Eu Eu Mesmo e Brandao

7/27/2019 Eu Eu Mesmo e Brandao

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PharmacologyBiochemistry and Behavior,Vo l. 44, pp. 119-125, 1993 0091-3057/93 $6.00 + .00Printed in the U.S.A . All rights reserved. Copyright1992 Pergam onPress Ltd.

A v e r s i v e a n d A n t i a v e r s i v e E f f e c t s o f M o r p h i n ein t h e D o r s a l P e r i a q u e d u c t a l G r a y o f R a t sS u b m i t t e d t o t h e E l e v a t e d P l u s - M a z e T e s t

V I T O R M O T T A A N D M A R C U S L . B R A N D , ~ O 1Laborat6rio de Psicobiologia, FFC LRP , Campus,A v Bandei rant es 3900, 14049 Ribeir~to Preto, S~to Paulo, Brasil

R e c e i v e d 1 7 M a r c h 1 9 92

M OTTA, V . AND M . L . BRAND, ~ O. Aversive and antiaversive effects of morphine in the dorsal periaqueductai grayof rats submitted to the elevatedplus-maze test. P HA RM ACO L B I OCHEM BEHAV 4 4 ( 1) 1 19 -1 25 , 1 9 9 3 . - Th e d o r s a lper iaqueductal gray (DP AG ) is a wel l -known region for processing defensive behavior in the bralnstem . Rats im planted withcannulae in the D PA G w ere submit ted to the e levated plus-maze tes t for 5 ra in . The effects of m orphine following systemic(0 .1 -1 .0 m g /kg) o r D PA G admin i s t r a t ion (5 -30 nm ol ) were com pared wi th the benzod iazep ine compound m idazo lam in jec tedsimilar ly (1-10 mg/ kg, IP, a nd 10-80 nM, DP AG ). Mo rphine and mida zolam caused dose-dependent increases in the num berof entr ies and t ime spent in the open arm s. A systemic inject ion of naloxone in doses that b lock/~-opioid receptors reversedthe effects of centra l ly adminis tered morphine. Higher doses of morphine (70 umol) induced a non-naloxone-revers ible"fearful" hyperreact ivi ty . I t is suggested that low doses of m orphine inhibi t the neural substra te of avers ion in the D PA G,prob ably through a ct ivat ion of /~-receptors , and that m icroinject ions of higher doses of mo rphine cause proaversive act ionsno t med ia ted by these op io id r ecep to r s .D PA G Opioids Naloxo ne Escape Aversion Elevated plus-maze

I T h a s b e e n e s t a b l i s h e d t h a t a s e t o f s t r u c t u r e s i n t h e C N Sc o m p r i s e d o f t h e d o r s a l p e r ia q u e d u c t a l g ra y ( D P A G ) , m e d i a lh y p o t h a l a m u s , a n d a m y g d a l a c o n s t i t u te s t h e m a i n n e u r a l s u b -s t r a te s f o r t h e i n t e g r a t i o n o f a v e r s iv e s t a te s i n t h e b r a i n( 1 , 1 3 , 2 4 ) . Th e s e s t r u c t u r e s h a v e b e e n n a m e d , a s a wh o l e , a st h e b r a i n a v e r s i v e s y s t e m ( BA S ) ( 1 2) . S e v e r a l l in e s o f e v i d e n c eh a v e c l e a r ly i m p l i c a t e d G A B A e r g i c , s e r o to n e r g i c , a n d a m i n oa c i d - m e d i a t e d m e c h a n i s m s i n t h e c o n t r o l o f t h e n e u r a l s u b -s t r a t e s c o m m a n d i n g d e f e n s i v e b e h a v i o r i n t h e B A S ( 4 , 6 ,1 2 , 29 ) . I t h a s b e e n s u g g e s t e d t h a t 5 - h y d r o x y t r y p t a m i n e 2 ( 5 -H T 2 ) m e c h a n i s m s e x e r t a p h a s i c m o d u l a t o r y r o l e o n t h i s s y s -t e m b e c a u s e a n i n c r e a s e i n 5 - H T a c t i v i t y i n t h e D P A G a t t e n u -a t e s th e a v e r s iv e c o n s e q u e n c e s o f D P A G s t i m u l a t i o n a n d t h i se f f e c t c an b e b l o c k e d b y 5 - H T 2 a n t a g o n i s t s s u c h a s r i t a n s e r ina n d k e t a n s e r i n l o c a l l y a p p l i e d w h i l e 5 - H T a n t a g o n i s t s b yt h e m s e l v e s d o n o t c a u s e a n y a p p a r e n t a v e r s i v e r e s p o n s e s( 1 2, 3 0) . S u p p o r t f o r a 5 - H T 2 p a r t i c i p a t i o n i n th e m o d u l a t i o no f a f f e c t i v e b e h a v i o r i n t h e D P A G h a s b e e n g i v e n b y r e c e n te l e c t r o p h y s i o l o g i c a l s t u d ie s s h o w i n g t h a t t h e m a j o r i t y o f 5 - H Tr e c e p t o r s i n t h i s r e g i o n b e l o n g s t o t h e 5 - HT2 t y p e ( 1 0 ) . On t h eo t h e r h a n d , G A B A i s s u p p os e d t o e x e r t a t o n i c i n h i b i t o r yc o n t r o l on t h e B A S b e c a u s e G A B A a g o n is t s a n d b e n z o d ia z e -p i n e c o m p o u n d s ( 6 ) a t t e n u a t e t h e a v e r s iv e co n s e q u e n c e s o fB A S e l e ct r i c a l s t i m u l a t i o n a n d G A B A ^ a n t a g o n i s t s , s u c h a s

1 To whom requests for repr ints should be addressed.

b i c u c u l l in e a n d p i c r o t o x i n , r e p r o d u c e t h e m . T h e s e r e s u l ts t o -g e t h e r l e d t o t h e s u g g e s t io n t h a t b e n z o d i a z e p i n e s m i g h t o w ep a r t o f t h e i r w e l l - k n o w n a n t i a v e r s i v e e f f e c ts t o t h e i n c r e a s e o ft h e e f f i c ie n c y o f G A B A e r g i c m e c h a n i s m s i n t h e s e s t ru c t u r e sc o m m a n d i n g d e f e n s i v e b e h a v i o r .I n t e r e s t in t h e p o s s i b l e m o d u l a t o r y r o l e p l a y e d b y o p i o i d -m e d i a t e d m e c h a n i s m s i n t h e B A S h a s g r o w n l a t e ly . T h e e f f e ct so f o p i o i d a g o n i s t s d e p e n d u p o n t h e t y p e o f r e c e p t o r s w i t hw h i c h t h e y i n t e r a c t . I t h a s b e e n s h o w n t h a t ~ - a g o n i s t s f u n c -t i o n a s p o s i t i v e r e i n f o r c e r s a n d ~ - a g o n i s t s c a u s e a v e r s i v e s t a t e s( 2 ,5 , 2 2) . M o r p h i n e p r e s e n ts m o r e a f f i n i t y f o r / ~ - t h a n f o r ~ -r e c e p t o r s ( 2 1 ) . I n t h i s r e s p e c t , t h e DP A G h a s s i g n i f i c a n t le v e l so f / ~ - a n d g - r e c ep t o r s ( 2 0) . A s a m a t t e r o f f a c t , i t h a s b e e nr e p o r t e d t h a t m i c r o i n j e c t i o n s o f l o w d o s e s o f m o r p h i n e i n t ot h e D P A G a t t e n u a t e i n a d o s e - d e p e n d e n t m a n n e r t h e a v e r si v ec o n s e q u e n c e s o f e l e c t r i c s t i m u l a t i o n a t t h i s s i t e ( 7 , 8 , 1 6 , 1 7 ) .H i g h d o s e s o f m o r p h i n e , h o w e v e r , w h e n l o c a l l y i n j e c t e d i n t ot h e D P A G c a u s e a b e h a v i o r a l ac t i v a t io n t o g e t h e r w i t h j u m p s ,w h i c h s h o w s a great s i m i l a r i ty to t h e r e a c t i o n o b s e r v e d f o l l o w -i n g e l e c t ri c a l s t i m u l a t i o n o f o r m i c r o i n j e c t i o n s o f G A B Ab l o c k e r s in t o t h i s s t r u c t u r e 0 4 , 1 5 ) . H o w e v e r , u n t i l n o w n os t u d y h a s b e e n c o n d u c t e d t o e x a m i n e t h e in v o l v e m e n t o f o p i -o i d m e c h a n i s m s u s i n g a t e s t w i t h a n a t u r a l i s ti c a p p r o a c h s u c ha s t h e e l e v a t e d p l u s - m a z e ( E P M ) . I t w a s t h e r e f o r e o f i n t e r e s t

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120 M O T T A A N D B R A N D ~ Ot o v e r i fy w h e t h e r a n d h o w m i c r o i n j e c ti o n s o f m o r p h i n e i n t ot h e D P A G w o u l d a f f e c t t h e e x p l o r a t o r y a c t iv i t y o f r a t s s u b -m i t t e d t o t h i s t e s t.

METHODAnimals

T w o - h u n d r e d e i g h ty m a l e W i s t a r ra t s w e i g h in g 2 2 0 - 2 8 0 gw e r e h o u s e d i n g r o u p s o f 6 w i t h f r e e a c c e s s t o f o o d a n d w a t e r .T h e y w e r e k e p t i n t h e e x p e r i m e n t a l r o o m f o r 4 8 h p r i o r t o t h ee x p e r i m e n t o n a 1 2 L : 1 2 D c y c l e ( l ig h t s o n a t 7 : 0 0 a . m . ) a t2 5 - 2 7 o C .Surgery

O n e - h u n d r e d f i f t y - f o u r a n i m a l s w e r e a n e s t h e t i z e d w i th s o -d i u m p e n t o b a r b i t a l (4 5 m g / k g , I P ) a n d f i x e d i n a s t e r e o t a x i cf r a m e ( D a v i d K 6 p f , T o p a n g a , C A ) . A c h e m i t ro d e m a d e o f as t a in l e s s s t e el g u i d e c a n n u l a ( o . d . 0 . 6 r a m , i . d . 0 . 4 r a m ) g l u e dt o a b r a i n e l e c t r o d e (1 6 0 ~ m ) w a s im p l a n t e d i n t h e m i d b r a l n ,a i m e d a t t h e D P A G . T h e e l e c t r o d e w a s m a d e o f s ta i n le s s s te e lw i r e , 1 60 ~ ,m i n d i a m e t e r , i n s u l a t e d e x c e p t a t t h e c r o s s - s e c t i o no f t h e t i p re a c h i n g 1 m m b e l o w t h e l o w e r e n d o f t h e c a n n u i a .T h e u p p e r i n c i s o r b a r w a s s e t a t 3 . 3 m m b e l o w t h e i n t e r a u r a ll i n e s u c h t h a t t h e s k u l l w a s l e v e l e d b e t w e e n b r e g m a a n dl a m b d a . T h e c h e m i t r o d e w a s i n t r o d u c e d v e r t i c a ll y u s i n g t h ef o l l o w i n g c o o r d i n a t e s , w i t h l a m b d a s e r v i n g a s t h e r e f e r e n c ef o r e a c h p l a n e : p o s t e r o a n t e r i o r , 0 . 3 m m ; m e d i o l a t e r a l , 0 .5r a m ; a n d d o r s o v e n t r a l , 4. 5 m m ( 2 5 ). T h e c h e m i t r o d e w a s fi x e dt o t h e s k u l l b y m e a n s o f a c r y l i c r e s i n a n d t h r e e s t a i n l e ss s te e ls c re w s . T h e e l e c t r o d e w i r e w a s c o n n e c t e d t o a m a l e p i n , p a r a l -l e l t o t h e o u t e r e n d o f th e c a n n u l a . T o g e t h e r , t h e y c o u l d b ep l u g g e d i n t o a n a m p h e n o l s o c k e t a t th e e n d o f a f l e x i b le e l e c -t r ic a l c a b le a n d u s e d f o r b r a i n s t im u l a t i o n . A t t h e e n d o f t h es u r g e r y , e a c h g u i d e c a n n u l a w a s s e a l e d w i t h a s t a i n l e s s s t e e lw i r e t o p r o t e c t i t f r o m c o n g e s t i o n .

ApparatusO n e w e e k a f t e r s u r g e r y , r a t s w e r e p l a c e d i n a n a r e n a ( c ir c u -l a r , 6 0 c m i n d i a m e t e r a n d 5 0 c m h i g h ) w i t h a f l o o r d i v i d e di n t o 1 2 s e c t io n s . T h i s a r e n a w a s s i t u a t e d i n a n e x p e r i m e n t a lc o m p a r t m e n t i l lu m i n a t e d w i t h a 4 0 - W f l u o re s c e n t l a m p ( 3 50l u x a t t h e a r e n a f l o o r l e v e l) . R a t s w e r e a l l o w e d a 1 5 - ra i n p e r i o do f h a b i t u a t i o n i n t h e e n c l o s u r e , a f t e r w h i c h t h e b r a i n w a ss t i m u l a t e d e l e c t r i c a l ly b y m e a n s o f a s i n e - w a v e s t i m u l a t o r( 1 9) . T h e s t i m u l a t i o n c u r r e n t w a s m o n i t o r e d b y m e a s u r i n g t h ev o l t a g e d r o p a c r o s s a 1 - K r e s i s t o r w i t h a n o s c i l l o s c o p e ( L a b o ,B r a s i l ) . B r a i n s t i m u l i ( A C , 6 0 H z , 1 5 s ) w e r e p r e s e n t e d a tl - r a i n i n t e r v a l s w i t h t h e c u r r e n t i n t e n s i t y in c r e a s i n g b y s t e p so f 5 ~ A f o r m e a s u r e m e n t s o f t h e a v e r s iv e t h r e s h o ld . E s c a p et h r e s h o l d w a s o p e r a t i o n a l l y d e f i n e d a s th e l o w e s t i n t e n s i typ r o d u c i n g r u n n i n g ( g a l lo p ) o r ju m p i n g i n t w o c o n s e c u ti v e a s -c e n d i n g s e r i e s o f e l e c t r i c a l s t i m u l a t i o n . A n i m a l s w i t h a n e s -c a p e t h r e s h o l d a b o v e 2 0 0 ~ A ( p e a k t o p e a k ) w e r e d i s c a r d e df r o m t h e e x p e r i m e n t .Procedure

I n t h e d a y f o l l o w i n g th r e s h o l d d e t e r m i n a t i o n , e a c h r a t w a si n d i v i d u a l l y p l a c e d i n a w o o d e n a r e n a (6 0 x 6 0 3 0 c m ) f o r5 m i n . I m m e d i a t e l y a f t e rw a r d , t h e a n i m a l w a s t r a n s f e rr e d t oa n E P M s i m i l a r t o t h a t p r e v i o u s l y d e s c r i b e d b y P e l l o w e t a l .( 2 6 ,2 7 ) . B r i e fl y , t h e m a z e w a s 5 0 c m f r o m t h e f l o o r a n d c o n -s is te d o f t w o o p e n a r m s a n d t w o c l o s e d a r m s ( w i t h n o r o o f )a r r a n g e d s u c h t h a t l i k e a r m s w e r e o p p o s i t e e a c h o t h e r .

T h e r a t w a s p l a c e d i n th e c e n t e r o f th e E P M f a c in g o n e o ft h e c l o s e d a r m s . T h e n u m b e r o f e n t r i e s i n t o e a c h t y p e o f a r mw a s r e c o r d e d f o r 5 r a in ( a l l f o u r p a w s d e f i n i n g a n e n t r y ) . T h er a t i o o f t h e n u m b e r o f e n t r ie s a n d t i m e s p e n t in t h e o p e n a r m so v e r t h e t o t a l n u m b e r o f e n t r i e s a n d t i m e s p e n t i n a l l a r m s 1 0 0 w e r e c a l c u l a t e d . T h e s e r a t i o s a r e c o n s i d e r e d t o r e f l e c t t h ef e a r - i n d u c e d i n h i b i t i o n t o e n t e r t h e o p e n a r m s a n d c a n b er e l a t e d t o t h e l e v e l o f " a n x i e t y " e x p e r i e n c e d b y t h e t e s t a n i m a l( 2 6 ,2 7 ) . T o t a l e x p l o r a t o r y a c t i v i t y w a s m e a s u r e d a s t h e t o t a ln u m b e r o f a r m e n t r i e s . T h e m e a s u r e s w e r e t a k e n b y a n o b -s e r v e r st a n d i n g s t i l l i n t h e s a m e r o o m f o r 5 r a i n u s i n g s t a n d a r de l e c t r o m e c h a n i c a l G r a s o n - S t a d l e r e q u i p m e n t . R a t s t h a t r e -c e i v e d I P i n j e c t i o n s d i d n o t r e c e i v e a n y k i n d o f s u r g e r y a n dw e r e h o u s e d i n g r o u p s o f s i x f o r 2 d a y s b e f o r e t h e t e s t .R a t s w e r e r a n d o m l y a l l o c a t e d t o t h e f o l l o w i n g g r o u p s : a )v e h i c l e c o n t r o l a n d I P m i d a z o l a m ( 1 . 0, 3 . 0 , 5 . 6 , a n d 1 0 r a g /k g ) ; b ) v e h i c l e c o n t r o l a n d m o r p h i n e I P ( 0 .1 , 0 . 3 , 0 . 5 6 , a n d1 .0 m g / k g ) ; c ) v e h ic l e c o n t r o l a n d D P A G m i d a z o l a m ; d ) v e h i-c l e c o n t r o l a n d D P A G m o r p h i n e ( 5 , 1 0 , 3 0 , a n d 7 0 n m o l ) .D r u g s a n d v e h i c l e s a s c o n t r o l s w e r e in j e c t e d I P i n a v o l u m e o f1 m l / k g . I n t r a c e r e b r a l in j e c t i o n s w e r e m a d e a t t h e d o s e s g i v e na b o v e i n a v o l u m e o f 0 . 2 ~ 1 . T e n m i n u t e s a f t e r i n j e c t i o n s , r a t sw e r e p l a c e d in a w o o d e n a r e n a f o r 5 r a i n a n d i m m e d i a t e l ya f t e r w a r d i n t h e E P M . E a c h r a t r e c e i v e d o n l y o n e i n j e c t io n .A n i m a l s r e c e i v i n g 1 0 n m o l m o r p h i n e w e r e a l s o i n j e c t e d w i t he i t h e r s a l in e o r n a l o x o n e ( 1 m g / k g , I P ) 5 m i n a f t e r m o r p h i n ea d m i n i s t r a t i o n . T h e r e w e r e 1 0 r a t s p e r d r u g d o s e , s e l e c te d a tr a n d o m f r o m t h e v a r i o u s c a g e s.T h e i n t e n s e b e h a v i o r a l a c t i v a t i o n t o g e t h e r w i t h j u m p s i n -d u c e d b y 7 0 u m o l m o r p h i n e p r e c l u d e d t h e a c c o m p l i s h m e n t o ft h e E P M t e s t . F o r s t u d y i n g th e e x c i t a t o r y e ff e c t s i n d u c e d b yt h i s d o s e o f m o r p h i n e , a n i m a l s w e r e p l a c e d i n t h e m i d d l e o ft h e a r e n a i m m e d i a t e l y a f t e r m i c r o i n j e c t i o n s fo r r e c o r d i n g l o -c o m o t o r a c t i v i ty . T h e f o l l o w i n g b e h a v i o r a l r e s p o n s e s w e r e re -c o r d e d e v e r y m i n u t e f o r 1 h : n u m b e r o f c r o s s i n g s ( i . e ., n u m -b e r o f f l o o r s e c t io n s t r a v e r s e d ) , n u m b e r o f r e a r i n g s , ju m p s ,a n d r o t a t i o n s . T o e x a m i n e t h e n a t u r e o f t h e e x c i t a t o r y e f f e c tc a u s e d b y m o r p h i n e i n a n a d d i t i o n a l g r o u p o f a n i m a l s , n a l o x -o n e (1 m g / k g , I P ) w a s in j e c te d 5 r a in b e f o r e D P A G m o r p h i n em i c r o i n j e c t i o n . E i g h t a n i m a l s p e r g r o u p w e r e u s e d i n t h e s ee x p e r i m e n t s .Intracranial Injection Procedures

A n i m a l s w e re g e n t ly w r a p p e d i n a c l o t h , h a n d h e l d , a n d at h i n d e n t a l n e e d l e (o . d . 0 . 3 r a m ) i n t r o d u c e d t h r o u g h t h e g u i d ec a n n u i a e u n t i l i t s l o w e r e n d w a s 1 m m b e l o w t h e g u i d e c a n n u -l a e , r e a c h i n g t h e s a m e d e p t h a s t h e e l e c t r o d e t i p s . T h e i n j e c -t i o n n e e d l e w a s l i n k e d t o a 5 - ~1 H a m i l t o n s y r i n g e ( H a m i l t o nC o . , R e n o , N V ) b y m e a n s o f a p o l y e t h y le n e t u b i n g . A v o l u m eo f 0 . 2 ~ 1 w a s i n j e c t e d d u r i n g 2 0 s a n d t h e n e e d l e w a s h e l d i np l a c e f o r a n a d d i t i o n a l 1 0 s . T h e d i s p l a c e m e n t o f a n a i r b u b b l ei n s i d e t h e p o l y e t h y l e n e ( P E - 1 0 ) c a t h e t e r c o n n e c t i n g t h e s y r i n g en e e d l e t o t h e i n t r a c e r e b r a l n e e d l e w a s u s e d t o m o n i t o r m i c r o -i n j e c t i o n .Drugs

M o r p h i n e s u lp h a t e (R o c h e P r o d u c t s L t d . , B r a m p t o n , O n -t a r i o , C a n a d a ) , n a l o x o n e ( E n d o L a b o r a t o r i e s , G a r d e n C i t y ,N Y ) , a n d m i d a z o l a m ( R o c h e ) w e re e a c h d i ss o l v e d i n p h y s i o -l o g i c a l s a l in e ( 0 . 9 % ) s h o r t l y b e f o r e u s e . P h y s i o l o g i c a l s a l i n ea l s o s e r v e d a s v e h i c l e c o n t r o l .Histology

U p o n c o m p l e t i o n o f e x p e r i m e n t s, a n i m a l s w e r e d e e p l y a n -e s th e t iz e d w i t h s o d i u m p e n t o b a r b i t a l a n d p e r f u s e d i n t r a c a r d i-


3/7

E F F E C T S O F M O R P H I N E I N T H E P L U S - M A Z E T E S T 121a l l y w i t h s a l i n e f o l l o w e d b y f o r m a l i n s o l u t i o n ( 1 0 % ) . T h r e ed a y s l a t e r , b r a i n s w e r e r e m o v e d a n d f r o z e n . S e r i a l 5 0- ~, mb r a i n s e c t i o n s w e r e c u t u s i n g a m i c r o t o m e a n d s t a i n e d w i t hn e u t r a l r e d t o l o c a l i z e t h e p o s i t i o n s o f t h e e l e c t r o d e t ip s a c -c o r d i n g to P a x i n o s a n d W a t s o n ( 2 5 ). O n l y d a t a f r o m t h o s ea n i m a l s w i t h h i s t o l o g i c a l l y c o r r e c t c a n n u l a e p l a c e m e n t s w e r eu s e d f o r s u b s e q u e n t s t a t i s t i c a l a n a l y s i s .Stat is tical Ana lys is

A n a l y s i s o f v a r i a n c e ( A N O V A ) w a s p e r f o r m e d o n t h e p e r -c e n t a g e o f o p e n a r m e n t ri e s a n d t i m e s p e n t i n t h e o p e n a r m sw i t h d r u g t r e a tm e n t a s th e f a c t o r . A N O V A w a s a l s o p e r -f o r m e d o n t h e t o t a l n u m b e r o f a r m e n t r ie s . W h e n e v e r a d r u gi n c r e a se d o r d e c r e a s e d b o t h t o t a l a r m e n t ri e s a n d p e r c e n t a g eo f o p e n a r m e n t r i e s, a c o r r e l a t i o n c o e f f i c i e n t ( p r o d u c t x m o -m e n t c o r r e l a ti o n ) w a s c a l c u l a te d t o d e t e r m i n e t o w h a t e x t e n t

t h e a l t e r a ti o n i n o p e n a n n e n t ri e s w a s in d e p e n d e n t o f a n ye f f e c t o n c l o s e d a r m e n t r ie s . P o s t h o c c o m p a r i s o n s b e t w e e nm e a n s a n d c o n t r o l s w e r e p e r f o r m e d u s i n g D u n n e t t ' s t - te s t .T h e h y p e r e x c i t a b i l it y f o l l o w i n g 7 0 - n m o l m o r p h i n e m i c r o -i n j ec t i o n s in t o t h e D P A G i s e x p r es s ed a s t h e t o t a l n u m b e r o fc r o ss i ng s a n d j u m p s r e c o r d e d i n th e a r e n a . T h e s e d a t a w e r en o t n o r m a l l y d i s tr i b u t e d s o a n a ly s is w a s b y t h e M a n n - W h i t -n e y u - t e s t.

RESULTST h e e l e c t r o d e t ip s w e r e s i t u a t e d m o s t l y i n t h e d o r s o l a t c r a ia s p e c t s o f t h e D P A G a s i ll u s t r a t e d i n F i g . I . B e c a u s e b o t h t h el o w e r e n d o f t h e i n j e c t i o n n e e d le a n d t h e e l e c t r o d e t i p r e a c h e d1 m m b e l o w t h e g u i d e c a n n u l a o f t h e c h e m i t ro d e , b r a i n i n j e c -t i o n s w e r e m a d e c l o s e t o t h e s e e l e c t ro d e s .G r a d u a l i n c r e a se s i n D P A G e l e c tr ic a l s t im u l a t i o n o f r a t s

7

" x x l ! Ii " ~ '1 Is t" / X

B r e g m a - 6 . 3 0 m m

I I ~_--- ~J l-"~"

\B r e g m a - 6 . 8 0 m m

/ /I

I//

I'%

\ l/

. - - \ ; / -@I

~ t\ , , /

B r e g m a - 7 . 3 0 m mFIG . 1 . Locat ion o f e l ec t rode s it es on cross -sect ions f rom P axinos and Wat son 's r a t br a in ar i as (25) . F iguresr epresent t he a t l as coordinat es in /~m po s t er ior t o br egm a. CG D, cen t r a l gr ay; DR, dor sa l r aphe nucleus ; ml ,me dial lemniscus; aq, ce rebral aque duct .


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122 MOTT A AND BRANDAO

504O.J

o 30L.~~ 2o

lO

Ip

Xl

C 3 5.6 10DOSE (mg/kg)

DPAG

C 1 0 2 0 40 8 0D O SE ( n r n o l )

FIG. 2. Mean (+ SEM) percentage of entries into and time spent inthe open arms of the elevated plus-maze by rats tested with midazolamadministered IP (left) or directly into the dorsal periacqueductal gray(DPAG) (right) 10 rain before the test. Open columns represent num-ber of entries and hatched columns indicate time spent in the arms.n = 1 0 . *p < 0.05 and **p < 0.01, Dunnett's t-test.

tested in the arena caused a state of a rousal followed by freez-ing behavior. Animals stopped their ongoing behavior as ifthey oriented themselves toward an en vironm ental stimulus.With f urther increases in the inten sity of electrical stimula tion,freezing behavior gave way to a clear behavioral activationexpressed by running and/o r jum ping that stopped as soon asthe bra in stimul ation was switched off.Figure 2 shows the effects of systemic 0eft) and DPAG(righ0 administration of midazolam on the number of entriesinto and time spent in the EPM. The effects of systemic ad-ministration of midazolam (1-10 mg/kg) on the overall ex-

ploratory activity of rats in the EPM are shown in Table 1.ANOVA revealed a nonsignifica nt effect on the total num berof arm entrie s, F(4, 45) = 1.89, p > 0.05. As shown in Fig.2, these doses of midazola m caused a signifi cant effect on thepercentage of entries into, F(4, 45) = 3.12, p < 0.05, andtime spent, F(4, 45) = 4.15, p < 0.01, in the open arms.Posthoc analyses showed that 3, 5.6, and 10 mg/k g produceda signifi cant increase in the percent of numb er of entries(p < 0.05 in all cases) and time spent (p < 0.05 for 3 and 10mg/ kg an dp < 0.01 for 5.6 mg/kg) in the open arms. DPAGmicroin jectio n of rats with midaz olam also caused a nonsig-nifica nt effect on the total arm entries, F(4, 45) = 1.55, p >0.05 (see Table 1). In Fig. 2, it can be seen that this treatmentcaused a signifi cant increase in the percentage of op en armentries, F(4, 45) = 3.26, p < 0.05, and percentage of timespent in the open arms, F(4, 45) = 4.19, p < 0.01. Posthocanalysis posthoc showed that these effects were main ly due tothe doses of 20 (p < 0.05 for entries and time spent in theopen arms) and 40 nmol (p < 0.05 for entries and p < 0.01for time spent in the open arms).Morphine (0.1-1.0 mg /kg) had a significant effect on thetotal number of arm entries, F(4, 45) = 2.78, p < 0.05. Post-hoc analysis showed that at 0.3 mg/k g there was a significantincrease in this measure (p < 0.05, see Tabl e 1). As illustrate din Fig. 3 (left), systemic morphin e did si gnificantly alter thepercentage of open arm entries, F(4, 45) = 2.61, p < 0.05,and time spent in the open arms, F(4, 45) = 3.50, p < 0.05.Posthoc analysis showed that this effect was only due to 0.3mg/kg regarding the percentage of open arm entries (p


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124 MOTT A AND BRAND.~Ocannot be discarded, earlier studies have indicated that wheninjected in the approxi mate volume used in these experimentsthe maximal effective spread of drugs is probably not morethan 1.0 mm (3,18,23). This effect of morphine seems to heopioid in nature because it was antagonized by previous ad-minist ratio n of nalo xone. So, besides GABA mechanisms,opioid processes may also take part in the modulation of de-fensive behavior at bralns tem structures.In the present work, mi croinjec tions of high doses of mor-phine into the DPAG caused fearful hyperactivity. In thiswork, we presented evidence that high doses of morphi ne ap-plied to this mesencephalic area cause proaversive effects tha tare not blocked by naloxone in doses reported to block /~-receptors (21,22,28). Based upon these data, we suggest thathigh doses of morphine act locally in the DPAG, causingaversive effects that are not mediated by/~-opioid receptors.Other workers have ob taine d results consistent with this sug-gestion 04,15). As bicucuilin e mimicks and GABA itselfblocks this nonnaloxone-reversi ble action of mo rphine , Jac-quet et ai. (14) suggested that this morphine excitation wasdue in part to GABAA receptor blockade.Another possibility that remains to be investigated is thatmorphi ne causes its proaversive effects in the DPA G by acting

on other opioid receptors, for example, k-receptors. Increas-ing evidence suggests that opioid agonists can produce rein-forcing or aversive effects depending upon the types of recep-tors with which they interact. Thus, ~- and &receptor agonistsfunction as positive reinforcers and k-agonists produce aver-sive states as shown in several experimental paradigms(2,5,22,31). The DPA G of rats contains significant concentra-tions of ~- and k-receptors (20). These data, along with thewell-known fact that morphine has much higher affinity for~- than for k-receptors (21,28), could explain the differentialeffects caused by morph ine depending upon the dose injected.Low doses, which are supposed to activate/~-receptors, pro-duce antiaversive effects and proaversive effects may resultfrom the activation of k-receptors by high doses of morphi ne.It still remains to use selective agonists and a ntagonists of thevarious opioid receptors to support the suggestions derivedfrom this work.

ACKNOWLEDGEMENTSThis work was supported by CNPq (Proc. 50.0482/90) andFAPESP (Proc. 90/3474-0). V.M. was the recipient of a Master schol-arship from CNPq. The authors thank Roche Produces Ltd. for

kindly providing midazolam.

REFERENCESI. Adams, D. B. Brain mechanisms for offense, defense and submis-sion. Behav. Brain Sci. 2:201-241; 1979.2. Bals-Kubik, R.; Herz, A.; Shippenberg, T. S. Evidence that theaversive effects of opinid antagonists and k-agonists are centrallymediated. Psychopharmacology (Berl.) 98:203-206; 1989.3. Bandier, R.; Carrive, P. Integrated defence reaction elicited byexcitatory amino acid microinjection in the midbrain periaque-ductal grey region of the unrestrained cat. Brain Res. 432:95-106;1988.4. Bandier, R.; DePaulis, A.; Vernes, M. Identification of midbrainneurones mediating defensive behavionr in the rat by microinjec-

tions of excitatory amino acids. Behav. Brain Res. 15:107-I19;1985.5. Bechara, A.; Vanderkooy, D. Opposite motivational effects ofendogenous opioids in brain and periphery. Nature 314:533-534;1987.6. Brandio, M. L.; Agniar, J. C.; Graeff, F. G. GABA mediationof the antiaversive action of the minor tranquilizers. Pharmacol.Biochem. Behav. 16:397-402; 1982.7. Brandto, M. L.; Cabral, A. M.; Vasquez, E. C.; Schmitt, P.Chlordiazepoxide and morphine reduce pressor response to brainstimulation in awake rats. Pharmacol. Biochem. Behav. 23:1069-1071; 1985.8. Brand,o, M. L.; Coimbra, N. C.; Le~o Borges, P. C. Effects ofmorphine and midazolam on reactivity to peripheral noxious andcentral aversive stimuli. Nenrosci. Biobehav. Rev. 14:495--499;1990.9. Brandio, M. L.; DiScala, G.; Bouchet, M. J.; Schmitt, P. Escapebehavior produced by the blockade of glutamic acid decarboxyl-ase (GAD) in mesencephalic central gray or medial hypothala-mus. Pharmacol. Biochem. Behav. 24:497-501; 1986.10. Brand,o, M. L.; Lopez-Garcia; J. A.; Graeff, F. G.; Roberts,M. H. T. Electrophysinlogical evidence for excitatory 5-I-IT2anddepressant 5-HTIA eceptors on neurones of the rat midbrain tec-tum. Brain Res. 556:259-266; 1991.11. Fanselow, M. S.; Calcagnetti, D. J.; Helmstetier, F. J. The roleof ~ and K opioid receptors in conditional-fear induced analgesia:The antagonistic actions of nor-binaltorphimine and the cyclicsomatostatin octapeptide, Cys Tyr Orn Pen-amide. J. Pharmacol.Exp. Ther. 250:825-830; 1989.12. Graeff, F. G. Brain defense systems and anxiety. In: Roth, M.;

Burrow, G. D.; Noyes, R., eds. Handbook of anxiety, vol. 3.New York: Elsevier Science Publishers; 1990:307-354.13. Hunsperger, R. W. Affektreaktionen auf electriscbe reizung inhirnstamm der katze. Helv. Physiol. Pharmacni. Acta 14:70-92;1956.14. Jacquet, Y. F.; Saedrup, E.; Squires, R. F. Nonstereospecificexcitatory actions of morphine may be due to GABAAreceptorblockade. Enr. J. Pharmacol. 138:285-288; 1987.15. Jacquet, Y. F.; Squires, R. F. Excitatory amino acids: Role inmorphine excitation in rat periaqueductal gray. Behav. Brain Res.31:85-88; 1988.16. Jenck, F.; Schmitt, P.; Karll, P. Morphine applied to the mes-ensephalic central gray suppresses brain stimulation escape. Phar-macol. Biocbem. Behav. 19:301-308; 1983.17. Jenck, F.; Schmitt, P.; Karli, P. Morphine injected into the peri-aqueductal gray attenuates brain stimulation-induced effects: Anintensity discrimination study. Brain Res. 378:274-284; 1986.18. Lomax, P. The distribution of morphine following intracerebralmicroinjection. Experientia 22:249-250; 1966.19. Marseillan, R. F. A solid state sine wave stimulator. Physiol.Behav. 19:339-340; 1977.20. Mansour, A.; Khachaturian, H.; Lewis, M. E.; Akil, H.; Wat-son, S. J. Anatomy of CNS opioid receptors. Trends Neurosci.11:308-314; 1988.21. Martin, W. R. Pharmacology of opioids. Pharmacol. Rev. 35:283-323; 1983.22. Mucha, R . F . ; H e r z , A . Motivational properties of kappa and muopioid receptor agonists studied with place and taste preference

conditioning. Psychopharmacology (Bcrl.) 86:274-280; 1985.23. Myers, R. D. Injection of solutions into cerebral tissue: Relationbetween volume and diffusion. Physiol. Behav. 1:171-174; 1966.24. Olds, M. E.; Olds, J. Approach avoidance analysis of rat dien-cephalon. J. Comp. Neurol. 120:259-295; 1963.25. Paxinos, G.; Watson, P. The rat brain in stereotaxic coordinates.New York: Academic Press; 1986.26. Pellow, S.; Chopin, P.; File, S. E. Brlley, M. Validation of open:closed arm entries in the elevated plus-maze as a measure of anxi-ety in the rat. J. Neurosci. Meth. 14:149-167; 1985.27. Peliow, S.; File, S. E. Anxiolytic and anxiogenic drug effects onexploratory activity in an elevated plus-maze: A novel test of anxi-ety in the rat. Pharmacol. Biochem. Behav. 24:525-529; 1986.


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EFFECTS OF MORPH INE IN THE PLUS-MAZE TEST 12528. Robson, L. E.; Pasterson, S. J.; Kosterlitz, H. W. Opiate recep-tor. In: Iversen, L. L.; Snyder, S. H., eds. Handbook of psycho~pharmacology, vol. 17. New York: Plenum Press; 1983:13-80.29. Schmitt, P.; Carrive, P.; DiScala, G.; Jenck, F.; Brand,to, M.L.; Bagri, A.; Moreau, J. L.; Sandner, G. A neuropharmacologi-cal study of the periventricular neural substrate involved in flight,Behav. Brain Res. 22:181-190; 1986.

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