diazepam and discriminative motor control

8/18/2019 Diazepam and Discriminative Motor Control

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Pharmacology Biochemistry Behavior.Vo l. 35, pp. 419--427. © Pergamon Press plc, 1990. Printed in the U.S .A. 0091-3057/90 3.00 + .00

Diazepam and Discr iminat ive Motor Contro l :

A cu te Ch ronic and W ithdrawal Ef fect s

J O H N W . C U L B E R S O N , M A I S Y T A N G , 1 C H Y A N E . L A U A N D J O H N L . F A L K 2

D e p a r t m e n t o f P s y c h o l o g y - - B u s c h , R u t g e r s U n i v e rs it y , N e w B r u n s w i c k , N J 0 8 9 0 3

R e c e i v e d 1 5 S e p t e m b e r 1 9 8 9

CULBERS ON, J . W. , M. TA NG, C . E . LAU AND J . L . F ALK. Diazepam and discriminative motor control: Acute, chronic and

withdrawal effects. PHA RM ACO L BIOCHE M BE HAV 35(2) 419--427, 1990 .- -Rats were t rained to hold a force t ransducer operated

with a paw so that i t remained between upper and low er limits of a force band for a continuous 1.5-sec period to deliv er each food

pellet. Acute doses o f diazepam impaired measures o f this performance. Separate groups received chronic diazepam injections (6

mg/kg, IP) either presession (Before Group) or postsession (After Group), or presession vehicle (Vehicle Group). Th e After and

Vehicle Groups demonstrated that neither chronic postsession diazepam, nor time alone, altered motor performance. The performance

of the Before Group was affected by the daily diazepam, and although tolerance to the impairment developed, i t was incomplete. Late

in the chronic-administration phase (after 75 days) a toxic effec t of the suspending agent became evident in all groups as a decrease

in work rate, although the other performance indices were not affected. The withdrawal of diazepam from the Before Group led to

improved performance which returned to the original baseline level.

Mo tor performance Diazepam Chronic benzodiazepine Diazepam withdrawal

T H E i n v e s t i g a t i o n o f d r u g e f f e c t s o n m o t o r b e h a v i o r h a s e m p h a -

s i z e d t h e a n a l y s i s o f u n l e a r n e d , r e l a t i v e l y g l o b a l b e h a v i o r ( e . g . ,

gene r a l ac t i v i t y , r o t o r od ) w i t h l i t t l e a t t en t i on pa i d t o quan t i f i ab l e

m e a s u r e s o n s p e c i f i c a l l y t r a i n e d d i s c r i m i n a t i v e m o t o r t o p o g r a -

p h i e s . E a r l y s t u d ie s o f d r u g e f f e c t s o n c o n t i n u o u s l e v e r ( 8 ) a n d

head ( 5 ) pos i t i on i ng s t ead i ness t a sks wer e p r om i s i ng , bu t l i t t l e

p u r s u e d . W e h a v e u s e d a s i m i l a r t a s k w i t h r a ts , a d i s c r i m i n a t i v e

m o t o r c o n t r o l t e c h n i q u e , i n w h i c h h o l d i n g a f o r c e t r a n s d u c e r f o r a

s h o r t , c o n t i n u o u s p e r i o d w i t h i n a f o r c e b a n d i s r e i n f o r c e d b y t h e

d e l i v e r y o f a f o o d p e l l e t . T h e a c u t e , c h r o n i c a n d w i t h d r a w a l

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

h a v e b e e n s t u d i e d in ra t s ( 1 1 , 2 2 , 2 3 , 3 7 , 4 0 , 4 1 ) , m o n k e y s ( 1 , 1 0 ,

2 0 , 3 5 ) a n d h u m a n s ( 3 , 4 6 ) .

A l t h o u g h t h e b e n z o d i a z e p i n e s h a v e v a r i o u s b e h a v i o r a l e f f e c t s ,

t h e m u s c l e - r e l a x a n t a n d s e d a t i v e - h y p n o t i c c o m p o n e n t s r a i s e t h e

q u e s t i o n o f t h e e x t e n t t o w h i c h t h i s c l a s s o f a g e n t s m i g h t a f f e c t

m o t o r b e h a v i o r . A p r e v i o u s s t u d y e x p l o r e d t h e a c u te , c h r o n i c a n d

w i t h d r a w a l e f f e c t s o f t h e u l t r a s h o r t -a c t i n g b e n z o d i a z e p i n e a g e n t

m i d a z o l a m o n d i s c r i m i n a t i v e m o t o r c o n t r o l ( 4 1 ) . I n t h e p r e s e n t

s t u d y , t h e a i m w a s t o e x p l o r e t h e g e n e r a l i t y o f t h e e f fe c t s o b s e r v e d

w i t h m i d a z o l a m b y u s i n g d i a z e p a m , a n o t h e r b e n z o d i a z e p i n e

w h i c h h a s b e e n e m p l o y e d e x t e n s i v e l y a s a t h e r a p e u t i c a g e n t .

METHOD

Animals

N i n e m a l e , a l b i n o , a d u l t ra t s o f t h e H o l t z m a n s t r ai n w i t h a

m e a n i n i ti a l b o d y w e i g h t o f 3 8 2 . 2 g ( r a n g e : 3 7 7 - 3 8 8 g ) w e r e u s e d .

T h e y w e r e h o u s e d i n d i v i d u a l l y i n s t a i n l e s s - s t e e l c a g e s i n a

t e m p e r a t u r e - r e g u la t e d r o o m w i t h a d a i l y c y c l e o f i l l u m i n a t i o n

f r o m 7 0 0 - 1 9 0 0 h r . A n i m a l s w e r e n a i v e a n d w e r e r e d u c e d t o 8 0 %

o f t h e i r a d l i b b o d y w e i g h t s b y l i m i t i n g d a il y f o o d r a t i o n s. F o o d

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

l i v i n g c a g e s i m m e d i a t e l y a f t e r d a il y e x p e r i m e n t a l s e s s i o n s . W a t e r

w a s a v a i l a b l e c o n t i n u o u s ly i n t h e l i v i n g c a g e s .

D r u g

D i a z e p a m w a s s u s p e n d e d i n a v e h i c l e c o n s i s t in g o f d i s t i ll e d

w a t e r a n d 2 m g / m l o f A g e n t K ( B i o S e r v , I n c . , F r e n c h t o w n , N J ) ,

u l t r a son i ca t ed i n an i ce ba t h f o r 5 m i n , and i n j ec t ed ( I P ) i n a

v o l u m e o f 1 m l / k g b o d y w e i g h t . [ T h r e e a n im a l s , Y 1 6 , Y 1 7 a n d

Y 2 2 r e c e i v e d d i a z e p a m a n d v e h i c l e i n j e c t io n s u s i n g a n i n a d e q u a t e

c o n c e n t r a t i o n o f A g e n t K ( 1 r n g / m l ) d u r i n g t h e d o s e - e f f e c t

d e t e r m i n a t i o n s a n d f o r t h e f i r s t 2 w e e k s o f t h e c h r o n i c - a d m i n i s -

t r a t i on phase . ]

Serum Diazepam Analys i s

Bl oo d sam p l es ( 100 i x l ) wer e t aken f r o m t he t a i l t i p . S am pl es

w e r e c e n t r i f u g e d f o r 5 r a i n a t 1 3 , 7 0 0 x g . T h e c l e a r s e r u m l a y e r

w a s a n a l y z e d f o r d i a z e p a m a n d i t s m e t a b o l i t e s u s i n g a s i n g le

ex t r ac t i on p r oce du r e ( 21 ) . F o r ca l i b r a t i on s t anda r ds , 25 i x l o f t he

i n t e r na l s t anda r d ( 1 ~g / m l de m o xepa m ) and 50 I L l o f t he d r ug

s t anda r d wer e p i pe t t ed i n t o a 15 - m l con i ca l cen t r i f uge t ube and

1Present address: Department of Neuropathology (Neuroscience), Harvard M edical School, Boston, M A 02115.

2Requests for reprints should be addressed to John L. Falk.

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4 2 0 C U L B E R S O N

ET AL

e v a p o r a t e d t o d r y n e s s u n d e r a s t r e a m o f n i t r o g e n . A 5 0 - ~ 1 b l a n k

s e r u m s a m p l e , 1 0 0 Ix l o f 1 M b o r a t e b u f f e r p H 9 . 0 ) a n d 2 . 5 m i o f

d i e t h y l e th e r w e r e t h e n a d d e d t o t h e s a m p l e , v o r t e x - m i x e d f o r 3 0

s e c , f o l l o w e d b y c e n t r i f u g i n g a t r o o m t e m p e r a t u r e f o r 5 m i n a t

1 1 0 0 × g . T h e e t h e r l a y e r w a s c a r e f u l ly t r a n s f e r re d t o a 5 - m l

c o n i c a l c en t r i f u g e t u b e a n d e v a p o r a t e d u n d e r n i t r o g e n . T h e r e s i d u e

w a s r e s u s p e n d e d i n 5 0 ~ 1 o f t h e m o b i l e p h a s e a n d t h e n 1 0 0 I xl o f

n - h e x a n e w a s a d d e d i n o r d e r t o r e m o v e t h e c o l l o i d a l l ip i d s . T h e

m i x t u r e w a s v o r t e x - m i x e d f o r 2 s e c a n d t h e h e x a n e l a y e r w a s

r e m o v e d i m m e d i a t e l y w i t h a d i s p o s a b l e p i p e tt e . S a m p l e s f o r d r u g

s e r u m a n a l y s i s w e r e p r e p a r e d i d e n t i c a l l y e x c e p t d r u g s t a n d a r d s

we re not a dde d . Ins te a d , s e ru m sa m ple s 50 Ix l) we re a dde d a f t e r

t h e i n t e r n a l s t a n d a r d w a s i n i t i a l l y e v a p o r a t e d t o d r y n e s s . S e p a r a -

t i o n w a s p e r f o r m e d o n a n U l t r a sp h e r e C 1 8 c o l u m n 5 I x m p a r t i cl e

s i z e, 1 5 0 × 2 . 0 n u n i . d . , A l t e x , S a n R a m o n , C A ) , a n d t h e m o b i l e

p h a s e c o n s i s t e d o f m e t h a n o l , a c e t o n i t r i l e a n d 0 . 0 5 6 M s o d i u m

a c e ta t e b u f f e r w h i c h h a d b e e n a d j u s te d t o p H 4 . 0 4 7 . 5 : 9 : 4 3 . 5 ) .

T h e f l o w r a t e w a s s e t a t 0 . 3 m l / m i n a n d n o r m a l l y o p e r a te d a t a

p r e s s u re o f 2 0 0 0 p . s . i .

Apparatus

T h e e x p e r i m e n t a l s p a c e f o r e v a l u a t i n g d i s c r i m i n a t i v e m o t o r

c o n t r o l p e r f o r m a n c e w a s a P l e x i g l a s c h a m b e r 2 5 × 3 0 x 3 0 c m )

w i t h s t a i n l e s s -s t e e l f r o n t a n d r e a r p a n e l s a n d a f l o o r c o n s i s t i n g o f

p a r a l l e l - m o u n t e d , s p a c e d , s t a i n l e s s- s t e e l r o d s . T h e o p e r a n d u m

w a s a s t a i n le s s - s te e l l e v e r m o u n t e d 2 . 5 c m f r o m t h e f l o o r . I t w a s

s u r r o u n d e d b y a P le x i g l a s s h i e l d w i t h a 1 c m w i d e × 4 c m h i g h

s lo t so tha t a c c e ss to i t wa s l im i te d to a s ingle pa w. The f ro nt e dge

o f t h e o p e r a n d u m w a s r e c e s s e d 1 . 2 c m f r o m t h e f r o n t s u r fa c e o f

t h e s h i e l d t o p r e v e n t n o s e - p o k i n g o r r e s p o n s e s o t h e r t h a n p a w

a c t u a t io n f r o m o p e r a t i n g t h e le v e r . T h e o p e r a n d u m w a s s u s p e n d e d

b y a p h o s p h o r - b r o n z e l e a f s p r i n g 0 . 2 0 m m t h i c k ) , a n d i t s s h a f t

r e s t ed o n a d r i v e r o d c o n n e c t e d t o a f o r c e t r a n s d u c e r m o d e l U C 3

s t r a in g a u g e , S t a t h a m I n s t r u m e n t s , O x n a r d , C A ) t h r o u g h a l o a d

c e l l S t a t h a m m o d e l U L 4 ) . T h e v o l t a g e o u t p u t f r o m t h e f o r c e

t r a n s d u c e r w a s c o n v e y e d t o a c u s t o m i z e d s i g n a l c o n t r o l b o x

T r i - T e c h S e r v i c e s , H a m i l t o n S q u a r e , N J ) a n d s o r t e d i n t o o n e o f

t h r e e s ig n a l r e g io n s : a b o v e , b e l o w o r w i t h i n a w i n d o w d e f i n e d b y

p r e s e t l o w e r a n d u p p e r v o l t a g e l i m i t s . T h e s e l i m i t s c o rr e s p o n d e d

t o a p p l ie d f o r c e s o f 0 .1 4 7 N 1 5 g fo r c e ) a n d 0 . 2 6 5 N 2 7 g f o rc e ) ,

r e s p e c t iv e l y , i n c i d e n t a t t h e p a w - p l a c e m e n t r e g i o n o f t h e o p e r a n -

d u m . A b u f f e r w a s s e t s o th a t a m i n i m u m f o r c e o f 0 . 0 1 5 N 1 . 5 g

f o r c e ) w a s r e q u i r e d f o r s i g n a l r e c o g n i t i o n . A C o m m o d o r e P e t

4 0 1 6 m i c r o c o m p u t e r w as p r o g r a m m e d i n a s s e m b l y l a n g u a g e t o

s a m p l e s i g n a l i n p u t o n c e e v e r y 1 0 m s e c . W h e n t h e f o r c e a p p l i e d

b y t h e a n i m a l w a s w i t h i n t h e 0 . 1 4 7 t o 0 . 2 6 5 N b a n d , a n a u d i o

f e e d b a c k s i g n a l S o n a l e r t S C 6 4 8 H , P . R . M a l l o r y , I n d i a n a p o l i s ,

I N ) w a s t u r n e d o n .

Discriminative Motor Control Measures

A c o n t i n u o u s l y a p p l i e d i n - b a n d f o r c e w i t h i n t h e 0 . 1 4 7 - 0 . 2 6 5

N w i n d o w ) l a s t i n g t .5 s e e w a s r e q u i r e d fo r t h e d e l i v e ry o f a 4 5 - m g

f o o d p e l l e t B i o S e r v , I n c . ) . I f t h e a p p l i e d f o r c e le f t t h e b a n d b e f o r e

1 . 5 s e c h a d e l a p s e d , t h e n t h i s t i m e r w a s r e s e t . T h u s , t h e b e h a v i o r

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

s t e a d il y o p e r a te d w i t h i n t h e f o r ce b a n d f o r a c o n t i n u o u s , s e t p e r i o d

o f t i m e . O r d i n a r i l y , a s e s s io n w a s t e r m i n a t e d w h e n t h e 5 0 t h p e l l e t

h a d b e e n d e l i v e r e d , b u t a s e s s i o n w a s a l s o t e r m i n a t e d i f 3 0 m i n

h a d e l a p s e d w i t h o u t o p e r a t i o n o f t h e t r a n s d u c e r . T h i s h a p p e n e d

r a r e ly a n d w a s d r u g - d o s e r e l a t ed . T h e s e o c c u r r e n c e s a r e n o t e d i n

t h e r e l e v a n t f i g u r e l e g e n d s .

I n e a c h s e s s i o n , t h e r a w m e a s u r e s o f m o t o r b e h a v i o r w e r e :

S e s s i o n T i m e t h e t im e t a k e n t o e a rn 5 0 f o o d p e l l e ts ) , T o t a l

R e s p o n s e T i m e t h e a m o u n t o f th e s e s s i o n t i m e t h a t t h e t r an s d u c e r

w a s h e l d o p e r a t ed a b o v e t h e m i n i m u m r e c o g n i t i o n t h r e s h o l d o f

0 . 0 1 5 N ) , I n - B a n d T i m e t h e a m o u n t o f t h e s e s s i o n t i m e t h at t h e

t r a n s d u c e r w a s h e l d o p e r a t e d w i t h i n t h e f o r c e - b a n d w i n d o w ) a n d

E n t r a n c e s t h e n u m b e r o f ti m e s d u r i n g a s e s s i o n t h a t a p p l ie d f o r ce

e n t e r e d t h e b a n d f r o m e i t h e r t h e l o w e r o r u p p e r s e t l im i t s ) . E x c e p t

i n t h e c a s e o f E n t r a n c e s , t h e s e r a w m e a s u r e s a r e n o t u s e f u l

c h a r a c te r i z at i o n s o f m o t o r p e r f o r m a n c e . F o r e x a m p l e , t h e I n - B a n d

T i m e m e a s u r e i s m o r e i n f o r m a t i v e w h e n c o m p a r e d w i t h t h e

m i n i m u m t o ta l I n - B a n d T i m e t h at w o u l d p r o d u c e t h e d e l i v e ry o f

5 0 f o o d p e ll e t s 1 . 5 s e c / p e ll e t x 5 0 p e l le t s = a M i n i m u m P o s s i b l e

I n - B a n d T i m e o f 7 5 s ec ) . T h e r e f o r e, a m e a s u r e o f I n - B a n d

E f f i c i e n c y i s c a l c u l at e d b y t a k i n g t h e r a t i o o f t h e s e t w o v a l u e s :

, ~ l i i ,~ r r ; c ; e n c = M i n i m u m P o s s ib l e I n - B an d T i m e

I n - B a n d

I n - B a n d T i m e

A l s o , r a w I n - B a n d T i m e c a n b e v i e w e d i n r e la t i o n to t h e T o t a l

R e s p o n s e T i m e i n a s e s s i o n . T h u s , T o n i c A c c u r a c y m e a s u r e s t h e

p r o p o r t i o n o f t h e t o t a l r e s p o n s e t i m e o f a s e s s i o n t h at i s s p e n t i n

b a n d :

Toni c Acc ura cy = In BandTime

Total Response Time

W o r k R a t e i s s i m p l y t h e p r o p o r t i o n o f t h e S e s s i o n T i m e t h a t t h e

a n i m a l s p e n t o p e r a t i n g t h e t r a n s d u c e r :

W o r k R a t e - T o t a l R e s p o n s e T i m e

S e s s i o n T i m e

A s i n d i c a t e d a b o v e , t h e E n t r a n c e s m e a s u r e i s s i m p l y a c o u n t o f t h e

n u m b e r o f t i m e s d u r i n g a s e s s i o n t h at t h e a p p l i e d f o r c e e n t e rs t h e

b a n d f r o m e i t h e r i ts u p p e r o r l o w e r l i m i t .

E n t r a n c e s = t o t a l n u m b e r o f e n t r a n c e s i n t o th e f o r ce b a n d

A p e r f e c t l y e f f ic i e n t p e r f o r m a n c e w o u l d y i e l d a n I n - B a n d

E f f i c i e n c y o f 1 . 0 0 . T h e T o n i c A c c u r a c y a p p r o a c h e s 1 . 0 0 a s t h e

t o t a l ti m e s p e n t r e s p o n d i n g a p p r o a c h e s t h e t i m e s p e n t i n b a n d . I t

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

w h a t d i f f e r e n t th a n t h a t m e a s u r e d b y I n - B a n d E f f i ci e n c y : A l -

t h o u g h a h i g h p r o p o r t i o n o f s e s s i o n o p e r a n d u m h o l d i n g m i g h t b e

w i t h i n t h e a p p r o p r i a te f o r c e b a n d , i f t h e h o l d i n g t i m e s a r e

f r e q u e n t l y o f t o o s h o r t a d u r a t i o n t o p r o d u c e p e l l e t d e l i v e r y , t h e n

T o n i c A c c u r a c y c o u l d b e h i g h a l t h o u g h I n - B a n d E f f i c i e n c y is l o w .

B e c a u s e W o r k R a t e c a n a p p r o a c h a v a l u e o f 1 . 0 0 o r z e r o , t h e

p r e v i o u s m e a s u r e s c a n a p p r o x i m a t e 1 . 0 0 o r z e r o i n c o m p l e t e

i n d e p e n d e n c e o f W o r k R a t e . A l t h o u g h t h e y o f t e n c o v a r y , E n -

t r a n c e s a n d I n - B a n d E f f i c i e n c y a r e i n d e p e n d e n t m e a s u r e s . F o r

e x a m p l e , r e l a ti v e i n e f f i c i e n c y c o u l d i n d i c a t e t h a t t h e i n - b a n d

h o l d i n g t i m e s o f t e n f a l l j u s t s h o r t o f t h e a p p r o p r i a t e h o l d t i m e ;

s u c h a p e r f o r m a n c e w o u l d n o t y i e l d a h i g h E n t r a n c e s m e a s u r e .

Procedure

A n i m a l s w e r e t r a in e d t o h o l d t h e f o r c e t r a n s d u c e r o p e ra t e d

w i t h i n t h e f o rc e b a n d b y t h e m e t h o d o f s u c c e s si v e a p p r o x i m a t i o n

t o t h e r e q u i r e d r e s p o n s e t o p o g r a p h y . D u r i n g t h e i n i t i a l t r a i n i n g

p e r i o d , a n i m a l s w e r e p e r m i t t e d t o e a r n u p t o 1 0 0 f o o d p e l l e ts p e r

s e s s io n . A s s e s s i o n - t o - se s s i o n p e r f o r m a n c e s t a b i li z e d , t h e n u m b e r

o f p e l l e ts w a s r e d u c e d t o 5 0 p e r s e s s i o n . D a i l y s e s s i o n s w e r e g i v e n

f o r t h e d u r a t i o n o f t h e e x p e r i m e n t . A f t e r a p p r o x i m a t e l y 5 m o n t h s ,

s e s s i o n p e r f o r m a n c e r e a c h e d a s t a b le b a s e l i n e w i t h r e s p e c t t o t h e

m e a s u r e s c a l c u l a t e d . T h e n , a c u t e d o s e - e f fe c t f u n c t i o n s f o r d i a z -

e p a m 1 . 5 - -6 . 0 m g / k g ) w e r e d e t e r m i n e d . A n i m a l s w e r e i n j e c t ed IP

3 0 r a i n b e f o r e a s e s s io n . D o s e s w e r e a d m i n i s t e r e d i n t h e f o l l o w i n g

s e q u e n c e : 0 , 1 . 5 , 3 . 0 , 6 . 0 , 0 m g / k g . D o s e s w e r e s e p a ra t e d b y

7 - 1 0 d a y s .


3/9

DIAZEPAM AND MOTOR CONTROL 421

After the determination of the acute dose-effect funct ion,

animals were assigned to one o f 3 groups (N = 3 each) for the

duration of the experiment. The Before Group received a daily

injection of 6.0 mg/kg diazepam 30 min before each session and

an injection of Agent K vehicle immediately after each session.

The After Group received a daily injection of Agent K vehicle 30

rain before each session and an injection of 6.0 mg/kg diazepam

immediately after each session. The Vehicle Group received a

daily injection of Agen t K vehicle before and immediately after

each session. As animals completed the ini tial acute dose-effect

determination, they were assigned sequentially to treatment groups

in order to insure that each group received an equal number of

animals. Immediately following assignment to a treatment group,

each animal began the chronic-injectionphase of the experiment.

A blood sample was taken from each animal 1 hr after the 6.0

mg/kg diazepam injection for the determination of serum diaz-

epam and its metabolites. This was the last drug dose given in the

determination of the acute dose-effect relation. A second blood

sample was taken from each animal 1 hr after receiving an

inject ion of 6.0 mg/kg diazepam after they had been on the

chronic-injection regimen for about 70 days. Animals in the

Before and After Groups also had occasional blood samples taken

just before a session to determine whether there was any residual

diazepam or metabolite remaining from the previous day' s injec-

tion.

After about 4 months of chronic diazepam injection, animals in

the Before and After Groups had injections of Agent K substituted

for the drug to assess the effects of withdrawal from diazepam.

R E S U L T S

Acute Effects of Diazepam

Figure 1 shows the diazepam dose-effect relations for 6 animals

on the two measures of motor behavior most sensitive to drug-

produced performance decrements. With the exception of rats D18

and D4, In-Band Efficiency showed clear decrements at either 3 or

6 mg/kg, or both doses of diazepam. Except for D18, Entrances

were increased by diazepam at the 3 and 6 mg/kg doses, usual ly in

a dose-related manner. Data from only 6 animals are shown since

analysis of serum diazepam levels indicated that the initial drug

solution suspensions for Y16, Y17 and Y22 were inadequate. As

noted in the above section (cf., see the Drug section), 1 mg/ml of

Agent K provided insufficient suspension of the drug. This

concentration was used for these animals during the initial dose-

effect de termina tion phase, as well as during the first 2 weeks o f

the chronic-administrationphase. Consequently, these data are not

presented. The inadequacy of the suspension was determined by

analyz ing the serum samples taken 1 hour after the 6 mg/kg dose

given in the initia l phase. These 3 animals showed diazepam levels

of 34 ng/ml or less. As other animals had not yet been phased into

the experiment, we were able to correct the suspension. The

corresponding serum samples for the subsequent 6 animals, for

which the corrected suspension concent ration was used, revealed a

much greater diazepam level (94-255 ng/ml ; mean = 190 ng/ml) as

shown in the left side of Table 1. These latter values agree with the

levels reported for IP injection of 5 mg/kg diazepam in the rat (13).

One animal (D18) failed to complete the session following the

6 mg/kg dose (Fig. 1, point a). This dose was selected as the

maximum to be administered during the acute phase of the

experiment, and the daily dose to be used in the chronic-

administration phase.

Effects of Chronic Administration and Withdrawal

Figures 2 and 3 show the values for individual animals of the

four performance measures. At points A, the means of the last 10

1 0

° I

o °

+ f I

0 .8 D8 ~ I 6

0 6

0 4

0 2

- 4 - ' - 4 - - - 4 - - - - + - - : ' t

i |

1

2 _

- t - : : : ,

• 0 1 5 3 6

D 5 I D 1 8

• • e , . . . . e

- I Z

D o s e o f d la z e p a m m g / k o )

FIG. 1. Dose-effect relation for diazepam (IP, 30 min postinjection) or

two measures of motor performance. Each drug point based on data from

single determinations,except 0 mg/kg (vehicle), which is the mean of 2

injectionsgiven at the beginningand end of the dose-effect determination.

B = mean (SEs are small and lie within he plotted point dimensions)of 10

baseline sessions, 2 precedingeach dose. a = animal did not finish session

at this dose.

baseline sessions before the start of the chronic phase are shown.

During the chronic phase, animals received two injections daily,

one 30 min before the session and the other immediately after:

Before Group (Diazepam-Vehicle), After Group (Vehicle-Diaz-

epam) and Vehicle Group (Vehicle-Vehicle). For the chronic-

administration phase, session values are plotted for the first 14

days; mean values for each animal for the last 10 days of this

chronic phase are plotted at points B. During the withdrawal

phase, all animals received the Vehicle-Vehicle inject ion regimen.

The drug was withdrawn from the Before and After Groups after

100-136 days of chronic administration (mea n= 120--_6 days).

Indiv idual session values are plotted for 10-14 days after with-

drawal of the drug. Animals in the Vehicle Group received

injections for the number of days indicated on each axis. The

length of the chronic regimen for this group was limited by the

toxicity of the vehicle as explained below.

Vehicle Group The

Vehicle Group was stable on all measures

(D6 revealed a few deviant sessions) with the exception of Work

Rate over the course of the experiment. Although Work Rate

remained unaffected during the initial 75 days of the chronic

phase, at poin t B Y17 had decreased to 77 of baseline point A;

D4 and D6 were affected at about chronic days 77 and 79,

respectively.

Before Group The Before Group animal D5 decreased to 76

of its baseline on In-Band Efficiency with the first chronic

inject ion and remained at that level throughout most of the chronic

phase (cf., point B). D3 was similar, although values remained

near baseline for the first 3 days of chronic drug administrationand

were at 88 of baseline at point B. Following correction of the

inadequate suspending agent on chronic day 17, serum drug values


4/9

4 2 2 C U L B E R S O N

ET AL

T A B L E 1

SERUM LEVELS (ng/ml) OF DIAZEPAM DZ) AND

N-DESMETHYLDIAZEPAM DesDZ) 1 HR AFTER P INJECTIONOF 6.0 mg/

kg DIAZEPAM N THE ACUTEAND CHRONIC (AFTERABOUT70 DAILY

IP INJECTIONSOF EITHER 6.0 m g/kg DIAZEPAMOR VEHICLE) PHASES

Acute Phase Determ. Chronic Phase Determ.

Rat Group DZ DesDZ DZ DesDZ

Y17 Vehicle i i 146 43

D4 Vehicle 248 156 180 58

D6 Vehicle 170 71 159 --

Y22 Before i i 274 52

D5 Before 128 60 151 37

D3 Before 94 61 104 42

Y16 After i i 210 80

D18 Afte r 255 120 267 51

D8 After 244 143 262 24

i = inadequate vehicle (sample not counted); - - = value undetectable.

f o r Y 2 2 w e r e w i t h i n t h e e x p e c t e d r a n g e . S u b s e q u e n t l y , c h r o n i c

d i a z e p a m a d m i n i s tr a t io n w a s c o n t i n u e d f o r 1 2 0 d a y s . A d e c r e m e n t

i n I n - B a n d E f f i c i e n c y p e r s i s te d d u r i n g t h e 4 m o n t h s o f c h r o n i c

a d m i n i s t ra t i o n at 7 6 o f b a s e l i n e ( c f . , p o in t B ) . U p o n w i t h d r a w a l ,

I n - B a n d E f f i c i e n c y r e t u rn e d t o t h e o r i g i n a l b a s e l i n e l e v e l o f p o i n t

A e i t h e r i m m e d i a t e l y ( Y 2 2 , D 3 ) o r s l o w l y ( D 5 ) .

T h e E n t r a n c e s m e a s u r e p r e s e n t e d a s i m i l a r p i c t u re . T h e r e w a s

an inc r ease i n sess ion E n t r ances du r ing the i n i t i a l ch r on ic days

w h i c h c o r r e l a t e d w i t h t h e d e c r e a s e d I n - B a n d E f f i c i e n c y ( D 3 , D 5 ) .

T h i s i nc r ease pe r s i s t ed t h r oughou t t he ch r on ic admin i s t r a t i on

per iod , so t ha t a t po in t s B the va lues wer e 188 and 118 o f

b a s e l i n e p o i n t s A f o r D 5 a n d D 3 , r e s p e c t i v e l y . F o r Y 2 2 , t h e

c o r r e s p o n d i n g v a l u e w a s 1 7 9 o f b a s e l i n e . A l l 3 a n im a l s i n t h e

B e f o r e G r o u p r e t u rn e d t o t h e b a s e l i n e l e v e l o f E n t r a n c e s i m m e d i -

a t e l y a ft e r w i t h d r a w a l o f d i a z e p a m . N o s i g n s o f a n a b s t i n e n c e

e f f e c t f r o m w i t h d r a w a l w e r e e v i d e n t i n m o t o r p e r f o r m a n c e .

T h e T o n i c A c c u r a c y m e a s u r e d i f f e r e d s o m e w h a t f r o m t h e

a b o v e m e a s u r e s i n b o t h t h e t i m e c o u r s e a n d e x t e n t o f t h e d ru g

e f f e c t s o n m o t o r p e r f o r m a n c e . W i t h t h e f i rs t c h r o n i c -p h a s e i n j e c -

t i o n , D 5 d e c r e a s e d i n T o n i c A c c u r a c y to 8 4 o f b a s e l i n e ;

s u b s e q u e n t s e s s io n v a l u e s w e r e v a r i a b l e , b u t s ta b i l iz e d t o 8 6 o f

b a s e l i n e a t p o i n t B . D 3 e x h i b i t e d d e c r e m e n t s i n T o n i c A c c u r a c y

f o r s e s s io n s 4 - 6 o f t h e c h r o n i c p h a s e a n d t h e n r e t u rn e d t o b a s e l i n e

p e r f o r m a n c e . Y 2 2 s t a b i l iz e d a t 8 0 o f b a s e l i n e ( p o i n t B ) . U p o n

d r u g w i t h d r a w a l , D 5 a n d Y 2 2 r e t u r n e d t o b a s e l i n e . D 3 h a d

r e c o v e r e d b a s e l i n e p e r f o rm a n c e p r i o r to w i t h d r a w a l a n d r e m a i n e d

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

W o r k R a t e r e m a i n e d a p p r o x i m a t e l y a t b a s e l i n e l e v e l s d u ri n g

the i n i ti a l 2 wee ks o f t he ch r on ic phase f o r t he 2 an imal s ( D3 , D5 )

f o r w h i c h t h e s e d a t a w e r e a v a i l a b le . H o w e v e r , W o r k R a t e f o r Y 2 2

a n d D 3 d u r i n g t h e 4 m o n t h s o f c h r o n i c a d m i n i s t ra t i o n d i s p l a y e d a

d e c r e m e n t s i m i l a r to t h a t s e e n i n th e V e h i c l e G r o u p . A l l 3 a n i m a l s

s h o w e d a n a d d i t io n a l d e c r e a s e i n W o r k R a t e d u r i n g t h e w i t h d r a w a l

phase .

After Group

O c c a s i o n a l s e r u m s a m p l e s t a k e n a p p r o x i m a t e l y

2 4 h r a f t e r d i a z e p a m i n j e c t io n f r o m t h e s e a n i m a l s a n d t h o s e i n t h e

B e f o r e G r o u p r e v e a l e d n o d e t e c t a b l e l e v e l s o f d i a z e p a m o r

m e t a b o l i t e . H e n c e , a n y c h r o n i c p e r f o r m a n c e d e c r e m e n t s c a n n o t b e

e x p l a i n e d b y a n a c c u m u l a t i o n o f d r u g o r a c t i v e m e t a b o l i t e i n t h e

se r um as a r esu l t o f da l l y dos ing .

T h e I n - B a n d E f f i c i e n c y m e a s u r e r e v e a l e d s o m e d e c r e m e n t s i n

t h e p e r f o r m a n c e o f D 8 w h i c h p e r s i s t e d t h r o u g h o u t t h e c h r o n ic

p h a s e ( 8 9 o f b a s e l i n e a t p o i n t B ) . Y 1 6 a n d D 1 8 s h o w e d n o

d e c r e a s e i n t h is m e a s u r e a s a r e s u lt o f c h r o n i c d o s i n g . U p o n

w i t h d r a w a l o f t h e d r u g , I n - B a n d E f f i c i e n c y fo r D 8 r e t u r n e d t o t h e

b a s e l i n e l e v e l f o r a b o u t a w e e k a n d t h e n d e c l i n e d . B y t h e 1 t h d a y

of wi thd r awal , t he an im al f a i l ed t o comple t e t he sess ion . Wi thd r aw -

a l p ro d u c e d n o n o t a b le c h a n g e i n t h i s m e a s u r e f o r Y 1 6 o r D 1 8 .

T h e E n t r a n c e s m e a s u r e d i s p l a y e d a s i m i l a r p i c t u re . A t p o i n t B ,

D 8 w a s at 1 3 0 o f t h e b a s e l i n e l e v e l , w h i l e Y 1 6 a n d D 1 8 s h o w e d

n o d e c r e m e n t i n p e r f o r m a n c e . W i t h d r a w a l p r o d u c e d e f f e c t s s i m -

i l a r t o t h o s e o b s e r v e d f o r I n - B a n d E f f i c i e n c y : a t e m p o r a r y i m -

p r o v e m e n t f o r D 8 a n d n o n o t a b l e c h a n g e f o r Y 1 6 o r D 1 8 .

T o n i c A c c u r a c y r e m a i n e d q u i t e s t a b le f o r t h is g r o u p , s h o w i n g

l i t t l e o r n o d e c r e m e n t a t p o i n t B . L i k e w i s e , t h e v a l u e s a f t e r

w i t h d r a w a l r e m a i n e d r e l a t i v e ly s t a b l e .

F o r t h e 2 a n i m a l s m e a s u r e d d u r i n g t h e i n i t i al 2 w e e k s o f t h e

c h r o n i c p h a s e ( D 1 8 , D 8 ) , W o r k R a t e re m a i n e d s t a b l e . H o w e v e r ,

a s w a s t h e c a s e f o r s o m e a n i m a l s i n t h e o th e r g r o u p s , W o r k R a t e

began to dec r ease a f t e r abou t 85 days o f ch r on ic admin i s t r a t i on ,

d r o p p i n g t o 7 5 a n d 6 7 o f b a s e l i n e , r e s p e c t i v e l y ( p o i n t B ) . Y 1 6

s h o w e d a 5 1 d e c r e m e n t . U p o n w i t h d ra w a l , W o r k R a t e e i t h e r

r e m a i n e d s t a b l e , b u t l o w ( Y 1 6 ) , o r r e m a i n e d l o w a n d u n s t a b l e

( D18 , D8) .

Vehicle Toxicity

A s n o t e d i n t h e a b o v e r e s u l t s , m o s t a n i m a l s l a t e i n t h e

c h r o n i c - a d m i n i s tr a t i o n p h a s e , o r i n t h e w i th d r a w a l p h a s e , s h o w e d

a m a r k e d d e c r e a s e i n W o r k R a t e . I n a s m u c h a s t h e s e la t e d e c r e as e s

occur r ed i n a l l g r oups i t sugges t ed t he poss ib l e t ox i c i t y o f t he

s u s p e n d i n g a g e n t . W e i g h t g a i n s a t t h e s a m e t i m e i n d i c a t e d a n

e d e m a t o u s c o n d i t i o n s i n c e f o o d r a t i o n s w e r e c o n t r o l l e d w i t h i n

n a r r o w l i m i t s . T h e r e d u c t i o n s i n W o r k R a t e d i d n o t a p p e a r i n a n y

a n i m a l e a r l i e r t h a n t h e 7 5 t h d a y o f t h e c h r o n i c p h a s e . O t h e r

m e a s u r e s o f p e r f o r m a n c e w e r e n o t a d v e r s e l y a f fe c t e d . O n e a n i m a l

d i ed and 6 o the r s wer e sac r i f i ced in a mor ibund cond i t i on a t t he

e n d o f t h e e x p e r i m e n t . D i s c o u n t i n g t h e 2 0 - 3 0 d a y s f o r t h e 3

a n i m a l s t h a t r e c e i v e d t h e l o w e r c o n c e n t r a ti o n o f A g e n t K ( 1

mg /ml ) i n i t i a l l y , t he hea l th o f a l l bu t 2 an imal s w as se r ious ly

c o m p r o m i s e d a f t e r 8 7 - 1 3 7 d a y s o f c h r o n i c A g e n t K ( 2 m g / m l )

a d m i n i s tr a t io n . P o s t m o r t e m e x a m i n a t i o n s h o w e d e x c e s s i v e f l u i d

i n t h e p e r i t o n e a l c a v i t i e s . E l e v a t e d b l o o d u r e a n i t r o g e n v a l u e s

i n d i c a te d r e n a l f a i l u r e . H i s t o l o g i c a l e x a m i n a t i o n o f l i v e r , s p l e e n

a n d k i d n e y s e c t io n s r e v e a l e d a n o c c l u s i o n o f g l o m e r u l a r c a p i l l a r ie s

a n d a n a c c u m u l a t i o n o f f o r e ig n m a t e r i a l w i t h i n th e s p l e e n . L i v e r

d a m a g e w a s m i n i m a l . I n o r d e r t o c o n f i r m t h a t A g e n t K b y t h e I P

r ou te o f admin i s t r a t i on was t he t ox i c f ac to r , two add i t i ona l

a n i m a l s w e r e r e d u c e d t o 8 0 b o d y w e i g h t a n d g i v e n 2 in j e c t i o n s

d a l l y ( s e p a ra t e d b y 0 . 5 h r ) o f A g e n t K ( 2 m g / m l ) . O n e d i e d a f t e r

101 days and the o the r a f t e r 115 days ; s im i l a r pa tho log ica l changes

w e r e n o t e d .

Serum Levels o f Diazepam and D esmethyldiazepam

T a b l e 1 sh o w s t h e s e r u m d r u g l e v e l s in t h e a c u t e a n d c h r o n i c

p h a s e s o f t he e x p e r i m e n t . D i a z e p a m l e v e l s w e r e c o m p a r a b l e i n th e

t w o p h a s e s . A l t h o u g h m e t a b o l i t e l e v e l s v a r i e d g re a t l y , t h e s e d a t a

s u g g e s t t h a t t h e d i a z e p a m / d e s m e t h y l d i a z e p a m r a t i o m a y i n c r e a s e

a f t e r ch r on ic t r ea tmen t .

DISCUSSION

Acute Effects of Diazepam

I n t he p r esen t d i sc r imina t ive moto r con t r o l p r ocedur e , pe r f o r -

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

e f f e c t r e l a t io n w a s d e t e r m i n e d . M o s t a n i m a l s s h o w e d i m p a i r e d

p e r f o r m a n c e i n t h e 3 - 6 m g / k g d i a z e p a m d o s e r a n g e . A c u t e


5/9

D I A Z E P A M A N D M O T O R C O N T R O L 4 2 3

L0

0 . 8

0 . 6

0 . 4

0 . 2

0.0

¢~ 1.0

C

.~- ) 0 .8

° ~

~

0.6

0 . 4

ID

t - 0.2

m

m

o.o '

i

e -

~ . 0

Vehicle Group

Y17

..+- ::: :: :::-:.:: --e - i;;:;;:;,:::;;

1 3 7 1 5 0

D6

5 8 8 2

0 . 6

0 + 4 D 4

0 2

A

C h r o n i c C h r o n i c

D a y s D l y s

2 5 0 0

2 0 0 0 I

1 . ~ o [ Y 1 7

t 3 1 1 5 0

~ 2ooo

D6

j

r - ' 1 5 0 0

t13 500 a

~ . . t 0 ~

e -

2 ° °

D4

1500

C h r o n i c C h m n l o

D a y s D a y s

Before Group

Y22

After Group

Y16

D5

D 8

. . .p ~ . .~ , t, l lee w.~

T ' '

3 08

; F ~ , ~ ~ : :~ ::¢ i ̀ ~ ; 'T '~ , ~ i~ ::~ -';~ ~,

C h r o n i c W i t h d r a w a l C h r o n i c W i t h d r a w a l

D a y s O a y a D a y s D a y s

Y22

Y 6

• • w ¢ ~

• i.- q- - ll+4l++k l*~+

D 5

. L ~ IF . , , , . , .

D18

03 D8

, . , . . . . + ~ • , l ~ a , , I ~ '

1 S I 1 4 1 5 I ) 1 4

C h r o n c W i t h d r a w a l C h r o n c W l t h d r a w l l

D a y s D a y s D a y s D a y s

F I G . 2 . M e a n ( - + S E ) a n d s u c c e s s i v e - s e ss i o n v a l u e s f or m o t o r p e r f o r m a n c e m e a s u r e s ( I n - B a n d

E f f i c i e n c y , E n t r a n c e s ) o f i n d i v i d u a l r a t s i n 3 g r o u p s . B E F O R E G R O U P : D i a z e p a m ( 6 m g / k g , I P 3 0

m ain p r e s e s s i o n ) - - V e h i c l e I P p o s t s e ss i o n . A F T E R G R O U P : V e h i c l e 1 P 3 0 r a i n p r e s e s s i o n - - D i a z e p a m

( 6 m g / k g , I P p o s t s e s s io n ) . V E H I C L E G R O U P : V e h i c l e I P 3 0 m i n p r e s e s s i o n - - V e h i c l e I P p o s t s es s i o n .

A = m e a n ( -+ S E ) o f 1 0 s e s s i o n s b e f o r e s t a r t o f c h r o n i c , d a i l y i n j e c t i o n s . B = M e a n ( + S E ) o f l a s t 1 0

s e s s i o n s o f c h r o n i c p h a s e . ( S E s n o t v i s i b l e l ie w i t h i n p l o t t e d p o i n t b o r d e r s . )


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D I A Z E P A M A N D M O T O R C O N T R O L 42 5

adm ini s t r a t ion of d i azepa m to r a t s t es t ed on the ro torod 39 ,44) or

e l e v a t e d r u n w a y 4 ) t a sk s p r o d u c e d m o t o r p e rf o r m a n c e i m p a i r -

m e n t s w i t h i n t h e s a m e d o s e r a n g e . C o m p a r e d t o t h e e f f e c t s o f

m i d a z o l a m o n t h e s a m e d i s c r im i n a t i v e m o t o r c o n t r o l p ro c e d u r e

41) , d i aze pam y ie lded qual i t a t i ve ly s imi l ar r esul ts , wi th mo s t

a n i m a l s s h o w i n g d o s e - r e l a t e d im p a i r m e n t s m o r e c l e a r l y th a n w a s

t h e c a s e w i t h m i d a z o l a m .

I n h u m a n s , p s y c h o m o t o r p e rf o r m a n c e s s u c h a s r e a c t i o n ti m e ,

t r acking , d ig i t - symbol subs t i t u t ion and cancel l a t i on were af f ec t ed

b y d i a z e p a m , a s w e l l a s o t h e r b e n z o d i a z e p i n e s 4 7 ) . I n g e n e r a l ,

p e r f o r m a n c e o n t a s k s r e q u i ti n g r e p e t i t i v e - a c t s p e e d a n d r a p i d

r e a c t io n t i m e s w e r e m o r e r e a d i l y c o m p r o m i s e d b y a c u t e a d m i n i s -

t r a ti o n o f b e n z o d i a z e p i n e s t h a n w e r e t h o s e d e m a n d i n g a c c u r a c y

18, 36 , 43 , 45) . Th e acqui s i t i on of new beh avior i s a l so impai r ed

by acute d i aze pam admini s t r a t i on [ e .g . , 14) ] . The grea t er s ens i -

t i v i ty of acqu i s i t ion , as opp osed to s t eady- s t a t e per forma nce , t o

d i s r u p t i o n b y d i a z e p a m i n h u m a n s w a s s h o w n b y r e q u i t i n g a

subjec t to e i t her per form a wel l -pr ac t i ced r esponse s eque nce par t

of the t ime or acqui r e a new sequence 42) . On the acqui s i t i on

comp onent , d i aze pam IV in a 70 kg subjec t) i ncr eased the er ror

r a t e s li g h t ly w i t h a 5 m g d o s e , m o r e w i t h 1 0 m g , a n d m a r k e d l y

wi th 15 mg, but only a s l i ght e f f ec t on the per forman ce compo nent

f ina l ly occur r ed a t t he 15 mg dose . Fur ther , psyc hom otor per for -

m a n c e s w e r e m o r e f r e q u e n t l y a n d d e f i n i t i v e ly im p a i r e d w h e n

funct ions were as ses sed soon af t er drug adm ini s t r a t ion 24 ,47) .

Wi th the pr esent t echnique , f r ee-motor cont ro l , r a ther t han

s p e e d , w a s a s s e s s ed , b u t a c u t e - d o s e p e rf o r m a n c e d e c r e m e n t s w e r e

detec t ed . Animal s were t es t ed soon af t er drug admini s t r a t i on , t he

per iod dur ing which o ther s tudies a l so found impai r ed per for -

manc e . O ur anim al s were t r a ined to a l ong- t erm s t abi l i t y cr i t e r ion ,

never the l es s , t hey r emained s ens i t i ve to acute d i azepam dosage .

T h e a c q u i s it i o n o f n e w b e h a v i o r w a s n o t a n e c e s s a r y c o n d i t i o n fo r

t h e d e m o n s t r a t i o n o f i m p a i r m e n t b y d i a z e p a m . B u t p e r h a p s t h e

d o s e s e m p l o y e d w e r e g r e a t e r t h a n t h o s e c o m m o n i n h u m a n

therapeut i cs .

Chronic Effects o f Diazepam

T h r o u g h o u t t h e p e r i o d o f c h r o n ic a d m i n i s t r a ti o n u p t o a n d

i n c l u d in g p o i n t B o f F i g s . 2 a n d 3 ) , t h e V e h i c l e G r o u p m a i n t a i n e d

a d i s cr imina t ive moto r cont ro l per formance c lose to t he or ig inal

b a s e l i n e v a l u e c f . , p o i n ts A ) o n a l l 4 m e a s u r e s . A n i m a l Y 1 7 w a s

a n e x c e p t i o n o n o n e m e a s u r e w i th a d e c r e a s e d W o r k R a t e a t p o i n t

B . ) F o r t h e A f t e r G r o u p , b e h a v i o r c l o s e l y a p p r o x i m a t e d t h e

basel ine measures t hroughou t t he chronic- admin i s t r a t ion phase ,

e x c e p t f o r th e f i n a l W o r k R a t e s p o i n t B ) , w h i c h w i l l b e d i sc u s s e d

i n th e f o l l o w i n g se c t io n . A n i m a l D 8 , h o w e v e r , d i s p l a y e d a s m a l l

d e c r e m e n t i n I n - B a n d E f f i c ie n c y a n d E n t r a n c e s t h ro u g h o u t m o s t

of t he chronic- admini s t r a t i on phase . ) Sum mar iz ing the per for -

mance of t hese two groups : wi th the except ion of l a t e d i s rupt ions

in Work Rate , ne i ther t ime a lone , chronic vehic l e admini s t r a t i on ,

n o r t h e p o s t s e s si o n a d m i n i s t ra t i o n o f d i a z e p a m h a d a p p r e c i a b l e

ef f ec t s on per formance through poin t B .

The B efore Grou p d i sp l aye d a r a ther d i ff er ent p i c tur e . Dur ing

the f i r s t 2 week s of chronic adm ini s t r a t ion , t her e was a d i s rupt ion

i n I n - B a n d E f f i c i e n c y , E n t ra n c e s a n d T o n i c A c c u r a c y , b u t n o t i n

W o r k R a t e . T h e s e d i s r u p ti o n s w e r e m a x i m a l o n l y a f t e r a f e w d a y s

o f c h r o n i c d o s i n g . S o m e t o l e r a n c e o c c u r r e d to t h e s e d e c r e m e n t s ,

b u t i t w a s i n c o m p l e t e c f . , p o i n t s B ) . T w o o f t h e 3 a n im a l s s h o w e d

d e c r e m e n t s i n W o r k R a t e a t p o i n t B .

A s im i l ar ly des igne d s tudy us ing chronic adm ini s t r a t ion of

m i d a z o l a m , r a t h e r t ha n d i a z e p a m , y i e l d e d s i m i l a r re s u l ts w i t h

r e s p e c t t o I n - B a n d E f f i c i e n c y , E n t r a n c e s a n d T o n i c A c c u r a c y

per forman ces 41) . Per formance was d i s rupted in i t i a l l y by chronic ,

p r e s e s si o n a d m i n i s tr a t io n o f 3 m g / k g S C m i d a z o l a m . T o l e r a n c e t o

t h e s e p e r f o rm a n c e d e c r e m e n t s d e v e l o p e d , b u t i t w a s i n c o m p l e t e .

A s m e a s u r e d i n th e r a t , b o t h m i d a z o l a m a n d d i a z e p a m a r e h i g h l y

l i p o p n i li c a n d h a v e c o m p a r a b l e b r a i n u p ta k e s 2 ) . R a t b r a i n

hal f - l i ves i ncreased as a funct ion of dose and were roug hly

c o m p a r a b l e fo r m i d a z o l a m a n d d ia z e p a m 3 8 ) . A l t h o u g h m i d a z o -

l am i s u l t r ashor t - ac ting in both r a t s and human s , d i azepa m i s

long-act ing in humans , w i th a ha l f - l i f e of 20-70 hr 17) , but qui te

shor t - ac t ing in t he r a t . At t he 3 m g/kg do se , t he s erum hal f - l i f e of

m i d a z o l a m w a s 0 . 9 2 h r f o r t h e r a t 4 1 ) , w h i l e a t 5 m g / k g t h e

p l a s m a h a l f -l i fe o f d ia z e p a m w a s 0 . 8 8 h r 1 3 ) . T h e r e s p e c t i v e

metabo l i t es ar e a l so r apid ly e l iminated in t he r a t 12 , 13 , 31) . For

the r a t , t hen , t hese two benzodiaz epines ar e pharmac okinet i ca l ly

qui t e s imi l ar agent s . Fur ther , w i th l ong- t erm dos ing , t he benzo-

diazepine s r eveal no changes in t he i r k ine t i cs 16) , nor d id we f ind

a sys t emat i c change for d i azepam cf . , Table 1) . I n concer t wi th

t h e s i m i l a r it y o f a c u t e k i n e ti c s f o r m i d a z o l a m a n d d i a z e p a m , a n d

t h e l a c k o f a l t e r e d k in e t i c s b y l o n g - t e r m d o s i n g , t h e f i n d i n g o f

com parable ef f ec t s on d i s cr iminat ive motor cont ro l wi th chronic

admini s t r a t i on of t hese agent s might be expected .

In r a t s , t o l er ance to t he d i s rupt ion of ro torod per formance by

d i a z e p a m d e v e l o p e d a f t e r 6 d a y s o f 3 m g / k g / d a y d i a z e p a m

admini s t r a t i on 39) . I n humans , a l t hough acute doses of t he

b e n z o d i a z e p i n e s c a n i m p a i r p e r f o r m a n c e s , t o l e r a n c e t o i m p a i r -

m e n t c a n d e v e l o p w it h a c h ro n i c d o s i n g re g i m e n 2 6 ) . H o w e v e r ,

p s y c h o m o t o r p e r f o r m a n c e d e f i c it s m a y s t il l re m a i n d u r i n g l o n g -

t erm diazepam adm ini s t r a t ion a t t her apeut i c l evel s 9 ,25) , or

despi t e t he development of t o l er ance to t he subchronic admini s -

t r a t ion of n igher doses 36) . Fo r exam ple , i n t est i ng whe re in the

per forman ce l evel of a d ig i t - symb ol subs t i t u tion t ask had n ot ye t

r eached fu l l e f f i c i ency, da i ly t herapeut i c- l evel d i azep am doses for

6 w e e k s p r e v e n t e d t h e d e v e l o p m e n t o f t h e i m p r o v e d p e r f o r m a n c e

observ ed in a p l acebo group as a funct ion of pr ac t i ce 27) . These

r e p o r ts o f i n c o m p l e t e d e v e l o p m e n t o f t o l e r a n c e t o r e p e a t e d d o s e s

of d i azepam agree wi th the pr esent r esul t s and wi th the s imi l ar

o u t c o m e o f c h r o n i c a d m i n i s tr a t io n o f m i d a z o l a m 4 1 ) .

I n h u m a n s , g i v e n t h e l o n g p l a s m a h a l f - l iv e s o f d ia z e p a m a n d

desm ethyld i aze pam 30) , t he incom ple t e t o l er ance to t he per for -

m a n c e d e f i c it s p ro d u c e d b y d a i l y d i a z e p a m d o s i n g c o u l d p e r h a p s

be a t t r i buted to t he accumula t ion of ac t ive drug and metabol i t es

15) dur ing the chronic adm ini s t r a ti on phase . How ever , f or t he r a t ,

ne i ther pr eses s ion nor pos t s es s ion s ampl ing in t he pr esent exper -

i m e n t r e v e a l e d e v i d e n c e o f a n y p r o g r e s s iv e m e t a b o l i t e a c c u m u l a -

t i on in s erum which could be appealed to i n order t o expla in the

i n c o m p l e t e d e v e l o p m e n t o f t o l e ra n c e .

In one r espect , t he d i aze pam resul t s d i f f er f rom those obta ined

w i t h m i d a z o l a m o n t h e d i s c r i m i n a t iv e m o t o r c o n t r o l p r o c e d u r e .

M i d a z o l a m , i n c o m p a r i s o n t o d i a z e p a m , i s n o t a b le n o t o n l y f o r i t s

g e n e r a l l y g re a t e r p o t e n c y , b u t a l s o f o r i t s m o r e p r o n o u n c e d

sedat ive ef f ec t s 19) . Wi th midazo lam, the f ir s t chronic-phase

ses s ion in t he Before Group produced a cons iderable decrease in

W o r k R a t e ; t o l e r a n c e d e v e l o p e d w i t h s u c c e e d i n g s e s s i o n s , s o t h a t

by the four th chronic s es s ion Work Rate had r e turned to t he

basel ine level 41) . The cor r espon ding chronic phase in t he pr esent

exper im ent d id not show th i s i n i t i a l decrease in W ork Rate wi th a

p r o g r e s s i v e d e v e l o p m e n t o f t o l e r a n c e . A s s u g g e s t e d p r e v i o u s l y

4 1 ) , W o r k R a t e d e c r e m e n t m a y b e a n i n d e x o f s e d a t i o n f o r

sedat ive-hypn ot i c agent s , wh ereas t he o ther i ndices measure

a s p e c t s o f p e r f o rm a n c e t h a t a r e m o r e s p e c i f i c a l l y m o t o r i c . W o r k

Rate in t he chronic phase , t hen , co uld be in t erpre t ed as i ndica t ing

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

t o d i a z e p a m .

Chronic Effects Work Rate and Agent K

A s d e s c r i b e d a b o v e , A g e n t K a f f e c te d a n i m a l s i n a l l g ro u p s


8/9

4 2 6 C U L B E R S O N

ET AL

a d v e r s e l y , b u t n o t b e f o r e t h e 7 5 t h d a y o f t h e c h r o n i c - a d m i n i s t r a -

t i o n p h a s e o f t h e e x p e r i m e n t . T h e a d v e r s e e f f e c t b e c a m e e v i d e n t a s

a d e c r e a s e i n W o r k R a t e a t t h a t t i m e . O t h e r p e r f o r m a n c e m e a s u r e s

w e r e s p a r e d , a n d t h e e f f e c t o f A g e n t K c o u l d b e d i s t i n g u i s h e d

f r o m d i a z e p a m e f f e c t s s i n c e t h e t w o c o n t r o l g r o u p s ( V e h i c l e a n d

A f t e r G r o u p s ) w e r e i n c l u d e d i n t h e d e s i g n . F u r t h e r , t h e s p e c i f i c it y

a n d l a t e n c y o f A g e n t K a s th e t o x i c c o m p o n e n t i n t h is e x p e r i m e n t

w a s c o n f i r m e d b y t h e i n c l u s i o n o f t w o a d d i t i o n a l a n i m a l s s u b -

j e c t e d o n l y t o A g e n t K t r e a t m e n t s . I n a s m u c h a s W o r k R a t e w a s t h e

o n l y m e a s u r e a f f e c t e d d u r i n g t h e l a t t e r p a r t o f t h e c h r o n i c -

a d m i n i s t r a t i o n p h a s e a n d t h e w i t h d r a w a l p h a s e , t h e d e s i g n a n d t h e

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

d i a z e p a m d o s i n g a n d i t s w i t h d r a w a l o n t h e o t h e r m e a s u r e s o f

m o t o r f u n c t i o n .

Withdrawal of Diazepam

I t i s w e l l - k n o w n f r o m p l a c e b o - c o n t r o l l e d , d o u b l e - b l i n d s t u d i e s

t h a t w i t h d r a w a l f r o m p r o l o n g e d u s e o f b e n z o d i a z e p i n e s ( i n c l u d i n g

d i a z e p a m ) a t t h e r a p e u t i c d o s e l e v e l s c a n r e s u l t i n a n a b s t i n e n c e

s y n d r o m e , p a r t i c u l a r l y u p o n a b r u p t d r u g d i s c o n t i n u a n c e ( 7 , 3 3 ,

3 4 ) . W i t h p r o l o n g e d m i s u s e o f d i a z e p a m ( 6 0 - 1 2 0 m g d a i l y ) i n 1 0

p a t i e n ts , w i t h d r a w a l p r o d u c e d a n o r e x i a , i n s o m n i a a n d a g i t a t io n ,

a s w e l l a s a n u m b e r o f m o t o r e f f e c ts : p a l p a b l e t r e m o r , m y o c l o n u s

a n d Q u i n q u a u d s s i g n ( 3 2) . N o s u c h a d v e r s e m o t o r e f f e c t s w e r e

o b s e r v e d i n t h e p r e s e n t e x p e r i m e n t w h e n d i a z e p a m w a s w i t h -

d r a w n ; r a t h e r , t h e i m p a i r e d m o t o r p e r f o r m a n c e o f t h e B e f o r e

G r o u p a n i m a l s r e t u r n e d t o b a s e l i n e l e v e l s . S i m i l a r l y , i n n o r m a l

h u m a n s u b j e c t s , M c L e o d a n d h i s a s s o c i a t e s ( 2 7 ) f o u n d t h a t

d i s c o n t i n u a n c e a f t e r 6 w e e k s o f 1 5 m g d i a z e p a m / d a y l e d t o

i m p r o v e d p e r f o r m a n c e s o n a n u m b e r o f p s y c h o m o t o r te s t s. P a -

t i e n ts i n t h e c l i n ic a l s t u d ie s c i t e d a b o v e w h o s h o w e d t h e a b s t i n e n c e

s y n d r o m e g e n e r a l l y h a d t a k e n g r e a t e r d a i l y a m o u n t s o f d i a z e p a m

f o r a p e r i o d o f y e a rs . A l t h o u g h t h e a n i m a l s i n t h e p r e s e n t s tu d y

w e r e e x p o s e d t o d i a z e p a m f o r a c o m p a r a t i v e l y l o n g p e r i o d o f t im e ,

p e r h a p s t h e d o s e l e v e l , o r t h e d i f f e r e n c e b e t w e e n h u m a n a n d r a t

h a l f - l iv e s o f d i a z e p a m a n d i t s m e t a b o l i t e s , w e r e i m p o r t a n t f a c t o r s

p r e c l u d i n g t h e d e m o n s t r a t i o n o f d is t u r b e d m o t o r p e r f o r m a n c e

u p o n w i t h d r a w a l . C o n s i d e r i n g t h e s e p o s s i b l e e x p l a n a t i o n s , i t i s

m o s t l i k e l y t h a t d o s e , r a t h e r t h a n p h a r m a c o k i n e t i c s , w a s t h e

c r i t ic a l d e t e rm i n a n t . F i r st , w e h a v e s h o w n a w i t h d r a w a l e f f e c t o n

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

d o s i n g w i t h m i d a z o l a m ( 4 1 ) , a n a g e n t w h o s e p h a r m a c o k i n e t i c s ( a s

d i s c u s s e d a b o v e ) a r e q u i t e s i m i l a r t o t h o s e o f d i a z e p a m i n t h e r a t.

S e c o n d , m u c h g r e a t e r d a i l y d i a z e p a m d o s e s [ e . g . , 1 3 3 m g / k g / d a y

( 2 8 ) ] t h a n t h e o n e u s e d i n t h e p r e s e n t e x p e r i m e n t a r e a p p a r e n t l y

r e q u i r e d f o r t h e f ra n k d e m o n s t r a t i o n 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

r a t ( 6 , 2 9 ) .

ACKNOWLEDGEMENTS

This research was supported by grant DA 03117 f rom the National

Ins ti tu te on Drug Ab use. Th ese data were presented by the senior author in

par tial fulf i l lment of the requirements for the M .Sc. degree in the graduate

program in toxicology. The authors wish to thank Dr. Tetsuo Shim amura

of the Dept . of Pathology, The U nivers i ty of Medicine and Den tis try of

New Jersey, Piscataway, NJ for his obse rvatio ns on the pathological

change s produced in the animals , and Dr . Peter F . Sor ter of Hoffman n-La

Roche, Inc., N utley, NJ for a generous supply of diazepam.

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diazepam and discriminative motor control

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