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P e r g a m o n

Neuroscienceand BiohehavioralReviews,Vo l. 18, No. 3 , pp. 373-383, 1994Copyright©1994 ElsevierScienceLt d

Printed n the USA. All rio ts reserved0149-7634/94$6.00 + .00

0149-7634(93)E0009-B

Ph ysical DependenceProd uced by C entral M orph ine Infusions:

A n A natom ical M apping Study

M I C H A E L A . B O Z A R T H

D e p a r t m e n t o f P s y c h o l o g y, B e h a v i o r a l N e u r o s c ie n c e P r o g r a m ,S t a t e U n i v er s it y o f N e w Yo r k a t B u f f a l o , B u f f a l o , N e w Yo r k 1 4 2 6 0- 411 0

R e c e i v e d 21 A u g u s t 1 9 89

BO Z A RT H , M . A .Physical dependence produc ed by central morphine infusions: An anatomical mapping study. NEU -ROS CI B IOBEH AV REV 18(3) 373-383, 1 994 .-M orph ine sulfa te (1.5 nmoles/h) was chronically infused into var iousbrain regions in ra ts . A fter 72 h of con tinuous infusions, an intraper i toneal injec tion of naloxone hydroch lor ide (5 mg/kg )was given to determ ine if physical dependence h ad developed. V arious withdraw al signs were present following the nalox onechallenge in ra ts chronically infused w ith morph ine into the p er iventr icular gray region. These w ithdrawal signs includedescape behavior, wet-dog shakes, and teeth chattering. Several other brain regions (e.g. , amygdala, caudate nucleus, lateralhypothalam us, nucleus accumbens, tha lamus) were a lso tested, but m orphine infusions into these areas produced only sl ightphysical dependence. The m agnitude of p hysical dependence produced b y morph ine infusions into the rostra] aspect of the

per iventr icular gray was comp arable to that seen following repeated systemic morphine injec tions, while the physical depen-dence produ ced by caudal infusions into the vic inity of the locus coeruleus w as considerably less. These data conf irm theimportan ce of the per iventr icular gray region in the developm ent of physical dependence o n m orphine and reveal that a drugaction in the mo re rostra l aspect of this bra in region produces the strongest s igns of physical dependence.

Am ygda la Caud ate nucleus Chronic infusions Escape behavior Latera l hypothalamusMorph ine , naloxone Nucleus accumbens Osm otic minipumps Per iventr icular gray regionPhysical dependence Teeth chatter ing Thalamu s Ventra l tegmental area Wet-dog shakesWithdrawa l behavior

R E P E A T E D e x p o s u r e t o m o d e r a t e d o s es o f o p i at e s p r o d u c e sp h y s i c a l d e p e n d e n c e . W h e n t h e o p i a t e i n j e c t i o n s a r e d is -c o n t i n u e d o r w h e n a n a r c o t i c a n t a g o n i s t s u c h a s n a l o x o n ei s in j e c t e d , w i t h d r a w a l s i g n s e m e r g e t h a t a r e g e n e r a l l y p r o p o r -t i o n a l t o t h e d e g r e e o f o p i a t e e x p o s u r e ( i . e . , d o s a g e a d m i n i s -t e r e d , n u m b e r o f i n j e c t i o n s (s e e 3, 4 8) . T h e n a r c o t i c a n t a g o n i s tc h a l le n g e h a s t h e a d v a n t a g e o f p r e c i p i t a t in g w i t h d r a w a l b e -h a v i o r w i t h a p r e d e t e r m i n e d t i m e - c o u r s e a n d u s u a l l y o f s o m e -w h a t g r e a t e r i n te n s i t y t h a n s i m p l e d i s c o n t in u a n c e w i t h d r a w a l .T h u s , s t u d ie s i n v o lv i n g t h e b r a i n m e c h a n i s m s m e d i a t i n g p h y s -i c a l d e p e n d e n c e g e n e r a l l y u s e p r e c i p i t a t e d w i t h d r a w a l b e h a v -i o r r a t h e r t h a n s i m p l e d i sc o n t i n u a n c e w i t h d r a w a l .

I n e a r l y a t t e m p t s t o d e t e r m i n e b r a i n s i t es r e s p o n s i b le f o ro p i a t e d e p e n d e n c e , r a t s w e r e u s u a l l y m a d e p h y s i c a l l y d e p e n -d e n t b y s y s t em i c a d m i n i s tr a t io n o f m o r p h i n e a n d w i t h d r aw a lw a s p r e c i p i t a t e d b y c e n t r a l n a r c o t i c a n t a g o n i s t a d m i n i s t r a -t i o n . I n a n e x p e r i m e n t b y We i e t a l . ( 5 1 ) , r a t s w e r e m a d ed e p e n d e n t b y m o r p h i n e p e l l e t i m p l a n t a t i o n s a n d c r y s t a l l i n e

n a l o x o n e w a s a p p l i e d t o v a r i o u s b r a i n s i t e s t o a s s e s s t h e i r

c o n t r i b u t i o n s t o o p i a t e d e p e n d e n c e . S i t es in t h e t h a l a m i c a n dp e r i v e n t r i c u l a r g r a y r e g i o n s w e r e s h o w n t o b e i n v o l v e d i np h y s i c a l d e p e n d e n c e , w h i l e t h e a p p l i c a t i o n o f n a l o x o n e t o t h ec a u d a t e n u c l e u s o r o t h e r f o r e b r a i n s i t es s e l d o m p r e c i p i t a t e dw i t h d r a w a l b e h a v i o r s . Aw a r e o f t h e p r o b l e m o f e x t e n s iv e d ru gs p r e a d w i t h l i p o p h i l i c s u b s t a n ce s l i k e n a l o x o n e , L a s c h k a e t a l .( 2 3 ) u s e d i n t r a v e n t r i c u l a r i n j e c t i o n s o f a n a r c o t i c a n t a g o n i s tf o l l o w i n g t h e i n s e r t i o n o f " p l u g s " i n t o v a r i o u s p a r t s o f t h ev e n t r i c u l a r s y s t e m . T h i s p r o c e d u r e r e s t r i c t e d t h e e n t r y o f t h en a r c o t i c a n t a g o n i s t t o c e r t a in p a r t s o f t h e c e r e b r a l v e n t r ic u l a rs y s t e m . T h e s e s t u d i e s i d e n t i f i e d t h e c a u d a l v e n t r i c u l a r s y s t e m( i . e ., f o u r t h v e n t r i c le ) a s b e i n g p r o x i m a l t o t h e o p i a t e - r e c e p t o rf i e l d r e s p o n s i b l e f o r p h y s i c a l d e p e n d e n c e . T h e f a i l u r e t o f i n dp r e c i p i t a t e d w i t h d r a w a l f r o m n a r c o t i c a n t a g o n i s t i n j e c t i o n sr e s t ri c t e d t o t h e r o s t r a l v e n tr i c u l ar s y s t e m w o u l d a p p e a r t o b ei n c o n f l i c t w i t h We i c t a l . ' s ( 5 1 ) r e p o r t o f w i t h d r a w a l f r o mn a l o x o n e a p p l i c a t i o n in t h a l a m i c s i t es . T h is m a y b e d u e t oe i t h e r ( a ) t h e s p r e a d o f n a l o x o n e t o m o r e d i s t a l s it e s i n t h e

p e r i v e n t r i c u l a r g r a y r e g i o n i n W e i e t a l . ' s ( 5 1) s t u d y ; ( b ) o r t h e

37 3

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3 7 4 B O Z A RT H

f a i lu r e o f v e n t r i c u l a r n a r c o t i c a n t a g o n i s t i n f u s i o n s t o p r o d u c ee f f e c t i v e c o n c e n t r a t i o n s i n t h a l a m i c s i t e s i n L a s c h k a e t a l . ' s( 2 3) s tu d y. W h a t e v e r t h e c a s e , b o t h l i n e s o f e v i d e n c e e l i m i n a t ea f o r e b r a i n s i te o f a c t i o n f o r o p i a t e - i n d u c e d p h y s i c a l d e p e n -d e n c e a n d s u g g e s t t h e i m p o r t a n c e o f c a u d a l m i d b r a i n a n dp e r i v e n t r i c u l a r g r a y o p i a t e - r e c e p t o r f i e l d s .

A m o r e d i r e c t a p p r o a c h t o a s s e ss i n g w h i c h b r a i n s i t e s a r er e s p o n s i b le f o r t h e d e v e l o p m e n t o f p h y s i c a l d e p e n d e n c e o no p i a t e s i n v o l v e s t h e d i r e c t i n t r a c r a n i a l a p p l i c a t i o n o f m o r -p h i n e i n t o v a r i o u s b r a i n r e g io n s . We i ( 4 9) a n d W e i a n d L o h( 50 ) h a v e r e p o r t e d t h a t r a t s c h r o n i c a l l y a d m i n i s t e r e d m o r -p h i n e i n t o t h e p e r i a q u e d u c t a l g r a y - f o u r t h v e n t ri c l e r e g i o ns h o w p r o n o u n c e d w i t h d r a w a l s i g n s w h e n c h a l l e n g e d w i th s y s -t e m i c n a l o x o n e i n j e c t io n s . I n t h e s e s t u d i e s, m o r p h i n e w a s i n -f u s e d c h r o n i c a l ly u s i n g o s m o t i c m i n i p u m p s ( A l z e t C o r p . ; s e e4 5 ) a n d v a r i o u s w i t h d r a w a l s i g n s w e re o b s e r v e d f o l l o w i n g as y s t e m i c c h a l l e n g e d o s e o f n a l o x o n e . A l t h o u g h a v a r i e t y o fw i t h d r a w a l s i g n s w e r e d e m o n s t r a b l e a f t e r c e n t r a l m o r p h i n ei n f u s io n s , t h e m o s t c l e ar a p p e a r t o h a v e b e e n e s c a p e a tt e m p t sf r o m a c y l i n d r i c a l e n c l o s u r e , t e e t h c h a t t e r i n g , a n d w e t - d o g

s h a k e s .I n t h e p r e s e n t s tu d y, t h e p r o c e d u r e d e v e l o p e d b y We i

( 4 9, 50 ) w a s u s e d t o a n a t o m i c a l l y m a p v a r i o u s b r a i n r e g i o n sf o r t h e i r a b i li t ie s t o p r o d u c e p h y s i c a l d e p e n d e n c e o n c h r o n i cm o r p h i n e i n f u s i o n s . Tw e l v e b r a i n s i t es w e r e s e l e c t e d f o r s t u d yb a s e d o n p r e v i o u s r e p o r t s o f t h e i r r o le s i n p h y s i c a l d e p e n d e n c eo r o n t h e i r p r o p o s e d i n v o l v e m e n t i n o t h e r o p i a t e e f f e c t s .T h e s e s i t es w e r e s y s t e m a t i c a ll y e x a m i n e d u s i n g a s t a n d a r d p r o -t o c o l s o t h a t t h e r e l a t iv e i m p o r t a n c e o f t h e s e b r a i n r e g i o n s int h e d e v e l o p m e n t o f m o r p h i n e d e p e n d e n c e c o u l d b e d e t e r -m i n e d . T h e p a r a m e t e r s s e l e c te d f o r th i s s t u d y w e r e t h e o p t i m a li n f u s i o n d o s e , d u r a t i o n o f i n f u s i o n , a n d n a l o x o n e c h a l l e n g ed o s e r e p o r t e d i n We i ' s ( 4 9) s t u d y.

METHODS

Subjects

M a l e , L o n g - E v a n s r a t s ( w e ig h i n g 30 0 - 37 5 g ) w e r e s te r e o -t a x i c a l l y i m p l a n t e d w i t h u n i l a t e r a l 2 1 - g a u g e s t a i n l e s s s te e l c a n -n u l a e a i m e d a t t h e v a r i o u s b r a i n r e g i o n s li s t ed in Ta b l e 1 .W h i l e t h e r a t s w e r e a n e s t h e t i z 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( 6 0 m g / k g , I P ) , c a n n u l a e t h a t h a d b e e n f i l l e d w i t h s t e ri l e m o r -p h i n e s u l f a te s o l u t i o n w e r e c o n n e c te d t o o s m o t i c m i n i p u m p s( A l z e t C o r p . , M o d e l 2 0 0 1 ), a n d t h e c a n n u l a t i p s w e r e l o w e r e dt o t h e b r a i n r e g io n s u n d e r s t u d y. T h e m i n i p u m p s d e l i v e r e d 1# l / h o f 0. 5 # g / # l m o r p h i n e s u l f a te (1 .5 n m o l e / h m o r p h i n eb a s e ) d i s s o l v e d i n R i n g e r ' s s o l u t i o n . So m e o f t h e v e n t r a l te g -m e n t a l c a n n u l a e w e r e a n g l e d a t 2 0 - 3 0 ° f r o m t h e m i d l i n e t o

a v o i d p e n e t r a t i o n o f t h e p e r i v e n t r i c u l a r g r a y r e g i o n w h i c h h a sp r e v i o u s l y b e e n s h o w n t o p r o d u c e p h y s i c a l d e p e n d e n c e . T h em i n i p u m p s w e r e i m p l a n t e d s u b c u t a n e o u s l y b e t w e e n t h e s c a p -u l a e o f t h e a n i m a l s , a n d p o l y e t h y l e n e t u b i n g ( P E 6 0 ) w a s u s e dt o c o n n e c t t h e m i n i p u m p s t o t h e i m p l a n t e d c a n n u l a e ( F o rd e t a i l s , se e 4 9 , 5 4 ). Pe n i d U i n G ( 3 0 , 00 0 u n i t s , i . m . ) w a s a d m i n -i s t er e d p r o p h y l a c t i c a l l y f o l l o w i n g s u r g e r y. A f t e r r e c o v e r yf r o m t h e a n e s t h e t i c , a n i m a l s w e r e h o u s e d i n a 1 2 - h l i g h t / 1 2 - hd a r k c y c l e o f i l l u m i n a t i o n . A l l b e h a v i o r a l t e s t i n g o c c u r r e dd u r i n g t h e f i g h t p h a s e o f t h i s c y c l e . F o o d a n d w a t e r w e r ef r e e l y a v a i l a b l e in t h e h o m e c a g e s . So m e r a t s u s e d i n t h i s s t u d yt o d e t e r m i n e t h e e f f e c t s o f o t h e r e x p e r i m e n t a l m a n i p u l a t i o n sd i d n o t r e c e iv e i m p l a n t e d m i n i p u m p s n o r u n d e r g o s u r g e r y.

B e c a u s e o f a l a t e n c y o f a b o u t f o u r h i n r e a c h in g t h e i rn o m i n a l f l o w r a t e o f I / z l / h ( 5 5 ,5 6 ), t h e m i n i p u m p s w e r e k e p to v e r n i g h t ( r a n g e 1 6 - 2 4 h ) i n i s o t o n i c s a l i n e a t 3 7 ° C p r i o r t o

TA B L E 1STEREOTAXIC COO RDINATES*USED FOR MORPH INE INFUSIONS

Anterior-Placement Posteriort Lateral~ Ve ntral§ Sub jects¶

FCX + 4.4 + 2.5 2.0 14NA S + 3.5 + 1.6 6.2 15CPU + 2.0 + 3.0 6.0 12AMY G +0.4 +4 .8 7 .8 16LH A - 0.6 ± 1.5 9.2 10TH AL - 1.0 ± 2.0 6.0 17D3V - 3.8 + 0.0 4.9 13VT A - 3.8 + 0.6 8.2 12VTA -S - 3.8 + 0.6 8.2 14VTA -30 ° - 3.8 + 0.6 8.8 19RF - 3.8 + 1.7 6.8 14SC - 3.8 + 2.8 5.8 10PVG -R - 3.8 ± 0.6 5.8 14PVG-C -6 .8 +0 .0 to ±0 .6 4 .5 20

Two groups of animals implanted with cannulae in the ventra ltegmental area had cannulae angled 20-30 ° f rom the sagit ta l plane toavoid the pc riventricula r gray region. Sup erior colliculus and reticularform ation placements were also angled 20-30 ° from the sag ittal planeso that they followed the same tra jec tory as the angled ventra l tegmen-tal cannulae. The d orsal third-ventricle placement was angled a t 15 ofrom the sagit ta l plane to a void the midsagit ta l s inus. A ll other cannu-lae were para lle l to the sagit ta l plane . Abb revia t ions: FCX , f ronta lcortex; NAS, nucleus accumbens; CPU, caudate nucleus; AMYG,amygdala ; LHA , la tera l hypothalamic area; THA L, thalamus; D3V,med ial aspect of the dorsa l third-ventricle; V TA , ventral tegmentalarea (naloxone challenged); VTA-S, ventra l tegmental area (physio-logical saline challenged); V TA-30 °, v entral tcgm ental area (eannulaeangled 20-30°); RF, reticular formation; SC, superior colliculus;PVG -R, per iventr icular gray substance ( rostra l aspect); PVG -C, ped-ventricu lar gray substance (c audal aspect including the f ourth ventri-cle and locus caeruleus). A dap ted from (33).

*Upper incisor bar 5 nan above the interaura l l ine ; tram frombregma; ~mm from the midline; §ram from dura; ¶num ber of subjectstested.

i m p l a n t a t i o n i n t h e r a t s . A l s o , b e c a u s e th e m i n i p u m p s d e l iv e r1 / ~ l / h f o r a m i n i m u m o f 2 0 0 h , e a c h w a s r e u s e d in a s e c o n ds u b j e c t. T h e m i n i p u m p s w e r e b a t h e d i n i s o t o n i c s al i ne a t 3 7 ° Ct o m a i n t a i n t h e i r e q u i l i b r a t e d f l o w r a t e b e t w e e n t h e f i r s t a n ds e c o n d g r o u p o f r a t s t o r ec e i ve t h e s a m e m i n i p u m p s . F l u o r o -m e t r i c d e t e r m i n a t i o n s o f f l o w r at e p e r f o r m e d b y A l z e t C o r p o -r a t i o n s h o w e d t h a t t h e m e a n i n f u s io n r a t e f o r t h i s l o t o f m i n i -

p u m p s w a s 0 . 99# l / h + 0 . 1 / z l(see 5 9 ) .A f t e r c o m p l e t i o n o f b e h a v i o r a l t e s t i n g , r a t s w e r e d e e p l y

a n e s t h e t i z 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 p p r o x i m a t e l y 9 0m g / k g , I P ) a n d i n t r a c a r d i a ll y p e r f u s e d w i t h i s o t o n i c s a l in ef o l l o w e d b y a 1 0 % f o r m a l i n s o l u t i o n . T h e i r b r a in s w e r e t h e nr e m o v e d a n d f'L Xe d n 1 0 % f o r m a l i n f o r a t l e a s t t w o d a y s , a n df r o z e n s e c t i o n s w e r e t a k e n a t 4 0 m i c r o n i n t e rv a l s . F o l l o w i n gf o r m a l - t h i o n i n s t a i n in g , t h e c a n n u l a p l a c e m e n t s w e r e e x a m -i n e d a t a p p r o x i m a t e l y 1 0 - ti m e s m a g n i f i c a t i o n , a n d t h e s u b -j e c t s w e r e g r o u p e d a c c o r d i n g t o t h e h i s t o lo g i c a l v e r i f i ca t i o n o fc a n n u l a p l a c e m e n t s .

Procedure

A f t e r 7 2 ( + 2 ) h o f c o n t in u o u s m o r p h i n e i n f u s i o n s, t h er a t s w e r e p l a c e d in a P l e x i g l a s c y l i n d e r (2 3 x 2 5 c m ) . Fo l l o w -

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ing a 5-min adaptation period, they were challenged with anintraperitoneal injection of naloxon e hydrochioride (5 mg/kg )and placed back in the Plexiglas cylinder. The presence ofteeth chattering and of wet-dog shakes and the number ofescapes from the cylinder were scored in 5-min segments forthe next 20 rain (see Table 2). Other withdrawal signs werealso observed, but no attempt was made to quantify thembecause they have been shown to be less reliable than themeasures selected in this study (see 51).

RESULTS

N T

Mesencephalic Placements

Unilateral morphine infusio ns into either the rostral or can-dal regions of the periventricular gray substance producedmarked physical dependence as quantified by escape behaviorfollowing naloxone challenge (see Fig. 1). The animals withcannulae in the ven tral tegmental area also demonstrated sig-nificant escape behavior, although at abou t one third the rateof subjects that received morphine into the rostral periventric-ular gray region. Morph ine infused into the medial aspect ofthe dorsal third-ventricle failed to produce appreciable escapebehavior, an d this suggests that the respondin g seen followingmorphin e infusions into the other brain regions was not theresult of ventricular diffusion to a distal site of action. Ananalysis of variance (53) revealed a significant effect for thefactor of cann ula placements (F (3,61) = 12.406, p < .001).A Newm an- Keu ls' test (53) further showed that the escapebehavior associated with the rostral pe riventr icular gray place-ment was significantly greater than th at foun d with the otherplacements shown in Fig. 1 (p~ < .01). Ten unoperated ratswere also tested for withdrawal signs following an injection ofnaloxone (5 mg/kg, IP). No ne of these rats demonstrated anysigns of physical dependence including escape behavior,

although several of the subjects did attempt to leave the cylin-

TABLE 2VARIOUS WITHDRAWAL SIGNS

USED TO SCORE PRECIPITATED WITHDRAWAL

Withdrawal sign Criterion

Escape behavior

Teeth-chattering

Wet-dog shakes

Diarrhea

Vocalization

Aggression

Weight-loss

Three or more paws on the top r im of thecylindrical enclosure.

An audible sound made by rapid mastica-tion-like movements of the jaws.

Rotational movements of the body whichresemble the mot ion of a dog shaking him-self when wet.

A soft, formless stool without definite shape.

Squealing upon touch or spontaneously.

Attack behavior directed toward the experi-menter when touching the subject.

More than 2 g loss of body weight within20 minutes of the naloxone challenge.

Aggression, diarrhea, and weight-loss were observed in only a fewcases of the 200 animals tested in the chronic in fusion study. Vocaliza-tion scores, initially quantified, were deleted from the study when itbecame apparent that what was rated as spontaneous vocalization wasfrequently produced by the animal being approached by the experi-menter.

~ " 5 O

EO

4 ow@e~

0 8 0MUJ'3

2O

E= 10Zc-a®~ o

V TA P V G - R D 3 V

BRAIN SITES OF MORPHI NE DEPENDENCE 375

P V G - C

FIG. 1. Escape behavior following chronic morphine infusions intomesencephalic placements. The figure shows the mean ( + SEM) num-ber of escapes in 20 rain. NT = unoperated control animals chal-lenged with naloxone. (See Table 1 for o ther abbreviations.)

der during the 5-rain adaptation period prior to nalo xone in-jections.

Other withdrawal signs were also seen in the rats infusedwith morphin e into the periventricular gray regions. As shownin Table 3, teeth chattering was seen with similar frequency inanimals infused in either the rostral or caudal periventriculargray regions, bu t wet-dog shakes were primarily observed fol-lowing infusions into the caudal periventricular gray region.In fact, this observation is even more apparent if a measurethat not only represents frequency but is also sensitive to theintensity of the withdr awal sign is used. Figur e 2 was derivedby computing the mean numb er of periods each sign was pres-ent duri ng testing. A su bject that displayed a given withdraw alsign during only one of the four 5-min periods was assigned ascore of on e while a subject displaying the withd rawal sign forthe entire 20 min of observation (i.e., all four 5-rain periods)received a score of four. These values were then conver ted to

TABLE 3PERCENTAGE OF ANIMALS SHOWING VARIOUS

WITHD RAWAL SIGNS FOR MORE THAN ONE 5-MINP E R IO D F O L L O WIN G N A L O X O N E C H A L L E N G E

Escape Teeth- Wet-DogPlacement Behavior Chattering Shakes

FCX 0 0 0NAS 33 27 7CPU 0 0 0AMYG 12 0 6LHA 40 0 0THAI, 41 0 0D3V 21 14 0VTA 76 0 0VTA-S 25 0 0VTA-30 ° 32 10 0RF 100 14 0SC 0 0 0PVG-R 100 43 14PVG-C 70 25 45

See Table 1 for abbreviations.

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B R A I N S IT E S O F M O R P H I N E D E P E N D E N C E 377

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FIG . 4. Escape behavior following chronic morphine infusions intoforebrain an d m idbrain regions. Th e figure shows the mean ( + SEM)num ber o f escapes in 20 rain. (See Table I fo r abbreviations.)

o f t he an i m a l s t h a t r ece i ved m orph i n e i n fu s i ons i n t o t he am yg-da l a , t ha l am us , an d l a t e ra l hy po t ha l am us d i s p l ayed a l owlevel of escape behavior, bu t no h i s to logical d i fferences wereapparen t am on g r e s po nder s and n on res p onder s i n t h es egroup s . I n general the level of respond ing seen af ter chronici n fu s io ns i n t o fo reb ra i n an d m i db ra i n r eg i on s was ve ry lowand t e s t s fo r v en t r i cu l a r d i f fu s i on were n o t un der t aken be -caus e the p r i m ary s i t e o f ac t i on wo u l d a ppea r t o be a s s oc i a t edwi t h t he m es en ceph a l i c reg i o ns wh i ch s uppo r t a m u c h h i ghe rlevel of escape behav ior. A n analys i s o f var iance (53) fa i l ed tos ho w any s i g n i f i can t d i f f erences fo l l owi ng m o rp h i ne i n fu si onsin to the brain reg ions i l lus t ra ted in F ig . 4 (F (95 ,78) = 1 .657,p > .05).

Characteristics o f Withdrawal ResponsesB l ~ i g e t a l . ( 3 ) have s ugges t ed t ha t t h e w i t hd rawa l s yn -

d rom e p rec i p i t a t ed a f t e r a na rco t i c an t agon i s t cha l l enge canbe d i v i ded i n t o d o m i n an t and r eces si ve si g ns. Th ey r epo r t edan i n ve rs e r el a t i on sh i p be t ween t he appe a rance o f dom i n an ts igns such as escape behavior and teeth chat ter ing and reces-s ive s igns such as we t -dog shakes . Th ey sugges ted th i s re la t ion-s h i p was a fu nc t i o n o f bo t h t he l evel o f p hys i ca l depen denceand th e t ime s ince the an tagon is t chal lenge. F ig ure 5 i llus t ra test he d i s t r ibu t i on o f e s cape behav i o r, t ee t h cha t t e r i n g , and we t -dog s h akes ove r t he 20 m i n ob s e rva t i on pe r i o d . There w as noev i dence t h a t t he i nc i d ence o f we t -dog s hakes i nc reas ed t o -ward t h e en d o f t h e obs e rva t i on pe r i od ( c f. 3 ; F i g . 6 ). Infac t , t h e p e rcen t age o f an i m a l s s howi ng t h es e t h ree s igns was

highes t dur ing the in i t i a l 5 -min per iod . The d i screpant f ind-ings of the present s tudy and Blas ig e t a l . (3) could be due tot he f ac t t h a t t h e l a t t e r s t udy qu an t i f i ed t he i n t ens i t y o f a l lth ree measures whi le the present s tudy quant i f i ed escape be-hav i o r b u t s i m p l y t ab u l a t ed t he pe rcen t age o f an i m a l s s h owi ngt ee t h cha t t e ri n g an d we t -d og s hakes . Thes e s i g ns m i gh t havei nc reas ed i n f r equency wi t h i n s u b j ec t s wh i l e t he n um ber o fan i m a l s s ho wi ng a g i ven s i gn dec reas ed . A n o t h e r and p ro ba -b l y m o re i m p o r t an t d i f f e rence is t ha t B l ~ i g e t a l. ( 3 ) p roducedphys i ca l d epen dence wi t h s y s t em i c d rug i n j ec t i o ns , and t hewi t hd rawa l s yn d rom e no t ed i n t he i r s t u dy p robab l y r e f l ec t edt he con cu r ren t i nvo l v em en t o f s eve ral op i a t e - r ecep t o r f i e l ds .Nonetheless , the present s tudy sugges t s that the re la t ionshipbe t ween d o m i nan t and r eces s i ve w i t h d rawa l s i g ns m ay bem o re com pl i ca t ed t han p rev i ous l y s uggest ed .

A nu m b er o f o t he r w i t hd rawa l s i gn s have been r epo r t ed by

other inves t igators . These include abnormal pos tur ing , d i -ar rhea, hyperact iv i ty, mas t icat ion , and vocal izat ion (e .g . , 3 ,27 ,29 ,51) . Al l o f these s igns except d iar rhea were seen in thepresent s tudy . Because there i s d i ff i cu l ty in the quant i f i ca t ionof these s igns and because they tend t o be less re l iab le than thewi thdrawal s igns measured in th i s s tudy, these o ther wi th-draw al s igns were not used to assess the in tens i ty of mo rphin ewi t hd rawa l .

Histological Analysis

Representa t ive h i s to logies are i l lus t ra ted in F ig . 6 . Cannu-l ae a im ed a t t he f ron t a l co r t ex , nuc leus accum b ens , a nd cau -da t e nuc l eus were d i s per s ed wi t h i n abou t a 1 m m rad i u s o fthe p lacements shown in the f igure . No appreciab le s igns ofwi thdrawal were seen in any of these ra t s in th i s range ofp lacements except in the case of several nucleus accumbensinfused an imals previous ly d i scussed . Ca nnu la p lacements int he t ha l am u s were genera l ly i n t he caud a l po r t i o n o f t he ven -t r a l nuc l eus , and s om e l a t e ra l hypo t ha l am i c cannu l ae ex -tended to the zona incer ta . S i tes t es ted in the amygdala in-c luded the cent ra l , cor t i ca l , and pars l a tera l i s amygdalo idnucle i . His to logical analyses were unable to d i scern any re la-t ionships between wi thdrawal behavior and wi th in s i t e var ia-t ions in cannula p lacements for these brain reg ions . Even i freg ional var ia t ions ex i s ted , i t would be unl ikely that such f inean a t om i ca l d i s t i nc t i ons cou l d be m ade becau s e m orph i ne i n -fused a t th i s hour ly ra te i s l ikely to d i ffuse m ore tha n a m i l li -meter f ro m th e infus io n s i t e (see (26)) .

A n um ber o f s ub j ec t s whos e da t a were i nc l uded i n t hecauda l pe r i ven tr i cu la r g ray g roup had cann u l ae a i m ed s pec i fi -cal ly a t the locus coeru leus . Because the s ize of th i s s t ructurei s about the same as the 21-gauge eannulae used to infuse themorphine, l es ions were f requent ly created by the inser t ion ofthe cannulae . Fur thermore, s ince the locus coeru leus i s jus t

vent ra l to the four th vent r ic le (see F ig . 6H) , in fus ions del iv-e red t o t h i s a r ea a re accom pan i ed by app rec i ab l e d i f fu s i onin to the vent r icu lar sys tem. Likewise , morphine infus ions in tothe caudal per ivent r icu lar gray reg ion readi ly reach the locuscoeru leus by vent r icu lar d i ffus ion . In addi t ion , several sub-ject s t ested ear ly in the s tudy had c annulae a im ed at the l a tera laspect of the caudal per ivent r icu lar gray reg ion . Even these

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FIG. 5. Distribution o f the various withdrawal signs over the 20 minobservation period. Only subjects that displayed the correspondingwithdrawal sign during at least one of the observation periods wereincluded in the computations. Means for escape behavior representthe num ber of escape responses in a given time interval wh ile bothwet-dog shakes and teeth chattering means represent the percentageof sub jects showing these signs in each time interval.

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3 7 8 B O Z A RT H

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FIG . 6. Representative histolog ical econs tructions of the various brain regions tested in this study. Each placement is indicate dby a c irc le and the angle of can nula penctra t ion by an arrow. I t should be no ted that the placements i l lustra ted in sections A- Fare appro ximate mo dal values an d ap preciable dispersion of cann ulae occurred around these sites. Ab brevia t ions: AL , la tera lam ygd aloid nucleus; BC, brach ium con junctivu m (superior cerebellar peduncle); ca, ante rior commissure; cc, corpus callosum;cci, commissure of the inferior colliculns; CI, internal capsule; CIF, inferior colliculus; CO, optic chiasm; CPU, caudatenuclcus-putamen; CS, sup erior colliculus; D3V, dorsa l third-ventricle; DM H, d orsom edial nucleus of the hypo thalam us; FX,fornix, G P, glo bns pallidus; IP, interpe dunc ular nucleus; LC , locus coeruleus; LM , medial lemniscus; LS, lateral septal nucleus;MD , dorsom edial nucleus o f the thalamus; M S, m edia l septa l nucleus; NAS , nucleus accumbens; O T, optic trac t; PO A, la tera lpreoptic area; PV F, p er iventr icular gray substance; RF, re t icular formation; SN, substantia nigra ; TO L, la tera l olfac tory trac t;V, ventric le ; VD , ce ntra l nucleus of the thalam us, do rsa l par t ; VE, ventra l nucleus o f the thalamus; V MH ,ventromedial nucleusof the h ypothalamus (ad apted f rom (33); ventr icular nomencla ture f rom (31) .

p l a c e m e n t s , h o w e v e r , u s u a l l y r e s u lt e d i n r u p t u r i n g t h e f o u r t hv e n t r ic l e a n d s u b s e q u e n t c a n n u l a e w e r e l o w e r e d t h r o u g h t h em i d i i n e . H i s t o l o g i c a l e x a m i n a t i o n d i d n o t r e v e a l a n y a n a t o m i -c a l f a c t o r t h a t c o u l d a c c o u n t f o r t h e v a r i a b i l i t y o f b e h a v i o r a lr e s p o n s e s a s s o c i a t e d w i t h t h i s b r a i n r e g i o n . H e n c e , t h e s e s u b -j e c t s w e r e t r e a t e d a s a s i n g l e g r o u p .

B e c a u s e t h e v e n t r a l t e g m e n t a l a r e a h a s b e e n i m p l i c a t e d i nt h e r e w a r d i n g e f f e c t s o f o p i a t e s [ f o r a r e v i e w, s e e ( 5 , 6 , 9 ) ] ,t h e p o s s i b l e i n v o l v e m e n t o f t h i s r e g i o n i n t h e d e v e l o p m e n t o fp h y s i c a l d e p e n d e n c e o n m o r p h i n e i s p a r t i c u l a r ly i n te r e s ti n g .To f u l ly c h a r a c t e ri z e t h e a n a t o m i c a l s p e c i fi c i ty o f t h e p h y s i c a ld e p e n d e n c e p r o d u c e d b y v e n t r a l t e g m e n t a l m o r p h i n e i n f u -s i o n s, t h e d e p e n d e n c e - p r o d u c i n g e f f e c t o f m o r p h i n e i n f u s io n s

i n t o t h e a r e a s u r r o u n d i n g t h e v e n t r a l t e g m e n t u m w a s e x a m -

i n e d i n d e t a i l ( s ee 5 ). So m e c a n n u l a e w e r e a n g l e d 2 0 - 3 0 ° f r o mt h e m i d l ln e t o a v o i d p e n e t r a t i o n o f t h e l a t e r a l a s p e c t o f t h er o s t r a l p e r i v e n t r i c u l a r g r a y s u b s t a n c e ; t h i s i s a n e x t r e m e l y i m -p o r t a n t c o n t r o l b e c a u s e u n a n g l e d v e n t r a l t e g m e n t a l c a n n u l a ep e n e t r a t e t h e l a t e r a l a s p e c t o f t h e m o s t s e n s i t iv e b r a i n s i t e f o rp r o d u c i n g p h y s i c a l d e p e n d e n c e ( s e e F i g . 6 ( 3) . O t h e r c a n n u l a ew e r e p l a c e d 2. 5 a n d 3 .5 m m d o r s a l t o t h e v e n t ra l t e g m e n t u ma l o n g t h e s a m e l i n e o f t r a j e c t o r y f o l l o w e d b y t h e a n g l e d c a n -n u l a e . T h i s r e s u l t e d i n p l a c e m e n t s t h a t i n c l u d e d t h e r e t i c u l a rf o r m a t i o n a n d t h e s u p e r i o r c o l li c u lu s . C a n n u l a e i n t h e r e t ic u -l a r f o r m a t i o n w e r e f r e q u e n t l y m o r e m e d i a l t h a n t h e p l a c e m e n ti l l u s t r a t e d i n F i g . 6 ( 3 . Ca n n u l a e i n t h e s u p e r i o r c o l l l c u l u sr a n g e d d o r s a l t o t h e d o r s a l t h i rd - v e n t r ic l e a n d a s f a r a n t e r i o r

a s t h e l a t e r a l n u c l e us o f t h e p o s t e r i o r th a l a m u s a n d t h e l a t e r a l

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B R A I N S IT E S O F M O R P H I N E D E P E N D E N C E 379

genicula te body. The vent ra l t egmental reg ion tes ted for i t sab i l i t y t o p ro du ce phys i ca l d ependen ce f rom m orph i ne i n fu -s i on s i nc l ud ed t h e zone s how n t o c on t a i n t he r eward - re l ev an top i a t e r ecep t o r s (7 , 8 ) an d r ang ed f rom abou t 3 . 2 t o 4 . 2 m mpos t e r i o r t o b regm a . The ro s t r a l pe r i ven t r i cu l a r g ray r eg i onincluded an an ter ior-pos ter ior d i spers ion s imi lar to the vent ra lt egm en t a l p l acem en t s .

F i g u re 7 i ll u s tr a te s t he r an ge o f t i s s ue dam ag e p roduced byi n fu s i o n o f m o rp h i n e s u l f a t e a t t he r a t e o f l l z l / h fo r 72 h .M any o f t h e h i s t o l og i es s ho wed nec ro s i s i n t he r ange i l l u s -t r a t ed i n b ra i nSection Abu t m o s t were i n t e rm ed i a t e be t weenthe low (SectionA ) a n d m o d e r a t e(Section B)l evel s of dam-age . S ev e ra l an i m a l s had ex t ens i v e d am age p rod u ced by t h i schronic infus ion reg imen, and these subject s were dele tedfrom the data analys i s ( i . e . , see F ig . 7-C) . The var iab i l i ty incel l loss was surpr i s ing bec ause a l l subject s received ident icalo s m o t i c m i n i p um ps an d becaus e ca re was exe rci s ed t o i n s u ret ha t t he no m i na l f l ow ra t e o f l # l / h had been r eached p r i o r t oimplanta t ion . Nonetheless , there were l arge var ia t ions in theex t en t o f t i ss ue dam ag e fo l l owi ng ch ron i c m o rph i ne i n fu s ions .

The m o d a l l eve l o f t i s su e d am ag e w as h i g he r t han u s u a l l y s eenin s tudies us ing a s imi lar infus ion volume del ivered over as ho r t e r t i m e i n t e rv a l (M . B o zar t h , unpu b l i s hed obs e rva ti o ns ) .

Peripheral Compo nent in Weight-Loss

Th e f a i l u re t o obs e rv e wi t hd rawa l d i a r rhea o r s i g n i f i can tweight - loss was surpr i s ing because (a) opia tes have both cen-t ra l ly and per ipheral ly mediated effect s on in tes t inal mot i l i ty(43 ,44) ; (b) in t ravent r icu la r c lonid in e inhib i t s wi thdraw al d iar-rhea (41) ; and (c) the sys temic adminis t ra t ion of a narcot ican t ag on i s t t ha t d oes n o t c ro s s t he b l o od -b ra i n ba r r i e r f a ll s t op rec ip i t a te w i t hd rawa l d i a r rh ea (A . Herz , unp ub l i s hed ob s e r-

FIG . 7. Range of tissue damage produced by the chronic infusions.The f'flled circles illustrate th e extent of necrotic tissue visible under10-times magnification in form al-thionin stained brain section. Ab-breviations: IP, interpeduncular nucleus; PVG, periventricular gray

substance; SN, substantia nigra.

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FIG . 8. W ithdrawal w eight-loss following systemic morphine in jec-tions. T he figure shows the mean ( + SEM ) weight-loss (g) in 20 min.The 95% confidence interval for animals injected with naloxone hy-drochloride is also shown.

vat ion , c i t ed in 22). The fact that m orph ine chronical ly in-fused in to the caud al per ivent r icu lar gray reg ion (and the sur-rounding four th vent r ic le) d id not resu l t in d iar rhea fo l lowingna l oxone cha l l en ge does no t s uppo r t t h e n o t i o n t ha t w i t h -d rawa l d i a r rhea i s m ed i a t ed by an op i a t e ac t i o n a t t he l ocuscoern leus (cf . 14). Cons iderat ion o f these data prom pted ad i r ec t eva l ua t i on o f t he i nvo l vem en t o f pe r i p he ra l op i a t e -receptors in wi thdrawal d iar rhea and weight - loss .

To tes t the poss ib le cont r ibut ion of per ipheral op ia te-receptors to wi thdraw al d iar rhea and weight - loss , 37 ra t s werem ad e phys i ca l ly dependen t f ro m s ys tem i c m o rp h i ne i n j ec ti onsand w i t h d rawa l b ehav i o r was quan t i fi ed fo l l o wi ng t he adm i n -i s t ra t ion of a per ipheral ly act ing narcot ic an tagonis t . Nalox-one m e t h i od i de i s a qua t e rna ry de r iva t ive o f n a l oxo ne wh i chdoes n o t c ro s s t he b l o od -b ra i n ba r r i e r. The re fo re , w i t hd rawa ls i gns p rec i p i ta t ed by t h i s com p oun d a re p res u m ab l y m ed i a t edby per ipheral op ia te- receptors . Fo ur group s of ra t s (n = 8-12/group) were in jected twice dai ly wi th morphine su l fa te (80m g/k g, IP every 12 + 2 h) for 3 days . Rats then received anin t raper i toneal in ject ion of nalox one meth iodid e (0 .0 , 0 .4 , 2 ,or 10 m g/k g) d i sso lved in phys io logical sa l ine . A ddi t io nal ra t sreceived the same morphine in ject ion reg imen and were tes tedfo l l owi n g an i n t r ap er i t on eal i n j ect i on o f na l oxo n e hyd roch l o -r ide (5 m g/k g , n = 14). This las t g ro up served as a pos i t ivecont ro l to assess phys ical dependence fo l lowing th i s reg imeno f s y s t em i c m orph i ne i n j ec t i ons . B od y we i gh ts were m eas u red

and t h e p res ence o f d i a r rhea no t ed 20 r a i n fo l l o wi ng t he na l ox -one in ject ions .F igure 8 shows weight - loss fo l lowing var ious doses of nal -

oxone m e t h i od i de . Th i s pe r i ph e ra ll y ac t i ng na rco t i c an t ago -nis t p recip i ta ted s igni f icant weight - loss (F (3 , 33 )= 8 .767,p < .001), a l though d iarrhea w as seen in only a few subjects .I t i s poss ib le that d iar rhea emerged some t ime la ter, bu t thefai lure to observe d iarrhea shor t ly af ter adminis t ra t ion of theper ipheral ly act ing narcot ic an tagonis t i s cons i s ten t wi th theear l i er observat ion by Herz (see 22) . Surpr i s ingly, on ly 21%of t he r a t s cha l lenged wi t h na l oxone hy d ro ch l o r i de d i s p l ayedd i a r rhea wi t h i n 20 r a i n , and m an y r a t s s howed s pon t aneouswi thd rawal as quant i f i ed by the weight - loss in subject s noti n j ec ted wi t h na l oxone . None t he l es s , na l oxon e m e t h i od i de ap -pea red t o r ap i d l y d i s i nh i b i t f eca l e l i m i na t i o n and p roduced

s igni f icant weight -loss . These da ta sugges t a per ipheral cont r i -

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3 8 2 B O Z A RT H

a f t e r s y s t e m i c n a r c o t i c a n t a g o n i s t c h a l l e n g e . T h e o b s e r v a t i o n( in t h e p r e s e n t s t u d y ) th a t i n f u s i o n s o f m o r p h i n e i n t o t h e p e r i -v e n t r i c u l ar g r a y r e g i o n p r o d u c e d t h e g r e a t e s t d e g re e o f p h y s i -c a i d e p e n d e n c e i s c o n c o r d a n t w i t h t h e H e r z e t a l . ( 1 8 ) a n dL a s c h k a e t a l . ( 2 3 ) s t u d i e s . T h e r e i s , h o w e v e r , o n e i m p o r t a n td i f f e re n c e a m o n g t h e s e st u d i es : t h e i r p r o c e d u r e m a y h a v e p r e -v e n t e d d r u g d i f f u s i o n t o t h e m o s t s e n s i ti v e p a r t o f t h e p e r i v e n -t r i c u la r g r a y s u b s t a n c e . T h e e x a c t a n a t o m i c a l l o c a t i o n o f t h ep e r i v e n t r i c u l a r "p l u g " u s e d b y H e r z e t a l . (1 8 ) a n d L a s c h k a e ta l . ( 2 3 ) i s d i f f i c u l t t o d e t e r m i n e b a s e d o n t h e i r p u b l i s h e d r e -p o r t s . I n a n o t h e r e x p e r i m e n t u s i n g t h e s a m e p r o c e d u r e , t h ea p p r o x i m a t e l o c a t i o n o f t h e " p l u g " w a s i l l u s t r a te d ( s ee 4 2 , F i g .1 ). I t w o u l d a p p e a r t h a t t h e v e n t r i c u l a r "p l u g " u s e d t o r e s t r i c tt h e v e n t r i c u la r d i f f u s i o n o f m i c r o i n j e c t e d n a r c o t i c a n t a g o n i s tw o u l d e f f e c ti v e l y b l o c k d r u g d i f f u s i o n t o t h e r o s t r a l p o r t i o no f t h e p e r i v e n t r i c u l a r g r a y r e g i o n . T h u s , t h e f o u r t h v e n t r i c lei n j e c ti o n s o f L a s c h k a e t a l . ( 2 3 ) w o u l d r e a c h t h e c a u d a l p e r i -v e n t r i c u l a r g r a y s u b s t a n c e t e s t e d i n t h e p r e s e n t e x p e r i m e n t ,h u t t h e " p l u g " w o u l d p r o b a b l y p r o h i b i t a p p r e c i a b l e d r u g d i f -f u s i o n t o t h e r o s t r a l p e r i v e n t r i c u l ar g r a y r e g i o n f o u n d i n t h e

p r e s e n t s t u d y t o b e t h e m o s t s e n s i t iv e a r e a t o t h e c h r o n i ci n f u s i o n o f m o r p h i n e . S i m i l a r l y, i n j e c ti o n s i n t o t h e l a t e r a lv e n t r i c l e w o u l d f a i l t o r e a c h t h e r o s t r a l p e r i v e n t r i c u l a r g r a yr e g i o n . T h e r e f o r e , t h e s t u d i e s u s i n g t h e v e n t r i c u l a r "p l u g "p r o b a b l y m i s s e d w h a t m a y h a v e b e e n t h e m o s t e f f e ct i v e s i te inp r o d u c i n g t h e o p i a t e w i t h d r a w a l s y m p t o m o f e s ca p e b e h a v i o r .

T h e l e v el o f r e s p o n d i n g s e e n a f t e r n a i o x o n e c h a l le n g e i na n i m a l s c h r o n i c a l l y i n f u s e d w i t h m o r p h i n e i n t o t h e r o s t r a lp e r i v e n t r ic u l a r g r a y r e g i o n c o m p a r e s f a v o r a b l y w i t h t h a t s e e ni n o t h e r p h y s i c a l d e p e n d e n c e s t u d i e s. Ta b l e 4 s u m m a r i z e s v a r i-o u s s t u d i e s u s i n g s e v e r a l m e t h o d s o f d e m o n s ~ c a t i n g p h y s i c a ld e p e n d e n c e . T h e i n t e n s i t y o f t h e e s c a p e b e h a v i o r s e en i n t h ep r e s e n t s t u d y ( s e e F i g u r e 1 , PV G - R) i s h i g h e r t h a n t h a t r e -p o r t e d f r o m s y s te m i c d r u g a d m i n i s t r a t i o n s u g g e st i n g t h a t t h e

d e g r e e o f p h y s i c a l d e p e n d e n c e i n t h e s e s u b j e c t s i s a s g r e a t a st h a t o b t a i n a b l e w i t h s y s t em i c m o r p h i n e a d m i n i s t r a t i o n . T h i sw o u l d b e e x p e c t e d i f t h e r o s t r a l p e r i v e n t r i c u l a r g r a y r e g i o nw e r e a p r i m a r y s i t e o f a c t i o n f o r p h y s i c a l d e p e n d e n c e p r o -d u c e d b y s y s t e m i c o p i a t e in j e c t i o n s .

W h e t h e r t h e p e r i v e n t r i c u l a r g r a y r e g i o n i s h e t e r o g e n e o u sw i t h r e g a r d t o i ts a b i l i t y t o p r o d u c e p h y s i c a l d e p e n d e n c e o nm o r p h i n e h a s n o t b e e n c l e a r l y e st a b l is h e d . B e c a u s e v e n t r ic u l a rf l o w i n t h i s b r a i n r e g i o n i s i n a r o s t r a l t o c a u d a l d i r e c t i o n ( 1 2 ),i t is p o s s i b l e t h a t t h i s s t r u c t u r e is h o m o g e n e o u s a n d t h a t t h eg r e a t e r l e v e l o f e s c a p e b e h a v i o r a f t e r r o s t r a l p e r i v e n t r i c u l a rg r a y i n f u s i o n s i s th e r e s u l t o f d r u g d i f f u s i o n ( v i a t h e c e r e b r a la q u e d u c t ) t o a l a r g e r p r o p o r t i o n o f p e r i v e n t r i c n ia r g ra y o p i a t er e c e p t o rs . R e g a r d l es s o f t h e e x p l a n a t i o n f o r t h e s t r o n g p h y s i -c a l d e p e n d e n c e p r o d u c e b y r o s t r a ] p e r i v e n t ri c u l a r g r a y i n f u -s i o n s, t h i s b r a i n r e g io n a p p e a r s t o h a v e a n i m p o r t a n t r o l e int h e d e v e l o p m e n t o f o p i a t e p h y s i c a l d e p e n d e n c e . A l t h o u g ho t h e r b r a i n r e g i o n s a l so c o n t r i b u t e t o t h e n e t w i t h d r a w a l s y n -d r o m e f o l l o w i n g a b s t i n e n c e f r o m s y s t e m i c a l l y d e l i v e r e d o p i -a t e s , t h e s t r o n g e s t s i g n s o f p h y s i c a l d e p e n d e n c e a r e a s s o c i a t e d

w i t h a d r u g a c t i o n i n a r e l a t i v e l y w e l l d e f i n e d b r a i n r e g i o n .

ACKNOWLEDGEMENTS

Ma rtha A sselin is thanked fo r anim al surgery and behaviora l test-ing. C indy M. P udiak is thanked for conducting the study with naiox-one methiodide . Roy A. W ise is thanked for comm ents o n an ear l ierversion of the paper. A nd En do Laborator ies (Garden City, N J) isthanked for their generous don ation of naioxone hydroch lor ide . Thisresearch was supported by grants f rom the Nationa l Insti tute on D rugAbuse (DA02285) and f rom the Natura l Sciences and Engineer ingResearch Council of Canada (NSERC). Some data for this repor twere collected while the author was an NSERC University ResearchFellow at the Center for Studies in Behavioral Neu robiolo gy, Con -cord ia University, Montr~ai.

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