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Conditioned defensive burying : a new paradigm for the study of anxiolytic agents Treit, Dallas R. 1981

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CONDITIONED DEFENSIVE BURYING: A NEW PARADIGM FOR THE STUDY OF ANXIOLYTIC AGENTS DALLAS ROBERT TREIT B.A. The U n i v e r s i t y of B r i t i s h Columbia, 1975 M.A. The U n i v e r s i t y of B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF by Doctor of Philosophy in THE FACULTY OF GRADUATE STUDIES Department of Psychology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March 1981 © D a l l a s Robert T r e i t , 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of / ^ t / ^ / ) l£r( The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 DE-6 (2/79) i i ABSTRACT In g e n e r a l , t r a d i t i o n a l animal behaviour paradigms have not p r o v i d e d a r e l i a b l e b a s i s f o r i d e n t i f y i n g compounds with p o t e n t i a l a n x i o l y t i c a c t i o n , or f o r d e l i n e a t i n g the mechanisms of t h e i r a c t i o n s . However, the p o s s i b i l i t y that s p e c i e s - s p e c i f i c defense r e a c t i o n s c o u l d serve as a b a s i s f o r the study of a n x i o l y t i c agents had been overlooked. The r e s u l t s of the present i n v e s t i g a t i o n s showed that the r a t ' s p r o c l i v i t y to bury o b j e c t s a s s o c i a t e d with a v e r s i v e s t i m u l a t i o n i s p a r t i c u l a r l y s e n s i t i v e to the e f f e c t s of a n x i o l y t i c agents. The d e f e n s i v e burying behaviour of r a t s was r a p i d l y suppressed by a s i n g l e i n j e c t i o n of the a n x i o l y t i c drug, diazepam, and as the dose of diazepam was i n c r e a s e d , the magnitude of i t s suppressive e f f e c t was i n c r e a s e d . In a d d i t i o n , the r e l a t i v e p o t e n c i e s of the a n x i o l y t i c s diazepam, c h l o r d i a z e p o x i d e , and p e n t o b a r b i t a l in the c o n d i t i o n e d burying paradigm compared f a v o r a b l y with t h e i r r e l a t i v e p o t e n c i e s i n c l i n i c a l s e t t i n g s . Furthermore, c o n d i t i o n e d d e f e n s i v e burying seemed to be s e l e c t i v e l y s e n s i t i v e to the e f f e c t s of a n x i o l y t i c s : four n o n a n x i o l y t i c compounds, p i c r o t o x i n , p e n t y l e n e t e t r a z o l , d-amphetamine, and morphine, d i d not r e l i a b l y suppress b u r y i n g . One n o n a n x i o l y t i c compound, chlorpromazine, d i d suppress c o n d i t i o n e d d e f e n s i v e burying; however, i t s e f f e c t s c o u l d be r e l i a b l y d i s s o c i a t e d from the e f f e c t s of diazepam by v a r y i n g the s e v e r i t y of the a v e r s i v e s t i m u l u s . Chlorpromazine suppressed burying at both low and high shock i n t e n s i t i e s ; whereas, diazepam suppressed burying only at the low shock i n t e n s i t y . Thus, the c o n d i t i o n e d d e f e n s i v e burying paradigm i i i appears to f u l f i l the three major c r i t e r i a of a v a l i d animal t e s t of a n x i o l y t i c agents: s e n s i t i v i t y , r e l a t i v e potency, and s e l e c t i v i t y . In the remaining s t u d i e s , the c o n d i t i o n e d d e f e n s i v e burying t e s t was used as a model to study some of the p u t a t i v e mechanisms of a c t i o n of a n x i o l y t i c agents. The o b s e r v a t i o n that diazepam suppressed the uncondi t i o n e d d e f e n s i v e burying e l i c i t e d in the absence of p a i n f u l s t i m u l a t i o n r u l e d out the p o s s i b i l i t y that the suppressive e f f e c t of diazepam was due to a n a l g e s i c , r a t h e r than a n x i o l y t i c , a c t i o n . In subsequent experiments, a l t e r i n g the f u n c t i o n s of GABA e r g i c neurons with p i c r o t o x i n was found to have no e f f e c t on e i t h e r c o n d i t i o n e d or unconditioned d e f e n s i v e b u r y i n g . However, p i c r o t o x i n d i d reverse the suppr e s s i v e e f f e c t of diazepam, suggesting that the mechanism whereby diazepam e x e r t s i t s e f f e c t on burying may involve' an i n t e r a c t i o n between diazepam and GABA e r g i c n e u r a l systems. The systematic and robust nature of the present r e s u l t s suggests that the c o n d i t i o n e d d e f e n s i v e burying paradigm c o u l d prove to be a v a l u a b l e t o o l f o r sc r e e n i n g p o t e n t i a l a n x i o l y t i c agents and f o r studying t h e i r mechanisms of a c t i o n . iv TABLE OF CONTENTS ABSTRACT i i LIST OF FIGURES . v i i ACKNOWLEDGEMENTS i x INTRODUCTION 1 A n x i o l y t i c agents and b e h a v i o u r a l pharmacology 3 Animal t e s t s of a n x i o l y t i c agents 8 E f f e c t s of a n x i o l y t i c s on u n t r a i n e d r e a c t i o n s 9 E f f e c t on plasma c o r t i c o s t e r o i d s 9 E f f e c t on muscle tone 9 E f f e c t on p e n t y l e n e t e t r a z o l - i n d u c e d s e i z u r e s 10 E f f e c t on a g g r e s s i v e b e h a v i o u r 11 E f f e c t on consummatory b e h a v i o u r s 11 E f f e c t on e x p l o r a t o r y b e h a v i o u r 13 E f f e c t s of a n x i o l y t i c s on t r a i n e d responses 15 E f f e c t s on c o n d i t i o n e d e m o t i o n a l r e s p o n d i n g 16 E f f e c t s on c o n d i t i o n e d a v o i d a n c e r e s p o n d i n g 17 E f f e c t s on pu n i s h e d operant b e h a v i o u r : The G e l l e r ' c o n f l i c t ' t e s t 18 The a r b i t r a r y n a t u r e of c o n v e n t i o n a l a n i m a l t r a i n i n g paradigms 23 The c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm: A new model f o r the study of a n x i o l y t i c agents 26 I n i t i a l d e m o n s t r a t i o n 26 Ev i d e n c e f o r a s s o c i a t i v e l e a r n i n g 27 Robustness of the c o n d i t i o n e d d e f e n s i v e b u r y i n g phenomenon '. 28 B u r y i n g as a d e f e n s i v e response i n r o d e n t s 30 V Purpose 34 GENERAL METHOD 35 Subject s 35 Apparatus 35 Procedures 35 H a b i t u a t i o n 35 Drug a d m i n i s t r a t i o n 36 Shock a d m i n i s t r a t i o n 36 Beh a v i o u r a l o b s e r v a t i o n and q u a n t i f i c a t i o n 37 S t a t i s t i c a l a n a l y s i s 38 PART I Burying as a s c r e e n i n g t e s t f o r a n x i o l y t i c agents .. 39 Experiment 1 39 Method 39 Re s u l t s and D i s c u s s i o n 40 Experiment 2 47 Method 47 R e s u l t s and D i s c u s s i o n 48 Experiment 3 55 Method 55 R e s u l t s and D i s c u s s i o n 55 Experiment 4 58 Method 58 R e s u l t s and D i s c u s s i o n 59 PART II Mechanisms of a c t i o n 67 Experiment 5 69 Method t 69 R e s u l t s and D i s c u s s i o n 70 Experiment 6 77 v i Method ..'. .. . 78 R e s u l t s and d i s c u s s i o n 79 Experiment 7 80 Method ...... 80 R e s u l t s and d i s c u s s i o n 80 Experiment 8 82 Method 82 R e s u l t s and d i s c u s s i o n 82 GENERAL DISCUSSION 88 I C o n d i t i o n e d d e f e n s i v e b u r y i n g and the i d e n t i f i c a t i o n of a n x i o l y t i c compounds ......... 88 I I Mechanisms of a c t i o n 93 REFERENCES 98 v i i LIST OF FIGURES 1. Mean d u r a t i o n of b u r y i n g by d i a z e p a m - i n j e c t e d and v e h i c l e - i n j e c t e d r a t s i n Experiment 1 42 2. Mean h e i g h t of m a t e r i a l a c c umulated by diazepam-i n j e c t e d and v e h i c l e - i n j e c t e d r a t s i n Experiment 1 45 3. Mean d u r a t i o n of b u r y i n g by r a t s i n j e c t e d w i t h diazepam, c h l o r d i a z e p o x i d e , p e n t o b a r b i t a l , or v e h i c l e i n Experiment 2 50 4. Mean h e i g h t of m a t e r i a l accumulated by r a t s i n j e c t e d w i t h diazepam, c h l o r d i a z e p o x i d e , p e n t o b a r b i t a l , or v e h i c l e i n Experiment 2 53 5. Mean d u r a t i o n of b u r y i n g by d i a z e p a m - i n j e c t e d r a t s shocked w i t h 1 mA or 10 mA, and by c h l o r p r o m a z i n e - i n j e c t e d r a t s shocked w i t h 1 mA or 10 mA i n Experiment 4 61 6. Mean h e i g h t of m a t e r i a l a c c u m u l a t e d by diazepam-i n j e c t e d r a t s shocked w i t h 1 mA or 10 mA, and by c h l o r p r o m a z i n e - i n j e c t e d r a t s shocked w i t h 1 mA or 10 mA i n Experiment 4 64 7. Mean d u r a t i o n of b u r y i n g by d i a z e p a m - i n j e c t e d and v e h i c l e - i n j e c t e d r a t s i n Experiment 5 72 8. Mean h e i g h t of m a t e r i a l a c c u m u l a t e d by diazepam-i n j e c t e d and v e h i c l e - i n j e c t e d r a t s i n Experiment 5 75 9. Mean d u r a t i o n of b u r y i n g by r a t s i n the v e h i c l e , diazepam, p i c r o t o x i n , o r diazepam p l u s p i c r o t o x i n c o n d i t i o n s of Experiment 8 84 Mean h e i g h t of m a t e r i a l accumulated by r a t s i n the v e h i c l e , diazepam, p i c r o t o x i n , or diazepam p l u s p i c r o t o x i n c o n d i t i o n s of Experiment 8 i x ACKNOWLEDGEMENTS I would l i k e to thank Dr. John P i n e l f o r h i s guidance and support throughout the conduct of t h i s r e s e a r c h . In a d d i t i o n , I am g r a t e f u l to Drs. Don W i l k i e , B o r i s G o r z a l k a , Merry B u l l o c k , and H.C. F i b i g e r , whose comments and suggestions a i d e d i n the w r i t i n g of the manuscript. F i n a l l y , the a s s i s t a n c e of M. Spetch, L. T e r l e c k i , D. Gray, R. S k e l t o n , and C. Ludeman i s g r a t e f u l l y acknowledged. 1 INTRODUCTION During the past 20 years, a number of animal t e s t s have been developed to i d e n t i f y compounds that have p o t e n t i a l a n x i o l y t i c a c t i o n s and to study the mechanisms by which they achieve t h e i r a n t i a n x i e t y e f f e c t s . However, these t e s t s have proven to be f a r from adequate. The r e s u l t s of the present i n v e s t i g a t i o n s suggested that the " c o n d i t i o n e d d e f e n s i v e b u r y i n g " paradigm w i l l prove to be a v a l u a b l e a l t e r n a t i v e to the b e h a v i o u r a l procedures a l r e a d y a v a i l a b l e f o r the study of a n x i o l y t i c agents. A c c o r d i n g l y , the f i r s t p a r t of the I n t r o d u c t i o n i s comprised of a b r i e f d e s c r i p t i o n of the major a n x i o l y t i c agents and t h e i r c l i n i c a l a p p l i c a t i o n s , f o l l o w e d by a d i s c u s s i o n of the r a t i o n a l e u n d e r l y i n g the b e h a v i o u r a l t e s t i n g of these agents and the c r i t e r i a a g a i n s t which the adequacy of these t e s t s i s t y p i c a l l y judged. In the second part of the I n t r o d u c t i o n , the e x i s t i n g "animal t e s t s " of a n x i o l y t i c e f f e c t s are d e s c r i b e d and then e v a l u a t e d i n terms of the aforementioned c r i t e r i a . I t was concluded on the b a s i s of t h i s review that each of the t r a d i t i o n a l t e s t i n g paradigms i s inadequate at l e a s t i n some r e s p e c t s . It was suggested that ' s p e c i e s - t y p i c a l ' defense r e a c t i o n s , such as the d e f e n s i v e burying response, c o u l d serve as a b a s i s f o r d e v e l o p i n g a new, e f f e c t i v e animal t e s t of a n x i o l y t i c drug e f f e c t s . Thus, a review of the d e f e n s i v e burying l i t e r a t u r e comprises the t h i r d s e c t i o n of the I n t r o d u c t i o n . The unique advantages of the c o n d i t i o n e d d e f e n s i v e burying paradigm as a 2 t e s t of a n x i o l y t i c e f f e c t s , i . e . , i t s speed, s i m p l i c i t y , and r e l i a b i l i t y , are the f o c a l p o i n t s f o r t h i s review and represent the r a t i o n a l e f o r the present i n v e s t i g a t i o n s . In the f o u r t h and f i n a l s e c t i o n of the I n t r o d u c t i o n , the purpose of the present i n v e s t i g a t i o n s i s o u t l i n e d . 3 A n x i o l y t i c a g ents and b e h a v i o u r a l pharmacology A n x i o l y t i c s have been d e f i n e d i n the g e n e r a l sense as a c l a s s of t h e r a p e u t i c compounds used i n the t r e a t m e n t of ' a n x i e t y ' ( c f . L e a v i t t , 1974); they a r e t y p i c a l l y used by p h y s i c i a n s t o t r e a t p a t i e n t s who c o m p l a i n of such symptoms as a p p r e h e n s i o n , t e n s i o n , g a s t r o i n t e s t i n a l d i s t r e s s , d i z z i n e s s , and i n s o m n i a . The f i r s t c h e m i c a l agent t h a t was used f o r the s p e c i f i c t r e a t m e n t of such symptoms was meprobamate, a carbamate compound ( H i l l & T e d e s c h i , 1971). However, the most p o t e n t and w i d e l y used a n x i o l y t i c agents are the b e n z o d i a z e p i n e s : c h l o r d i a z e p o x i d e , diazepam, and oxazepam (tradenamed L i b r i u m , V a l i u m , and Serax, r e s p e c t i v e l y ) . The c l i n i c a l i mportance of a n x i o l y t i c a g e n t s i s i n d i c a t e d by a r e c e n t e s t i m a t e t h a t i n 1977 t h e r e were over 8000 tons of b e n z o d i a z e p i n e s p r e s c r i b e d i n N o r t h America ( T a l l m a n , P a u l , S k o l n i c k , & G a l l a g e r , 1980). T h i s r e p r e s e n t s a p p r o x i m a t e l y h a l f of a l l p r e s c r i p t i o n s f o r p s y c h o t h e r a p e u t i c drugs (Weise & P r i c e , 1975). In a d d i t i o n t o the widespread use of b e n z o d i a z e p i n e s as a n x i o l y t i c s , they a r e f r e q u e n t l y p r e s c r i b e d as muscle r e l a x a n t s , a n t i c o n v u l s a n t s , or h y p n o t i c s (Goodman & G i l m a n , 1975). These a d d i t i o n a l t h e r a p e u t i c p r o p e r t i e s of b e n z o d i a z e p i n e s , i n c o m b i n a t i o n w i t h t h e i r h i g h t h e r a p e u t i c p o t e n c y , low t o x i c i t y , and moderate a d d i c t i v e p o t e n t i a l , p r o b a b l y account f o r t h e i r w i d e s p r e a d use i n p h a r m a c o t h e r a p e u t i c s ( G r e e n b l a t t & Shader, 1974). A l t h o u g h a n x i o l y t i c s , p a r t i c u l a r l y the b e n z o d i a z e p i n e s , a r e p r o b a b l y among the s a f e s t p h a r m a c o t h e r a p e u t i c a g e n t s , they can 4 have a number of a d v e r s e e f f e c t s i n c l u d i n g a t a x i a , nausea, h y p o t e n s i o n , headache, c o n f u s i o n , and d e p r e s s i o n (Rotenburg & Hughes, 1978). Of p a r t i c u l a r c oncern i s t h e i r p o t e n t i a l f o r abuse; e v i d e n c e of p s y c h o l o g i c a l and p h y s i c a l dependence i s sometimes apparent a f t e r the t e r m i n a t i o n of b e n z o d i a z e p i n e t h e r a p y ( G r e e n b l a t t & Shader, 1974). However, t h e r e have been thousands of b e n z o d i a z e p i n e d e r i v a t i v e s s y n t h e s i z e d i n the past few y e a r s , some of which may be a b l e t o i n h i b i t ' a n x i e t y ' w i t h o u t a d v e r s e s i d e - e f f e c t s (Gschwend, 1979). Because of the g r e a t p r o l i f e r a t i o n of new, p o t e n t i a l a n x i o l y t i c a g e n t s , t h e r e has been need f o r e f f i c i e n t p r e c l i n i c a l " a n i m a l s c r e e n i n g t e s t s " of t h e i r e f f e c t s . A n i m a l s c r e e n i n g t e s t s a r e used i n i t i a l l y t o p r e d i c t the s a f e t y and e f f i c a c y of any n o v e l compound because of the o b v i o u s dangers i n v o l v e d i n s t u d y i n g the e f f e c t s of u n t e s t e d compounds on humans. Another a p p l i c a t i o n of a n i m a l s c r e e n i n g t e s t s i s i n the e l u c i d a t i o n of the mechanisms of drug a c t i o n . Only a n i m a l t e s t s can p r o v i d e the degree of e x p e r i m e n t a l c o n t r o l n e c e s s a r y t o conduct t h i s t y p e of r e s e a r c h . Knowledge of the mechanisms of d r u g a c t i o n can h e l p the r e s e a r c h b i o c h e m i s t t o s y n t h e s i z e drugs t h a t a c t more s e l e c t i v e l y , thus i m p r o v i n g the s a f e t y and e f f i c a c y of p h a r m a c e u t i c a l compounds ( L i p p a , Nash, & G r e e n b l a t t , 1978). An a n i m a l s c r e e n i n g t e s t must f u l f i l a number of w i d e l y r e c o g n i z e d c r i t e r i a i f i t i s t o be s u c c e s s f u l y used t o p r e d i c t the e f f e c t s of drugs i n humans and t o u n d e r s t a n d t h e i r mechanisms of a c t i o n ( G l i c k , 1976). F i r s t , the a n i m a l t e s t must be s e n s i t i v e i n a dose dependent f a s h i o n t o s t a n d a r d s u b s t a n c e s t h a t a r e known t o p o s s e s s the d e s i r e d t h e r a p e u t i c p r o p e r t y . For 5 example, i n the t a i l - f l i c k t e s t of a n a l g e s i a , s y s t e m a t i c i n c r e a s e s i n the dose of morphine a d m i n i s t e r e d t o a r a t produce s y s t e m a t i c i n c r e a s e s i n the r a t ' s l a t e n c y t o withdraw i t s t a i l from a t h e r m a l s t i m u l u s (D'Amour & Smith, 1941). Second, the r e l a t i v e potency of known t h e r a p e u t i c agents i n the a n i m a l s c r e e n i n g t e s t s h o u l d be comparable t o t h e i r r e l a t i v e potency i n humans. Thus, the r a n k - o r d e r potency of a s p i r i n , c o d e i n e , and demerol i n the t a i l - f l i c k t e s t i s the same as t h a t found i n c l i n i c a l t r i a l s i n humans ( e . g . , G r o t t o & Sulman, 1967). T h i r d , the s c r e e n i n g t e s t s h o u l d be s e l e c t i v e , i . e . , the a c t i v i t y of the c l a s s of compounds b e i n g d e v e l o p e d s h o u l d be d i s t i n g u i s h a b l e from the a c t i v i t y of compounds t h a t be l o n g t o o t h e r c l a s s e s . In the t a i l - f l i c k t e s t f o r example, agents w i t h o u t any c l i n i c a l a n a l g e s i c a c t i v i t y a r e u s u a l l y i n a c t i v e ( c f . W i n t e r , O r a h o v a t s , & Lehman, 1956). I t s h o u l d be emphasized t h a t o n l y the c o r r e l a t i o n a l r e l a t i o n s h i p between the e f f e c t of s t a n d a r d drugs i n the s c r e e n i n g t e s t and t h e i r e f f e c t i n humans d e t e r m i n e s the p r e d i c t i v e v a l i d i t y of the t e s t ; t h e r e need be no l o g i c a l or f u n c t i o n a l c o n n e c t i o n between the phenomena t h a t a r e the i n d i c e s of drug a c t i o n i n the a n i m a l t e s t and the phenomena t h a t a r e the t a r g e t of p h a r m a c o l o g i c a l i n t e r v e n t i o n i n humans ( G l i c k , 1976). For example, the i n h i b i t i o n of M e t r a z o l - i n d u c e d s e i z u r e s i n r o d e n t s i s a f a i r l y r e l i a b l e c h a r a c t e r i s t i c of s u b s t a n c e s t h a t have a n x i o l y t i c a c t i v i t y i n humans (Swinyard & C a s t e l l i o n , 1966), and on t h i s b a s i s i t has become an i m p o r t a n t i n i t i a l s c r e e n i n g t e s t . N o t h i n g more has t o be e s t a b l i s h e d about the r e l a t i o n s h i p between s e i z u r e s i n r o d e n t s and a n x i e t y i n humans 6 i n o r d e r f o r the t e s t t o have p r e d i c t i v e v a l i d i t y . I t i s more d i f f i c u l t t o judge the adequacy of a s c r e e n i n g t e s t when i t i s used as an 'animal model' f o r s t u d y i n g the mechanisms t h a t govern the t h e r a p e u t i c a c t i o n s of a dr u g . The b a s i c purpose of an 'animal model' of human d i s o r d e r s i s t o study d i s e a s e mechanisms i n a r e l a t i v e l y s i m p l e , e x p e r i m e n t a l l y a c c e s s i b l e system ( I n g l e & S h e i n , 1975). A c e n t r a l problem i n the i n i t i a l development of an a n i m a l model i s d e c i d i n g what a n i m a l p r o c e s s might be r e l e v a n t t o u n d e r s t a n d i n g the human c l i n i c a l c o n d i t i o n t h a t i s the o b j e c t of s t u d y . In most a r e a s of b i o m e d i c a l r e s e a r c h , the b a s i s on which a n i m a l models a r e chosen i s c l e a r : t hey a r e chosen on the b a s i s of s t r u c t u r a l or f u n c t i o n a l s i m i l a r i t i e s between the a n i m a l p r o c e s s and the human c o n d i t i o n ( I n g l e & S h e i n , 1975). Thus, f o r example, the r e s e a r c h e r who wants t o i d e n t i f y the v a r i a b l e s t h a t c o n t r o l the growth of tumours i n humans may choose t o study the growth of tumours i n mice. U n f o r t u n a t e l y , i n some ca s e s i t i s d i f f i c u l t t o determine the q u a l i t a t i v e s i m i l a r i t i e s of human and a n i m a l p r o c e s s e s . For example, a l t h o u g h i t i s r e l a t i v e l y easy t o measure tumorous growths i n mice and compare them t o those found i n human c a n c e r p a t i e n t s , i t i s not so easy t o measure a n x i e t y i n r o d e n t s ( S k i n n e r , 1953), l e t a l o n e compare i t t o human a n x i e t y . Because of the d i f f i c u l t i e s i n h e r e n t i n d e v e l o p i n g a n i m a l models t h a t c o r r e s p o n d i n some manner t o p s y c h o p a t h o l o g i c a l s t a t e s i n humans, some r e s e a r c h e r s ( e . g . , C a r l t o n , 1978; L i p p a , G r e e n b l a t t , & Pelman, 1977) have op t e d f o r what might be c a l l e d a c o r r e l a t i o n a l approach t o a n i m a l models ( c f . C a r l t o n , 1978). 7 In g e n e r a l , a c o r r e l a t i o n a l model i s based on a s e t of e m p i r i c a l r e l a t i o n s h i p s between the e f f e c t s of a v a r i e t y of t h e r a p e u t i c i n t e r v e n t i o n s i n the c l i n i c and t h e i r e f f e c t s i n the a n i m a l l a b o r a t o r y . I t can be seen t h a t t h i s concept of an a n i m a l model i s e q u i v e l a n t t o t h a t of an a n i m a l s c r e e n i n g t e s t ( c f . L i p p a , Nash, & G r e e n b l a t t , 1979). In b oth c a s e s , a l l t h a t i s r e q u i r e d i s t h a t the e f f e c t s of t h e r a p e u t i c i n t e r v e n t i o n s on a l a b o r a t o r y measure a r e c o r r e l a t e d w i t h t h e i r e f f e c t s i n humans. The c r i t e r i a of an a c c e p t a b l e c o r r e l a t i o n a l model are the same as those of an a n i m a l s c r e e n i n g t e s t ( i . e . , s e n s i t i v i t y , s e l e c t i v i t y , and r e l a t i v e p o t e n c y ) . There i s a problem a s s o c i a t e d w i t h t h i s c o r r e l a t i o n a l approach t h a t can be i l l u s t r a t e d by a d i s c u s s i o n of the M e t r a z o l t e s t of a n x i o l y t i c drug e f f e c t s . The M e t r a z o l t e s t c l e a r l y f u l f i l s most of the r e q u i r e m e n t s of a c o r r e l a t i o n a l model (Swinyard & C a t e l l i o n , 1966). However, most r e s e a r c h e r s would agree t h a t u s i n g the M e t r a z o l t e s t t o study the mechanisms by which a n x i o l y t i c s reduce a n x i e t y i n humans i s not as l i k e l y t o be s u c c e s s f u l as u s i n g the same t e s t t o study the mechanisms by which a n x i o l y t i c s i n h i b i t c l i n i c a l s e i z u r e s . The reason i s t h a t i n the l a t t e r a p p l i c a t i o n t h e r e i s a r e l a t i v e l y c l e a r isomorphism between the a n i m a l model and the c l i n i c a l c o n d i t i o n t h a t i t i s supposed t o r e p r e s e n t ; whereas, i n the former a p p l i c a t i o n , t h i s isomorphism i s f a r l e s s e v i d e n t . G i v e n the s u c c e s s of i s o m o r p h i c models i n most a r e a s of b i o m e d i c a l r e s e a r c h ( I n g l e & S h e i n , 1975), i t i s not u n r e a s o n a b l e t o d e c i d e between d i f f e r e n t c o r r e l a t i o n a l models on the b a s i s of t h e i r r e l a t i v e s i m i l a r i t y t o the human p r o c e s s t h a t i s the o b j e c t of 8 s t u d y , p a r t i c u l a r l y when mechanisms of a c t i o n a r e of s p e c i a l c o n c e r n . A n i m a l t e s t s of a n x i o l y t i c agents A l t h o u g h a n x i o l y t i c compounds have a v a r i e t y of r e a s o n a b l y d i s t i n c t i v e b i o b e h a v i o u r a l e f f e c t s ( D a n t z e r , 1977; F i e l d i n g & L a i , 1979), o n l y a few of these a r e used r o u t i n e l y t o i d e n t i f y a n x i o l y t i c ^compounds or t o study t h e i r mechanisms of a c t i o n . The b e h a v i o u r a l phenomena t h a t have been used as i n d i c e s of a n x i o l y t i c drug a c t i o n can be grouped i n t o two c a t e g o r i e s , based on whether the e f f e c t s a r e measured i n t r a i n e d or u n t r a i n e d a n i m a l s . U n t r a i n e d a n i m a l s a r e used t o measure the e f f e c t s of a n x i o l y t i c s on: 1) s t r e s s - r e l a t e d i n c r e a s e s i n plasma c o r t i c o s t e r o i d s , 2) M e t r a z o l - i n d u c e d s e i z u r e s , 3) muscle t o n e , 4) e x p e r i m e n t a l l y - i n d u c e d a g g r e s s i v e b e h a v i o u r , 5) consummatory b e h a v i o u r s , and 6) e x p l o r a t o r y b e h a v i o u r . T r a i n e d a n i m a l s must be used i n o r d e r t o measure the e f f e c t s of a n x i o l y t i c s on: 1) c o n d i t i o n e d a v o i d a n c e b e h a v i o u r , 2) c o n d i t i o n e d s u p p r e s s i o n ( c o n d i t i o n e d e m o t i o n a l r e s p o n d i n g ) , and 3) p u n i s h e d operant b e h a v i o u r . Each of t h e s e a n i m a l t e s t s w i l l be r e v i e w e d and e v a l u a t e d i n terms of the t h r e e a f o r e m e n t i o n e d c r i t e r i a of an o p t i m a l s c r e e n i n g system ( i . e . , dose-dependent s e n s i t i v i t y , r e l a t i v e p o t e n c y , and s e l e c t i v i t y ) . In c a s e s where the a n i m a l t e s t i s a l s o b e i n g used t o study the mechanisms of a c t i o n of a n x i o l y t i c d r u g s , the c r i t e r i o n of isomorphism w i l l a l s o be taken i n t o c o n s i d e r a t i o n . However, because none of t h e s e a n i m a l t e s t s c o n s i s t e n t l y f u l f i l s the r e q u i r e m e n t s of even a p r e d i c t i v e s c r e e n i n g system ( c f . L i p p a , Nash, & G r e e n b l a t t , 1979; S e p i n w a l l 9 & Cook, 1978), the i s s u e of isomorphism i s of secondary importance here. E f f e c t s of a n x i o l y t i c s on 'untrained' r e a c t i o n s E f f e c t on plasma c o r t i c o s t e r o i d s • L a h t i and Barsuhn (1974) found that s t r e s s - i n d u c e d i n c r e a s e s i n plasma c o r t i c o s t e r o i d s c o u l d be blocked by a number of a n x i o l y t i c agents in a dose-dependent manner. The r e l a t i v e potency of a n x i o l y t i c s i n t h i s t e s t compared f a v o r a b l y with t h e i r r e l a t i v e potency in b l o c k i n g human a n x i e t y , and the t e s t a l s o appeared to be s e l e c t i v e because a v a r i e t y of n o n a n x i o l y t i c agents were i n e f f e c t i v e . However, other s t u d i e s have shown that the s t r e s s - i n d u c e d i n c r e a s e i n plasma c o r t i c o s t e r o i d s a l s o can be blocked by a n t i -d epressants and n e u r o l e p t i c s (Bassett & C a i r n c r o s s , 1974; Keim & Sigg, 1977; Pekkarinen, 1970). The r e l i a b i l i t y of the t e s t i s a l s o d o u b t f u l i n view of the o b s e r v a t i o n that small v a r i a t i o n s i n the method of ind u c i n g s t r e s s can produce l a r g e f l u c t u a t i o n s in b a s e - l i n e l e v e l s of plasma c o r t i c o s t e r o i d s ( L a h t i & Barsahn, 1974). E f f e c t on muscle tone. A r e l i a b l e p r o p e r t y of a n x i o l y t i c s i s t h e i r a b i l i t y to induce muscle r e l a x a t i o n ( G l i c k , 1976). A simple v e r s i o n of a m u s c l e - r e l a x a t i o n t e s t of a n x i o l y t i c drugs i n v o l v e s p l a c i n g a rodent on an i n c l i n e d plane and e s t a b l i s h i n g the dose of the drug that produces the t a r g e t e f f e c t i n 50% of the animals ( i . e . , the ED50), which i n t h i s paradigm i s the dose at which 50% of the animals s l i d e o f f the plane. I f t h i s dose i s below the n e u r o t o x i c range f o r that drug, then a p o t e n t i a l a n x i o l y t i c a c t i o n i s suggested. More s o p h i s t i c a t e d procedures 10 i n v o l v e r e c o r d i n g the muscle c o n t r a c t i o n s of l a r g e r animals such as the c a t and then observing the i n h i b i t o r y e f f e c t s of a n x i o l y t i c s . (Ngai, Tseng, & Wang, 1966). Although the muscle-r e l a x a n t t e s t s s a t i s f y the c r i t e r i a of s e n s i t i v i t y and r e l a t i v e potency (Zbinden & R a n d a l l , 1967), the e f f e c t s of a n x i o l y t i c s i n these t e s t s cannot be c l e a r l y d i f f e r e n t i a t e d from those of n e u r o l e p t i c s , even at comparable doses ( c f . L i p p a , Nash, & G r e e n b l a t t , 1979). E f f e c t s on Metrazol-induced s e i z u r e s . One of the most widely used p r e l i m i n a r y i n d i c a t o r s of a n x i o l y t i c a c t i o n i s the i n h i b i t i o n of M e t r a z o l - i n d u c e d ( p e n t y l e n e t e t r a z o l - i n d u c e d ) s e i z u r e s i n rodents ( H i l l & Tedesehi, 1971). T h i s M e t r a z o l t e s t i s very s e n s i t i v e to a wide v a r i e t y of a n x i o l y t i c agents, with the ED50 f o r some agents (diazepam) as low as 1 mg/kg (Zbinden & Randall,. 1967). Furthermore, the r e l a t i v e potency of v a r i o u s a n x i o l y t i c s i n b l o c k i n g M e t r a z o l - i n d u c e d s e i z u r e s i s s i m i l a r to the potency of t h e i r a n t i a n x i e t y e f f e c t s i n humans, although the rank-order potency of oxazepam i n t h i s t e s t i s at v a r i a n c e with i t s t h e r a p e u t i c potency ( C h i l d r e s s & Gluckman, 1964; R a n d a l l , S checkel, & Banzinger, 1965). A n t i e p t i l e p t i c agents can be d i f f e r e n t i a t e d from a n x i o l y t i c s i n t h i s t e s t on the b a s i s of which component ( t o n i c or c l o n i c ) of the b e h a v i o u r a l s e i z u r e i s i n h i b i t e d . However there are a few n o n a n x i o l y t i e s (e.g., muscle r e l a x a n t s such as. c a r i s o p r o d o l ) that have e f f e c t s on M e t r a z o l -induced s e i z u r e s i n d i s t i n g u i s h a b l e from those of a n x i o l y t i c s (Irwin, 1967), and thus the s e l e c t i v i t y of t h i s procedure i s q u e s t i o n a b l e . Furthermore, the u t i l i t y of the M e t r a z o l t e s t i s somewhat l i m i t e d because there i s no apparent isomorphism 11 between the i n h i b i t i o n of s e i z u r e s i n r o d e n t s and the i n h i b i t i o n of a n x i e t y i n humans. E f f e c t s on a g g r e s s i v e b e h a v i o u r . One of the major reasons f o r t e s t i n g the b e n z o d i a z e p i n e s i n human c l i n i c a l p o p u l a t i o n s was t h a t t h e s e a gents appeared t o i n h i b i t v a r i o u s forms of a g g r e s s i o n i n e x p e r i m e n t a l a n i m a l s ( c f . G l i c k , 1976; H a e f e l e y , 1978). However, i t i s now apparent t h a t t h e s e a n t i a g g r e s s i o n e f f e c t s may not be r e l i a b l e i n d i c a t o r s of a n x i o l y t i c e f f i c a c y ( L i p p a , Nash, & G r e e n b l a t t , 1979). A l t h o u g h i t was i n i t i a l l y r e p o r t e d t h a t some a n x i o l y t i c s c o u l d i n h i b i t a g g r e s s i o n induced by i s o l a t i o n ( H o f f m e i s t e r & Wottke, 1969), f o o t shock ( V a l z e l l i , 1967), s e p t a l l e s i o n s ( R a n d a l l , H e i s e , S c h a l l e k , Bagdon, B a n z i g e r , B o r i s , Moe, & Abrams, 1961), h y p o t h a l a m i c s t i m u l a t i o n ( M a l i c k , 1970), or h a n d l i n g ( H e i s e & B o f f , 1961); i t has a l s o been shown t h a t n o n a n x i o l y t i c a g e n t s , e s p e c i a l l y the n e u r o l e p t i c s , have s i m i l a r or even more pronounced i n h i b i t o r y e f f e c t s on a g g r e s s i v e b e h a v i o u r i n e x p e r i m e n t a l a n i m a l s ( H o f f m e i s t e r & Wottke, 1969; S c r i a b i n e & B l a k e , 1962; T e d e s c h i , F o w l e r , M i l l e r , & Macko, 1969; V a l z e l l i , 1967). F u r t h e r m o r e , many known a n x i o l y t i c s e i t h e r have no e f f e c t on a g g r e s s i v e b e h a v i o u r (Gluckman, 1965), or they may a c t u a l l y i n c r e a s e a g g r e s s i o n a f t e r r e p e a t e d a d m i n i s t r a t i o n (Fox, Webster, & G u e r r i e r o , 1972; G u a i t o n i , M a r c u c c i , & G a r a t t i n i , 1971). Thus, a n t i a g g r e s s i o n e f f e c t s a r e c l e a r l y u n r e l i a b l e i n d e x e s of a n x i o l y t i c a g ents ( c f . G l i c k , 1976; H i l l & T e d e s c h i , 1971). E f f e c t s on consummatory b e h a v i o u r s . One of the f i r s t e f f e c t s of b e n z o d i a z e p i n e s t o be r e p o r t e d was t h e i r a b i l i t y t o 12 i n c r e a s e the amount of food e a t e n by r a t s and dogs ( R a n d a l l , S c h a l l e k , H e i s e , K e i t h , & Bagdon, 1960). T h i s e f f e c t has s i n c e been c o n s i s t e n t l y r e p l i c a t e d i n b o t h s a t i a t e d ( e . g . , M argules & S t e i n , 1967) and d e p r i v e d a n i m a l s ( e . g . , Hanson & Stone, 1964), and has s e r v e d as a s i m p l e b a s i s f o r s c r e e n i n g p o t e n t i a l a n x i o l y t i c agents (Hanson & Stone, 1964). The r a n k - o r d e r p o t e n cy of a n x i o l y t i c s i n t h i s t e s t i s e q u i v a l e n t t o t h a t found i n the t r e a t m e n t of human a n x i e t y ( P o s c h e l , 1971). However, a major problem i s t h a t a l t h o u g h n e u r o l e p t i c s , a n t i d e p r e s s a n t s , and a n t i c o n v u l s a n t s a re not e f f e c t i v e i n t h i s t e s t , some known a n x i o l y t i c s ( e . g . , meprobamate and p e n t o b a r b i t o l ) a l s o do not i n c r e a s e consummatory b e h a v i o u r s ( P o s c h e l , 1971). Thus, the consummatory t e s t has a number of l i m i t a t i o n s as a b e h a v i o u r a l assay of a n x i o l y t i c a g e n t s ; however t h e f a c t t h a t some a n x i o l y t i c s do i n c r e a s e consummatory b e h a v i o u r s has i m p o r t a n t i m p l i c a t i o n s f o r the assessment of o t h e r p o t e n t i a l s c r e e n i n g p r o c e d u r e s t h a t i n v o l v e consummatory r e s p o n s e s . For example, n a t u r a l consummatory b e h a v i o u r s t h a t a r e s u p p r e s s e d by a v a r i e t y of e x p e r i m e n t a l c o n d i t i o n s ( e . g . , a d m i n i s t r a t i o n of a v e r s i v e e l e c t r i c shock) can be r e s t o r e d when the a n i m a l i s t r e a t e d w i t h a n x i o l y t i c s ( B a i n b r i d g e , 1968; F a l k & B u r n i d g e , 1970; L i p p a e t a l . , 1979; M a r g u l e s & S t e i n , 1967; Naess & Rasmussen, 1958; V o g e l & P r i n c i p i , 1971). V o g e l and P r i n c i p i (1971) and L i p p a e t a l . (1979) have c o n f i r m e d and extended t h i s g e n e r a l f i n d i n g by showing: 1) t h a t a v a r i e t y of known a n x i o l y t i c s can r e s t o r e s u p p r e s s e d consummatory b e h a v i o u r i n a dose-dependent manner, 2) t h a t t h e r a n k - o r d e r potency of a n x i o l y t i c s i n r e s t o r i n g s u p p r e s s e d consummatory b e h a v i o u r s i s 13 comparable t o t h a t found i n the c l i n i c , and 3) t h a t t h e e f f e c t i s r e a s o n a b l y s p e c i f i c t o a n x i o l y t i c a g e n t s . However, i t s h o u l d be noted t h a t t h i s e f f e c t may s i m p l y r e f l e c t the mechanisms t h a t u n d e r l i e the i n c r e a s e i n consummatory b e h a v i o u r t h a t a n x i o l y t i c s produce i n the absence of a v e r s i v e s t i m u l i ( c f . Wise & Dawson, 1974). I f the i n c r e a s e i n consummatory b e h a v i o u r o b s e r v e d i n a shocked, but drugged a n i m a l r e p r e s e n t s n o t h i n g more than an i n c r e a s e i n i t s a p p e t i t e f o r a p a r t i c u l a r s u b s t a n c e , then the i n t r o d u c t i o n of a v e r s i v e s t i m u l i o f f e r s no p a r t i c u l a r advantage over s i m p l e r s c r e e n i n g p r o c e d u r e s t h a t employ consummatory b e h a v i o u r s by t h e m s e l v e s . T h i s i s s u e i s i l l u s t r a t e d by the r e s u l t s of a study by M i c z e k & Lau (1975). They found t h a t c h l o r d i a z e p o x i d e i n c r e a s e d d r i n k i n g i n a group of r a t s shocked f o r d r i n k i n g , but t h a t the drug a l s o produced a p a r a l l e l i n c r e a s e i n t h i s b e h a v i o u r i n r a t s t h a t were not shocked. The p o i n t here i s t h a t u n l e s s the a d d i t i o n of an a v e r s i v e s t i m u l u s t o a s i m p l e consummatory t e s t can be shown t o i n c r e a s e the p r e d i c t i v e v a l i d i t y of the t e s t , i t s a d d i t i o n i s of q u e s t i o n a b l e v a l u e . T h i s i s s u e w i l l be d e v e l o p e d f u r t h e r i n the s e c t i o n d e a l i n g w i t h t e s t paradigms t h a t employ b e h a v i o u r s p o s i t i v e l y r e i n f o r c e d w i t h f o o d . E f f e c t s on e x p l o r a t o r y b e h a v i o u r . A number of s t u d i e s have shown t h a t a n x i o l y t i c s t e n d t o i n c r e a s e the e x p l o r a t o r y b e h a v i o u r of r o d e n t s i n an u n f a m i l i a r environment ( e . g . , B o i s s i e r & Simon, 1964; C h r i s t m a s & M a x w e l l , 1970; M a r r i o t t & Spencer, 1965). In some of the s t u d i e s ( e . g . , B o i s s i e r e t a l . , 1976) the e f f e c t of a n x i o l y t i c s on e x p l o r a t o r y b e h a v i o u r appeared t o be q u i t e s e l e c t i v e ; i t was not o b s e r v e d w i t h 14 n e u r o l e p t i c s , a n t i h i s t a m i n e s , a n t i c o n v u l s a n t s , a n t i c h o l i n e r g i c s , a n t i d e p r e s s a n t s , a n a l g e s i c s , or B - a d r e n e r g i c b l o c k e r s . The most, w i d e l y known procedure t h a t u t i l i z e s i n c r e a s e s i n e x p l o r a t o r y b e h a v i o u r as an index of a n x i o l y t i c agents i s the s o - c a l l e d f o u r - p l a t e t e s t ( B o i s s i e r , Simon, & Aron, 1968). In t h i s p r o c e d u r e r a t s a r e p l a c e d on f o u r m e t a l p l a t e s t h r o u g h which e l e c t r i c c u r r e n t can be passed. When r a t s a r e shocked f o r c r o s s i n g between the f o u r p l a t e s , the i n c i d e n c e of t h i s b e h a v i o u r i s d r a m a t i c a l l y reduced. B o i s s i e r e t a l . found t h a t the r e d u c t i o n of c r o s s i n g s c o u l d be r e v e r s e d by a number of known a n x i o l y t i c s ; whereas, n e u r o l e p t i c s , a n t i d e p r e s s a n t s , s t i m u l a n t s , and a n a g e s i c s were i n e f f e c t i v e . However, t h e s e f i n d i n g s were c o n t r a d i c t e d by the r e s u l t s of a subsequent st u d y (Aron, S i n o n , L a r o u s s e , & B o i s s i e r , 1971) i n which some s t i m u l a n t s ( e . g . , amphetamine) produced an i n c r e a s e i n p u n i s h e d p l a t e c r o s s i n g s and some known a n x i o l y t i c s ( e . g . , amobarbitone) d i d n o t . In summary, i t can be seen t h a t none of the a n i m a l t e s t s r e v i e w e d i n t h i s s e c t i o n c o m p l e t e l y s a t i s f i e s the t h r e e c r i t e r i a of dose-dependent s e n s i t i v i t y , r e l a t i v e p o t e n c y , and s e l e c t i v i t y . However, because these p r o c e d u r e s do not r e q u i r e e x t e n s i v e p e r i o d s of t r a i n i n g b e f o r e an a n i m a l can be used t o t e s t a d r u g , and because the t e s t s t hemselves t e n d t o be q u i t e s h o r t , some of t h e s e t e s t s have found wide use among p h a r m a c o l o g i s t s . W i t h o u t the c o n s t r a i n t s of e x t e n s i v e p r e t r a i n i n g and l e n g t h y t e s t s e s s i o n s , the r e s e a r c h e r i s a b l e t o s c r e e n a l a r g e number of compounds i n a r e l a t i v e l y s h o r t p e r i o d of t i m e , d e s p i t e the f a c t t h a t the r e s u l t s of t h e s e t e s t s may i n 15 some cases be d i f f i c u l t to i n t e r p r e t ( c f . H a e f e l y , 1978; Sepinwall & Cook, 1978). E f f e c t s of a n x i o l y t i c s on t r a i n e d responses In s p i t e of the p r a c t i c a l advantages of using u n t r a i n e d s u b j e c t s to study a n x i o l y t i c agents, many r e s e a r c h e r s have chosen to use experimental animals t r a i n e d i n t r a d i t i o n a l a v e r s i v e l e a r n i n g paradigms (e.g., c o n d i t i o n e d suppression, c o n d i t i o n e d a voidance). In these t r a d i t i o n a l paradigms, a v e r s i v e e l e c t r i c shock i s t y p i c a l l y used to c o n d i t i o n the t e s t response. T h i s c o n d i t i o n i n g may take hundreds of t r i a l s , and even then the t e s t response may not occur r e l i a b l y ( B o l l e s , 1970) . There appear to be two major reasons f o r the p o p u l a r i t y of a v e r s i v e l e a r n i n g paradigms among pharmacologists i n t e r e s t e d i n the study of a n x i o l y t i c agents. The f i r s t i s the b e l i e f that by using w e l l t r a i n e d animals, v a r i a b i l i t y w i t h i n the t e s t system can be reduced, thereby p r o v i d i n g a more r e l i a b l e t e s t of a n x i o l y t i c e f f e c t s ( S e p i n w a l l & Cook, 1978). I t w i l l be shown in the f o l l o w i n g s e c t i o n t h a t t h i s b e l i e f , with one e x c e p t i o n ; i s unfounded. In most i n s t a n c e s , t r a d i t i o n a l a v e r s i v e l e a r n i n g paradigms are u n r e l i a b l e t e s t s of a n x i o l y t i c e f f e c t s . The second reason i s the b e l i e f that there may be some concordance, e i t h e r i n terms of antecedent s t i m u l i or subsequent responses, between some a v e r s i v e l e a r n i n g s i t u a t i o n s and c l i n i c a l a n x i e t y ( c f . E s t e s & Skinner, 1941; Wolpe, 1958). Whether or not such a b e l i e f i s j u s t i f i e d , the c r i t e r i o n of isomorphism has c l e a r l y guided the s e l e c t i o n of r e s e a r c h paradigms by pharmacologists whose main i n t e r e s t has been to study the mechanisms of a c t i o n 16 of a n x i o l y t i c a g ents ( e . g . , S e p i n w a l l & Cook, 1978). E f f e c t s on c o n d i t i o n e d e m o t i o n a l r e s p o n d i n g . C o n d i t i o n e d e m o t i o n a l r e s p o n d i n g (CER) can be produced by p r e s e n t i n g the s u b j e c t w i t h an u n a v o i d a b l e e l e c t r i c shock t h a t i s r e l i a b l y preceded by a s i g n a l , i . e . , a c o n d i t i o n e d s t i m u l u s (CS). The CS i s then superimposed upon an ongoing b e h a v i o u r t h a t i s b e i n g p o s i t i v e l y r e i n f o r c e d ( i . e . , an o p e r a n t ) . The s u p p r e s s i o n of operant r e s p o n d i n g t h a t o c c u r s i n the presence of the CS i s c a l l e d a c o n d i t i o n e d e m o t i o n a l response (Brady & Hunt, 1955) o r , a l t e r n a t i v e l y , c o n d i t i o n e d s u p p r e s s i o n ( E s t e s & S k i n n e r , 1941). CER, because i t was i n i t i a l l y thought t o be a good a n i m a l model of human a n x i e t y ( c f . E s t e s & S k i n n e r , 1941), a t t r a c t e d the a t t e n t i o n of p h a r m a c o l o g i s t s i n t e r e s t e d i n the stu d y of a n x i o l y t i c a g ents ( c f . G l i c k , 1976). However, the r e s u l t s of s t u d i e s of the e f f e c t s of a n x i o l y t i c s on CER have be.en i n c o n s i s t e n t ( H a e f e l y , 1978; K e l l e h e r & Morse, 1968). For example, a n x i o l y t i c agents have been r e p o r t e d t o a t t e n u a t e (Lauener, 1963; Tenen, 1967), f a c i l i t a t e ( S t e i n & B e r g e r , 1969), or have no e f f e c t (Ray, 1965; S c o b i e & Gar s k e , 1970) on CER. N o n a n x i o l y t i c a gents have a l s o had i n c o n s i s t e n t e f f e c t s on CER, s u g g e s t i n g t h a t the s e l e c t i v i t y of the p r o c e d u r e i s q u e s t i o n a b l e . For example, Tenen (1967) r e p o r t e d t h a t amphetamine had no e f f e c t on CER; whereas, C a p e l l , G i n s b e r g , and Webster (1972) r e p o r t e d t h a t amphetamine a t t e n u a t e d CER. S i m i l a r l y , some s t u d i e s showed t h a t r e s e r p i n e and morphine a t t e n u a t e d CER, ( B e l l e v i l l e & W i k l e r , 1957; Brady, 1956; H i l l , P e s c o r , B e l l e v i l l e , & W i k l e r , 1957; Ray, 1964;1965); whereas, i n o t h e r s t u d i e s , t h e s e agents had no e f f e c t ( K i n n a r d , A c e t o , & 17 B u c k l e y , 1962; Lauener, 1963; Yamahiro, B e l l , & H i l l , 1961). A number of a u t h o r s ( D a n t z e r , 1977; Huppert & I v e r s e n , 1975; K e l l e r & Morse, 1968; M e l l e n s o n & L e s l i e , 1974) have a t t r i b u t e d t h e s e i n c o n s i s t e n t r e s u l t s t o p r o c e d u r a l v a r i a t i o n s between d i f f e r e n t e x p e r i m e n t s . However, D a n t z e r (1977) has argued t h a t even when p r o c e d u r a l f a c t o r s a r e h e l d c o n s t a n t , the e f f e c t s of a n x i o l y t i c agents on CER are s t i l l v a r i a b l e . Thus, t r y i n g t o use the CER paradigm as a d e v i c e f o r s c r e e n i n g a n x i o l y t i c agents appears t o be f u t i l e . By the same t o k e n , i t i s improbable t h a t the CER paradigm c o u l d be used t o s t u d y the mechanisms through which a n x i o l y t i c s have t h e i r t h e r a p e u t i c a c t i o n . E f f e c t s on c o n d i t i o n e d a v o i d a n c e r e s p o n d i n g . C o n d i t i o n e d a c t i v e a v o i d a n c e r e s p o n d i n g (CAR) i s s a i d t o have o c c u r r e d when a s u b j e c t a v o i d s an imminent a v e r s i v e s t i m u l u s or reduces the p r o b a b i l i t y of i t s o c c u r r e n c e by e m i t t i n g a l e a r n e d response ( F a n t i n o , 1973). A n i m a l s may be t r a i n e d t o respond i n e i t h e r a d i s c r e t e t r i a l s p r o c e d u r e ( e . g . , s i g n a l l e d s h u t t l e a v o i d a n c e ) or i n a c o n t i n u o u s a v o i d a n c e procedure ( e . g . , Sidman a v o i d a n c e ) . The r e s u l t s of s t u d i e s t h a t have employed CAR i n o r d e r t o s t u d y the e f f e c t s of a n x i o l y t i c a g e n t s have g e n e r a l l y been i n c o n s i s t e n t . For example, a n t i a n x i e t y agents have been shown t o e i t h e r f a c i l i t a t e (Sansone, 1975), i n h i b i t ( T a k a o r i , Yada, & M o r i , 1969), or have no e f f e c t on the a c q u i s i t i o n of CAR ( G o l d b e r g , H e f n e r , Robichaud, & D u b i n s k y , 1973). B i g n a m i , de A c e t i s , and G a t t i c (1971) have shown t h a t the same dose of an a n x i o l y t i c drug can improve the a v o i d a n c e b e h a v i o u r of poor p e r f o r m e r s ( r a t s r e c e i v i n g h i g h r a t e s o f . s h o c k ) and d i s r u p t the b e h a v i o u r of good p e r f o r m e r s ( r a t s r e c e i v i n g low r a t e s of 18 s h o c k ) . A n x i o l y t i c s have been r e p o r t e d t o i m p a i r the a v o i d a n c e b e h a v i o u r of g u i n e a p i g s (Sansone & M a r i o , 1969), and t o improve the a v o i d a n c e b e h a v i o u r of mice (Sansone, 1975). However, the a v o i d a n c e b e h a v i o u r of more a c t i v e s t r a i n s of mice i s i n h i b i t e d by a n x i o l y t i c s , whereas the same be h a v i o u r i s f a c i l i t a t e d i n l e s s a c t i v e s t r a i n s ( G a r a t t i n i , M u s s i n i , & R a n d a l l , 1973). Thus, many f a c t o r s , i n c l u d i n g s p e c i e s , s t r a i n , and response r a t e may modulate the e f f e c t s of a n x i o l y t i c s on a v o i d a n c e b e h a v i o u r , t h e r e b y c o n t r i b u t i n g t o the i n c o n s i s t e n c i e s i n the l i t e r a t u r e . Whatever the s o u r c e of these i n c o n s i s t e n c i e s , however, i t i s c l e a r t h a t CAR does not at p r e s e n t c o n s t i t u t e a r e l i a b l e method f o r s c r e e n i n g a n x i o l y t i c a g e n t s . E f f e c t s on p u n i s h e d operant b e h a v i o u r : The G e l l e r  ' c o n f l i c t ' t e s t . Punishment r e f e r s t o a p r o c e d u r e i n which a response ( e . g . , bar p r e s s i n g ) i s suppressed by the subsequent c o n t i n g e n t p r e s e n t a t i o n of an a v e r s i v e s t i m u l u s ( e . g . , e l e c t r i c s h o c k ) . Thus, by f o l l o w i n g each b a r p r e s s w i t h an e l e c t r i c f o o t -shock, the f r e q u e n c y of b a r p r e s s i n g i s d r a m a t i c a l l y reduced. T h i s p r o c e d u r e i s the f o u n d a t i o n f o r the G e l l e r ' c o n f l i c t ' t e s t of a n x i o l y t i c agents ( G e l l e r & S e i f t e r , 1960). U n l i k e o t h e r t r a d i t i o n a l a v e r s i v e l e a r n i n g p r o c e d u r e s , however, the G e l l e r c o n f l i c t t e s t and i t s v a r i a n t s ( e . g . , Howard & P o l l a r d , 1977) have been shown t o be r e a s o n a b l y r e l i a b l e i n d i c e s of a n x i o l y t i c a c t i v i t y ( c f . L i p p a , Nash, & G r e e n b l a t t , 1979; S e p i n w a l l & Cook, 1978). In the c o n f l i c t t e s t s , r a t s a r e t r a i n e d t o b a r p r e s s on a s c h e d u l e of food reward t h a t has two components. In one component of the s c h e d u l e , r e s p o n s e s a r e t y p i c a l l y r e i n f o r c e d 19 with food a f t e r a v a r i a b l e i n t e r v a l of time ( V I ) , and i n the second component every response i s r e i n f o r c e d . A s t i m u l u s i s i n t r o d u c e d at the beginning of the second component that s i g n a l s a ' c o n f l i c t ' p e r i o d d u r i n g which every response i s both r e i n f o r c e d with food and punished with e l e c t r i c shock. F o l l o w i n g f a i r l y e x t e n s i v e p e r i o d s of t r a i n i n g , r a t s r e l i a b l y c o n f i n e most of t h e i r responding to the VI component of the schedule, responding f a r l e s s d u r i n g the c o n f l i c t p e r i o d s . However, when these r a t s are i n j e c t e d with a n x i o l y t i c s such as c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l , t h e i r responding d u r i n g the c o n f l i c t phase of the schedule d r a m a t i c a l l y i n c r e a s e s . Most n o n a n x i o l y t i c s , such as a n t i p s y c h o t i c s , a n a l g e s i c s , or s t i m u l a n t s e i t h e r have no e f f e c t on t h i s task or they suppress responding even f u r t h e r (Cook & S e p i n w a l l , 1975). T h i s a n t i c o n f l i c t e f f e c t of a n x i o l y t i c s has been shown in a v a r i e t y of s p e c i e s , i n c l u d i n g r a t s (e.g., Cook & Davidson, 1973; G e l l e r & S e i f t e r , 1960), p i g s (Dantzer, 1975; Dantzer & Roca, 1974), c a t s (Jacobsen, 1957), pigeons (McMillan, 1973; Morse, 1964; Wuttke & K e l l e h e r , 1970), g o l d f i s h ( G e l l e r , Croy, & Ryback, 1974), s q u i r r e l monkeys (e.g., Beer & M i g l e r , 1975; Cook & C a t a n i a ; 1964), rhesus monkeys (Holtzman & V i l l a r r e a l , 1973), and humans (Beer & M i g l e r , 1975; Lehmann, 1969; Lehmann & Ban; 1971). In a d d i t i o n , the r e l a t i v e potency of v a r i o u s a n x i o l y t i c agents i n a number of s p e c i e s t e s t e d i n the c o n f l i c t paradigms compares f a v o r a b l y with t h e i r r e l a t i v e potency i n humans (Cook & Davidson, 1973). Thus, the c o n f l i c t procedures appear to f u l f i l the major c r i t e r i a of a v a l i d b e h a v i o u r a l t e s t of a n x i o l y t i c agents. 20 There are a few problems with the c o n f l i c t t e s t s , however. The f i r s t problem i s that the e f f e c t of a n x i o l y t i c drugs on responses punished i n the G e l l e r paradigm seems to vary depending upon whether or not the animal has had p r i o r experience with these drugs. Margules and S t e i n (1968) and Cook and Davidson (1973) have noted that the i n i t i a l e f f e c t of known a n x i o l y t i c agents on the punished responses of drug-naive animals i s o f t e n an a d d i t i o n a l suppression of punished responses, and only a f t e r s e v e r a l t e s t s e s s i o n s with the drug w i l l the response-enhancing e f f e c t of the drug appear. Thus, the i n i t i a l e f f e c t of an unknown agent i n the G e l l e r c o n f l i c t t e s t may be opp o s i t e to i t s . l a t e r e f f e c t . T h e r e f o r e any c o n c l u s i o n about a drug's a n x i o l y t i c p o t e n t i a l based on i t s i n i t i a l e f f e c t i n the c o n f l i c t t e s t c o u l d be erroneous ( c f . Ha e f e l y , 1978). The problem cannot be so l v e d by simply basing the e v a l u a t i o n of a drug on i t s e f f e c t s d u r i n g the l a t e r phases of t e s t i n g because c e r t a i n n o n a n x i o l y t i c s can a l s o have a response-enhancing e f f e c t a f t e r c h r o n i c a d m i n i s t r a t i o n ( H i l l & Te d e s c h i , 1971). A second problem a l r e a d y mentioned i s that s i g n i f i c a n t a n t i c o n f l i c t e f f e c t s have been produced by n o n a n x i o l y t i c s such as amphetamine (Lehmann Sc Ban, 1971; McKearney & B a r r e t t , 1975; M c M i l l a n , 1973; Miezek, 1973) and r e s e r p i n e ( H i l l & Tedeschi, 1971). Such e f f e c t s have u s u a l l y been small and commonly occur at only one dose l e v e l , but they do show that the c o n f l i c t t e s t s are not t o t a l l y s e l e c t i v e . The t h i r d problem i s that the a d d i t i o n of an a v e r s i v e s t i m u l u s (e.g., shock) to an a n x i o l y t i c s c r e e n i n g procedure that 21 i n v o l v e s consummatory responses must be j u s t i f i e d b o t h . i n terms of p r e d i c t i v e v a l i d i t y and i n terms of p r a c t i c a l u t i l i t y . I t has a l r e a d y been shown that many a n x i o l y t i c s (e.g., benzodiazepines) have a powerful s t i m u l a t o r y e f f e c t on food-motivated behaviour (e.g., Wise & Dawson, 1974) and i n f a c t t h i s s t i m u l a t o r y e f f e c t alone has been used to p r e d i c t the t h e r a p e u t i c e f f i c a c y of a n x i o l y t i c agents (Hanson & Stone, 1964). In the c o n f l i c t t e s t s , where responses are both r e i n f o r c e d with food and punished with e l e c t r i c shock, i t i s d i f f i c u l t to determine whether the i n c r e a s e i n responding observed a f t e r the a d m i n i s t r a t i o n of a n x i o l y t i c agents i s due to a s p e c i f i c a n t i c o n f l i c t e f f e c t or to a d i r e c t e f f e c t on food-motivated behaviour. Although some authors have argued that the s t i m u l a t i o n of food-motivated behaviours does not c o n t r i b u t e s i g n i f i c a n t l y to the a n t i c o n f l i c t e f f e c t of a n x i o l y t i c agents (Cook & Davidson, 1973; Sepinwall & Cook, 1978), others have expressed r e s e r v a t i o n s i n t h i s regard (Dantzer, 1977; Wise & Dawson, 1974) . Regardless of the i n t e r p r e t a t i o n of the e f f e c t s of a n x i o l y t i c agents i n the c o n f l i c t t e s t s , i t has not been shown that the i n t r o d u c t i o n of an a v e r s i v e shock stimulus i n the c o n f l i c t procedures adds s i g n i f i c a n t l y to the p r e d i c t i v e v a l i d i t y of these t e s t s . I t may be the case, f o r example, that the s l i g h t i n c r e a s e i n responding (e.g., 20%) o f t e n observed d u r i n g the n o n c o n f l i c t p e r i o d s of the c o n f l i c t t e s t s a f t e r moderate doses of a n x i o l y t i c agents i s j u s t as v a l i d a p r e d i c t o r of a n x i o l y t i c p o t e n t i a l as the i n c r e a s e s observed d u r i n g the shock p e r i o d s . Furthermore, even i f a s i g n i f i c a n t i n c r e a s e i n p r e d i c t i v e v a l i d i t y were shown to r e s u l t from the a d d i t i o n of 22 e l e c t r i c shock, t h i s i n c r e a s e would have t o compensate f o r the r e s u l t a n t d e c r e a s e i n the speed and s i m p l i c i t y of the t e s t . Thus, a l t h o u g h the c o n f l i c t p r o c e d u r e g e n e r a l l y s a t i s f i e s the c r i t e r i a of a v a l i d s c r e e n i n g t e s t of a n x i o l y t i c a g e n t s , i t i s not c l e a r t h a t i t r e p r e s e n t s a s i g n i f i c a n t improvement over s i m p l e consummatory t e s t s t h a t a r e f a r more r a p i d and e c o n o m i c a l t o a d m i n i s t e r . In summary, i t can be seen t h a t none of the t h r e e t r a d i t i o n a l a v e r s i v e l e a r n i n g paradigms t h a t has been used t o study the a n x i o l y t i c e f f e c t s of drugs i s e n t i r e l y s a t i s f a c t o r y . C o n d i t i o n e d s u p p r e s s i o n and c o n d i t i o n e d a v o i d a n c e , a l t h o u g h they have been c h a r a c t e r i z e d as good a n i m a l models of human a n x i e t y ( E s t e s & S k i n n e r , 1941) a r e not r e l i a b l e i n d i c e s of s t a n d a r d a n x i o l y t i c compounds. In c o n t r a s t , the c o n f l i c t t e s t s a r e r e a s o n a b l y s e n s i t i v e t o a n x i o l y t i c compounds, but i t i s not c l e a r t h a t they r e p r e s e n t i s o m o r p h i c models f o r s t u d y i n g t h e mechanisms of a c t i o n of a n x i o l y t i c compounds. Whereas, most r e s e a r c h e r s would agree t h a t a 'pure' a n t i p u n i s h m e n t e f f e c t might be a s u i t a b l e b e h a v i o u r a l model f o r s t u d y i n g the mechanisms of a n x i o l y t i c a c t i o n , they would not agree t h a t a 'pure' p o s i t i v e r e i n f o r c e m e n t e f f e c t c o u l d s e r v e the same f u n c t i o n ( c f . Wise & Dawson, 1974). T h i s p r e s e n t s a problem f o r the G e l l e r c o n f l i c t t e s t because i t i s u n c l e a r whether a n x i o l y t i c a g ents i n t h i s t e s t a r e r e d u c i n g the p u n i s h i n g e f f e c t of e l e c t r i c shock, i n c r e a s i n g the r e i n f o r c i n g e f f e c t of f o o d , or both (Wise & Dawson, 1974). U n l e s s t h i s a m b i g u i t y can be r e s o l v e d , i t i s d i f f i c u l t t o see how the c o n f l i c t t e s t can be used as a model t o study the mechanisms of a n x i o l y t i c a c t i o n . 23 The a r b i t r a r y n a t u r e of c o n v e n t i o n a l a n i m a l t r a i n i n g paradigms Because a v e r s i v e s t i m u l a t i o n , e x p e r i e n c e d or a n t i c i p a t e d , i s a p i v o t a l c o n s t r u c t i n almost a l l t h e o r e t i c a l a c c o u n t s of c l i n i c a l a n x i e t y ( e . g . , M a n d l e r , 1960; M i l l e r , 1948; Mowrer, 1940; S k i n n e r , 1953; Wolpe, 1958), i t i s not s u r p r i s i n g t h a t many a n i m a l models of c l i n i c a l a n x i e t y have i n v o l v e d a v e r s i v e s t i m u l a t i o n i n some form ( e . g . , Cook & D a v i d s o n , 1973;Estes & S k i n n e r , 1942; G e l l e r & S e i f t e r , 1 9 6 0 ) . These a n i m a l models have, f o r the most p a r t , been m o d i f i c a t i o n s of a v e r s i v e l e a r n i n g paradigms d e v e l o p e d by p s y c h o l o g i s t s i n t e r e s t e d i n the mechanisms of l e a r n i n g . However, c o n s i d e r a t i o n of the a s s umptions on which p s y c h o l o g i s t s based the o r i g i n a l development of t h e s e c o n d i t i o n i n g paradigms s u g g e s t s t h a t such an a p p l i c a t i o n i s i n a p p r o p r i a t e . The f o l l o w i n g b r i e f d i s c u s s i o n of the assumptions u n d e r l y i n g the o r i g i n a l development of t r a d i t i o n a l c o n d i t i o n i n g paradigms r e v e a l s some of the problems a s s o c i a t e d w i t h t h e i r use i n p h a r m a c o l o g i c a l r e s e a r c h (see ) I n t r o d u c t i o n ) and s u g g e s t s a more f r u i t f u l approach t o the b e h a v i o u r a l t e s t i n g of a n x i o l y t i c d r u g s . Sometimes we f o r g e t why p s y c h o l o g i s t s ever t r a i n e d w h i t e r a t s t o p r e s s b a r s f o r l i t t l e p e l l e t s of f l o u r or sounded metronomes f o l l o w e d by meat powder f o r domestic dogs. A f t e r a l l , when i n the r e a l w o r l d do r a t s e ncounter l e v e r s which t h e y l e a r n t o p r e s s i n o r d e r t o e a t , and when do our pet dogs ever come a c r o s s metronomes whose c l i c k i n g s i g n a l s meat powder? ( S e l i g m a n , 1970., p. 24 406). One of the major reasons why p s y c h o l o g i s t s d e v e l o p e d a v e r s i v e l e a r n i n g paradigms was t o d i s c o v e r the g e n e r a l p r i n c i p l e s or laws t h a t govern the l e a r n i n g of a s s o c i a t i o n s between s t i m u l i (respondent c o n d i t i o n i n g ) or between responses and t h e i r consequences (operant c o n d i t i o n i n g ) . Because t h e i r purpose was t o d i s c o v e r the g e n e r a l laws of l e a r n i n g , p s y c h o l o g i s t s tended t o study a r b i t r a r y c o m b i n a t i o n s of s t i m u l i ( e . g . , l i g h t s , t o nes) and responses ( e . g . , bar p r e s s e s , key pecks) i n a r b i t r a r y s u b j e c t s ( e . g . , r a t s , p i g e o n s ) . The v e r y a r b i t r a r i n e s s of these l e a r n i n g paradigms was i n t e n d e d t o m i n i m i z e the i n f l u e n c e s of s t i m u l u s - s p e c i f i c , r e s p o n s e - s p e c i f i c , and s p e c i e s - s p e c i f i c f a c t o r s and t h e r e b y i n s u r e the g e n e r a l i t y of the r e s u l t s t h a t were o b t a i n e d from these paradigms ( c f . S c h w a r t z , 1978; Seligman, 1970). I t i s i m p o r t a n t f o r p h a r m a c o l o g i s t s t o r e c o g n i z e not o n l y t h a t t h e s e paradigms were d e s i g n e d t o meet the s p e c i f i c needs of p s y c h o l o g i s t s , but t h a t they have not been unambiguously s u c c e s s f u l i n l e a d i n g t o the d i s c o v e r y of g e n e r a l laws of l e a r n i n g ( c f . S e l i g m a n , 1970). In f a c t , t h i s e n t i r e approach t o the s t u d y of a n i m a l b e h a v i o u r has been the f o c u s of a d i v i s i v e c o n t r o v e r s y among p s y c h o l o g i s t s f o r s e v e r a l y e a r s ( c f . B o l l e s , 1970; G a r c i a , C l a r k e , & H a n k i n s , 1973; S c h w a r t z , 1978; S e l i g m a n , 1970; S h e t t l e w o r t h , 1972). Some p s y c h o l o g i s t s have argued t h a t the study of a n i m a l s i n a r b i t r a r y l e a r n i n g s i t u a t i o n s imposes s e r i o u s c o n s t r a i n t s on the a n i m a l ' s a b i l i t y t o p e r f o r m a d a p t i v e r e s p o n s e s , so t h a t t h e i r . a c t u a l l e a r n i n g c a p a c i t i e s a r e not unambiguously r e v e a l e d ( e . g . , B o l l e s , 1970; 25 G a r c i a , C l a r k e , & H a n k i n s , 1973; P i n e l & T r e i t , 1980). An a n i m a l may be c o n s t r a i n e d by h a v i n g t o l e a r n a s s o c i a t i o n s between s t i m u l i t h a t have l i t t l e r e l e v a n c e t o i t s n a t u r a l environment or by h a v i n g t o make responses t h a t a r e o n l y i n d i r e c t l y r e l a t e d t o those t h a t h e l p i t s u r v i v e i n i t s n a t u r a l h a b i t a t ( B o l l e s , 1970). These c o n s t r a i n t s on the a n i m a l ' s s p e c i e s - t y p i c a l a d a p t a t i o n s may have c o n t r i b u t e d t o a l a r g e number of the i n c o n s i s t e n c i e s found i n the a n i m a l l e a r n i n g l i t e r a t u r e ( B o l l e s , 1970; B r e l a n d & B r e l a n d , 1961; P i n e l & T r e i t , 1980). For example, many a n i m a l s f a i l t o l e a r n an a p p a r e n t l y s i m p l e t a s k l i k e p r e s s i n g a bar t o a v o i d s i g n a l l e d e l e c t r i c shock (D'Amato & S c h i f f , 1964), y e t they can l e a r n t o jump out of a chamber t o a v o i d shock i n one t r i a l (Maatch, 1959). These k i n d s of i n c o n s i s t e n c i e s i m p ly t h a t o r d e r l y r e l a t i o n s h i p s between s t i m u l i and res p o n s e s may be found i n some s i t u a t i o n s (jump-out a v o i d a n c e ) but not i n o t h e r s ( b a r - p r e s s a v o i d a n c e ) . In view of these o b s e r v a t i o n s , p h a r m a c o l o g i s t s who study the e f f e c t s of a n x i o l y t i c agents e x c l u s i v e l y i n t r a d i t i o n a l a v e r s i v e l e a r n i n g paradigms might e x p e c t t o encounter two major problems. The f i r s t problem i s v a r i a b i l i t y . The v a r i a b i l i t y of the e f f e c t s of a n x i o l y t i c s i n t h e s e paradigms may sometimes r e f l e c t an i n s t a b i l i t y of the a r b i t r a r y c o n d i t i o n i n g paradigms r a t h e r than v a r i a b i l i t y a s s o c i a t e d w i t h the pharmacology of a n x i o l y t i c a g e n t s . The second problem i s c o m p l e x i t y . A r b i t r a r y forms of a v e r s i v e c o n d i t i o n i n g may have a p a r t i c u l a r l y complex n e u r a l b a s i s . Forms of a v e r s i v e l e a r n i n g t h a t a r e the d i r e c t r e s u l t of hundreds of thousands of y e a r s of e v o l u t i o n a r y 26 p r e s s u r e a r e not o n l y l i k e l y t o be more r o b u s t and r e l i a b l e , but the n e u r a l b a s i s of the s e t y p e s of l e a r n i n g may be l e s s complex and more d i r e c t l y r e l a t e d t o t h e n e u r a l s u b s t r a t e s of a n x i e t y . Thus, the n e u r o p h a r m a c o l o g i s t who i s a t t e m p t i n g t o d i s c o v e r the n e u r o c h e m i c a l c o r r e l a t e s of a n x i e t y by s t u d y i n g a r b i t r a r y forms of a v e r s i v e c o n d i t i o n i n g may be u s i n g paradigms t h a t a re not p a r t i c u l a r l y a p p r o p r i a t e f o r t h i s purpose ( c f . P i n e l & T r e i t , 1980). The c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm: A new model f o r the study of a n x i o l y t i c agents In a r e c e n t review paper, P i n e l and T r e i t (1980) d e s c r i b e d a new a v e r s i v e c o n d i t i o n i n g paradigm, the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm, whose rema r k a b l e r o b u s t n e s s appears t o be d e r i v e d from the f a c t t h a t i t was d e s i g n e d t o mimic a form of l e a r n i n g t h a t i s im p o r t a n t f o r the s u r v i v a l of r o d e n t s i n the w i l d r a t h e r than t o m i n i m i z e the c o n t r i b u t i o n s of such a d a p t i v e p r e d i s p o s i t i o n s . The speed, r e l i a b i l i t y , and s i m p l i c i t y of t h i s p a r t i c u l a r ' n o n a r b i t r a r y ' a v e r s i v e l e a r n i n g paradigm suggested t h a t i t would prove u s e f u l f o r s t u d y i n g the e f f e c t s of a n x i o l y t i c a g e n t s . A c c o r d i n g l y , the f o l l o w i n g s e c t i o n i s c o m p r i s e d of a b r i e f summary of the c o n d i t i o n e d d e f e n s i v e b u r y i n g l i t e r a t u r e . See P i n e l and T r e i t (1980) f o r a more complete r e v i e w . I n i t i a l D e m o n s t r a t i o n . P i n e l and T r e i t (1978) found t h a t r a t s shocked once t h r o u g h a s t a t i o n a r y , wire-wrapped pro d mounted on the w a l l of the t e s t chamber r e t u r n e d t o the prod and b u r i e d i t w i t h bedding m a t e r i a l from the f l o o r of the chamber. 27 Almost a l l of the r a t s sprayed bedding m a t e r i a l over the prod when the i n t e r v a l between the shock and the t e s t was s h o r t , and s i g n i f i c a n t l e v e l s of burying were observed even when the shock-t e s t i n t e r v a l was as long as 20 days. . Evidence f o r A s s o c i a t i v e L e a r n i n g . On the b a s i s of the i n i t i a l demonstration of the burying phenomenon ( P i n e l & T r e i t , 1978), i t might be assumed that shocked r a t s b u r i e d the prod because they had l e a r n e d the a s s o c i a t i o n between the shock and the prod. However, these i n i t i a l r e s u l t s d i d not r u l e out the p o s s i b i l i t y that burying behaviour i s an u n c o n d i t i o n e d response of r a t s to e l e c t r i c shock r e g a r d l e s s of i t s source, and that shocked r a t s b u r i e d the prod more than d i d unshocked c o n t r o l s simply because i t was the only novel o b j e c t i n the t e s t chamber ( c f . P i n e l & T r e i t , 1978). However, three subsequent o b s e r v a t i o n s confirmed the view that the b u r i a l of shock prods by r a t s i s a c o n d i t i o n e d response, c o n t r o l l e d by the a s s o c i a t i o n of the shock and the prod. F i r s t , r a t s shocked by a prod i n t h e i r home cages b u r i e d the prod when i t was subsequently encountered i n the t e s t chamber, whereas those that had been shocked through a g r i d f l o o r i n s t e a d d i d not ( P i n e l & T r e i t , 1978). Second, changing e i t h e r the b r i g h t n e s s or p o s i t i o n of the prod d u r i n g a 1-min i n t e r v a l between the shock and the t e s t produced systematic decreases i n burying behaviour ( P i n e l , T r e i t , & W i l k i e , 1980). And t h i r d , r a t s shocked from one of two prods mounted on opposite w a l l s of the t e s t chamber b u r i e d only the shock prod ( P i n e l & T r e i t , 1978; T e r l e c k i et a l . , 1979; P i n e l & T r e i t , 1979). Although these three o b s e r v a t i o n s leave l i t t l e doubt that 28 r a t s bury, shock prods because of the c o n d i t i o n e d a s s o c i a t i o n of the shock and the prod, T e r l e c k i et a l . (1979) have shown that d e f e n s i v e burying can a l s o occur as an unconditioned component of the r a t ' s neophobic r e a c t i o n to some novel o b j e c t s . T e r l e c k i et a l . found that when r a t s f i r s t encountered a mouse-trap or a f l a s h b u l b i n a f a m i l i a r t e s t chamber, they b u r i e d these novel o b j e c t s ; whereas r a t s p r e v i o u s l y h a b i t u a t e d to the t r a p or the f l a s h b u l b d i d not. In c o n t r a s t , r a t s d i d not bury a wooden prod or a l e n g t h of p o l y e t h y l e n e t u b i n g , even on f i r s t encounter. However when these o b j e c t s were subsequently p a i r e d with a v e r s i v e s t i m u l a t i o n the r a t s r e a d i l y d i s p l a y e d c o n d i t i o n e d d e f e n s i v e b u r y i n g ; i . e . , they s e l e c t i v e l y b u r i e d the source of a v e r s i v e s t i m u l a t i o n but not a comparable c o n t r o l o b j e c t . Thus, r a t s appear to enter the experimental environment with an a l r e a d y e s t a b l i s h e d tendency to bury some novel o b j e c t s (unconditioned d e f e n s i v e burying) but not o t h e r s , and they r e a d i l y l e a r n to s e l e c t i v e l y bury an o b j e c t that has been the source of a v e r s i v e s t i m u l a t i o n ( c o n d i t i o n e d d e f e n s i v e b u r y i n g ) . Robustness of the c o n d i t i o n e d d e f e n s i v e burying phenomenon. In c o n t r a s t to t r a d i t i o n a l forms of a v e r s i v e c o n d i t i o n i n g , c o n d i t i o n e d d e f e n s i v e burying i s an extremely robust phenomenon. In a l a r g e number of s t u d i e s i n t h i s l a b o r a t o r y and o t h e r s , there has been l i t t l e i f any o v e r l a p between the burying scores of unshocked c o n t r o l r a t s and those r e c e i v i n g a s i n g l e prod shock (e.g., Davis & Rossheim, 1980; P i n e l & T r e i t , 1978; P i n e l , T r e i t , Ladak, & MacLennan, 1980; P i n e l , T r e i t , & W i l k i e , 1980; T e r l e c k i et a l . , 1979; T r e i t , P i n e l , & T e r l e c k i , 1980). In f a c t , i t i s not uncommon f o r burying behaviour to be completely 29 absent in unshocked controls and to appear in every shocked subject (e.g., Terlecki et a l . , 1979). Investigators from other laboratories have also commented on the remarkable robustness of t h i s phenomenon (Davis & Rossheim, 1980). The robustness of the conditioned defensive burying paradigm appears to be derived from the fact that i t was designed to mimic a form of learning that i s important for the survival of rats in the wild rather than to minimize the contribution of such adaptive predispositions. There are two features of the paradigm that were s p e c i f i c a l l y designed to accommodate the defensive p r o c l i v i t i e s of rats and thus dis t i n g u i s h i t from more t r a d i t i o n a l paradigms in which aversive learning i s not as robust. F i r s t , the aversive stimulus i s not a d i f f u s e e l e c t r i c shock administered through a g r i d f l o o r , as i s common in t r a d i t i o n a l paradigms; i t i s a l o c a l i z e d e l e c t r i c shock administered from a well-defined source, the prod. Pinel, T r e i t , and Wilkie (1980) have argued that i t easier for a rat to learn the association between a shock and a cue that s i g n i f i e s shock when the two are s p a t i a l l y contiguous than when they are not. In t r a d i t i o n a l learning experiments, rats must learn the association between s p a t i a l l y separated cues that are temporally paired with diffuse grid shock. Because aversive stimuli in the rat's natural environment are usually well-defined s p a t i a l l y and are contiguous with the cues that signal their occurrence, i t i s not unreasonable to assume that laboratory animals may be better able to learn about s p a t i a l l y contiguous events than about those that are not. The second modification of t r a d i t i o n a l aversive conditioning paradigms is that instead of the usual g r i d floor 30 there i s a l a y e r of commercial bedding m a t e r i a l that the r a t can manipulate to p r o t e c t i t s e l f from the source of a v e r s i v e s t i m u l a t i o n . Obviously d e f e n s i v e burying i s impossible i n t r a d i t i o n a l a v e r s i v e l e a r n i n g chambers where there are no movable m a t e r i a l s on the f l o o r . Burying as a Defensive Response i n Rodents. The f a c t that burying r e l i a b l y occurs i n a paradigm that was s p e c i f i c a l l y designed to accommodate s p e c i e s - t y p i c a l d e f e n s i v e behaviour does not i n i t s e l f prove that burying occurs as an a d a p t i v e response of rodents to n a t u r a l t h r e a t s . N e v e r t h e l e s s , there i s now a growing body of i n d i r e c t support f o r P i n e l and T r e i t ' s (1978) c h a r a c t e r i z a t i o n of burying as a d e f e n s i v e response ( c f . P i n e l & T r e i t , 1980) . F i r s t , l i k e a l l other rodent d e f e n s i v e behaviours (Blanchard & Blanchard, 1971; B o l l e s , 1970; Grossen & K e l l e y , 1972; U l r i c h , 1967), burying behaviour i s a r e l i a b l e response of rodents to p a i n f u l e l e c t r i c shock i n a v a r i e t y of s i t u a t i o n s (e.g., P i n e l & T r e i t , 1978; 1979; P i n e l , T r e i t , Ladak, & MacLennan, 1980; P i n e l , T r e i t , & W i l k i e , 1980; T e r l e c k i et a l . , 1979; T r e i t , T e r l e c k i , & P i n e l , 1980), and the amount of burying i n c r e a s e s with i n c r e a s e s i n the i n t e n s i t y of the shock ( T r e i t , P i n e l , & T e r l e c k i , 1980). Moreover, burying behaviour has a l s o been shown to occur i n response to a v e r s i v e s t i m u l i other than p a i n f u l e l e c t r i c shock, some of which are more comparable to those that rodents might encounter i n t h e i r n a t u r a l environments. For example, r a t s have been shown to bury o b j e c t s a s s o c i a t e d with a v e r s i v e s t i m u l i such as an a i r b l a s t , a l i g h t f l a s h , or a p h y s i c a l blow ( T e r l e c k i et a l . , 1979), and they w i l l 31 bury spouts containing saccharin previously associated with toxic o s i s (Wilkie, MacLennan, & P i n e l , 1979). A second source of support for the idea that burying serves a defensive function in the wild comes from experiments in which Pinel and T r e i t ' s standard test environment was altered in order to further mimic s p e c i f i c features of natural settings. It was found that rats were capable of using a variety of natural (e.g., sand) or cumbersome (e.g., blocks) materials to bury a shock prod, even when the materials f i r s t had to be transported to the prod (Pinel & T r e i t , 1979). These observations suggest that burying could be an e f f e c t i v e defensive response in a natural environment, where burying materials are not always readily available and where available materials may require a varied response topography for t h e i r d i s p o s i t i o n . Another important feature of natural settings i s that they may afford the rodent an opportunity to hide or to flee from the source of aversive stimuli (cf. Calhoun, 1962). Si g n i f i c a n t in t h i s regard are reports that rats display conditioned defensive burying in chambers large enough to permit a considerable degree of f l i g h t , and in chambers in which they could escape into a 'safe' compartment (Pi n e l , T r e i t , Ladak, & MacLennan, 1980). The t h i r d and most convincing source of evidence that burying by rodents serves a defensive function in the wild comes from several ethplogical studies. Calhoun (1962) maintained that wild rats exposed to " t e r r i t o r i a l threat" from other conspecifics would bury the entrance holes to their underground burrows, thus protecting themselves from intruders, and Hudson (1950) reported that burying behaviour i s a common response of 32 gophers t o r e p t i l e s or t o t r a p s s e t i n t h e i r burrows. S i m i l a r l y , Owings and Coss (1978) have found t h a t C a l i f o r n i a ground s q u i r r e l s d r i v e o f f a p p r o a c h i n g snakes by s p r a y i n g sand a t them w i t h r a p i d , f o r e l i m b s p r a y i n g movements a p p a r e n t l y i d e n t i c a l t o those used by r a t s t o bury a v e r s i v e o b j e c t s i n l a b o r a t o r y s t u d i e s ( c f . P i n e l & T r e i t , 1978). In a d d i t i o n t o showing t h a t ground s q u i r r e l s can h a r r a s s snakes from a s a f e d i s t a n c e by u s i n g t h i s b u r y i n g r e s p o n s e , Coss and Owings (1978) demonstrated t h a t ground s q u i r r e l s can s e a l t hemselves from an a p p r o a c h i n g snake by s p r a y i n g and p i l i n g sand over the e n t r a n c e t o the burrow ( c f . Calhoun, 1962). Taken t o g e t h e r w i t h the p r e v i o u s l a b o r a t o r y o b s e r v a t i o n s , these e t h o l o g i c a l r e p o r t s s t r o n g l y suggest t h a t b u r y i n g i s a complex, s p e c i e s - t y p i c a l d e f e n s i v e response of r o d e n t s t h a t can be c o n d i t i o n e d i n one t r i a l t o a v a r i e t y of ' n e u t r a l ' s t i m u l i ( c f . P i n e l & T r e i t , 1980). I t i s c l e a r from the p r e c e d i n g d i s c u s s i o n t h a t t h e r e a r e s e v e r a l f e a t u r e s of the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm i n a d d i t i o n t o i t s remarkable r o b u s t n e s s t h a t i n d i c a t e t h a t i t may be a p a r t i c u l a r l y e f f e c t i v e model f o r the study of a n x i o l y t i c a g e n t s . 1. Speed. A major advantage of the d e f e n s i v e b u r y i n g response i s t h a t i t can be r e a d i l y c o n d i t i o n e d i n o n l y a s i n g l e t r i a l . A l t h o u g h t h e r e a r e some t r a d i t i o n a l a v e r s i v e l e a r n i n g paradigms t h a t a r e r e l i a b l e enough t o be used t o d e t e c t p o t e n t i a l a n x i o l y t i c compounds, t h e i r r o u t i n e use as s c r e e n i n g d e v i c e s i s p r o h i b i t e d by the time and e f f o r t r e q u i r e d t o i n i t i a l l y c o n d i t i o n the t e s t a n i m a l s ( c f . H a e f e l e y , 1978; 33 L i p p a , Nash, & G r e e n b i a t t , 1979). 2. S i m p l i c i t y . Because c o n d i t i o n e d d e f e n s i v e burying appears to be a form of l e a r n i n g that i s important f o r the s u r v i v a l of r a t s i n the w i l d ( P i n e l & T r e i t , 1980), i t s n e u r a l b a s i s may be l e s s complex than that of a r b i t r a r y forms of a v e r s i v e l e a r n i n g , and more d i r e c t l y r e l a t e d to the n e u r a l s u b s t r a t e s of a n x i e t y . Thus, i t may be a p a r t i c u l a r l y u s e f u l paradigm f o r the neuropharmacologist who i s attempting to study the mechanisms of a c t i o n of a n x i o l y t i c agents. In a d d i t i o n , because the burying response can be produced by a v e r s i v e s t i m u l a t i o n alone, a n t i a n x i e t y e f f e c t s i n t h i s paradigm are not confounded with e f f e c t s on a p p e t i t i v e l y motivated behaviour, as they are i n other l e a r n i n g paradigms (e;g., the G e l l e r c o n f l i c t t e s t ) . 3. G e n e r a l i t y . Although the study of c o n d i t i o n e d d e f e n s i v e burying has only r e c e n t l y begun, i t has a l r e a d y been e s t a b l i s h e d that the response i s not s p e c i f i c to a p a r t i c u l a r l e v e l of shock ( T r e i t , T e r l e c k i , & P i n e l , 1980), a p a r t i c u l a r s t r a i n , s p e c i e s , sex, or age of r o d e n t ( T r e i t , T e r e c k i , & P i n e l , 1980), a p a r t i c u l a r type of t e s t e n v i r o n m e n t ( P i n e l , T r e i t , Ladak, & MacLennan, 1980), a p a r t i c u l a r type of a v e r s i v e stimulus ( T e r l e c k i et a l . , 1979; W i l k i e et a l . , 1979), a p a r t i c u l a r i n t e r v a l between c o n d i t i o n i n g and t e s t i n g ( P i n e l & T r e i t , 1978; P i n e l , T r e i t & W i l k i e , 1980), or a p a r t i c u l a r type of burying m a t e r i a l ( P i n e l & T r e i t , 1979). Thus r e s e a r c h e r s using t h i s paradigm to study a n x i o l y t i c e f f e c t s would not be c o n s t r a i n e d by the n e c e s s i t y of adhering to a s p e c i f i c experimental format; to a s u b s t a n t i a l degree, i t i s p o s s i b l e to 34 t a i l o r the paradigm to meet the demands of a p a r t i c u l a r , experiment. Purpose In view of the need f o r an animal model that can be used both to d e t e c t a n x i o l y t i c agents r a p i d l y and r e l i a b l y and to study t h e i r mechanisms of a c t i o n , a pharmacological i n v e s t i g a t i o n of the c o n d i t i o n e d d e f e n s i v e burying response was warranted. A c c o r d i n g l y , the f i r s t purpose of the present i n v e s t i g a t i o n s was to assess the extent to which the c o n d i t i o n e d d e f e n s i v e b urying paradigm f u l f i l s the three c r i t e r i a a s s o c i a t e d with a p r e d i c t i v e animal t e s t of a n x i o l y t i c agents, i . e . , dose-dependent s e n s i t i v i t y , r e l a t i v e potency, and s e l e c t i v i t y . The second purpose was to show that the c o n d i t i o n e d b u r y i n g t e s t of a n x i o l y t i c agents, once developed, c o u l d be used to help d e l i n e a t e t h e i r mechanisms of a c t i o n . The e i g h t experiments that comprise the present t h e s i s are grouped i n two s e c t i o n s that correspond to these two purposes. 35 GENERAL METHODS In each of the p r e s e n t e x p e r i m e n t s , the e f f e c t s of drugs on c o n d i t i o n e d or u n c o n d i t i o n e d d e f e n s i v e b u r y i n g were a s s e s s e d , and the same g e n e r a l methods were employed. S u b j e c t s . In each e x p e r i m e n t , a d u l t , 250 t o 450 g, n a i v e , male, hooded r a t s , purchased from Canadian B r e e d i n g Farm and L a b o r a t o r i e s , La P r a i r i e , Quebec s e r v e d as s u b j e c t s . The r a t s were housed i n groups of s i x i n 84 x 18 x 18 cm w i r e mesh cages under a 12-hr l i g h t / d a r k c y c l e , w i t h c o n t i n u o u s a c c e s s t o P u r i n a l a b o r a t o r y chow and water. R a t s were u s u a l l y t e s t e d d u r i n g the l i g h t p a r t of the 12-hr l i g h t / d a r k c y c l e . A p p a r a t u s . A l l t e s t i n g was done i n a 44 x 30 x 44 cm P l e x i g l a s t e s t chamber, the f l o o r of which was e v e n l y c o v e r e d w i t h 5 cm of S a n - i - c e l , a com m e r c i a l bedding m a t e r i a l made of ground c o r n cob (Paxton P r o c e s s i n g Co., Paxton, 1 1 1 . ) . In the c e n t r e of each of the f o u r w a l l s , 2 cm above the l e v e l of the S a n - i - c e l , was a s m a l l h o l e t h r o u g h which a 6.5 x .5 x .5 cm wire-wrapped wooden dowel ( i . e . , the shock prod) c o u l d be^ i n s e r t e d . E l e c t r i c c u r r e n t was a d m i n i s t e r e d t h r o u g h the two u n i n s u l a t e d w i r e s wrapped around the p r o d . In one e x p e r i m e n t , i n p l a c e of the p r o d , was a 9.8 x 4.5 x .5 cm wooden mousetrap ( V i c t o r , Woodstream Corp., L i l i t z , Pa.) f a s t e n e d h o r i z o n t a l l y on the c e n t r e of one w a l l , 2 cm above the l e v e l of the bedding. The b e h a v i o u r of each r a t i n each experiment was m o n i t o r e d f o r 15 min from a s e p a r a t e room v i a c l o s e d c i r c u i t t e l e v i s i o n . P r o c e d u r e s H a b i t u a t i o n . B e f o r e each of the e x p e r i m e n t s , t h e r a t s were 36 placed in the Plexiglas test chamber in groups of f i v e or six for 30-min periods on each of 4 consecutive days. Drug administration. On the f i f t h day of each experiment, shortly before they were tested, the rats were randomly assigned to drug or vehicle groups. The rats in the drug groups received an intraperitoneal i n j e c t i o n of the drug in solution with the vehicle; whereas, those in the vehicle control groups received an intraperitoneal in j e c t i o n of an equivalent volume of the appropriate vehicle. Shock administration. Shortly before testing on the f i f t h day, the shock prod was inserted 6 cm into the experimental chamber through the hole in one end wall and fixed there. Each animal was then placed i n d i v i d u a l l y into the centre of the chamber so that i t faced away from the prod. When the rat f i r s t touched the prod with i t s forepaw, i t received a brief e l e c t r i c shock from an 800-V power source. In some cases, the animals received a low l e v e l current (approximately 1 mA), which t y p i c a l l y e l i c i t e d a s l i g h t f l i n c h away from the prod followed by a slow withdrawal toward the back of the test chamber; whereas, in other cases they received a high l e v e l current (approximately 9 mA), which t y p i c a l l y e l i c i t e d a sudden, fullbody f l i n c h , immediately followed by a rapid withdrawal toward the back of the chamber. In both cases, the shock was i n i t i a t e d by the experimenter and terminated by the withdrawal of the subject. The intensity of the current administered from the 800-V power source was fixed by wiring either an 80k or 400k ohm r e s i s t o r in series with the subjects to produce the high- or the 37 low-shock l e v e l s , r e s p e c t i v e l y . The a c t u a l i n t e n s i t y and d u r a t i o n of shocks r e c e i v e d by r a t s from each of these c i r c u i t s was monitored by a storage o s c i l l o s c o p e i n a separate p i l o t study (n=20). The h i g h - l e v e l c u r r e n t was a c t u a l l y 9.4 mA i n i n t e n s i t y (SD=2.2) and 35.2 msec in d u r a t i o n (SD=10.4); whereas the l o w - l e v e l shock was .9 mA i n i n t e n s i t y (SD=.14) and 29.6 msec (SD=11.6) in d u r a t i o n ( c f . P i n e l & T r e i t , 1978; T e r l e c k i et a l . , 1979; T r e i t , P i n e l , & T e r l e c k i , 1980). B e h a v i o u r a l o b s e r v a t i o n and q u a n t i f i c a t i o n . Immediately a f t e r shock a d m i n i s t r a t i o n , the behaviour of each r a t was viewed over the t e l e v i s i o n monitor f o r 15 min, and the d u r a t i o n of each burying sequence was recorded on a c h a r t r e c o r d e r . These burying sequences are remarkably s t e r e o t y p e d when r a t s are shocked by a prod i n the presence of bedding m a t e r i a l ( c f . P i n e l £ T r e i t , 1979). The r a t t y p i c a l l y begins by f a c i n g the prod from a d i s t a n c e ; then i t moves d i r e c t l y toward the prod, pushing and s p r a y i n g a p i l e of bedding m a t e r i a l ahead with r a p i d a l t e r n a t i n g movements of i t s forepaws. I t i s the forward motion of the r a t s ' f o r e l i m b s , which d i r e c t s the bedding toward the prod, and thus d e f i n e s burying behaviour ( c f . P i n e l & T r e i t , 1980). The r e l i a b i l i t y of the d u r a t i o n - o f - b u r y i n g measure has been e s t a b l i s h e d i n s e v e r a l p r e v i o u s s t u d i e s . P i n e l , T r e i t , and W i l k i e (1980), P i n e l , Hoyer, and T e r l e c k i (1980), Davis and Rossheim (1980), and Davis, Whiteside, Dickson, Thomas, and Heck ( i n press) have found c o r r e l a t i o n s of .988, .990, .93, and .91, r e s p e c t i v e l y , between the scores compiled by independent o b s e r v e r s . In a d d i t i o n to t h i s b e h a v i o u r a l measure of burying, once 38 the a n i m a l was removed from the chamber a f t e r the 15-min t e s t , the h e i g h t of the bedding m a t e r i a l was measured a t the j u n c t i o n between t h e prod and the w a l l . Because t h i s h e i g h t measure tends t o be h i g h l y c o r r e l a t e d (r = .89; P i n e l & T r e i t , 1979) w i t h the d u r a t i o n measure, the r e s u l t s of i t s s t a t i s t i c a l a n a l y s i s u s u a l l y c o r r o b o r a t e the a n a l y s i s of the d u r a t i o n measure ( c f . P i n e l & T r e i t , 1980). S t a t i s t i c a l A n a l y s e s . The d e s i g n of the m a j o r i t y of e x p e r i m e n t s was c e n t r e d around one s e t of a p r i o r i c o m p a r i s o n s ; i . e . , the comparisons between the mean b u r y i n g s c o r e s of d r u g -i n j e c t e d and v e h i c l e - i n j e c t e d r a t s . The e f f e c t of d i f f e r e n t drug doses and d i f f e r e n t shock l e v e l s on b u r y i n g b e h a v i o u r were a s s e s s e d w i t h a n a l y s i s of v a r i a n c e , f o l l o w e d by a p o s t e r i o r i p a i r - w i s e c o m p a r i s o n s . 39 PART I_ C o n d i t i o n e d d e f e n s i v e b u r y i n g as a s c r e e n i n g t e s t f o r a n x i o l y t i c a g e n t s Experiment 1 The purpose of Experiment 1 was t o show t h a t the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm s a t i s f i e s t he f i r s t c r i t e r i o n of a u s e f u l s c r e e n i n g t e s t of a n x i o l y t i c a g e n t s ; i . e . , t h a t i t i s s e n s i t i v e i n a dose-dependent manner t o the e f f e c t s of a known a n x i o l y t i c (see I n t r o d u c t i o n ) . Thus, i n Experiment 1, the amount of c o n d i t i o n e d d e f e n s i v e b u r y i n g d i s p l a y e d by r a t s i n j e c t e d w i t h diazepam was compared t o t h a t of v e h i c l e - i n j e c t e d c o n t r o l s . Method On day 5, a f t e r the 4 c o n s e c u t i v e days of h a b i t u a t i o n , the 80 r a t s s e r v i n g as s u b j e c t s were randomly a s s i g n e d t o one of two c o n d i t i o n s . The r a t s i n one c o n d i t i o n ( d r u g - i n j e c t e d s u b j e c t s , n=40) were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h e i t h e r .1 mg/kg (n=10), .5 mg/kg (n=10), 1 mg/kg (n=10), or 2 mg/kg (n=10) of diazepam (Roche), 30 min b e f o r e they were p l a c e d i n the P l e x i g l a s t e s t chamber. When t h e r a t s f i r s t c o n t a c t e d the s t a t i o n a r y wire-wrapped p r o d w i t h a forepaw, they r e c e i v e d a 1-mA shock (see G e n e r a l Method). The r a t s (n=40) i n the second c o n d i t i o n ( v e h i c l e - i n j e c t e d s u b j e c t s ) were t r e a t e d i n e x a c t l y the same manner, except t h a t t h e r a t s i n each group (n=10) r e c e i v e d a volume of the v e h i c l e t h a t was e q u i v a l e n t t o t h a t r e c e i v e d by r a t s of e q u a l weight i n each of the r e s p e c t i v e e x p e r i m e n t a l groups. The b u r y i n g b e h a v i o u r of each r a t was r e c o r d e d d u r i n g the e n s u i n g 15-min t e s t p e r i o d . 40 R e s u l t s and D i s c u s s i o n I t i s apparent from F i g . 1 t h a t the amount of c o n d i t i o n e d d e f e n s i v e b u r y i n g d i s p l a y e d by the d i a z e p a m - t r e a t e d r a t s was w e l l below t h a t d i s p l a y e d by c o n t r o l r a t s . P l a n n e d o r t h o g o n a l comparisons between the d u r a t i o n of b u r y i n g i n e x p e r i m e n t a l and c o n t r o l r a t s c o n f i r m e d t h a t diazepam s i g n i f i c a n t l y s u p p r e s s e d b u r y i n g b e h a v i o u r a t every dose except .1 mg/kg {.1 mg/kg, t ( 1 8 ) = .11, p>.5 ; .5 mg/kg, t ( 1 8 ) = 2.18, p<.05; 1.0 mg/kg, t ( 1 8 ) = 3.78, p<.001; 2.0 mg/kg, t ( 1 8 ) = 6.22, p<.0001}. A s i m i l a r a n a l y s i s of the h e i g h t d a t a c o r r o b o r a t e d t h i s a n a l y s i s ; the h e i g h t of m a t e r i a l accumulated a t the prod by d i a z e p a m - i n j e c t e d r a t s was s i g n i f i c a n t l y l e s s than t h a t accumulated by v e h i c l e -i n j e c t e d r a t s a t every dose except .lmg/kg {.1 mg/kg, t ( 1 8 ) = .52, p>.5; .5 mg/kg, t ( 1 8 ) = 2.42, p<.03; 1.0 mg/kg, t ( 1 8 ) = 4.05, p<.0007; 2.0 mg/kg, t ( 1 8 ) = 4.80, p<.0001}. In o r d e r t o a s s e s s whether or not the s u p p r e s s i v e e f f e c t of diazepam on c o n d i t i o n e d d e f e n s i v e b u r y i n g was dose-dependent, the d u r a t i o n and h e i g h t d a t a were each s u b j e c t e d t o 2-way a n a l y s e s of v a r i a n c e . The 2 by 4 a n a l y s i s of v a r i a n c e of the d u r a t i o n d a t a c o n f i r m e d the s u p p r e s s i v e e f f e c t of diazepam on b u r y i n g b e h a v i o u r { F ( l , 7 2 ) = 25.84, p<.0001} and r e v e a l e d a s i g n i f i c a n t dose-by-drug i n t e r a c t i o n (F(3,72) = 3.64, p<.02}. The main e f f e c t of dose was not s i g n i f i c a n t (F(3,72) = .29, p>.50}. The dose-by-drug i n t e r a c t i o n was broken down i n t o i t s components by subsequent a p o s t e r i o r i p a i r - w i s e comparisons (Duncan's m u l t i p l e range t e s t ; p - .05). The r e s u l t s of the s e p a i r - w i s e comparisons showed t h a t the s u p p r e s s i o n of b u r y i n g b e h a v i o u r by 2 mg/kg of diazepam was s i g n i f i c a n t l y g r e a t e r than 41 F i g u r e 1. Mean d u r a t i o n ( S.E.M. ) of b u r y i n g at each of four doses f o r diazepam-injected ( d i a g o n a l l y - s t r i p e d bars) and v e h i c l e - i n j e c t e d r a t s (open bars) i n Experiment 1. mean durat ion of burying (sec) 43 t h a t produced by .1 mg/kg . S i m i l a r a n a l y s i s of the h e i g h t d a t a c o r r o b o r a t e d the b e h a v i o u r a l r e s u l t s . The 2 by 4 a n a l y s i s of v a r i a n c e of the h e i g h t s c o r e s r e v e a l e d a s i g n i f i c a n t main e f f e c t of diazepam { F ( l , 7 2 ) = 22.27, p<.0001}, no s i g n i f i c a n t main e f f e c t of dose {F(3.,72) = 1.73, p>.10}, and a s i g n i f i c a n t dose-by-drug i n t e r a c t i o n {F(3,72) = 4.75, p<.004}. Subsequent a p o s t e r i o r i p a i r - w i s e comparisons (Duncan's p=.05) c o n f i r m e d t h a t the amount of m a t e r i a l accumulated a t the prod by r a t s i n the .5 and 2 mg/kg c o n d i t i o n s was s i g n i f i c a n t l y l e s s than the amount acc u m u l a t e d by r a t s i n the .1 mg/kg c o n d i t i o n . No o t h e r d i f f e r e n c e s between the d r u g - i n j e c t e d groups were s i g n i f i c a n t . I t s h o u l d be emphasized t h a t the .5 mg/kg dose a t which diazepam was a b l e t o s i g n i f i c a n t l y s u p p r e s s c o n d i t i o n e d d e f e n s i v e b u r y i n g i s c o n s i d e r a b l y l e s s than the doses t h a t have been t y p i c a l l y r e q u i r e d t o r e v e a l an e f f e c t of diazepam i n o t h e r b e h a v i o u r a l paradigms ( e . g . , the G e l l e r c o n f l i c t t e s t ) . Thus, the c o n d i t i o n e d b u r y i n g response i s h i g h l y s e n s i t i v e t o the e f f e c t s of diazepam. F u r t h e r m o r e , the range of doses where diazepam produced a s i g n i f i c a n t s u p p r e s s i o n of c o n d i t i o n e d b u r y i n g was w e l l o u t s i d e of the range t h a t i s known t o produce o b v i o u s b e h a v i o u r a l t o x i c i t y ( L i p p a , Nash, & G r e e n b l a t t , 1979). Other than the m i l d a t a x i a shown by t h r e e of the a n i m a l s i n the h i g h e s t dose c o n d i t i o n , t h e g e n e r a l appearance of diazepam-t r e a t e d r a t s was not o b v i o u s l y d i f f e r e n t from t h a t of v e h i c l e -t r e a t e d c o n t r o l s . Most of the d r u g - i n j e c t e d a n i m a l s (30/40) s p r a y e d a t l e a s t some bed d i n g m a t e r i a l toward the p r o d , which was comparable t o the i n c i d e n c e of t h i s b e h a v i o u r i n c o n t r o l s 44 F i g u r e 2. Mean height ( S.E.M. ) of m a t e r i a l accumulated at each of four doses f o r diazepam-treated ( d i a g o n a l l y - s t r i p e d bars) and v e h i c l e - i n j e c t e d r a t s (open bars) i n Experiment 1. 45 mean height of material (cm) o m 3 U2> C P <o ho T " 1 I J H • 0 < o m A) x cz m 46 (38/40). Thus, the mean d i f f e r e n c e s i n b u r y i n g b e h a v i o u r o b s e r v e d between the e x p e r i m e n t a l and the c o n t r o l a n i m a l s d i d not seem t o occur because d r u g - i n j e c t e d a n i m a l s c o u l d not make the b u r y i n g r e s p o n s e . 47 Experiment 2 The f i r s t purpose of Experiment 2 was to s y s t e m a t i c a l l y r e p l i c a t e the f i n d i n g s of Experiment 1 using d i f f e r e n t a n x i o l y t i c agents. The second purpose was to assess the degree to which the c o n d i t i o n e d d e f e n s i v e burying paradigm f u l f i l s the second c r i t e r i o n of a v a l i d animal t e s t of a n x i o l y t i c agents, i . e . , the c r i t e r i o n of r e l a t i v e potency (see I n t r o d u c t i o n ) . A c c o r d i n g l y , three a n x i o l y t i c agents that are known to d i f f e r i n t h e i r r e l a t i v e a b i l i t y to suppress a n x i e t y i n humans ( i . e . , diazepam, c h l o r d i a z e p o x i d e , and p e n t o b a r b i t a l ) were assessed f o r t h e i r r e l a t i v e a b i l i t y to suppress c o n d i t i o n e d d e f e n s i v e burying in r a t s at four d i f f e r e n t doses: 0,1,3, and 6 mg/kg. In c l i n i c a l s e t t i n g s , the potency of diazepam i s s u b s t a n t i a l l y g r e a t e r than the potency of e i t h e r c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l ; whereas, the potency of c h l o r d i a z e p o x i d e i s only m a r g i n a l l y s u p e r i o r to that of p e n t o b a r b i t a l ( c f . Cook & Davidson, 1973). Method A f t e r the 4 days of h a b i t u a t i o n , each of the 120 r a t s was randomly a s s i g n e d to one of 12 groups (nine drug groups and three v e h i c l e groups) of 10 s u b j e c t s each. On day 5, 30 min before being p l a c e d i n the P l e x i g l a s chamber, r a t s i n each of the groups r e c e i v e d a .1 mg/kg, 3 mg/kg, or 6 mg/kg i n t r a p e r i t o n e a l i n j e c t i o n of e i t h e r diazepam, c h l o r d i a z e p o x i d e , or p e n t o b a r b i t a l , or .5 ml/kg of one of the r e s p e c t i v e v e h i c l e s ( i . e . , 0 dose groups). A f t e r each r a t r e c e i v e d the 1-mA shock from the prod, i t s behaviour was observed f o r 15 min 48 Results and Discussion It can be seen from F i g . 3 that conditioned defensive burying was suppressed by a l l three a n x i o l y t i c s , e s p e c i a l l y at the higher dose l e v e l s . Whereas, a l l 30 of the vehicle-injected (0 dose) control rats displayed some burying behaviour, burying behaviour was observed in 9, 24, and 20 of the subjects (n=30) treated with diazepam, chlordiazepoxide, and pentobarbital, respectively. Three sets of a p r i o r i comparisons (Dunnetts' p =.05) between the duration of burying in animals in each of the three control groups and animals in each of the respective experimental groups confirmed that drug-injected animals spent s i g n i f i c a n t l y less time burying. A similar analysis of the height data corroborated the analysis of the duration data (Fig. 4). Thus, the results of Experiment 2 confirmed the suppressive effect of diazepam found in Experiment 1 and extended the generality of these findings to two additional a n x i o l y t i c agents. In order to assess the r e l a t i v e potency of diazepam, chlordiazepoxide, and pentobarbital> the suppressive e f f e c t of each agent was compared at the 1, 3, and 6 mg/kg dose leve l s using a 3 by 4 analysis of variance followed by a p o s t e r i o r i comparisons. A drug is considered more potent than another i f i t produces a s i g n i f i c a n t l y greater effect at a given dose (Julien, 1975). The 3 by 4 analysis of the duration of burying resulted in a s i g n i f i c a n t main effect of the type of drug {F(2,108) = 6.15, p<.003}, a s i g n i f i c a n t main e f f e c t of dose {F(3,108) = 16.95, p<.0001} and a s i g n i f i c a n t dose-by-drug interaction (F(6,108) = 49 F i g u r e 3. Mean d u r a t i o n ( S.E.M. ) of burying at each of four doses f o r diazepam-injected ( d i a g o n a l l y - s t r i p e d b a r s ) , c h l o r d i a z e p o x i d e - i n j e c t e d (black b a r s ) , or p e n t o b a r b i t a l -i n j e c t e d r a t s (open bars) i n Experiment 2. 50 D O S E (mg/kg) 51 2.22, p<.05}. Subsequent Duncan's m u l t i p l e range t e s t s (p=.05) showed t h a t diazepam produced a s i g n i f i c a n t l y g r e a t e r s u p p r e s s i o n of c o n d i t i o n e d b u r y i n g b e h a v i o u r than e i t h e r c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l a t 1 mg/kg, and a s i g n i f i c a n t l y g r e a t e r s u p p r e s s i o n than c h l o r d i a z e p o x i d e a t 3 mg/kg. C h l o r d i a z e p o x i d e and p e n t o b a r b i t a l d i d not d i f f e r from each o t h e r a t e i t h e r the 1 mg/kg or the 3 mg/kg dose l e v e l s . At 6 mg/kg, t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the t h r e e a n x i o l y t i c s i n the amount they s u ppressed c o n d i t i o n e d b u r y i n g b e h a v i o u r . The 3 by 4 a n a l y s i s of the h e i g h t d a t a c o n f i r m e d the r e s u l t s of the a n a l y s i s of the d u r a t i o n d a t a . T h i s a n a l y s i s r e s u l t e d i n a s i g n i f i c a n t main e f f e c t of the type of drug {F(2,108) = 7.65, p<.0009}, a s i g n i f i c a n t main e f f e c t of dose {F(3,108) = 13.93, p<.0001}, and a s i g n i f i c a n t dose-by-drug i n t e r a c t i o n {F(6,108) = 2.83, p<.01}. The Duncan's m u l t i p l e range t e s t s (p=.05) of the mean h e i g h t s of bedding m a t e r i a l r e v e a l e d a p a t t e r n of s i g n i f i c a n t d i f f e r e n c e s t h a t was almost i d e n t i c a l t o the d i f f e r e n c e s between the d u r a t i o n s c o r e s . At 6 mg/kg, t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between the s u p p r e s s i v e e f f e c t s of each of the t h r e e d r u g s ; a t 3 mg/kg, diazepam was s i g n i f i c a n t l y more p o t e n t than e i t h e r c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l , and a t 1 mg/kg diazepam was a g a i n more p o t e n t than c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l . Thus, of the t h r e e a n x i o l y t i c s , diazepam was the most e f f e c t i v e i n s u p p r e s s i n g c o n d i t i o n e d d e f e n s i v e b u r y i n g ; whereas, the l a t t e r two drugs d i d not d i f f e r s i g n i f i c a n t l y . In c l i n i c a l s e t t i n g s , diazepam has been shown t o be 5 t o 10 52 F i g u r e 4. Mean height ( S.E.M. ) of m a t e r i a l accumulated at each of four doses f o r diazepam-injected ( d i a g o n a l l y - s t r i p e d b a r s ) , c h l o r d i a z e p o x i d e - i n j e c t e d (black b a r s ) , or p e n t o b a r b i t a l -i n j e c t e d r a t s (open bars) i n Experiment 2. 0 DIAZEPAM • CHLORDIAZEPOXIDE D PENTOBARBITAL 0 1 3 6 D 0 S E ( m g / k g ) 54 tim e s more p o t e n t than e i t h e r c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l ( c f . Cook & D a v i d s o n , 1973). A l t h o u g h the c l i n i c a l p o t e n c i e s of c h l o r d i a z e p o x i d e and p e n t o b a r b i t a l do not d i f f e r g r e a t l y , the potency of c h l o r d i a z e p o x i d e i s g e n e r a l l y thought t o be g r e a t e r than t h a t of p e n t o b a r b i t a l ( D e S i l v e r i o , R i c k l e s , Raab,& Jameson, 1969; Goodman & Gilman, 1975; Gore & McComiskey, 1961; Jenner & K e r r y , 1967; K i n g s t o n e , V e l l e n e u v e , & K o s s a t z , 1969; Lader & Wing, 1961;" L o r r , M cNair, W e i n s t e i n , Michaux, & R a s k i n , 1961; Wheatley, 1968). The f a i l u r e of the p r e s e n t study t o d i f f e r e n t i a t e the potency of c h l o r d i a z e p o x i d e from t h a t of p e n t o b a r b i t a l i s d i f f i c u l t t o i n t e r p r e t . I t i s p o s s i b l e t h a t the s l i g h t d i f f e r e n c e between the potency of p e n t o b a r b i t a l and c h l o r d i a z e p o x i d e i s o n l y d e t e c t a b l e when the s e d a t i v e - h y p n o t i c p r o p e r t i e s of the s e drugs a r e more prominent; i . e . , beyond the range of doses used i n the p r e s e n t s t u d y . However, the s i g n i f i c a n t d i f f e r e n c e s between the e f f e c t s of diazepam and c h l o r d i a z e p o x i d e observed i n the p r e s e n t s t u d y a t 1 mg/kg and 3 mg/kg, and between the e f f e c t s of diazepam and p e n t o b a r b i t a l a t 1 mg/kg a r e c o n s i s t e n t w i t h the r e l a t i v e c l i n i c a l e f f i c a c y of thes e d r u g s . 55 Experiment 3 The purpose of Experiment 3 was to pro v i d e p r e l i m i n a r y evidence that the c o n d i t i o n e d d e f e n s i v e burying t e s t i s d i f f e r e n t i a l l y s e n s i t i v e to a n x i o l y t i c agents; i . e . , that c o n d i t i o n e d d e f e n s i v e burying i s not a f f e c t e d i n the same way by other p s y c h o t r o p i c drugs ( c r i t e r i o n 3). If drugs not known f o r t h e i r a n x i o l y t i c a c t i v i t y a l s o produced a comparable suppression of conditoned burying, i t would be d i f f i c u l t to argue that the c o n d i t i o n e d d e f e n s i v e burying paradigm would be a u s e f u l t e s t of a n x i o l y t i c e f f e c t s . Method On day 5, 72 r a t s were randomly assi g n e d to one of s i x drug or s i x v e h i c l e c o n d i t i o n s . Rats i n the drug c o n d i t i o n s (n=6) r e c e i v e d an i n t r a p e r i t o n e a l i n j e c t i o n of e i t h e r p i c r o t o x i n i (.5 mg/kg), d-amphetamine (1 mg/kg), morphine s u l f a t e (1.5 mg/kg), chlorpromazine (4 mg/kg), or diazepam (1 mg/kg) 30 min before being p l a c e d i n the t e s t chamber; r a t s i n the p e n t y l e n e t e t r a z o l c o n d i t i o n were i n j e c t e d (20 mg/kg)l min before the t e s t . The i n t e r v a l s between i n j e c t i o n and t e s t i n g f o r each drug had been shown to produce r e l i a b l e p h a r m a c o l o g i c a l e f f e c t s at these doses in p r e v i o u s s t u d i e s ( G l i c k , 1976). Immediately a f t e r r e c e i v i n g a 1-mA shock, the d r u g - i n j e c t e d r a t s were observed f o r 15 min and t h e i r - burying behaviour was recorded. Rats i n the s i x v e h i c l e c o n d i t i o n s (n=6) were t r e a t e d i n e x a c t l y the same manner, except that i n s t e a d of drugs, these r a t s r e c e i v e d an e q u i v a l e n t volume of the a p p r o p r i a t e v e h i c l e . R e s u l t s and D i s c u s s i o n The r e s u l t s of Experiment 3 i n d i c a t e that the suppression 56 of c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm i s not a r e l i a b l e e f f e c t of a l l p s y c h o a c t i v e a g e n t s . P l a n n e d o r t h o g o n a l comparisons between the d u r a t i o n of b u r y i n g by the d r u g - i n j e c t e d and the v e h i c l e - i n j e c t e d r a t s showed t h a t t h e r e was no s i g n i f i c a n t s u p p r e s s i o n of b u r y i n g b e h a v i o u r by the CNS s t i m u l a n t s , p i c r o t o x i n {drug: M = 83.4 s e c , SD = 71.8 sec; v e h i c l e : M = 81.4 s e c , SD = 58.7 s e c ; t ( 1 0 ) = .06, p>.05}, p e n t y l e n e t e t r a z o l {drug: M = 68.9 s e c , SD = 56.3 s e c ; v e h i c l e : M = 108.7 s e c , SD = 66.0 s e c ; t ( 1 0 ) = 1.12, p>.20}, d-amphetamine {drug: M = 165.2 s e c , SD = 103.9 s e c ; v e h i c l e : M = 105.1 s e c , SD = 50.1 s e c ; t ( 1 0 ) = 1.27, p>.20}, or by the n a r c o t i c a n a l g e s i c , morphine {drug: M = 74.8 s e c , SD = 49.3 sec; v e h i c l e : M = 80.3 s e c , SD = 72.6 s e c ; t ( 1 0 ) = .15, p>.5}; whereas, the a n x i o l y t i c , diazepam {drug: M= 36.6 s e c , SD = 24.0 s e c ; v e h i c l e : M = 148.5 s e c , SD = 92.6 sec; t ( 1 0 ) = 2.86, p<.02}, and the 'major' t r a n q u i l i z e r , c h l o r p r o m a z i n e {drug: M = 19.9 s e c , SD = 30.0 sec-v e h i c l e : M = 132.0 s e c , SD = 88.2 s e c ; t ( 1 0 ) = 2.94, p<.01} both produced a s i g n i f i c a n t s u p p r e s s i o n of b u r y i n g b e h a v i o u r . O r t h o g o n a l comparisons of the h e i g h t of m a t e r i a l a ccumulated a t the p r o d c o n f i r m e d the b e h a v i o u r a l r e s u l t s . P i c r o t o x i n {drug: M = 8.4 cm, SD = 2.9 cm; v e h i c l e : M = 8.2 cm, SD = 2.5 cm; t ( 1 0 ) = .05, p>.5}, p e n t y l e n e t e t r a z o l {drug: M = 8.0 cm, SD = 2.2 cm; v e h i c l e : M = 8.2 cm, SD = 2.3 cm; t ( 1 0 ) = .19, p>.50}, d-amphetamine {drug: M = 8.5 cm, SD = 2.5 cm; v e h i c l e : M = 9.2 cm, SD = 1.2 cm; t ( 1 0 ) = .66, p>.5}, and morphine {drug: M = 9.0 cm, SD = 2.4 cm; v e h i c l e : M = 8.0 cm, SD = 2.2 cm; t ( 1 0 ) = .76, p>.5} d i d not produce a s i g n i f i c a n t s u p p r e s s i o n , whereas diazepam {drug: M = 6.2 cm, SD = .74 cm; 57 v e h i c l e : M = 8.6 cm, SD = 1.8 cm; t ( 1 0 ) = 3.10, p<.01} and c h l o r p r o m a z i n e {drug: M = 5.9 cm, SD = 1.5 cm; v e h i c l e : M = 8.6 cm, SD = 1.9 cm t ( 1 0 ) = 2.70, p<.02} d i d . The p r e s e n t r e s u l t s suggest t h a t the s u p p r e s s i o n of c o n d i t i o n e d d e f e n s i v e b u r y i n g i s not a r e l i a b l e e f f e c t of a l l p s y c h o a c t i v e a g e n t s ; o n l y c h l o r p r o m a z i n e and diazepam, r e s p e c t i v e l y , produced a s i g n i f i c a n t s u p p r e s s i o n of b u r y i n g . However, i n view of the s u p p r e s s i v e e f f e c t of c h l o r p r o m a z i n e , i t cannot be c o n c l u d e d t h a t b e n z o d i a z e p i n e s a r e the o n l y p s y c h o a c t i v e drugs t h a t d i s r u p t c o n d i t i o n e d d e f e n s i v e b u r y i n g . A l t h o u g h c h l o r p r o m a z i n e has been used c l i n i c a l l y t o t r e a t a c u t e a n x i e t y ( e . g . , Delay & D e n i t e r , 1952), and has e f f e c t s on a number of a n i m a l s c r e e n i n g t e s t s t h a t a r e q u a l i t a t i v e l y s i m i l a r t o t h o s e produced by s t a n d a r d a n x i o l y t i c compounds ( e . g . , L i p p a et a l . , 1979), i t i s p r i m a r i l y used t o t r e a t p sychoses (Goodman & G i l l m a n , 1975), and i t s e f f e c t s i n some a n i m a l t e s t s can be c l e a r l y d i s s o c i a t e d from th o s e of s t a n d a r d a n x i o l y t i c compounds ( e . g . , the G e l l e r ' c o n f l i c t ' t e s t ) . Thus, a more d e t a i l e d i n v e s t i g a t i o n of the s u p p r e s s i v e e f f e c t s of c h l o r p r o m a z i n e and diazepam on c o n d i t i o n e d d e f e n s i v e b u r y i n g was w a r r a n t e d . 58 Experiment 4 The r e s u l t s of a number of s t u d i e s have s u g g e s t e d t h a t n e u r o l e p t i c s such as c h l o r p r o m a z i n e can i m p a i r f e a r - m o t i v a t e d b e h a v i o u r by d i s r u p t i n g the a n i m a l s ' a b i l i t y t o p e r f o r m c o o r d i n a t e d motor responses;whereas, a n x i o l y t i c s such as diazepam appear t o modulate f e a r - m o t i v a t e d b e h a v i o u r w i t h o u t d i s r u p t i n g motor performance per se ( e . g . , B e n i n g e r , MacLennan, & P i n e l , 1980; S c h l e c h t e r & B u t c h e r , 1972; S e p i n w a l l & Cook, 1978). F u r t h e r m o r e , the motor d e f i c i t s t h a t a r e produced by moderate doses of n e u r o l e p t i c a gents a r e u s u a l l y not a f f e c t e d by i n c r e a s e s i n the s e v e r i t y of the u n c o n d i t i o n e d a v e r s i v e s t i m u l u s ; whereas, the e f f e c t s of moderate doses of a n x i o l y t i c agents may be s u b s t a n t i a l l y d i m i n i s h e d by i n c r e a s e s i n the s e v e r i t y of the u n c o n d i t i o n e d a v e r s i v e s t i m u l u s ( e . g . , S e p i n w a l l & Cook, 1978). Thus, i t seemed p o s s i b l e t h a t the e f f e c t s of diazepam and c h l o r p r o m a z i n e i n the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm c o u l d be d i s s o c i a t e d by e x p o s i n g r a t s t o shocks of d i f f e r e n t i n t e n s i t y . Experiment 4 was d e s i g n e d t o a s s e s s t h i s p o s s i b i l i t y . Method On day 5, 160 r a t s were randomly a s s i g n e d t o one of two b a s i c c o n d i t i o n s . Rats i n one c o n d i t i o n ( c h l o r p r o m a z i n e ) were i n j e c t e d w i t h one of t h r e e d i f f e r e n t doses of c h l o r p r o m a z i n e (1,2, or 3 mg/kg) or .5 ml of i t s v e h i c l e (0 mg/kg) 30 min b e f o r e r e c e i v i n g e i t h e r a 1-mA or a 10-mA shock from the p r o d . Rats i n the o t h e r c o n d i t i o n (diazepam) were t r e a t e d i n e x a c t l y the same manner, except t h a t they were i n j e c t e d w i t h 1,2, or 3 mg/kg of diazepam or .5 ml of i t s v e h i c l e . A l l r a t s i n each of 59 the 16 groups (n=10) were t e s t e d f o r 15 min. R e s u l t s and D i s c u s s i o n It can be seen from F i g . 5 and Fig . 6 that i n the low-i n t e n s i t y shock c o n d i t i o n , both diazepam and chlorpromazine produced a dramatic suppression of c o n d i t i o n e d d e f e n s i v e b u r y i n g ; whereas, i n the h i g h - i n t e n s i t y c o n d i t i o n , only chlorpromazine suppressed b u r y i n g . Thus, the suppressive e f f e c t s of chlorpromazine and diazepam on c o n d i t i o n e d d e f e n s i v e burying were d i s s o c i a t e d by v a r y i n g the i n t e n s i t y of the e l e c t r i c shock. A 3-way a n a l y s i s of v a r i a n c e of the d u r a t i o n data confirmed that the i n t e n s i t y of shock (F(l,144) = 20.84, p<.0001}, the type of drug {F(1,144) = 6.73, p<.01}, and the dose of the drug (F(3,144) = 15.91, p<.0001} each had a s i g n i f i c a n t e f f e c t on the amount of time r a t s spent burying the prod. In a d d i t i o n , there was a s i g n i f i c a n t 2-way i n t e r a c t i o n between the i n t e n s i t y of shock and the type of drug {F(l,144) = 10.76, p<.001}. None of the other i n t e r a c t i o n e f f e c t s reached the .05 l e v e l of s i g n i f i c a n c e . Subsequent Duncan's m u l t i p l e comparison t e s t s (p=.05) of the o v e r a l l e f f e c t s of drug, dose, and shock i n t e n s i t y showed that a c r o s s dose and shock the sup p r e s s i v e e f f e c t of chlorpromazine on burying behaviour was s i g n i f i c a n t l y g r e a t e r than that of diazepam, that a c r o s s drug and shock c o n d i t i o n s the 3 and 2 mg/kg dosages produced a s i g n i f i c a n t l y g r e a t e r suppression than d i d e i t h e r the 1 or 0 mg/kg dosages, and that a c r o s s dose and drug c o n d i t i o n s the 10-mA shock produced s i g n i f i c a n t l y more burying behaviour than d i d the 1-mA shock. 60 F i g u r e 5. Mean d u r a t i o n ( S.E.M. ) of burying by diazepam-i n j e c t e d r a t s shocked with 1 mA (upper l e f t panel) or 10 mA (upper r i g h t panel) and by c h l o r p r o m a z i n e - i n j e c t e d r a t s shocked with 1 mA (lower l e f t panel) or 10 mA (lower r i g h t panel) at each of four dose l e v e l s i n Experiment 4. S H O C K 61 1mA 1 OmA 0 1 2 3" d i a z e p a m (mg/kg) I n d l a z e p a m (mg/kg) x c h I orproma z i ne (mg/kg) chl orpromazi ne (mg/kg) D R U G 62 These o v e r a l l d i f f e r e n c e s were analysed i n t o t h e i r c o n s t i t u e n t p a r t s u s i n g a l l p o s s i b l e p a i r - w i s e comparisons (Duncan's p=.05). The r e s u l t s of t h i s a n a l y s i s showed that, there were no s i g n i f i c a n t d i f f e r e n c e s between mean d u r a t i o n s of burying behaviour of r a t s in the four c o n t r o l c o n d i t i o n s ; however, i n the 1-mA shock c o n d i t i o n , animals t r e a t e d with 1, 2, and 3 mg/kg of diazepam b u r i e d the prod s i g n i f i c a n t l y l e s s than d i d r a t s i n t h e i r v e h i c l e c o n t r o l group, as d i d r a t s t r e a t e d with 1, 2 and 3 mg/kg of chlorpromazine. In c o n t r a s t , r a t s i n the 10-mA c o n d i t i o n that were t r e a t e d with diazepam were not s i g n i f i c a n t l y d i f f e r e n t from t h e i r v e h i c l e - i n j e c t e d c o n t r o l group at any dose l e v e l , whereas every dose of chlorpromazine produced a s i g n i f i c a n t suppression of burying behaviour. Thus, the s i g n i f i c a n t drug-by-shock i n t e r a c t i o n was due to the f a c t that chlorpromazine but not diazepam d i s r u p t e d c o n d i t i o n e d d e f e n s i v e burying at hig h shock i n t e n s i t i e s . T h i s g e n e r a l i z a t i o n was a l s o supported by p a i r - w i s e comparisons (Duncan's, p=.05) between the means of the d r u g - i n j e c t e d experimental groups at each of the three drug-dose l e v e l s . As expected, the diazepam t r e a t e d r a t s in the 1-mA c o n d i t i o n b u r i e d s i g n i f i c a n t l y l e s s than d i d diazepam t r e a t e d r a t s i n the 10-mA c o n d i t i o n at each of the three dose l e v e l s ; whereas, there were no s i g n i f i c a n t d i f f e r e n c e s i n the burying behaviour of chlorpromazine t r e a t e d r a t s i n the 1-mA and 10-mA c o n d i t i o n s . The same p a t t e r n of r e s u l t s was obt a i n e d when comparisons were made between the d u r a t i o n scores i n both drug c o n d i t i o n s . Although there were no s i g n i f i c a n t d i f f e r e n c e s between the diazepam-treated r a t s and the c h l o r p r o m a z i n e - t r e a t e d r a t s i n the 63 F i g u r e 6. Mean height ( S.E.M. ) of m a t e r i a l accumulated by d i a z e p a m - i n j e c t e d r a t s shocked with 1 mA (upper l e f t panel) or 10 mA (upper r i g h t panel) and by c h l o r p r o m a z i n e - i n j e c t e d r a t s shocked with 1 mA (lower l e f t panel) or 10 mA (lower r i g h t panel) at each of four dose l e v e l s i n Experiment 4. S H O C K 64 1 m A 10 mA 54 I i i 0 1 2 3 d i a z e pa m (mg/kg) l I "0 r 5 5 d i a z e p a m (mg/kg) I x T X ch lo rp romaz ine (mg /kg ) c h l o r p r o m a z i ne(mg/kg) D R U G 65 suppression of r a t s ' burying behaviour at 1-mA, at 10-mA, chlorpromazine produced a s i g n i f i c a n t suppression compared to diazepam at every dose except 6 mg/kg. These r e s u l t s c l e a r l y show t h a t the suppressive e f f e c t s of chlorpromazine and diazepam on c o n d i t i o n e d d e f e n s i v e burying can be d i s s o c i a t e d by v a r y i n g the i n t e n s i t y of the e l e c t r i c shock. A s i m i l a r a n a l y s i s of the height data confirmed the a n a l y s i s of the d u r a t i o n data. The 3-way a n a l y s i s of v a r i a n c e of the height data showed that there was a s i g n i f i c a n t e f f e c t of drug {F(l,144) = 8.59, p<.004}, of dose l e v e l {F(3,144) = 15.13, p<.0001} of shock (F(l,144) = 11.91, p<.0009}, and a s i g n i f i c a n t drug by shock i n t e r a c t i o n {F(l,144) = 8.83, p<.004}. Subsequent Duncan's m u l t i p l e comparison t e s t s (p=.05) showed a p a t t e r n of s i g n i f i c a n t d i f f e r e n c e s between the mean h e i g h t • s c o r e s that was i d e n t i c a l i n every respect to the p a t t e r n of d i f f e r e n c e s i n the d u r a t i o n s c o r e s , with only one e x c e p t i o n ; the height of m a t e r i a l accumulated by r a t s i n j e c t e d with 1 mg/kg of chlorpromazine and shocked at 1-mA was not s i g n i f i c a n t l y d i f f e r e n t from the height accumulated by t h e i r v e h i c l e - i n j e c t e d c o n t r o l s . Taken together with the f i n d i n g s of Experiment 3, the present r e s u l t s suggest that the c o n d i t i o n e d d e f e n s i v e burying t e s t i s s e l e c t i v e l y s e n s i t i v e to a n x i o l y t i c agents ( c r i t e r i o n 3). In a d d i t i o n to p r o v i d i n g evidence f o r the s e l e c t i v i t y of the c o n d i t i o n e d d e f e n s i v e burying t e s t , these r e s u l t s i n d i c a t e t h at chlorpromazine and diazepam may be producing t h e i r suppressant e f f e c t s by a c t i n g on d i f f e r e n t mechanisms. Because chlorpromazine has been shown to have powerful d i s r u p t i v e e f f e c t s on motor behaviour at the doses used i n the present 66 study ( S c h l e c h t e r & B u t c h e r , 1972), i t i s p o s s i b l e t h a t such motor impairment was r e s p o n s i b l e f o r the d i s r u p t i o n of b u r y i n g b e h a v i o u r i n the r a t s i n j e c t e d w i t h c h l o r p r o m a z i n e . On the o t h e r hand, a n x i o l y t i c s such as diazepam do not g e n e r a l l y s u p p r e s s motor b e h a v i o u r except a t doses much h i g h e r than those used i n the p r e s e n t study ( c f . L i p p a , Nash, & G r e e n b l a t t , 1979), which i s c o n s i s t e n t w i t h the f a c t t h a t the diazepam-t r e a t e d r a t s i n the h i g h - i n t e n s i t y shock c o n d i t i o n b u r i e d the prod i n a manner t h a t was i n d i s t i n g u i s h a b l e from t h a t of v e h i c l e - i n j e c t e d c o n t r o l s . Thus i t seems more l i k e l y t h a t diazepam i n h i b i t e d b u r y i n g b e h a v i o u r by i n t e r f e r i n g w i t h p r o c e s s e s that may u n d e r l i e the r a t ' s r e a c t i o n s t o a v e r s i v e s t i m u l i , r a t h e r than by d i r e c t l y d i s r u p t i n g motor b e h a v i o u r . The p r e s e n t r e s u l t s suggest t h a t the e f f e c t s of c h l o r p r o m a z i n e on c o n d i t i o n e d d e f e n s i v e b u r y i n g can be r e a d i l y d i s s o c i a t e d from those of diazepam by v a r y i n g the i n t e n s i t y of the e l e c t r i c shock. However, whether or not the e f f e c t s of o t h e r n e u r o l e p t i c s can be s i m i l a r l y d i s s o c i a t e d from those of o t h e r a n x i o l y t i c s i n t h i s p a r t i c u l a r paradigm remains t o be d e t e r m i n e d . The r e s u l t s of the p r e s e n t i n v e s t i g a t i o n suggest t h a t t h i s s t r a t e g y c o u l d prove u s e f u l i n d e a l i n g w i t h comparable c a s e s . 67 P a r t I I , Mechanisms of A c t i o n E v i d e n c e was p r o v i d e d by the p r e v i o u s f o u r e x p e r i m e n t s t h a t the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm i s s e n s i t i v e i n a dose-dependent manner t o the e f f e c t s of a n x i o l y t i c a g ents ( c r i t e r i o n 1; Ex p e r i m e n t s 1,2,3, and 4 ) ; t h a t the r e l a t i v e p o t e n c i e s of a number of a n x i o l y t i c agents i n the b u r y i n g paradigm compare r e a s o n a b l y w e l l w i t h t h e i r r e l a t i v e p o t e n c i e s i n t he t r e a t m e n t of human a n x i e t y ( c r i t e r i o n 2; Experiment 2 ) ; and t h a t the c o n d i t i o n e d d e f e n s i v e b u r y i n g t e s t i s d i f f e r e n t i a l l y s e n s i t i v e t o a n x i o l y t i c agents ( c r i t e r i o n 3; Exper i m e n t s 3 and 4 ) . Thus, a l t h o u g h f u r t h e r r e s e a r c h i s c l e a r l y r e q u i r e d b e f o r e any u l t i m a t e judgments can be made, the da t a from the f i r s t f o u r e x p e r i m e n t s suggest t h a t the c o n d i t i o n e d d e f e n s i v e b u r y i n g t e s t may prove t o be a b l e t o meet a l l t h r e e of the major c r i t e r i a of a v a l i d p r e d i c t i v e t e s t of a n x i o l y t i c a g e n t s . In the second p a r t of t h i s t h e s i s , i n o r d e r t o p r o v i d e a f u r t h e r i l l u s t r a t i o n of the p o t e n t i a l of c o n d i t i o n e d d e f e n s i v e b u r y i n g i n the study of a n x i o l y t i c d r u g s , the b u r y i n g paradigm was used t o i n v e s t i g a t e the mechanisms by which diazepam might be p r o d u c i n g i t s a n x i o l y t i c e f f e c t s . Two h y p o t h e s i z e d mechanisms of a c t i o n s e r v e d as the f o c u s f o r t h e s e s t u d i e s . The f i r s t h y p o t h e s i s i s t h a t diazepam produces i t s e f f e c t s on shock-m o t i v a t e d b e h a v i o u r by r e d u c i n g the a n i m a l s ' r e s p o n s i v e n e s s t o p a i n ( i . e . , by p r o d u c i n g a n a l g e s i a ) . A n i m a l s i n j e c t e d w i t h diazepam and s u b s e q u e n t l y shocked from a prod may bury l e s s than v e h i c l e - i n j e c t e d c o n t r o l s because they may be l e s s r e s p o n s i v e t o p a i n f u l e l e c t r i c shock. An a n a l g e s i c mechanism might a l s o 68 e x p l a i n why a n i m a l s i n j e c t e d w i t h diazepam respond more f o r food d e s p i t e r e c e i v i n g c o n t i n g e n t s h o c k s . Thus, a s i m p l e a n a l g e s i c mechanism might e x p l a i n b oth d e c r e a s e s and i n c r e a s e s i n r e s p o n d i n g t h a t r e s u l t from a v e r s i v e s t i m u l a t i o n . The second h y p o t h e s i s c o n c e r n s the n a t u r e of the n e u r a l mechanism t h a t mediates the e f f e c t s of diazepam on b e h a v i o u r . There i s now a f a i r l y l a r g e body of e v i d e n c e t h a t s u g g e s t s t h a t b e n z o d i a z e p i n e s produce some of t h e i r p h a r m a c o l o g i c a l e f f e c t s by an enhancement of gama a m i n o b u t y r i c a c i d , (GABA) a p u t a t i v e i n h i b i t o r y n e u r o t r a n s m i t t e r , e i t h e r by 1) a c t i v a t i n g GABA r e c e p t o r d i r e c t l y , 2) s t i m u l a t i n g the r e l e a s e of GABA from p r e s y n a p t i c s i t e s , 3) i n h i b i t i n g the re u p t a k e or c a t a b o l i s m of GABA, or 4) f a c i l i t a t i n g the p o s t s y n a p t i c response t o GABA (Tallman e t a l . , 1980). A l t h o u g h the e x a c t l o c u s of the GABA-b e n z o d i a z e p i n e i n t e r a c t i o n i s unknown, p o s t - s y n a p t i c r e c e p t o r s i t e s a r e the most l i k e l y l o c a t i o n s f o r t h i s i n t e r a c t i o n . Thus, s u b s t a n c e s t h a t s p e c i f i c a l l y b l o c k GABA e r g i c r e c e p t o r s i t e s s h o u l d a l t e r the p h a r m a c o l o g i c a l a c t i o n s of b e n z o d i a z e p i n e s . 69 Experiment 5_ Because most r e l i a b l e a n i m a l models of a n x i e t y have i n v o l v e d p a i n f u l s t i m u l i ( e . g . , e l e c t r i c s h o c k ) , i t has o f t e n been argued t h a t a n x i o l y t i c s may produce t h e i r e f f e c t s s i m p l y by r e d u c i n g the a n i m a l s ' s e n s i t i v i t y t o p a i n . I f a n x i o l y t i c s do produce t h e i r e f f e c t s e x c l u s i v e l y t h rough an a n a l g e s i c mechanism, then they s h o u l d have no e f f e c t on an ' a n x i o l y t i c ' t e s t response t h a t i s e l i c i t e d i n the absence of p a i n f u l s t i m u l a t i o n . In o r d e r t o t e s t t h i s p r e d i c t i o n , a n i m a l s i n the p r e s e n t experiment were i n j e c t e d w i t h one of f o u r doses of diazepam and then exposed t o an unset mousetrap, a n o v e l o b j e c t t h a t has been shown t o e l i c i t d e f e n s i v e b u r y i n g i n the absence of p a i n f u l s t i m u l i ( T e r l e c k i e t a l . , 1979). Method On day 5, a f t e r 4 c o n s e c u t i v e days of h a b i t u a t i o n , 80 n a i v e r a t s were randomly a s s i g n e d t o one of f o u r diazepam c o n d i t i o n s (n=40) or one of f o u r v e h i c l e c o n d i t i o n s (n=40). R a t s i n each diazepam c o n d i t i o n (n=10) r e c e i v e d a .1, .5, 1, or 2 mg/kg i n t r a p e r i t o n e a l i n j e c t i o n of diazepam 30 min b e f o r e b e i n g i n d i v i d u a l l y p l a c e d i n the P l e x i g l a s t e s t chamber. F i x e d t o the w a l l a t one end of the t e s t chamber was an u n s e t , wooden mousetrap (see G e n e r a l Methods). The t e s t s e s s i o n began i m m e d i a t e l y a f t e r the r a t s were p l a c e d i n the chamber, and the amount of time they spent p u s h i n g or s p r a y i n g the bedding m a t e r i a l toward the t r a p was r e c o r d e d i n the manner p r e v i o u s l y d e s c r i b e d . A f t e r each t e s t , the h e i g h t of m a t e r i a l a t the t r a p was measured. Rats i n each of the f o u r v e h i c l e c o n d i t i o n s (n=10) were t r e a t e d i n the same manner, except t h a t they 70 r e c e i v e d a volume of the v e h i c l e t h a t was e q u i v a l e n t t o the volume of f l u i d r e c e i v e d by r a t s i n each of the f o u r drug c o n d i t i o n s . R e s u l t s and D i s c u s s i o n F i g . 7 and F i g . 8 show t h a t b u r y i n g t h a t i s e l i c i t e d i n the absence of p a i n f u l s t i m u l a t i o n can be s u p p r e s s e d by diazepam. P l a n n e d o r t h o g o n a l comparisons c o n f i r m e d t h a t the amount of b u r y i n g b e h a v i o u r d i s p l a y e d by d i a z e p a m - t r e a t e d r a t s was s i g n i f i c a n t l y lower than the amount d i s p l a y e d by v e h i c l e -t r e a t e d r a t s a t e v e r y dose except .1 mg/kg {.1 mg/kg, t ( 1 8 ) = 1.01, p>.5; .5 mg/kg, t ( 1 8 ) = 2.77, p<.01; 1 mg/kg, t ( 1 8 ) = 2.48, p<.02; 2 mg/kg t ( 1 8 ) = 3.21, p<.004}. S i m i l a r l y , the h e i g h t of bedding m a t e r i a l a c c u mulated over the t r a p by d i a z e p a m - i n j e c t e d r a t s was s i g n i f i c a n t l y l e s s than t h a t a c c u m ulated by v e h i c l e - i n j e c t e d r a t s , a t a l l but the .1 mg/kg dose {.1 mg/kg, t ( 1 8 ) = .68, p>.5; .5 mg/kg, t ( 1 8 ) = 3.17, p<.005; 1 mg/kg, t ( 1 8 ) = 2.62, p<.02; 2 mg/kg, t ( 1 8 ) = 3.53, p<.002}. In o r d e r t o show t h a t the s u p p r e s s i v e e f f e c t of diazepam on u n c o n d i t i o n e d d e f e n s i v e b u r y i n g was dose dependent, the d u r a t i o n and h e i g h t d a t a were s u b j e c t e d t o s e p a r a t e 2-way a n a l y s e s of v a r i a n c e , f o l l o w e d by a p o s t e r i o r i p a i r - w i s e comparisons (Duncan's, p=.05). The 2 by 4 a n a l y s i s of the d u r a t i o n d a t a r e v e a l e d a s i g n i f i c a n t main e f f e c t of drug { F ( l , 7 2 ) = 11.87, p<.001}, and a s i g n i f i c a n t main e f f e c t of dose (F(3,72) = 5.22, p<.003}. The i n t e r a c t i o n of drug and dose d i d not r e a c h s i g n i f i c a n c e {F(3,72) = 1.75, p>.10}. Subsequent Duncan's p a i r -wise c o m p a r i s o ns of the means of each group showed t h a t 71 F i g u r e 7. Mean d u r a t i o n ( S.E.M. ) of burying by diazepam-i n j e c t e d ( d i a g o n a l l y - s t r i p e d bars) and v e h i c l e - i n j e c t e d r a t s (open bars) at each of four doses i n Experiment 5. 72 o C 5 o a 60 AO 20 • DIAZEPAM • VEHICLE V DOSE (mg/kg) 73 the r e d u c t i o n i n the d u r a t i o n of b u r y i n g produced by .5, 1, and 2 mg/kg of diazepam was s t a t i s t i c a l l y homogeneous; and each of these c o n d i t i o n s produced a s i g n i f i c a n t l y g r e a t e r s u p p r e s s i o n than d i d the .1 mg/kg dose of diazepam. T h i s p a t t e r n of s i g n i f i c a n t d i f f e r e n c e s was mimicked e x a c t l y by the r e s u l t s of the a n a l y s i s of the h e i g h t d a t a . The 2 by 4 a n a l y s i s of v a r i a n c e of t h e h e i g h t d a t a r e v e a l e d s i g n i f i c a n t main e f f e c t s of drug { F ( l , 7 2 ) = 18.67, p<.0001}, and of dose (F(3,72) = 4.94, p<.004}, but no s i g n i f i c a n t drug-by-dose i n t e r a c t i o n {F(3,72) = .82, p>.4}. The a p o s t e r i o r i p a i r -wise comparison t e s t s (Duncan's, p=.05) showed t h a t the mean h e i g h t s of m a t e r i a l a c c u m u l a t e d by r a t s i n t h e .5, 1, and 2 mg/kg c o n d i t i o n s were s i g n i f i c a n t l y l e s s than the h e i g h t a ccumulated by r a t s i n the .1 mg/kg c o n d i t i o n , but were not s i g n i f i c a n t l y d i f f e r e n t from each o t h e r . Thus, b o t h measures of u n c o n d i t i o n e d d e f e n s i v e b u r y i n g r e f l e c t e d the p r o g r e s s i v e s u p p r e s s i v e e f f e c t s of i n c r e a s i n g the doses of diazepam. Because the s u p p r e s s i v e e f f e c t of diazepam on d e f e n s i v e b u r y i n g i s not r e s t r i c t e d t o s i t u a t i o n s t h a t i n v o l v e p a i n f u l s t i m u l i , i t i s c l e a r l y u n r e a s o n a b l e t o assume t h a t the e f f e c t s of . a n x i o l y t i c s on d e f e n s i v e b u r y i n g a r e mediated by a n a l g e s i c e f f e c t s , u n l e s s one i s w i l l i n g t o assume t h a t the mechanisms m e d i a t i n g the e f f e c t s of diazepam i n the p r e s e n t experiment a r e d i f f e r e n t from those t h a t mediate i t s e f f e c t s on p a i n - m o t i v a t e d b e h a v i o u r . There a r e t h r e e o t h e r o b s e r v a t i o n s t h a t support the view t h a t a n a l g e s i c e f f e c t s a r e not r e s p o n s i b l e f o r the e f f e c t s of a n x i o l y t i c s on t e s t r e s p o n s e s . F i r s t , i n Experiment 3, morphine had no s i g n i f i c a n t e f f e c t on the amount of b u r y i n g 74 F i g u r e 8. Mean h e i g h t ( S.E.M. ) of m a t e r i a l accumulated by d i a z e p a m - i n j e c t e d ( d i a g o n a l l y - s t r i p e d b a r s ) and v e h i c l e - i n j e c t e d r a t s (open b a r s ) a t each of f o u r doses i n Experiment 5. mean height of material (cm) 76 d i s p l a y e d by a n i m a l s shocked from a p r o d , and second, morphine i s a l s o w i t h o u t e f f e c t i n o t h e r a n i m a l t e s t s of a n x i o l y t i c e f f e c t s . T h i r d , i n most c a s e s , a n x i o l y t i c s produce e f f e c t s i n s t a n d a r d a n a l g e s i c t e s t s ( e . g . , the t a i l f l i c k t e s t ) o n l y a t doses t h a t i m p a i r motor performance ( S e p i n w a l l & Cook, 1978). 77 Experiment 6 I t has been hypothesized that benzodiazepines such as diazepam may exert t h e i r e f f e c t s by i n t e r a c t i n g with GABA ( c f . Sepinwall & Cook, 1978). A number of p r e l i m i n a r y p h a r m a c o l o g i c a l o b s e r v a t i o n s support t h i s h ypothesized r e l a t i o n s h i p . For example, substances such as p i c r o t o x i n that antagonize the f u n c t i o n i n g of GABA e r g i c neurons produce b e h a v i o u r a l c o n v u l s i o n s that can be blocked by GABA f a c i l i t a t o r s such as aminooxyacetic a c i d (AOAA). Because the a n t i c o n v u l s a n t a c t i o n of substances such as AOAA i s a l s o shared by benzodiazepines, i t has been argued that the a n t i c o n v u l s a n t a c t i o n of benzodiazepines may be due to an enhancement of GABA e r g i c f u n c t i o n (Haefely, K u l c s a r , Mohler, P i e r i , Pole, & Sc h a f f n e r , 1975). In a d d i t i o n , n e u r o p h y s i o l o g i c a l data have i n d i c a t e d t hat benzodiazepines enhance p r e s y n a p t i c i n h i b i t i o n i n the s p i n a l c o r d , a f u n c t i o n that i s presumably mediated by GABA ( S c h l o s s e r , 1971; Schmidt, V o g e l , & Zimmerman, 1967). Benzodiazepines a l s o mimic the a c t i o n of GABA i n that they produce decreased l e v e l s of c y c l i c guanosine monophosphate (cGMP) i n the cerebellum (Costa, G u i d o t t i , & Mao, 1976). Moreover, most of these GABA mimicking a c t i o n s of benzodiazepines can be s e l e c t i v e l y r e v e r s e d by GABA rec e p t o r blockade. The evidence r e l a t i n g an enhancement of GABA e r g i c f u n c t i o n to other a c t i o n s of benzodiazepines, such as t h e i r a n t i a n x i e t y e f f e c t s , i s f a r l e s s c l e a r . Attempts to f a c i l i t a t e the a n t i c o n f l i c t e f f e c t of diazepam by a d m i n i s t e r i n g the GABA ag o n i s t , AOAA have been l a r g e l y u n s u c c e s s f u l , and attempts to 78 i n h i b i t diazepam's a n t i c o n f l i c t a c t i o n by a d m i n i s t e r i n g GABA b l o c k e r s such as p i c r o t o x i n have produced c o n t r a d i c t o r y r e s u l t s ( c f . S e p i n w a l l & Cook, 1978). A l t h o u g h S t e i n , Wise, and B e l l u z z i (1975) found t h a t p i c r o t o x i n a n t a g o n i z e d the a n t i c o n f l i c t e f f e c t of diazepam, S e p i n w a l l and Cook (1978) f a i l e d t o f i n d a s i g n i f i c a n t e f f e c t of p i c r o t o x i n i n the a n t i c o n f l i c t paradigm a t doses below those t h a t i n h i b i t e d unpunished r e s p o n d i n g . Thus, a r e l a t i o n s h i p between GABA and the a n t i a n x i e t y a c t i v i t y of diazepam has not been f i r m l y e s t a b l i s h e d . I f diazepam s u p p r e s s e s d e f e n s i v e r e s p o n d i n g by f a c i l i t a t i n g GABA e r g i c n e u r a l f u n c t i o n , p i c r o t o x i n might enhance d e f e n s i v e b u r y i n g by i n h i b i t i n g GABA e r g i c n e u r a l f u n c t i o n . A l t h o u g h the r e s u l t s of Experiment 3 suggested t h a t GABA r e c e p t o r b l o c k a d e does not enhance c o n d i t i o n e d d e f e n s i v e b u r y i n g , the dose of p i c r o t o x i n a d m i n i s t e r e d i n t h i s experiment ( i . e . , .5 mg/kg) may have been i n s u f f i c i e n t t o f a c i l i t a t e b u r y i n g . T h e r e f o r e , r a t s i n t he p r e s e n t experiment were i n j e c t e d w i t h one of t h r e e d i f f e r e n t doses of p i c r o t o x i n b e f o r e b e i n g c o n d i t i o n e d . Method On day 5, 24 n a i v e r a t s were randomly a s s i g n e d t o one of fo u r c o n d i t i o n s (n=6). Rats i n the t h r e e e x p e r i m e n t a l c o n d i t i o n s were i n t r a p e r i t o n e a l l y i n j e c t e d w i t h e i t h e r .1, 1, or 2 mg/kg of p i c r o t o x i n 15 min b e f o r e b e i n g p l a c e d i n d i v i d u a l l y i n t o the P l e x i g l a s chamber. When t h e s e r a t s f i r s t c o n t a c t e d the p r o d , they r e c e i v e d a 1-mA shock, a f t e r which t h e i r b e h a v i o u r was o b s e r v e d and r e c o r d e d f o r 15 min. Rats i n the c o n t r o l c o n d i t i o n were t r e a t e d i n e x a c t l y the same manner, e x c e p t t h a t 79 they r e c e i v e d a .2-ml i n t r a p e r i t o n e a l i n j e c t i o n of the v e h i c l e ( 5 % a c a c i a gum s o l u t i o n ) . R e s u l t s and D i s c u s s i o n GABA r e c e p t o r b l o c k a d e had no c o n s i s t e n t e f f e c t on e i t h e r the d u r a t i o n of b u r y i n g or the h e i g h t of bedding m a t e r i a l a ccumulated over the p r o d , s u g g e s t i n g t h a t GABA does not p l a y a d i r e c t r o l e i n the i n h i b i t i o n of d e f e n s i v e b u r y i n g . Dunnett's p a i r - w i s e comparisons (p=.05) of the average d u r a t i o n of b u r y i n g by r a t s i n j e c t e d w i t h .1 (M=100.7 sec;SD=51.1 s e c ) , 1 (M=71.5 sec;SD=31.7 s e c ) , or 2 mg/kg (M=73.0 sec;SD=71.1 sec) of p i c r o t o x i n , and r a t s i n j e c t e d w i t h v e h i c l e (M=87.9 sec;SD=48.0 sec) showed t h a t p i c r o t o x i n had no s i g n i f i c a n t e f f e c t on the amount of time t h a t r a t s spent b u r y i n g the p r o d . S i m i l a r c omparisons (D u n n e t t ' s , p=.05) of the average h e i g h t of m a t e r i a l a ccumulated over the prod by v e h i c l e - i n j e c t e d r a t s (M=8.0 cm;SD= 1.3 cm) and p i c r o t o x i n - i n j e c t e d r a t s (.1 mg/kg, M=10.4 cm,SD= 1.5 cm; 1 mg/kg, M=8.1 cm,SD=2.6 cm; 2 mg/kg, M=8.0 cm,SD=1.9 cm) a l s o f a i l e d t o r e v e a l a s i g n i f i c a n t e f f e c t of p i c r o t o x i n . Thus, the r e s u l t s of Experiment 6 c o n f i r m and e x t e n d those of Experiment 3. P i c r o t o x i n - i n d u c e d GABA r e c e p t o r b l o c k a d e does not i n i t s e l f f a c i l i t a t e c o n d i t i o n e d d e f e n s i v e b u r y i n g . 80 Experiment 7. Although the r e s u l t s of the p r e v i o u s experiment suggested that GABA receptor blockade i s not a s u f f i c i e n t c o n d i t i o n f o r the enhancement of d e f e n s i v e b u r y i n g , i t i s p o s s i b l e that an enhancement e f f e c t might have been obscured by the high l e v e l s of b u r y i n g o r d i n a r i l y d i s p l a y e d by r a t s that r e c e i v e shock from the prod. The purpose of Experiment 7 was to extend the g e n e r a l i t y of the r e s u l t s of the p r e v i o u s experiment by examining the e f f e c t s of GABA receptor blockade on burying behaviour that i s e l i c i t e d by a novel t r a p , a stimulus that o r d i n a r i l y produces l e v e l s of burying that are c o n s i d e r a b l y below those produced by e l e c t r i c shock (e.g., T e r l e c k i et a l . , 1979). I f unconditioned t r a p burying were a l s o not f a c i l i t a t e d by. p i c r o t o x i n , i t would be more d i f f i c u l t to argue that the presumed e f f e c t s of GABA blockade were obscured by a " c e i l i n g e f f e c t " . Method The methods were the same as those used i n Experiment 6 except that i n s t e a d of being shocked from the prod, the animals were exposed to an unset mousetrap. R e s u l t s and D i s c u s s i o n The average amount of unconditioned d e f e n s i v e burying d i s p l a y e d by r a t s t r e a t e d with p i c r o t o x i n was not a p p r e c i a b l y d i f f e r e n t from the average amount d i s p l a y e d by r a t s t r e a t e d with the v e h i c l e ; at the same time, the o v e r a l l l e v e l of burying by r a t s i n t h i s experiment was c o n s i d e r a b l y below that seen i n p r e v i o u s experiments i n which r a t s were shocked from a prod ( c f . F i g . 1, 2, and 4). Thus, the f a i l u r e of p i c r o t o x i n to 81 f a c i l i t a t e d e f e n s i v e b u r y i n g cannot be e a s i l y a t t r i b u t e d t o a c e i l i n g e f f e c t . D u nnett's t e s t s (p=.05) c o n f i r m e d t h a t the mean d u r a t i o n s of b u r y i n g d i s p l a y e d by r a t s g i v e n .1 (M=39.5 sec;SD=44.2 s e c ) , 1. (M=13.1 sec;SD=16.8 s e c ) , or 2 mg/kg (M=35.9 sec;SD=59.4 sec) were not s i g n i f i c a n t l y d i f f e r e n t from the mean d u r a t i o n d i s p l a y e d by r a t s i n j e c t e d w i t h the v e h i c l e (M=29.1 sec;SD=35.3 s e c ) . S i m i l a r l y , the average h e i g h t of m a t e r i a l accumulated over the t r a p by v e h i c l e i n j e c t e d c o n t r o l s (M=6.7 cm;SD=2.5 cm) was not s i g n i f i c a n t l y d i f f e r e n t from the h e i g h t accumulated by r a t s i n each of the t h r e e p i c r o t o x i n groups (.1 mg/kg, M=6.5 cm,SD=1.9 cm; 1 mg/kg, M=6.1 cm,SD=1.3 cm; 2 mg/kg, M=6.8 cm,SD=3.0 cm). Thus, GABA r e c e p t o r b l o c k a d e by i t s e l f does not appear t o enhance c o n d i t i o n e d or u n c o n d i t i o n e d d e f e n s i v e b u r y i n g . 82 Experiment 8 Although Experiments 6 and 7 suggested that GABA receptor blockade does not a f f e c t d e f e n s i v e burying, they d i d not d i r e c t l y e x p l o r e the p o s s i b i l i t y that the e f f e c t s of diazepam on de f e n s i v e burying might be mediated by GABA e r g i c a c t i v i t y . The purpose of Experiment 8 was to pro v i d e support f o r the view that GABA e r g i c n e u r a l mechanisms are i n v o l v e d i n the a n t i a n x i e t y a c t i o n s of benzodiazepines by showing that p i c r o t o x i n can reverse the diazepam-induced suppression of c o n d i t i o n e d d e f e n s i v e b u r y i n g . Method On Day 5, the 40 naive r a t s were randomly a s s i g n e d to one of four c o n d i t i o n s (n=10). Rats r e c e i v e d an i n t r a p e r i t o n e a l i n j e c t i o n of e i t h e r p i c r o t o x i n (1 mg/kg), diazepam (1 mg/kg), p i c r o t o x i n p l u s diazepam (1 mg/kg; 1 mg/kg), or the v e h i c l e (1 ml/kg) before being p l a c e d i n t o the P l e x i g l a s chamber. A l l i n j e c t i o n s of diazepam or v e h i c l e were a d m i n i s t e r e d 30 min before the c o n d i t i o n i n g s e s s i o n ; whereas, p i c r o t o x i n was always i n j e c t e d 15 min before the s e s s i o n . A l l r a t s were shocked from the prod (1-mA) and t h e i r behaviour was observed f o r 15 min. R e s u l t s and D i s c u s s i o n F i g . 9 and F i g . 10 show that GABA re c e p t o r blockade by p i c r o t o x i n r e v e r s e d the usual suppression of c o n d i t i o n e d burying produced by diazepam, but had no s u b s t a n t i a l e f f e c t on burying by i t s e l f . A 1-way a n a l y s i s of v a r i a n c e of the d u r a t i o n data r e v e a l e d a s i g n i f i c a n t main e f f e c t of drug {F(3,36)=3.93, p< .01}. Subsequent p a i r - w i s e comparisons (Duncan's p=.05) confirmed that the r a t s i n j e c t e d with diazepam alone spent 83 F i g u r e 9. Mean d u r a t i o n ( S.E.M. ) of burying by r a t s i n the v e h i c l e (V), diazepam (DZ), p i c r o t o x i n (PX), or diazepam plus p i c r o t o x i n (DZ + PX) c o n d i t i o n s of Experiment 8. 84 O CD C • MM J Q O C o o 13 c o o £ V DZ PX DZ PX 85 s i g n i f i c a n t l y l e s s time b u r y i n g than d i d the a n i m a l s i n each of the o t h e r t h r e e groups. There were no s i g n i f i c a n t d i f f e r e n c e s i n the d u r a t i o n of b u r y i n g d i s p l a y e d by r a t s i n t h e s e l a t t e r t h r e e g r o u p s . A s i m i l a r a n a l y s i s of the h e i g h t d a t a r e v e a l e d a s i g n i f i c a n t main e f f e c t of drug {F(3,36)=6.11,p<.002}. Duncan's p a i r - w i s e comparisons (p=.05) showed t h a t the average h e i g h t of m a t e r i a l accumulated over the p r o d by the d i a z e p a m - i n j e c t e d r a t s was s i g n i f i c a n t l y l e s s than the h e i g h t a c c umulated by r a t s i n j e c t e d w i t h the v e h i c l e , p i c r o t o x i n , or p i c r o t o x i n p l u s diazepam, whereas the h e i g h t s of m a t e r i a l accumulated by the l a t t e r t h r e e groups were not s i g n i f i c a n t l y d i f f e r e n t from each o t h e r . These d a t a c l e a r l y show t h a t the s u p p r e s s i o n of c o n d i t i o n e d d e f e n s i v e b u r y i n g produced by diazepam can be r e v e r s e d by p i c r o t o x i n , thus s u g g e s t i n g t h a t a GABA e r g i c n e u r a l system i s i n v o l v e d i n m e d i a t i n g the a n t i a n x i e t y a c t i o n s of b e n z o d i a z e p i n e s . 86 F i g u r e 10. Mean height ( S.E.M. ) of m a t e r i a l accumulated by r a t s i n the v e h i c l e (V), diazepam (DZ), p i c r o t o x i n (PX), or diazepam p l u s p i c r o t o x i n (DZ + PX) c o n d i t i o n s of Experiment 8. 87 o o £ O x : c o o E 8 v T DZ PX J—, I I DZ + PX 88 GENERAL DISCUSSION The g e n e r a l d i s c u s s i o n i s c o m p r i s e d of two p a r t s . The f i r s t i s a summary and d i s c u s s i o n of the e v i d e n c e t h a t the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm can f u n c t i o n as an e f f i c i e n t b e h a v i o u r a l assay f o r the i d e n t i f i c a t i o n of a n x i o l y t i c compounds. In the second, the a p p l i c a t i o n of the c o n d i t i o n e d d e f e n s i v e b u r y i n g model t o the study of the mechanisms of a c t i o n of a n x i o l y t i c compounds i s d i s c u s s e d . I . C o n d i t i o n e d d e f e n s i v e b u r y i n g and the i d e n t i f i c a t i o n of  a n x i o l y t i c compounds. The purpose of the f i r s t f o u r e x p e r i m e n t s c o m p r i s i n g t h i s t h e s i s was t o p r o v i d e some i n i t i a l e v i d e n c e r e g a r d i n g the s u i t a b i l i t y of the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm as a t e s t of a n x i o l y t i c drug e f f e c t s . The degree t o which the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm meets each of the t h r e e major c r i t e r i a of a p r e d i c t i v e a n i m a l model of a n x i o l y t i c compounds; i . e . , dose-dependent s e n s i t i v i t y , r e l a t i v e p o t e n c y , and s e l e c t i v i t y (see I n t r o d u c t i o n ) , was a s s e s s e d . The r e s u l t s of Experiment 1 c l e a r l y demonstrated t h a t the c o n d i t i o n e d d e f e n s i v e b u r y i n g response i s s e n s i t i v e i n a dose-dependent manner t o the e f f e c t s of a known a n x i o l y t i c agent. Every dose of diazepam over .1 mg/kg produced a s i g n i f i c a n t s u p p r e s s i o n of c o n d i t i o n e d b u r y i n g , and the magnitude of t h i s s u p p r e s s i v e e f f e c t i n c r e a s e d s i g n i f i c a n t l y as the dose of diazepam was i n c r e a s e d , w i t h o u t r e n d e r i n g the a n i m a l s a t a x i c or somnolent. The f a c t t h a t most of the r a t s i n j e c t e d w i t h diazepam s p r a y e d a t l e a s t some b u r y i n g m a t e r i a l toward the p r o d seemed t o r u l e out the p o s s i b i l i t y t h a t the mean d i f f e r e n c e s i n 89 c o n d i t i o n e d burying observed between the experimental and c o n t r o l animals occurred because d r u g - t r e a t e d s u b j e c t s c o u l d not make the burying response, or that they d i d not remember that the prod had been the source of an a v e r s i v e s t i m u l u s . I t i s more l i k e l y t h a t the burying behaviour of d r u g - t r e a t e d r a t s was suppressed because diazepam i n t e r f e r e d with the r a t s ' m o t i v a t i o n to respond to an a v e r s i v e s t i m u l u s . F i n a l l y , i t should be noted that the minimum dose at which diazepam was able to r e l i a b l y suppress c o n d i t i o n e d burying ( i . e . , .5 mg/kg) was c o n s i d e r a b l y l e s s than the minimum amounts that have been r e q u i r e d to r e v e a l an e f f e c t of diazepam i n other b e h a v i o u r a l paradigms (e.g., the G e l l e r c o n f l i c t t e s t ; the four p l a t e t e s t ; the Met r a z o l t e s t ; the food i n t a k e t e s t ; the a n t i a g g r e s s i o n t e s t ) . Moreover, i n a subsequent burying t e s t (Experiment 5) i n which the same low dose of diazepam (.5 mg/kg) was employed, burying was again r e l i a b l y suppressed. Thus, the c o n d i t i o n e d d e f e n s i v e burying t e s t i s extremely s e n s i t i v e t o . t h e e f f e c t s of at l e a s t one known a n x i o l y t i c . Experiment 2 was designed to t e s t the g e n e r a l i t y of the r e s u l t s of Experiment 1. I t was found that three a n x i o l y t i c s , diazepam, c h l o r d i a z e p o x i d e , and p e n t o b a r b i t a l , each suppressed c o n d i t i o n e d b u r y i n g . Furthermore, at 1 mg/kg, diazepam produced a s i g n i f i c a n t l y g r e a t e r s u p p r e s s i o n of burying behaviour than d i d e i t h e r c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l , and at 3 mg/kg, a s i g n i f i c a n t l y g r e a t e r s u p p r e s s i o n than d i d c h l o r d i a z e p o x i d e . At 6 mg/kg, however, there was no s i g n i f i c a n t d i f f e r e n c e between the p o t e n c i e s of the three a n x i o l y t i c s . The r e l a t i v e p o t e n c i e s of these drugs i n the c o n d i t i o n e d 90 b u r y i n g paradigm compare r e a s o n a b l y w e l l w i t h the p o t e n c i e s of t h e s e agents i n the t r e atment of human a n x i e t y . In c l i n i c a l s e t t i n g s , diazepam has been shown t o be 5 t o 10 t i m e s more p o t e n t than e i t h e r c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l , and t h i s was r e f l e c t e d by the f a c t t h a t diazepam suppressed c o n d i t i o n e d d e f e n s i v e b u r y i n g s i g n i f i c a n t l y more than d i d e i t h e r c h l o r d i a z e p o x i d e or p e n t o b a r b i t a l . The f a i l u r e t o f i n d s i g n i f i c a n t d i f f e r e n c e s between p e n t o b a r b i t a l and c h l o r d i a z e p o x i d e i s more d i f f i c u l t t o i n t e r p r e t ; i n c l i n i c a l p r a c t i c e c h l o r d i a z e p o x i d e has proven t o be s l i g h t l y more e f f e c t i v e than p e n t o b a r b i t a l . A l t h o u g h t h e r e was no d i f f e r e n c e i n the p o t e n c i e s of c h l o r d i a z e p o x i d e and p e n t o b a r b i t a l i n the b u r y i n g t e s t a t 1, 3, or 6 mg/kg, i t i s p o s s i b l e t h a t a t a h i g h e r dose l e v e l a s i g n i f i c a n t d i f f e r e n c e between the p o t e n c i e s of t h e s e two drugs c o u l d be d e t e c t e d . C l e a r l y , t h i s work must be extended t o o t h e r doses and o t h e r a n x i o l y t i c s b e f o r e i t i s p o s s i b l e t o judge the degree t o which the c o n d i t i o n e d d e f e n s i v e b u r y i n g t e s t s a t i s f i e s the c r i t e r i o n of r e l a t i v e p otency but t h e s e i n i t i a l r e s u l t s a r e c l e a r l y p r o m i s i n g . . In Experiment 3, the s e l e c t i v i t y of the c o n d i t i o n e d d e f e n s i v e b u r y i n g t e s t was a s s e s s e d by o b s e r v i n g the e f f e c t s on c o n d i t i o n e d b u r y i n g of a n x i o l y t i c and n o n a n x i o l y t i c compounds. I f the a c t i v i t y of n o n a n x i o l y t i c compounds i n a t e s t can not be d i f f e r e n t i a t e d from those of a n x i o l y t i c compounds, t h a t t e s t would be of q u e s t i o n a b l e v a l u e f o r s e l e c t i v e l y d e t e c t i n g compounds w i t h p o t e n t i a l a n x i o l y t i c a c t i o n . The f i n d i n g t h a t n e i t h e r CNS s t i m u l a n t s ( i . e . , p i c r o t o x i n , p e n t y l e n e t e t r a z o l , d-amphetamine) nor a n a r c o t i c a n a l g e s i c , ( i . e . , m o r p h i n e ) had a 91 s i g n i f i c a n t e f f e c t on c o n d i t i o n e d d e f e n s i v e b u r y i n g i n d i c a t e d that the burying paradigm possesses some degree of s e l e c t i v i t y . However, i n Experiment 3 the e f f e c t s of a major t r a n q u i l i z e r ( i . e . , chlorpromazine) c o u l d not be d i f f e r e n t i a t e d from the e f f e c t s of a minor t r a n q u i l i z e r ( i . e . , diazepam). Both c l a s s e s of drug produced comparable suppression of c o n d i t i o n e d b u r ying. Thus, f u r t h e r i n v e s t i g a t i o n of the sup p r e s s i v e e f f e c t s of chlorpromazine and diazepam was warranted. I t i s w e l l known that n e u r o l e p t i c s such as chlorpromazine have powerful i n h i b i t o r y e f f e c t s on motor f u n c t i o n that may d i s r u p t the performance of l e a r n e d responses. A n x i o l y t i c s , such as diazepam on the other hand do not g e n e r a l l y impair motor f u n c t i o n to the same degree, although they a l s o a f f e c t learned responses ( S c h l e c h t e r & Butcher, 1972; Sepinwall & Cook, 1978). The motor d e f i c i t s that are produced by moderate doses of n e u r o l e p t i c agents are u s u a l l y not a f f e c t e d by changes i n the i n t e n s i t y of m o t i v a t i o n a l s t i m u l i such as e l e c t r i c shock; whereas the e f f e c t of moderate doses of a n x i o l y t i c agents may be dim i n i s h e d s u b s t a n t i a l l y by i n c r e a s e s i n the s e v e r i t y of the e l e c t r i c shock (Cook & S e p i n w a l l , 1973). Thus, i t seemed p o s s i b l e that the e f f e c t s of diazepam and chlorpromazine i n the c o n d i t i o n e d burying paradigm c o u l d be d i s s o c i a t e d by exposing r a t s to shocks of d i f f e r e n t i n t e n s i t y . A c c o r d i n g l y , Experiment 4 was designed to d i f f e r e n t i a t e the e f f e c t s of chlorpromazine and diazepam by v a r y i n g the s e v e r i t y of the a v e r s i v e s t i m u l u s . Both drugs were a d m i n i s t e r e d to r a t s exposed to one of two shock c o n d i t i o n s that d i f f e r e d i n s e v e r i t y . In the l e s s severe shock c o n d i t i o n ( i . e . , 1 mA), both 92 drugs a g a i n produced an e q u i v a l e n t s u p p r e s s i o n of c o n d i t i o n e d b u r y i n g , but i n the more se v e r e c o n d i t i o n (10 mA), o n l y c h l o r p r o m a z i n e produced the s u p p r e s s i o n . Thus even when a n x i o l y t i c s and n o n a n x i o l y t i c s have comparable e f f e c t s on b u r y i n g c o n d i t i o n e d a t moderate shock i n t e n s i t i e s , t h e i r e f f e c t s can be d i s s o c i a t e d by v a r y i n g the shock i n t e n s i t y . Whether or not t h i s s t r a t e g y w i l l be e f f e c t i v e i n i d e n t i f y i n g as n o n a n x i o l y t i c s o t h e r n o n a n x i o l y t i c compounds t h a t may d i s r u p t c o n d i t i o n e d d e f e n s i v e b u r y i n g remains t o be d e t e r m i n e d . Taken t o g e t h e r , the r e s u l t s of the f i r s t f o u r e x p e r i m e n t s suggest t h a t the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm may be a b l e t o f u l f i l the t h r e e major c r i t e r i a of a p r e d i c t i v e a n i m a l model f o r s c r e e n i n g a n x i o l y t i c a g e n t s ; i . e . , d o s e dependent s e n s i t i v i t y , r e l a t i v e p o t e n c y , and s e l e c t i v i t y . Many more t e s t s w i t h o t h e r a n x i o l y t i c and n o n a n x i o l y t i c compounds must be conducted b e f o r e any educated judgments can be made; however, the p r e s e n t d a t a a r e c e r t a i n l y p r o m i s i n g enough t o warrant the c o n t i n u e d s t u d y of the p o t e n t i a l of the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm i n the i n v e s t i g a t i o n of a n x i o l y t i c e f f e c t s . In a d d i t i o n t o i t s apparent a b i l i t y t o meet the c r i t e r i a of s e n s i t i v i t y , s e l e c t i v i t y , and r e l a t i v e p o t e n c y , the c o n d i t i o n e d d e f e n s i v e b u r y i n g t e s t p o s s e s s e s a number of p r a c t i c a l a t t r i b u t e s t h a t s h o u l d f a c i l i t a t e the s c r e e n i n g of p o t e n t i a l a n x i o l y t i c compounds, which now number i n the thousands ( H a e f e l e y , 1979). The speed and s i m p l i c i t y of the b u r y i n g t e s t make i t p o s s i b l e t o s c r e e n l a r g e numbers of compounds i n a r e l a t i v e l y s h o r t p e r i o d of t i m e . The t e s t response o c c u r s r e l i a b l y a f t e r o n l y a s i n g l e exposure t o an a v e r s i v e s t i m u l u s , 93 and i t i s suppressed min a f t e r a s i n g l e i n j e c t i o n of an a n x i o l y t i c compound. Although there are other animal t e s t s of a n x i o l y t i c compounds that are reasonably r e l i a b l e (e.g., the G e l l e r c o n f l i c t t e s t ) , they o f t e n r e q u i r e l e n g t h l y p e r i o d s of p r e t r a i n i n g , expensive i n s t r u m e n t a t i o n , and repeated exposures to the t e s t compound. Moreover, the complexity of these paradigms o f t e n complicates i n t e r p r e t a t i o n of a drug's e f f e c t . In c o n t r a s t , the suppression of c o n d i t i o n e d burying appears to be a r e a d i l y q u a n t i f i e d , unambiguous measure of a n x i o l y t i c a c t i o n . I t must be emphasized, however, that although the c o n d i t i o n e d d e f e n s i v e burying t e s t appears to have a number of p r a c t i c a l advantages over c u r r e n t b e h a v i o u r a l t e s t s of a n x i o l y t i c a c t i o n , any meaningful comparisons must wait u n t i l the burying paradigm has been more e x t e n s i v e l y s t u d i e d . 11 Mechanisms of act ion In the f i n a l four experiments of t h i s t h e s i s , the mechanisms by which a n x i o l y t i c s e x e r t t h e i r e f f e c t on c o n d i t i o n e d burying were s t u d i e d . One p o s s i b i l i t y which was i n v e s t i g a t e d i n Experiment 5 was that a n x i o l y t i c s produce t h e i r e f f e c t s i n shock-motivated paradigms simply by s u p p r e s s i n g the animals' responsiveness to p a i n f u l s t i m u l i ( i . e . , by producing a n a l g e s i a ) . I f a n x i o l y t i c s exert t h e i r e f f e c t s e x c l u s i v e l y through an a n a l g e s i c mechanism, they should have no e f f e c t on responsiveness to n o n p a i n f u l s t i m u l i . In Experiment 5, r a t s i n j e c t e d with diazepam d i d not d i s p l a y the d e f e n s i v e burying u s u a l l y e l i c i t e d by t h e i r f i r s t exposure to an unset, wooden mousetrap. The a b i l i t y of diazepam to block 'unconditioned' d e f e n s i v e burying i s c l e a r l y i n c o n s i s t e n t with the view that the 94 s u p p r e s s i v e e f f e c t s of a n x i o l y t i c s on behaviour are mediated by a n a l g e s i a . In t r a d i t i o n a l paradigms where the same t e s t response cannot be e l i c i t e d i n the presence of e i t h e r p a i n f u l or no n p a i n f u l a v e r s i v e s t i m u l i , i t has proven d i f f i c u l t to r e f u t e the ' a n a l g e s i a ' hypothesis (Sepinwall & Cook, 1978). In Experiment 6, the p u t a t i v e r e l a t i o n s h i p between GABA neuronal f u n c t i o n s and the a n x i o l y t i c a c t i o n s of diazepam was examined. I t was i n i t i a l l y hypothesized on the b a s i s of neuropharamcological data that GABA e r g i c mechanisms are d i r e c t l y i n v o l v e d i n the suppression of c o n d i t i o n e d d e f e n s i v e burying; i . e . , that an enhancement of GABA f u n c t i o n suppresses c o n d i t i o n e d burying, and a l t e r n a t i v e l y t h a t i n h i b i t i o n of GABA f u n c t i o n f a c i l i t a t e s b u r y i n g . T h i s hypothesis was t e s t e d i n Experiment 6 by a d m i n i s t e r i n g a GABA rec e p t o r b l o c k e r ( i . e . , p i c r o t o x i n ) and observing i t s e f f e c t s on c o n d i t i o n e d d e f e n s i v e b u r y i n g . GABA receptor blockade by .1, 1, and 2 mg/kg of p i c r o t o x i n d i d not produce a s i g n i f i c a n t enhancement of c o n d i t i o n e d b u r y i n g . Thus, the i n i t i a l h ypothesis that GABA e r g i c mechanisms are d i r e c t l y i n v o l v e d i n the suppression of c o n d i t i o n e d burying was not supported. The p o s s i b i l i t y remained, however, that the e f f e c t s of p i c r o t o x i n i n Experiment 6 were masked by the high l e v e l s of burying that are normally produced when r a t s are shocked from a prod. In order to reduce the l i k e l i h o o d of such a c e i l i n g e f f e c t , r a t s i n Experiment 7 were exposed to an unset mousetrap, an a v e r s i v e stimulus that c h a r a c t e r i s t i c a l l y e l i c i t s l e v e l s of burying that are we l l below those produced by the prod. P i c r o t o x i n at .1, 1, and 2 mg/kg again f a i l e d to enhance burying 95 behaviour in spite of the fact that the ove r a l l l e v e l s of burying were only half of those in Experiment 6. Thus, i t i s unlikely that GABA. i s d i r e c t l y involved in the suppression of conditioned defensive burying. Recent studies of the binding of {H3} diazepam to high-a f f i n i t y receptor s i t e s in the brain have shown that diazepam binding can be f a c i l i t a t e d when GABA i s included in the binding assay (e.g., Tallman, Thomas, & Gallager, 1978). These data, taken together with other supportive pharmacological data (e.g., Haefely et a l . , 1975), strongly suggest that GABA and benzodiazepines interact, which implies that these substances may not exert their e f f e c t s independently of one another; i . e . , that perhaps i t is the synergistic interaction of GABA and diazepam that produces a n x i o l y t i c e f f e c t s . Accordingly, rats in Experiment 8 were injected with picrotoxin, a potent GABA blocker, in combination with diazepam. This manipulation e f f e c t i v e l y reversed the suppressive effect of diazepam, without producing an enhancement of burying behaviour. Thus, i t i s apparent that GABA, although i t may not play a primary role in the control of defensive behaviour, forms a necessary substrate upon which diazepam exerts i t s e f f e c t s . Studies that have examined the binding properties of {H3} diazepam to h i g h - a f f i n i t y receptor s i t e s in the brain suggest that diazepam may exert some o f i t s effects by acting on the putative benzodiazepine receptor (e.g., Tallman, Thomas, & Gallager, 1978). In p a r t i c u l a r , i t now seems possible that the an x i o l y t i c e f f e c t s of diazepam, in addition to being modulated by GABA, primarily result from i t s interaction with the putative 96 b e n z o d i a z e p i n e r e c e p t o r (Tallman et a l . , 1980). The p o s s i b l e b e h a v i o u r a l s i g n i f i c a n c e of these n e u r o p h a r m a c o l o g i c a l d a t a i s i n d i c a t e d by the c o r r e l a t i o n between t h e r e l a t i v e p o t e n c i e s of b e n z o d i a z e p i n e s i n the b i n d i n g assay and t h e i r r e l a t i v e p o t e n c i e s i n c l i n i c a l s e t t i n g s . More d i r e c t e v i d e n c e of the b e h a v i o u r a l s i g n i f i c a n c e of b e n z o d i a z e p i n e r e c e p t o r s has come from a number of r e c e n t s t u d i e s t h a t have examined the r e l a t i o n s h i p between r e c e p t o r d e n s i t y and e m o t i o n a l i t y i n r o d e n t s ( R o b e r t s o n , M a r t i n , & Candy, 1978). F i r s t , i t has been found t h a t the d e n s i t y of b e n z o d i a z e p i n e r e c e p t o r s may d i f f e r i n s t r a i n s of r a t s t h a t a l s o d i f f e r i n e m o t i o n a l i t y ( i . e . , ' t e a r f u l n e s s ' ; R o b e r t s o n , M a r t i n , & Candy, 1978). A l t h o u g h t h i s f i n d i n g by i t s e l f does not demonstrate a c a u s a l r e l a t i o n s h i p between r e c e p t o r d e n s i t y and e m o t i o n a l i t y , a f u n c t i o n a l r e l a t i o n s h i p between the two t r a i t s i s s u g g e s t e d by the f a c t t h a t b o t h were produced c o n c o m i t a n t l y by s e l e c t i v e b r e e d i n g . Second, b e n z o d i a z e p i n e r e c e p t o r d e n s i t y i n the "nervous" s t r a i n of mouse i s s i g n i f i c a n t l y l e s s than t h a t found i n o t h e r s t r a i n s ( e . g . , L i p p a , Sano, Coupet, K l e p n e r , & Beer, 1978), which a g a i n s u g g e s t s t h a t t h e r e i s a r e l a t i o n between e m o t i o n a l i t y and r e c e p t o r d e n s i t y i n r o d e n t s . T h i r d , s i g n i f i c a n t d e c r e a s e s i n b e n z o d i a z e p i n e r e c e p t o r b i n d i n g have been produced i n r a t s by the a d m i n i s t r a t i o n of a v e r s i v e f o o t s h o c k ( L i p p a , K l e p n e r , Yunger, Sano, S m i t h , & Beer, 1978). These d e c r e a s e s o c c u r r e d w i t h i n min of the f o o t s h o c k , s u g g e s t i n g t h a t t h e r e g u l a t i o n of e x p e r i m e n t a l l y - i n d u c e d ' a n x i e t y ' may be mediated t h r o u g h d i f f e r e n c e s i n the number- of b e n z o d i a z e p i n e r e c e p t o r s . 97 In summary, the l a s t f o u r e x p e r i m e n t s i n the p r e s e n t t h e s i s made two k i n d s of c o n t r i b u t i o n s . F i r s t , they r e v e a l e d t h a t the e f f e c t s of diazepam on c o n d i t i o n e d d e f e n s i v e b u r y i n g appear t o be n o n a n a l g e s i c i n n a t u r e and r e q u i r e GABA e r g i c m e d i a t i o n . Second, th e s e e x p e r i m e n t s i l l u s t r a t e d t h a t the c o n d i t i o n e d d e f e n s i v e b u r y i n g paradigm can be used e f f e c t i v e l y t o i n v e s t i g a t e the mechanisms of a n x i o l y t i c a c t i o n . 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