - o n e male a l b i n o rats (M/511-Wistar) weighing 240-280g at t i m e of surgery were used. T h e y were h o u s e d i n d i v i d u a l l y in c o n v e n t i o n a l rat cages and had food a n d w a t e r available ad lib. Testing was p e r f o r m e d d u r i n g the light p h a s e o f a 12 h r light-dark cycle.
J The authors gratefully acknowledge Miss Nina Holmelid and Mrs. May-Elin Figueroa for their excellent technical assistance. This research was supported in part by Norwegian Research Council for Science and the Humanities and Andssvakesakens Forskningsfond. 205
206
Surgery A K o p f s t e r e o t a x i c i n s t r u m e n t was used w i t h t h e incisor bar set 2.4 m m a b o v e the i n t e r a u r a l plane. U n d e r KETALAR anesthesia (100-200mg/kg), 5,7-DHT (5,7-dihydroxytryptamine c r e a t i n e sulfate, Regis) dissolved in 0.9% NaC1 c o n t a i n i n g a s c o r b i c acid (0,1 m g / m l ) was i n j e c t e d s t e r e o t a c t i c a l l y w i t h a 10 ul H a m i l t o n syringe in t h e region of t h e a s c e n d i n g 5 - H T b u n d l e s [ 15] in t h e mesencephalon. T h e c o o r d i n a t e s were for t h e m e d i a l 5 - H T b u n d l e (group Med, n = 8): p o s t e r i o r to b r e g m a 5.6, lateral 0.0, v e n t r a l t o t h e surface o f c o r t e x 7.5 r a m ; a n d for t h e c o m b i n e d lesions o f m e d i a l a n d lateral b u n d l e s (group Med + Lat, n = 7): p o s t e r i o r 5.6, lateral +- 0.6, a n d v e n t r a l 7.0 m m . At e a c h i n j e c t i o n site 4 ug, 5 , 7 - D H T ( c a l c u l a t e d as t h e base) in 4 ul was i n j e c t e d (1 u l / m i n ) . T h e n e e d l e was left in situ for 1 m i n a f t e r c o m p l e t i o n o f e a c h i n j e c t i o n . T w e n t y rain b e f o r e t h e i n t r a c e r e b r a l i n j e c t i o n s p r o t r i p t y l i n e HCI (Merck, S h a r p a n d D o h m e ) 25 m g / k g was i n j e c t e d IP [ 1 5 ] . C o n t r o l rats were t r e a t e d in t h e same m a n n e r as t h e 5 , 7 - D H T rats, e x c e p t t h a t t h e y were i n j e c t e d i n t r a c e r e b r a l l y w i t h t h e vehicle o n l y (group Veh, n - - 8 ) , or t h e c a n n u l a was n o t l o w e r e d i n t r a c r a n i a l l y (group S h a m , n = 8).
Histological and Bioehemical Analyses F o r t y - s i x d a y s a f t e r surgery t h e a n i m a l s were sacrificed b y d e c a p i t a t i o n . T h e b r a i n s t e m s of t h e 5 , 7 - D H T a n d vehicle i n j e c t e d rats were k e p t in 10% F o r m a l i n for one week. F r o z e n sections t h e n were cut at 30 u m and every t h i r d s e c t i o n saved a n d s t a i n e d by t h e eosin t e c h n i q u e . T h e in vitro u p t a k e o f 3H-5-HT a n d 3 H - N A was s t u d i e d in p u n c h e d - o u t slices ( d i a m e t e r 3 or 2 m m and t h i c k n e s s a b o u t 0,6 r a m ) f r o m the c o r t e x cerebri and t h e h y p o t h a l a m u s . T h e i n c u b a t i o n m e d i u m was a m o d i f i e d K r e b s - R i n g e r b i c a r b o n a t e b u f f e r [ 1 1 ] , pH 7.4, c o n t a i n i n g ascrobic acid 0.2 m g / m l , Pargyline 20 u g / m l , E D T A 46.5 m g / 1 , a n d s a t u r a t e d w i t h 95% O2 a n d 5% COs. The slices were p r e i n c u b a t e d in 2 ml of this b u f f e r at + 37°C for 5 m i n in a m e t a b o l i c shaker. T h e n 3H-5-HT ( 2 1 . 4 C i / m m o l ) or 3 H - N A (10.3 C i / m m o l , New E n g l a n d Nuclear) was a d d e d t o t h e m e d i u m t o give a final c o n c e n t r a t i o n o f 2 . 1 0 -SM, a n d t h e i n c u b a t i o n c o n t i n u e d for 10 min. A f t e r t e r m i n a t i o n of the i n c u b a t i o n , t h e slices were dissolved in S O L U E N E 3 5 0 ( P a c k a r d I n s t r u m e n t s ) , a n d scintillation fluid ( t o l u e n e / P E R M A B L E N D III, P a c k a r d ) was added. T h e s a m p l e s were c o u n t e d in a NuclearChicago Mark I liquid s c i n t i l l a t i o n s p e c t r o m e t e r .
Mouse Killing Behavior Nine days a f t e r surgery an a d u l t a l b i n o m o u s e was i n t r o d u c e d i n t o t h e r a t ' s h o m e cage. T h e b e h a v i o r of the rat was o b s e r v e d for 30 rain, a n d b i t i n g a t t a c k s on t h e m o u s e were scored.
Competition Tests Twelve days a f t e r surgery, a f t e r 36 h r o f w a t e r deprivat i o n , c o m p e t i t i o n for a w a t e r b o t t l e was tested. F o l l o w i n g this test session a 5 , 7 - D H T i n j e c t e d rat and a c o n t r o l rat lived t o g e t h e r in t h e test cage for 48 hr. At t h e e n d of this period, a f t e r a n o t h e r 36 h r p e r i o d o f w a t e r deprivat i o n , c o m p e t i t i o n for w a t e r was again tested. F i f t e e n days a f t e r surgery a t u b e fighting test ( a t t e m p t i n g to force
HOLE, JOHNSON AND BERGE a n o t h e r rat o u t o f a t u b e ) was p e r f o r m e d . T h e r e were n o significant d i f f e r e n c e s b e t w e e n groups in a n y of these c o m p e t i t i o n tests, a n d t h e y are n o t f u r t h e r r e p o r t e d .
Habituation to Acoustic Stimulation Testing was p e r f o r m e d o n t h e 1 6 t h , 1 7 t h a n d 1 8 t h day p o s t o p e r a t i v e l y in a G r a s o n - S t a d l e r (Model E 3 1 2 5 - A 100) S k i n n e r b o x (19 × 29 × 23 c m ) c o n t a i n e d w i t h i n a s o u n d a t t e n u a t e d c h a m b e r ( M o d e l E 3 1 2 5 A A - 3 ) . Backg r o u n d w h i t e noise was p r o v i d e d by t h e v e n t i l a t i o n fan. The s t i m u l u s was a w h i t e noise (85 dB for 3 sec) delivered f r o m a G r a s o n Stadler Noise G e n e r a t o r Model 9 0 l B . The rat was placed in the a p p a r a t u s 10 rain b e f o r e testing. The s t i m u l u s was p r e s e n t e d w i t h at least 5 sec intervals, and o n l y w h e n the rat was lying or sitting m o t i o n l e s s . T h e r a t ' s r e s p o n s e to the s t i m u l u s was rated in 5 grades as d e s c r i b e d previously [ 14]. The rat a t t a i n e d the h a b i t u a tion c r i t e r i o n w h e n n o response was o b s e r v e d to 3 successive stimuli.
Habituation to Touch Testing was p e r f o r m e d 1 7 - 1 8 days a f t e r surgery, in the h o m e cage. The t o u c h was applied with a stiff hair a t t a c h e d to a plastic rod, on a m a r k on the b a c k of the rat, in the midline, 3 cm f r o m the tail ( e l i m i n a t i n g visual stimuli). Care was t a k e n to apply the s t i m u l u s in a c o n s t a n t way each time, w h e n t h e rat was n o t moving. The s t i m u l u s was t e r m i n a t e d w h e n the hair was visibly b e n t . Intervals b e t w e e n stimuli were at least 15 sec. The rat's r e s p o n s e to the t o u c h was scored in 4 grades. Criterion for h a b i t u a t i o n was a t t a i n e d w h e n the rat s h o w e d n o visible r e s p o n s e for 3 successive trials.
Activity in Home Cage O b s e r v a t i o n of h o m e cage activity was p e r f o r m e d 20 days a f t e r surgery in the animal quarters. The floor of the h o m e cage was divided b y i m a g i n a r y lines i n t o 6 squares, 11 c m × 11 c m each. N u m b e r of squares e n t e r e d w i t h b o t h forepaws, n u m b e r of rearings, and n u m b e r of rearings of d u r a t i o n 10 sec or m o r e were c o u n t e d . The rat was observed for t w o 5 m i n periods. During the first period the cage was in the rack. T h e n the cage was gently m o v e d to a table in t h e same r o o m , and the s e c o n d o b s e r v a t i o n period started immediately.
Shock Elicited Fighting Testing was p e r f o r m e d 20 days a f t e r surgery. The testing a p p a r a t u s was the same as the one used for h a b i t u a t i o n to acoustic s t i m u l a t i o n . S c r a m b l e d shocks ( 4 0 shocks, 0.8 m A , d u r a t i o n 0.5 sec w i t h 2.5 sec intervals) were delivered t o the grid floor f r o m a G r a s o n Stadler (Model E 1 0 0 4 G S ) s h o c k generator. T w o rats f r o m the same e x p e r i m e n t a l group were t e s t e d t o g e t h e r a n d the o c c u r r e n c e o f a b o x i n g p o s i t i o n [1] was scored for e a c h shock. F o r statistical p u r p o s e s all c o n t r o l rats ( S h a m and V e h ) were t r e a t e d as one group.
Statistical Analysis W h e n n o t s t a t e d o t h e r w i s e s t a t e m e n t s of statistical significance are based on S t u d e n t s t-test, two-tailed, w i t h the c o n f i d e n c e limit set at t h e 5% level. The p-values refer to c o m p a r i s o n s of the 5 , 7 - D H T i n j e c t e d groups or the sham o p e r a t e d g r o u p w i t h the vehicle group.
5,7-DHT
LESIONS AND BEHAVIOR
207
RESULTS
T h e animals a p p e a r e d h e a l t h y , a n d t h e r e were n o significant g r o u p d i f f e r e n c e s in b o d y w e i g h t t h r o u g h o u t the e x p e r i m e n t . B i o c h e m i c a l a n d b e h a v i o r a l d a t a for the rats i n j e c t e d w i t h vehicle in the m i d l i n e (n = 4) a n d bilaterally ( n = 4) were a p p r o x i m a t e l y the same, a n d all vehicle i n j e c t e d rats t h e r e f o r e were t r e a t e d as o n e g r o u p in the data p r e s e n t a t i o n . G r o u p V e h was n o t significantly d i f f e r e n t from g r o u p S h a m for a n y o f the b i o c h e m i c a l or b e h a v i o r a l data.
Histology
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In the 5 , 7 - D H T i n j e c t e d a n d vehicle i n j e c t e d rats visible damage to the b r a i n tissue was o b s e r v e d o n l y in the n e e d l e track and its i m m e d i a t e s u r r o u n d i n g s ( n o t e x c e e d i n g 2 0 0 u r n in d i a m e t e r in a n y rat). With the histological t e c h n i q u e used, t h e r e was n o d e t e c t a b l e d i f f e r e n c e b e t w e e n 5 , 7 - D H T i n j e c t e d and vehicle i n j e c t e d rats. F l u o r e s c e n c e h i s t o c h e m i s t r y o f b r a i n s with this t y p e o f 5 , 7 - D H T lesions has b e e n d e s c r i b e d in a p r e v i o u s p a p e r [ 15].
In Vitro Uptake o f 3H - 5 - H T
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Figure 1 s h o w s t h a t 5 , 7 - D H T i n j e c t i o n s in the m e d i a l 5 - H T b u n d l e r e d u c e d 3 H - 5 - H T u p t a k e to 51% of c o n t r o l values in the c o r t e x cerebri, and to 44% in the h y p o t h a l a m u s , ~ H - N A u p t a k e in the c o r t e x was n o t c h a n g e d . 5 , 7 - D H T lesions of the medial plus lateral 5 - H T b u n d l e s r e d u c e d 3 H - 5 - H T u p t a k e to 42% o f c o n t r o l values in t h e c o r t e x a n d to 27% in t h e h y p o t h a l a m u s ; a r e d u c t i o n in H - N A u p t a k e in the c o r t e x t o 75% o f c o n t r o l in this group was n o t statistically significant. In a p r e v i o u s s t u d y [ 1 5 ] , we o b s e r v e d t h a t similar bilateral 5 , 7 - D H T lesions p r o d u c e d s o m e d a m a g e to the dorsal N A b u n d l e in some of the rats. T h e b e h a v i o r a l data o f the t h r e e rats of the Med + Lat g r o u p w i t h t h e l o w e s t cortical ~ H - N A u p t a k e therefore was c o m p a r e d to t h e data o f the rest o f the group, b u t n o clear d i f f e r e n c e s were f o u n d . Vehicle i n j e c t i o n s did n o t significantly affect 3 H - 5 - H T or 3 H - N A u p t a k e .
Mouse Killing Behavior N o n e of t h e rats in the t w o c o n t r o l groups killed the m o u s e d u r i n g the o b s e r v a t i o n period. H o w e v e r , 4 of the 7 rats in g r o u p Med ( p < 0 . 0 2 5 , Fisher-test, t w o tailed) a n d 6 of the 8 rats in g r o u p Med + Lat ( p < 0 . 0 0 5 ) killed the mouse.
Habituation to Acoustic Stimulation The Med + Lat g r o u p r e q u i r e d significantly m o r e trials to r e a c h the h a b i t u a t i o n c r i t e r i o n t h a n the c o n t r o l s ( p < 0 . 0 2 ) and group Med ( p < 0 . 0 5 ) . G r o u p Med was n o t significantly d i f f e r e n t f r o m the c o n t r o l s ( p > 0 . 1 0 ) (Fig. 2).
Habituation to Touch B o t h 5 , 7 - D H T i n j e c t e d groups r e q u i r e d significantly m o r e trials t h a n t h e c o n t r o l g r o u p s to r e a c h the h a b i t u a t i o n c r i t e r i o n ( p < 0 . 0 1 ) (Fig. 3).
Activity in Home Cage W h e n the cage was in the rack, the activity was very low
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FIG. 1. In vitro 3 H - 5 - H T uptake (mean and SEM), given as percent of the mean uptake in group Sham. Number of rats in (). * = p<0.001 (compared to group Veh). CX = Cortex. HT = Hypothalamus.
in all g r o u p s (0 or 1 crossing for m o s t rats), and t h e r e was n o significant d i f f e r e n c e b e t w e e n the groups. A f t e r t h e cage was m o v e d to the table, l o c o m o t o r activity was 25% l o w e r in the group Med + Lat t h a n in group Veh, h o w e v e r , this d i f f e r e n c e was n o t statistically significant. N u m b e r o f rearings, a n d n u m b e r o f rearings o f d u r a t i o n 10 sec or m o r e , were n o t significantly c h a n g e d b y the 5 , 7 - D H T injections.
Shock Elicited Fighting B o t h in g r o u p Med and group Med + Lat the s h o c k s elicited a significant h i g h e r n u m b e r of b o x i n g p o s i t i o n s t h a n in the c o n t r o l s ( p < 0 . 0 5 ) (Fig. 4). DISCUSSION
T h e 5 , 7 - D H T i n j e c t i o n s i n t o the a s c e n d i n g 5-HT p a t h ways r e s u l t e d in extensive lesions o f these p a t h w a y s , as i n d i c a t e d b y the r e d u c e d in vitro u p t a k e of 3 H - 5 - H T in the t e r m i n a l areas in the c o r t e x and the h y p o t h a l a m u s . B o t h the histological e x a m i n a t i o n o f the i n j e c t i o n site and the d a t a for the 3 H _ N A u p t a k e in the c o r t e x suggested t h a t the lesions were specific for the 5-HT n e u r o n s . In a p r e v i o u s s t u d y [ 15], f l u o r e s c e n c e h i s t o c h e m i c a l analysis o f brains w i t h similar 5 , 7 - D H T lesions i n d i c a t e d t h a t the unspecific lesions p r o d u c e d is small c o m p a r e d to the lesion of the 5 - H T p a t h w a y s . The f l u o r e s c e n c e h i s t o c h e m i c a l
208
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FIG. 3. Habituation to touch. Number of trials to criterion (mean and SEM). * = p<0.01.
FIG. 4. Shock elicited fighting. Number of shocks (mean and SEM) that elicited a boxing position, Number of pairs of rats in (). Each pair received 40 shocks. * = p<0.05, compared to all control rats (group Sham + group Veh). analysis also s h o w e d that the medial lesion mainly damaged the medial 5 - H T p a t h w a y , while the medial plus lateral lesion affected both the medial and the lateral 5 - H T pathways. In this e x p e r i m e n t , the medial lesion reduced the rate o f habituation to touch, and the medial plus lateral lesion both to t o u c h and to acoustic stimulation. This e x p e r i m e n t therefore supports previous studies indicating that lesion o f the ascending 5 - H T neurons or reduced synthesis o f 5 HT reduce the rate o f h a b i t u a t i o n or increase sensitization [3, 4, 5 ] . Several other studies, h o w e v e r , have failed to observe changes in the h a b i t u a t i o n rate in rats w i t h impaired f u n c t i o n o f the 5 HT neurons [8, 9, 2 5 ] . M e t h o d o l o g i c a l differences b e t w e e n the tests m a y underlie some of these differences. This is also suggested by the present results, since the group Med e v i d e n c e d a reduced rate o f habituation only w i t h regard to the response to touch, but not to audtiory stimulation. A general increase in reactivity m a y result in prolonged habituation. Furthermore, it should be p o i n t e d out that e l e c t r o l y t i c lesions in the brainstem m a y produce a reduced rate o f h a b i t u a t i o n [ 1 9 ] . The necrosis at the site o f injection was very small both after 5 , 7 - D H T and vehicle injections, and there was no clear difference b e t w e e n the t w o groups in the size o f this unspecific lesion. However, cell c o u n t i n g o f the lesion area was n o t performed. The possibility that there m a y exist small differences b e t w e e n the t w o groups in the damage to n o n - 5 - H T neurons therefore has n o t been e x c l u d e d . Lesions b o t h o f the medial and o f the medial plus the lateral 5 - H T bundles increased m o u s e killing behavior, and also increased n u m b e r of times the electric shocks elicited a b o x i n g position. These results are in accordance w i t h
5,7-DHT LESIONS AND BEHAVIOR
209
several p r e v i o u s studies. B o t h e l e c t r o l y t i c lesions in the r a p h e n u c l e i [ 1 7 , 2 6 ] , i n h i b i t i o n of 5 - H T s y n t h e s i s w i t h PCPA [6, 22, 2 3 ] , or w i t h p a r a c h l o r o - N - m e t h y l a m p h e t a m i n e [ 1 3 ] , a n d general lesion of 5 - H T t e r m i n a l s w i t h i n t r a c i s t e r n a l i n j e c t i o n o f 5 , 7 - D H T [2] p r o d u c e similar b e h a v i o r a l changes. This e x p e r i m e n t f u r t h e r s u p p o r t s the a s s u m p t i o n t h a t c e n t r a l 5 - H T n e u r o n s are i n v o l v e d in m u r i c i d e a n d s h o c k elicited fighting behavior. F u r t h e r m o r e , the e x p e r i m e n t i n d i c a t e s t h a t lesion of t h e m e d i a l 5 - H T b u n d l e is s u f f i c i e n t to p r o d u c e these b e h a v i o r a l changes. Everi~t, F u x e a n d J o n s s o n [7] o b s e r v e d b i t i n g a t t a c k s on males b y female rats w i t h 5 , 7 - D H T lesions o f t h e m e d i a l 5 - H T b u n d l e . Thus, in rats t h e a s c e n d i n g m e d i a l 5 - H T p a t h w a y seems to be i m p o r t a n t in c o n t r o l of agonistic b e h a v i o r b o t h in males a n d females, while in mice it has b e e n suggested t h a t o n l y female aggressive b e h a v i o r is c o n t r o l l e d b y s e r o t o n e r g i c m e c h a n i s m s [21 ]. T h e s h o c k elicited f i g h t i n g m o d e l has b e e n t h e m o s t f r e q u e n t l y used p a r a d i g m for aggressive behavior. B l a n c h a r d and B l a n c h a r d [ 1 ] , h o w e v e r , h a v e s h o w n t h a t the pain i n d u c e d b o x i n g p o s i t i o n m a i n l y is a defensive p o s t u r e . This test t h e r e f o r e s h o u l d be r e g a r d e d m a i n l y as a test for defensive b e h a v i o r . In the m o u s e killing b e h a v i o r t h e r e seems to be an i m p o r t a n t c o m p o n e n t of c o n s p e c i f i c a t t a c k , in a d d i t i o n to p r e d a t i o n and possible defensive c o m p o n e n t s [ 1 ] . It is possible t h e r e f o r e t h a t b o t h c o n s p e c i f i c a t t a c k and defensive b e h a v i o r are increased by the lesion of the medial 5 - H T p a t h w a y . In a p r e v i o u s s t u d y it was s h o w n t h a t o p e n field l o c o m o t o r activity was r e d u c e d in rats w i t h 5 , 7 - D H T lesions o f the m e d i a l plus lateral 5 - H T b u n d l e s [ 1 5 ] , in c o n t r a s t to the increase in activity a f t e r e l e c t r o l y t i c lesion
of t h e m e d i a n r a p h e n u c l e u s [ 1 8 , 2 4 ] . O n e possible r e a s o n for a d e c r e a s e d o p e n field l o c o m o t o r a c t i v i t y c o u l d b e a n increase in r e a c t i v i t y or e m o t i o n a l i t y in the 5 , 7 - D H T lesioned rats, p a r t i c u l a r l y since the field was b r i g h t l y i l l u m i n a t e d [ 15]. In the p r e s e n t s t u d y the rats t h e r e f o r e were o b s e r v e d in t h e h o m e cage (in a s i t u a t i o n w i t h o u t a n y n e w stimuli), a n d in a s i t u a t i o n w i t h a r a t h e r weak s t i m u l u s (gently m o v i n g the h o m e cage to a n o t h e r part of the r o o m , w i t h o u t h a n d l i n g o f the rat). H y p e r a c t i v i t y , as in the e l e c t r o l y t i c r a p h e l e s i o n e d rats, was n o t o b s e r v e d in a n y of these situations. On t h e c o n t r a r y , rats w i t h c o m b i n e d lesions in the m e d i a l a n d the lateral 5 - H T b u n d l e s t e n d e d to s h o w a r e d u c t i o n in t h e l o c o m o t o r activity also in this test, h o w e v e r , this r e d u c t i o n was n o t statistically significant. In c o n c l u s i o n , p r o l o n g e d h a b i t u a t i o n and increase in m o u s e killing b e h a v i o r a n d in s h o c k elicited defensive b e h a v i o r can be o b s e r v e d n o t o n l y a f t e r e l e c t r o l y t i c lesions of t h e 5 - H T n e u r o n s or p h a r m a c o l o g i c a l a l t e r a t i o n s o f the f u n c t i o n o f these n e u r o n s , b u t also a f t e r selective c h e m i c a l lesions o f t h e 5 - H T n e u r o n s . It seems likely t h e r e f o r e , t h a t these b e h a v i o r a l c h a n g e s are due to lesion o f the a s c e n d i n g 5 - H T p a t h w a y s . C o m p e t i t i v e and d o m i n a n c e b e h a v i o r s in o t h e r tests were n o t c h a n g e d , a n d lesion o f the a s c e n d i n g 5 - H T p a t h w a y s does n o t seem to result in a general increase in agonistic b e h a v i o r , b u t r a t h e r in p a r t i c u l a r c o m p o n e n t s o f this behavior. F u r t h e r m o r e , t h e s t u d y s u p p o r t s p r e v i o u s studies [ 1 5 , 2 0 ] , c o n c l u d i n g t h a t the increased a m b u l a t o r y activity o b s e r v e d in rats w i t h electrolytic lesions in the m e d i a n r a p h e n u c l e u s is n o t o n l y due to lesion o f the 5 - H T n e u r o n s a s c e n d i n g f r o m this nucleus.
REFERENCES 1. Blanchard, R. J. and D. C. Blanchard. Aggressive behavior in the laboratory rat. Behav. BioL, in press. 2. Breese, G. R. and B. R. Cooper. Behavioral and biochemical interactions of 5,7-dihydroxytryptamine with various drugs when administered intracisternally to adult and developing rats. Brain Res. 98: 5 1 7 - 5 2 7 , 1975. 3. Carlton, P. L. and C. Advokat. Attenuated habituation due to parachlorophenylalanine. Pharmac. Biochem. Behav. 1: 6 5 7 - 6 6 3 , 1973. 4. Conner, R. L., J. M. Stolk, J. D. Barchas and S. Levine. Parachlorophenylalanine and habituation to repetitive auditory startle stimuli in rats. Physiol. Behav. 5: 1215-1219, 1970. 5. Davis, M. and M. H. Sheard. Habituation of the rat startle response: Effects of raphe lesions. Physiol. Behav. 12: 4 2 5 - 4 3 1 , 1974. 6. Eichelman, B. S., Jr. and N. B. Thoa. The aggressive monoamines. Biol. Psychiat. 6: 143-164, 1973. 7. Everitt, B. J., K. Fuxe and G. Jonsson. The effects of 5,7-dihydroxytryptamine lesions of ascending 5-hydroxytryptamine pathways on the sexual and aggressive behaviour of female rats. J. Pharmac. 6: 2 5 - 3 2 , 1975. 8. File, S. E. Effects of parachlorophenylalanine and amphetamine on habituation of orienting. Pharmac. Biochem. Behav. 3: 9 7 9 - 9 8 3 , 1975. 9. Geyer, M. A., A. Puerto, D. B. Menkes, D. S. Segal and A. S. Mandell. Behavioral studies following lesions of the mesolimbic and mesostriatal serotonergic pathways. Brain Res. 106: 2 5 7 - 2 7 0 , 1976. 10. Grant, L. D., D. V. Coscina, S. P. Grossman and D. X. Freedman. Muricide after serotonin depleting lesions of midbrain raphe nuclei. Pharmac. Biochem. Behav. 1: 7 7 - 8 0 , 1973. 11. Hamberger, B. Reserpine-resistent uptake of catecholamines in isolated tissues of the rat. Acta physiol, scand. Suppl 295: 1-64, 1967.
12. Harvey, J. A. and C. E. Lints. Lesions in the medial forebrain bundle: Relationship between pain sensitivity and telencephalic content of serotonin. J. comp. physioL Psychol. 74: 2 8 - 3 6 , 1971. 13. Hodge, G. K. and L. L. Butcher. 5-Hydroxytryptamine correlates of isolation-induced aggression in mice. Eur. J. Pharrnac. 28: 326-337, 1974. 14. Hole, K. Behavior and brain growth in rats treated with p-cblorophenylalanine in the first weeks of life. Devl. Psychobiol. 5: 157-173, 1972. 15. Hole, K., K. Fuxe and G. Jonsson. Behavioral effect of 5,7-dihydroxytryptamine lesions of ascending 5-hydroxytryptamine pathways. Brain Res. 107: 385-399, 1976. 16. Hole, K. and S. A. Lorens. Response to electric shock in rats: Effects of selective midbrain raphe lesions. Pharrnac. Biochem. Behav. 3: 9 5 - 1 0 2 , 1975. 17. Jacobs, B. L. and A. Cohen. Differential behavioral effects of lesions of the median or dorsal raphe nuclei in rats: Open field and pain-elicited aggression. J. comp. physiol. Psychol. 90: 102-108, 1976. 18. Jacobs, B. L., W. D. Wise and K. M. Taylor. Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats. Brain Res. 79: 353-361, 1974. 19. Le Moal, M., L. Stinus and D. Galey. Radiofrequency lesions of the ventral mesencephalic tegmentum: neurological and behavioral considerations. ExplNeurol. 50: 5 2 1 - 5 3 5 , 1976. 20. Lorens, S. A., H. C. Guldberg, K. Hole, C. K6hler and B. Srebro. Activity, avoidance learning and regional 5-hydroxytryptamine following intrabrainstem 5,7-dihydroxytryptamine and electrolytic midbrain raphe lesions in the rat. Brain Res. 108: 9 7 - 1 1 3 , 1976.
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21. Neckers, L. M., M. X. Zarrow, M. M. Myers and V. H. Denenberg. Influence of olfactory bulbectomy and the serotonergic system upon intermale aggression and maternal behavior in the mouse. Pharmac. Biochem. Behav. 3: 545-550, 1975. 22. Sheard, M. M. and M. Davis. Shock elicited fighting in rats: Importance of intershock interval upon the effect of p-chlorophenylalanine (PCPA). Brain Res. 111: 433-437, 1976. 23. Sheard, M. H. Brain serotonin depletion by p-chlorophenylalanine or lesions of raphe neurons in rats. Physiol. Behav. 10: 809-811, 1973.
HOLE, J O H N S O N AND B E R G E 24. Srebro, B. and S. A. Lorens. Behavioral effects of selective midbrain raphe lesions in the rat. Brain Res. 89: 303-325, 1975. 25. Swonger, A. K. and R. H. Rech. Serotonergic and cholinergic involvement in habituation of activity and spontaneous alteration of rats in a Y maze. J. comp. physiol. Psychol. 81: 509-522, 1972. 26. Vergnes, M. and C. Penot. Aggression intrasp~ciflque induite par chocs ~lectriques et r~activite apr~s l~sion du raph~ chez le rat.. Effets de la physostigmine. Brain Res. 104: 107-119, 1976.
5,7-Dihydroxytryptamine Lesions of the Ascending 5-Hydroxytryptamine Pathways: Habituation, Motor Activity and Agonistic Behavior I KJELL
HOLE, GEORG E S P O L I N J O H N S O N A N D O D D - G E I R
BERGE
Institute o f Physiology, University o f Bergen, Norway ( R e c e i v e d 19 J a n u a r y 1 9 7 7 ) HOLE, K., G. E. JOHNSON AND O . - G . BERGE. 5,7-Dihydroxytryptamine lesions of the ascending 5-hydroxymotor activity and agonistic behavior. PHARMAC. BIOCHEM. BEHAV. 7(3) 2 0 5 - 2 1 0 , 1 9 7 7 . - Rats were injected stereotactically in mesencephalon with 5,7-dihydroxytryptamine (5,7-DHT) in the medial 5-hydroxytryptamine (5-HT) pathway (n = 8) and in the medial plus the lateral 5 - H T pathways (n = 7) or injected with vehicle (n = 8), or sham-operated (n = 8). The 5,7-DHT lesions reduced the in vitro 3H-5-HT uptake in the hypothalamus and the cortex cerebri to 2 7 - 5 1 % of control values, 3H-noradrenaline uptake was not significantly changed. 5,7-DHT lesions of the medial, and of the medial plus the lateral, 5 - H T pathways induced mouse killing behavior and increased number of boxing positions in the shock elicited fighting test. Both lesions also reduced the rate of habituation to touch, but only the lesion of the medial plus the lateral 5 - H T pathway significantly reduced the rate of habituation to acoustic stimulation. Activity in the home cage was not significantly changed by the lesions. It was concluded that selective chemical lesions of the ascending 5 - H T pathways result in prolonged habituation of the orienting response and increase in particular components of agonistic behavior. The increase in locomotor activity observed after electrolytic lesions of nucleus raphe medianus seems not to be due only to lesion of the 5 - H T neurons ascending from this nucleus.
tryptamine pathways: habituation,
5-Hydroxytryptamine Motor activity
5,7 ~Dihydroxytryptamine
Habituation of orienting
Agonistic behavior
5 - H T p a t h w a y s are due to d a m a g e o f o t h e r n e u r o n s t h a n the 5 - H T n e u r o n s . The b e h a v i o r a l effects of selective c h e m i c a l lesions o f the 5 - H T p a t h w a y s t h e r e f o r e s h o u l d be f u r t h e r investigated. In t h e p r e s e n t s t u d y the e f f e c t o f 5 , 7 - D H T lesions o f the a s c e n d i n g 5 - H T p a t h w a y s o n h a b i t u a t i o n o f t h e o r i e n t i n g response, agonistic b e h a v i o r , a n d m o t o r activity in t w o d i f f e r e n t s t i m u l u s s i t u a t i o n s in the h o m e cage, were studied.
IT HAS b e e n suggested t h a t the 5 - h y d r o x y t r y p t a m i n e ( 5 - H T ) n e u r o n s o r i g i n a t i n g in the m i d b r a i n r a p h e n u c l e i are i m p o r t a n t in c e n t r a l c o n t r o l of pain sensitivity [ 1 2 ] , m o t o r activity [9, 15, 18, 2 4 ] , h a b i t u a t i o n or sensitization to s e n s o r y s t i m u l a t i o n [3, 4, 5 ] , a n d aggressive b e h a v i o r [6, 17, 2 2 ] . In the m a j o r i t y of p r e v i o u s studies the f u n c t i o n o f the 5 - H T n e u r o n s was altered b y electrolytic lesions, p a r a c h l o r o p h e n y l a l a n i n e (PCPA) t r e a t m e n t , or o t h e r p h a r m a c o l o g i c a l m a n i p u l a t i o n s . These m e t h o d s affect o t h e r t y p e s o f n e u r o n s as well as t h e 5 - H T n e u r o n s . R e c e n t l y it was o b s e r v e d t h a t w h e n the 5 - H T n e u r o n s are l e s i o n e d selectively with, 5 , 7 - d i h y d r o x y t r y p t a mine ( 5 , 7 - D H T ) i n j e c t e d s t e r e o t a c t i c a l l y i n t o t h e 5 - H T p a t h w a y s , o p e n field l o c o m o t o r activity is r e d u c e d a n d pain sensitivity is u n c h a n g e d [ 1 5 , 1 6 ] , in c o n t r a s t to t h e increase in l o c o m o t o r activity [24] a n d increase in pain sensitivity [12] o b s e r v e d a f t e r e l e c t r o l y t i c lesions. Possibly some o f the b e h a v i o r a l changes o b s e r v e d a f t e r electrolytic lesions o f the m i d b r a i n r a p h e nuclei or the a s c e n d i n g
METHOD
Animals T h i r t y - o n e male a l b i n o rats (M/511-Wistar) weighing 240-280g at t i m e of surgery were used. T h e y were h o u s e d i n d i v i d u a l l y in c o n v e n t i o n a l rat cages and had food a n d w a t e r available ad lib. Testing was p e r f o r m e d d u r i n g the light p h a s e o f a 12 h r light-dark cycle.
J The authors gratefully acknowledge Miss Nina Holmelid and Mrs. May-Elin Figueroa for their excellent technical assistance. This research was supported in part by Norwegian Research Council for Science and the Humanities and Andssvakesakens Forskningsfond. 205
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Surgery A K o p f s t e r e o t a x i c i n s t r u m e n t was used w i t h t h e incisor bar set 2.4 m m a b o v e the i n t e r a u r a l plane. U n d e r KETALAR anesthesia (100-200mg/kg), 5,7-DHT (5,7-dihydroxytryptamine c r e a t i n e sulfate, Regis) dissolved in 0.9% NaC1 c o n t a i n i n g a s c o r b i c acid (0,1 m g / m l ) was i n j e c t e d s t e r e o t a c t i c a l l y w i t h a 10 ul H a m i l t o n syringe in t h e region of t h e a s c e n d i n g 5 - H T b u n d l e s [ 15] in t h e mesencephalon. T h e c o o r d i n a t e s were for t h e m e d i a l 5 - H T b u n d l e (group Med, n = 8): p o s t e r i o r to b r e g m a 5.6, lateral 0.0, v e n t r a l t o t h e surface o f c o r t e x 7.5 r a m ; a n d for t h e c o m b i n e d lesions o f m e d i a l a n d lateral b u n d l e s (group Med + Lat, n = 7): p o s t e r i o r 5.6, lateral +- 0.6, a n d v e n t r a l 7.0 m m . At e a c h i n j e c t i o n site 4 ug, 5 , 7 - D H T ( c a l c u l a t e d as t h e base) in 4 ul was i n j e c t e d (1 u l / m i n ) . T h e n e e d l e was left in situ for 1 m i n a f t e r c o m p l e t i o n o f e a c h i n j e c t i o n . T w e n t y rain b e f o r e t h e i n t r a c e r e b r a l i n j e c t i o n s p r o t r i p t y l i n e HCI (Merck, S h a r p a n d D o h m e ) 25 m g / k g was i n j e c t e d IP [ 1 5 ] . C o n t r o l rats were t r e a t e d in t h e same m a n n e r as t h e 5 , 7 - D H T rats, e x c e p t t h a t t h e y were i n j e c t e d i n t r a c e r e b r a l l y w i t h t h e vehicle o n l y (group Veh, n - - 8 ) , or t h e c a n n u l a was n o t l o w e r e d i n t r a c r a n i a l l y (group S h a m , n = 8).
Histological and Bioehemical Analyses F o r t y - s i x d a y s a f t e r surgery t h e a n i m a l s were sacrificed b y d e c a p i t a t i o n . T h e b r a i n s t e m s of t h e 5 , 7 - D H T a n d vehicle i n j e c t e d rats were k e p t in 10% F o r m a l i n for one week. F r o z e n sections t h e n were cut at 30 u m and every t h i r d s e c t i o n saved a n d s t a i n e d by t h e eosin t e c h n i q u e . T h e in vitro u p t a k e o f 3H-5-HT a n d 3 H - N A was s t u d i e d in p u n c h e d - o u t slices ( d i a m e t e r 3 or 2 m m and t h i c k n e s s a b o u t 0,6 r a m ) f r o m the c o r t e x cerebri and t h e h y p o t h a l a m u s . T h e i n c u b a t i o n m e d i u m was a m o d i f i e d K r e b s - R i n g e r b i c a r b o n a t e b u f f e r [ 1 1 ] , pH 7.4, c o n t a i n i n g ascrobic acid 0.2 m g / m l , Pargyline 20 u g / m l , E D T A 46.5 m g / 1 , a n d s a t u r a t e d w i t h 95% O2 a n d 5% COs. The slices were p r e i n c u b a t e d in 2 ml of this b u f f e r at + 37°C for 5 m i n in a m e t a b o l i c shaker. T h e n 3H-5-HT ( 2 1 . 4 C i / m m o l ) or 3 H - N A (10.3 C i / m m o l , New E n g l a n d Nuclear) was a d d e d t o t h e m e d i u m t o give a final c o n c e n t r a t i o n o f 2 . 1 0 -SM, a n d t h e i n c u b a t i o n c o n t i n u e d for 10 min. A f t e r t e r m i n a t i o n of the i n c u b a t i o n , t h e slices were dissolved in S O L U E N E 3 5 0 ( P a c k a r d I n s t r u m e n t s ) , a n d scintillation fluid ( t o l u e n e / P E R M A B L E N D III, P a c k a r d ) was added. T h e s a m p l e s were c o u n t e d in a NuclearChicago Mark I liquid s c i n t i l l a t i o n s p e c t r o m e t e r .
Mouse Killing Behavior Nine days a f t e r surgery an a d u l t a l b i n o m o u s e was i n t r o d u c e d i n t o t h e r a t ' s h o m e cage. T h e b e h a v i o r of the rat was o b s e r v e d for 30 rain, a n d b i t i n g a t t a c k s on t h e m o u s e were scored.
Competition Tests Twelve days a f t e r surgery, a f t e r 36 h r o f w a t e r deprivat i o n , c o m p e t i t i o n for a w a t e r b o t t l e was tested. F o l l o w i n g this test session a 5 , 7 - D H T i n j e c t e d rat and a c o n t r o l rat lived t o g e t h e r in t h e test cage for 48 hr. At t h e e n d of this period, a f t e r a n o t h e r 36 h r p e r i o d o f w a t e r deprivat i o n , c o m p e t i t i o n for w a t e r was again tested. F i f t e e n days a f t e r surgery a t u b e fighting test ( a t t e m p t i n g to force
HOLE, JOHNSON AND BERGE a n o t h e r rat o u t o f a t u b e ) was p e r f o r m e d . T h e r e were n o significant d i f f e r e n c e s b e t w e e n groups in a n y of these c o m p e t i t i o n tests, a n d t h e y are n o t f u r t h e r r e p o r t e d .
Habituation to Acoustic Stimulation Testing was p e r f o r m e d o n t h e 1 6 t h , 1 7 t h a n d 1 8 t h day p o s t o p e r a t i v e l y in a G r a s o n - S t a d l e r (Model E 3 1 2 5 - A 100) S k i n n e r b o x (19 × 29 × 23 c m ) c o n t a i n e d w i t h i n a s o u n d a t t e n u a t e d c h a m b e r ( M o d e l E 3 1 2 5 A A - 3 ) . Backg r o u n d w h i t e noise was p r o v i d e d by t h e v e n t i l a t i o n fan. The s t i m u l u s was a w h i t e noise (85 dB for 3 sec) delivered f r o m a G r a s o n Stadler Noise G e n e r a t o r Model 9 0 l B . The rat was placed in the a p p a r a t u s 10 rain b e f o r e testing. The s t i m u l u s was p r e s e n t e d w i t h at least 5 sec intervals, and o n l y w h e n the rat was lying or sitting m o t i o n l e s s . T h e r a t ' s r e s p o n s e to the s t i m u l u s was rated in 5 grades as d e s c r i b e d previously [ 14]. The rat a t t a i n e d the h a b i t u a tion c r i t e r i o n w h e n n o response was o b s e r v e d to 3 successive stimuli.
Habituation to Touch Testing was p e r f o r m e d 1 7 - 1 8 days a f t e r surgery, in the h o m e cage. The t o u c h was applied with a stiff hair a t t a c h e d to a plastic rod, on a m a r k on the b a c k of the rat, in the midline, 3 cm f r o m the tail ( e l i m i n a t i n g visual stimuli). Care was t a k e n to apply the s t i m u l u s in a c o n s t a n t way each time, w h e n t h e rat was n o t moving. The s t i m u l u s was t e r m i n a t e d w h e n the hair was visibly b e n t . Intervals b e t w e e n stimuli were at least 15 sec. The rat's r e s p o n s e to the t o u c h was scored in 4 grades. Criterion for h a b i t u a t i o n was a t t a i n e d w h e n the rat s h o w e d n o visible r e s p o n s e for 3 successive trials.
Activity in Home Cage O b s e r v a t i o n of h o m e cage activity was p e r f o r m e d 20 days a f t e r surgery in the animal quarters. The floor of the h o m e cage was divided b y i m a g i n a r y lines i n t o 6 squares, 11 c m × 11 c m each. N u m b e r of squares e n t e r e d w i t h b o t h forepaws, n u m b e r of rearings, and n u m b e r of rearings of d u r a t i o n 10 sec or m o r e were c o u n t e d . The rat was observed for t w o 5 m i n periods. During the first period the cage was in the rack. T h e n the cage was gently m o v e d to a table in t h e same r o o m , and the s e c o n d o b s e r v a t i o n period started immediately.
Shock Elicited Fighting Testing was p e r f o r m e d 20 days a f t e r surgery. The testing a p p a r a t u s was the same as the one used for h a b i t u a t i o n to acoustic s t i m u l a t i o n . S c r a m b l e d shocks ( 4 0 shocks, 0.8 m A , d u r a t i o n 0.5 sec w i t h 2.5 sec intervals) were delivered t o the grid floor f r o m a G r a s o n Stadler (Model E 1 0 0 4 G S ) s h o c k generator. T w o rats f r o m the same e x p e r i m e n t a l group were t e s t e d t o g e t h e r a n d the o c c u r r e n c e o f a b o x i n g p o s i t i o n [1] was scored for e a c h shock. F o r statistical p u r p o s e s all c o n t r o l rats ( S h a m and V e h ) were t r e a t e d as one group.
Statistical Analysis W h e n n o t s t a t e d o t h e r w i s e s t a t e m e n t s of statistical significance are based on S t u d e n t s t-test, two-tailed, w i t h the c o n f i d e n c e limit set at t h e 5% level. The p-values refer to c o m p a r i s o n s of the 5 , 7 - D H T i n j e c t e d groups or the sham o p e r a t e d g r o u p w i t h the vehicle group.
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RESULTS
T h e animals a p p e a r e d h e a l t h y , a n d t h e r e were n o significant g r o u p d i f f e r e n c e s in b o d y w e i g h t t h r o u g h o u t the e x p e r i m e n t . B i o c h e m i c a l a n d b e h a v i o r a l d a t a for the rats i n j e c t e d w i t h vehicle in the m i d l i n e (n = 4) a n d bilaterally ( n = 4) were a p p r o x i m a t e l y the same, a n d all vehicle i n j e c t e d rats t h e r e f o r e were t r e a t e d as o n e g r o u p in the data p r e s e n t a t i o n . G r o u p V e h was n o t significantly d i f f e r e n t from g r o u p S h a m for a n y o f the b i o c h e m i c a l or b e h a v i o r a l data.
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In the 5 , 7 - D H T i n j e c t e d a n d vehicle i n j e c t e d rats visible damage to the b r a i n tissue was o b s e r v e d o n l y in the n e e d l e track and its i m m e d i a t e s u r r o u n d i n g s ( n o t e x c e e d i n g 2 0 0 u r n in d i a m e t e r in a n y rat). With the histological t e c h n i q u e used, t h e r e was n o d e t e c t a b l e d i f f e r e n c e b e t w e e n 5 , 7 - D H T i n j e c t e d and vehicle i n j e c t e d rats. F l u o r e s c e n c e h i s t o c h e m i s t r y o f b r a i n s with this t y p e o f 5 , 7 - D H T lesions has b e e n d e s c r i b e d in a p r e v i o u s p a p e r [ 15].
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Figure 1 s h o w s t h a t 5 , 7 - D H T i n j e c t i o n s in the m e d i a l 5 - H T b u n d l e r e d u c e d 3 H - 5 - H T u p t a k e to 51% of c o n t r o l values in the c o r t e x cerebri, and to 44% in the h y p o t h a l a m u s , ~ H - N A u p t a k e in the c o r t e x was n o t c h a n g e d . 5 , 7 - D H T lesions of the medial plus lateral 5 - H T b u n d l e s r e d u c e d 3 H - 5 - H T u p t a k e to 42% o f c o n t r o l values in t h e c o r t e x a n d to 27% in t h e h y p o t h a l a m u s ; a r e d u c t i o n in H - N A u p t a k e in the c o r t e x t o 75% o f c o n t r o l in this group was n o t statistically significant. In a p r e v i o u s s t u d y [ 1 5 ] , we o b s e r v e d t h a t similar bilateral 5 , 7 - D H T lesions p r o d u c e d s o m e d a m a g e to the dorsal N A b u n d l e in some of the rats. T h e b e h a v i o r a l data o f the t h r e e rats of the Med + Lat g r o u p w i t h t h e l o w e s t cortical ~ H - N A u p t a k e therefore was c o m p a r e d to t h e data o f the rest o f the group, b u t n o clear d i f f e r e n c e s were f o u n d . Vehicle i n j e c t i o n s did n o t significantly affect 3 H - 5 - H T or 3 H - N A u p t a k e .
Mouse Killing Behavior N o n e of t h e rats in the t w o c o n t r o l groups killed the m o u s e d u r i n g the o b s e r v a t i o n period. H o w e v e r , 4 of the 7 rats in g r o u p Med ( p < 0 . 0 2 5 , Fisher-test, t w o tailed) a n d 6 of the 8 rats in g r o u p Med + Lat ( p < 0 . 0 0 5 ) killed the mouse.
Habituation to Acoustic Stimulation The Med + Lat g r o u p r e q u i r e d significantly m o r e trials to r e a c h the h a b i t u a t i o n c r i t e r i o n t h a n the c o n t r o l s ( p < 0 . 0 2 ) and group Med ( p < 0 . 0 5 ) . G r o u p Med was n o t significantly d i f f e r e n t f r o m the c o n t r o l s ( p > 0 . 1 0 ) (Fig. 2).
Habituation to Touch B o t h 5 , 7 - D H T i n j e c t e d groups r e q u i r e d significantly m o r e trials t h a n t h e c o n t r o l g r o u p s to r e a c h the h a b i t u a t i o n c r i t e r i o n ( p < 0 . 0 1 ) (Fig. 3).
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in all g r o u p s (0 or 1 crossing for m o s t rats), and t h e r e was n o significant d i f f e r e n c e b e t w e e n the groups. A f t e r t h e cage was m o v e d to the table, l o c o m o t o r activity was 25% l o w e r in the group Med + Lat t h a n in group Veh, h o w e v e r , this d i f f e r e n c e was n o t statistically significant. N u m b e r o f rearings, a n d n u m b e r o f rearings o f d u r a t i o n 10 sec or m o r e , were n o t significantly c h a n g e d b y the 5 , 7 - D H T injections.
Shock Elicited Fighting B o t h in g r o u p Med and group Med + Lat the s h o c k s elicited a significant h i g h e r n u m b e r of b o x i n g p o s i t i o n s t h a n in the c o n t r o l s ( p < 0 . 0 5 ) (Fig. 4). DISCUSSION
T h e 5 , 7 - D H T i n j e c t i o n s i n t o the a s c e n d i n g 5-HT p a t h ways r e s u l t e d in extensive lesions o f these p a t h w a y s , as i n d i c a t e d b y the r e d u c e d in vitro u p t a k e of 3 H - 5 - H T in the t e r m i n a l areas in the c o r t e x and the h y p o t h a l a m u s . B o t h the histological e x a m i n a t i o n o f the i n j e c t i o n site and the d a t a for the 3 H _ N A u p t a k e in the c o r t e x suggested t h a t the lesions were specific for the 5-HT n e u r o n s . In a p r e v i o u s s t u d y [ 15], f l u o r e s c e n c e h i s t o c h e m i c a l analysis o f brains w i t h similar 5 , 7 - D H T lesions i n d i c a t e d t h a t the unspecific lesions p r o d u c e d is small c o m p a r e d to the lesion of the 5 - H T p a t h w a y s . The f l u o r e s c e n c e h i s t o c h e m i c a l
208
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FIG. 4. Shock elicited fighting. Number of shocks (mean and SEM) that elicited a boxing position, Number of pairs of rats in (). Each pair received 40 shocks. * = p<0.05, compared to all control rats (group Sham + group Veh). analysis also s h o w e d that the medial lesion mainly damaged the medial 5 - H T p a t h w a y , while the medial plus lateral lesion affected both the medial and the lateral 5 - H T pathways. In this e x p e r i m e n t , the medial lesion reduced the rate o f habituation to touch, and the medial plus lateral lesion both to t o u c h and to acoustic stimulation. This e x p e r i m e n t therefore supports previous studies indicating that lesion o f the ascending 5 - H T neurons or reduced synthesis o f 5 HT reduce the rate o f h a b i t u a t i o n or increase sensitization [3, 4, 5 ] . Several other studies, h o w e v e r , have failed to observe changes in the h a b i t u a t i o n rate in rats w i t h impaired f u n c t i o n o f the 5 HT neurons [8, 9, 2 5 ] . M e t h o d o l o g i c a l differences b e t w e e n the tests m a y underlie some of these differences. This is also suggested by the present results, since the group Med e v i d e n c e d a reduced rate o f habituation only w i t h regard to the response to touch, but not to audtiory stimulation. A general increase in reactivity m a y result in prolonged habituation. Furthermore, it should be p o i n t e d out that e l e c t r o l y t i c lesions in the brainstem m a y produce a reduced rate o f h a b i t u a t i o n [ 1 9 ] . The necrosis at the site o f injection was very small both after 5 , 7 - D H T and vehicle injections, and there was no clear difference b e t w e e n the t w o groups in the size o f this unspecific lesion. However, cell c o u n t i n g o f the lesion area was n o t performed. The possibility that there m a y exist small differences b e t w e e n the t w o groups in the damage to n o n - 5 - H T neurons therefore has n o t been e x c l u d e d . Lesions b o t h o f the medial and o f the medial plus the lateral 5 - H T bundles increased m o u s e killing behavior, and also increased n u m b e r of times the electric shocks elicited a b o x i n g position. These results are in accordance w i t h
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several p r e v i o u s studies. B o t h e l e c t r o l y t i c lesions in the r a p h e n u c l e i [ 1 7 , 2 6 ] , i n h i b i t i o n of 5 - H T s y n t h e s i s w i t h PCPA [6, 22, 2 3 ] , or w i t h p a r a c h l o r o - N - m e t h y l a m p h e t a m i n e [ 1 3 ] , a n d general lesion of 5 - H T t e r m i n a l s w i t h i n t r a c i s t e r n a l i n j e c t i o n o f 5 , 7 - D H T [2] p r o d u c e similar b e h a v i o r a l changes. This e x p e r i m e n t f u r t h e r s u p p o r t s the a s s u m p t i o n t h a t c e n t r a l 5 - H T n e u r o n s are i n v o l v e d in m u r i c i d e a n d s h o c k elicited fighting behavior. F u r t h e r m o r e , the e x p e r i m e n t i n d i c a t e s t h a t lesion of t h e m e d i a l 5 - H T b u n d l e is s u f f i c i e n t to p r o d u c e these b e h a v i o r a l changes. Everi~t, F u x e a n d J o n s s o n [7] o b s e r v e d b i t i n g a t t a c k s on males b y female rats w i t h 5 , 7 - D H T lesions o f t h e m e d i a l 5 - H T b u n d l e . Thus, in rats t h e a s c e n d i n g m e d i a l 5 - H T p a t h w a y seems to be i m p o r t a n t in c o n t r o l of agonistic b e h a v i o r b o t h in males a n d females, while in mice it has b e e n suggested t h a t o n l y female aggressive b e h a v i o r is c o n t r o l l e d b y s e r o t o n e r g i c m e c h a n i s m s [21 ]. T h e s h o c k elicited f i g h t i n g m o d e l has b e e n t h e m o s t f r e q u e n t l y used p a r a d i g m for aggressive behavior. B l a n c h a r d and B l a n c h a r d [ 1 ] , h o w e v e r , h a v e s h o w n t h a t the pain i n d u c e d b o x i n g p o s i t i o n m a i n l y is a defensive p o s t u r e . This test t h e r e f o r e s h o u l d be r e g a r d e d m a i n l y as a test for defensive b e h a v i o r . In the m o u s e killing b e h a v i o r t h e r e seems to be an i m p o r t a n t c o m p o n e n t of c o n s p e c i f i c a t t a c k , in a d d i t i o n to p r e d a t i o n and possible defensive c o m p o n e n t s [ 1 ] . It is possible t h e r e f o r e t h a t b o t h c o n s p e c i f i c a t t a c k and defensive b e h a v i o r are increased by the lesion of the medial 5 - H T p a t h w a y . In a p r e v i o u s s t u d y it was s h o w n t h a t o p e n field l o c o m o t o r activity was r e d u c e d in rats w i t h 5 , 7 - D H T lesions o f the m e d i a l plus lateral 5 - H T b u n d l e s [ 1 5 ] , in c o n t r a s t to the increase in activity a f t e r e l e c t r o l y t i c lesion
of t h e m e d i a n r a p h e n u c l e u s [ 1 8 , 2 4 ] . O n e possible r e a s o n for a d e c r e a s e d o p e n field l o c o m o t o r a c t i v i t y c o u l d b e a n increase in r e a c t i v i t y or e m o t i o n a l i t y in the 5 , 7 - D H T lesioned rats, p a r t i c u l a r l y since the field was b r i g h t l y i l l u m i n a t e d [ 15]. In the p r e s e n t s t u d y the rats t h e r e f o r e were o b s e r v e d in t h e h o m e cage (in a s i t u a t i o n w i t h o u t a n y n e w stimuli), a n d in a s i t u a t i o n w i t h a r a t h e r weak s t i m u l u s (gently m o v i n g the h o m e cage to a n o t h e r part of the r o o m , w i t h o u t h a n d l i n g o f the rat). H y p e r a c t i v i t y , as in the e l e c t r o l y t i c r a p h e l e s i o n e d rats, was n o t o b s e r v e d in a n y of these situations. On t h e c o n t r a r y , rats w i t h c o m b i n e d lesions in the m e d i a l a n d the lateral 5 - H T b u n d l e s t e n d e d to s h o w a r e d u c t i o n in t h e l o c o m o t o r activity also in this test, h o w e v e r , this r e d u c t i o n was n o t statistically significant. In c o n c l u s i o n , p r o l o n g e d h a b i t u a t i o n and increase in m o u s e killing b e h a v i o r a n d in s h o c k elicited defensive b e h a v i o r can be o b s e r v e d n o t o n l y a f t e r e l e c t r o l y t i c lesions of t h e 5 - H T n e u r o n s or p h a r m a c o l o g i c a l a l t e r a t i o n s o f the f u n c t i o n o f these n e u r o n s , b u t also a f t e r selective c h e m i c a l lesions o f t h e 5 - H T n e u r o n s . It seems likely t h e r e f o r e , t h a t these b e h a v i o r a l c h a n g e s are due to lesion o f the a s c e n d i n g 5 - H T p a t h w a y s . C o m p e t i t i v e and d o m i n a n c e b e h a v i o r s in o t h e r tests were n o t c h a n g e d , a n d lesion o f the a s c e n d i n g 5 - H T p a t h w a y s does n o t seem to result in a general increase in agonistic b e h a v i o r , b u t r a t h e r in p a r t i c u l a r c o m p o n e n t s o f this behavior. F u r t h e r m o r e , t h e s t u d y s u p p o r t s p r e v i o u s studies [ 1 5 , 2 0 ] , c o n c l u d i n g t h a t the increased a m b u l a t o r y activity o b s e r v e d in rats w i t h electrolytic lesions in the m e d i a n r a p h e n u c l e u s is n o t o n l y due to lesion o f the 5 - H T n e u r o n s a s c e n d i n g f r o m this nucleus.
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