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The effect of neonatal testosterone propionate (TP) injections in male rats on active and passive avoidance… Deol, Gurcharn Singh 1974

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i THE EFFECT OF NEONATAL TESTOSTERONE PROPIONATE (TP) INJECTIONS IN MALE RATS ON ACTIVE AND PASSIVE AVOIDANCE TASKS DURING THE PREPUBESCENT AND ADULT PERIODS OF LIFE by GURCHARN SINGH DEOL B.A., University of B r i t i s h Columbia, 19?2 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of Psychology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 197k 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 o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 a n d s t u d y . I f u r t h e r a g r e e 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d 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 n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8 , C a n a d a D e p a r t m e n t o f ABSTRACT The e f f e c t of neonatal testosterone propionate (TP) ( 1 0 0 ug/day f o r the f i r s t 5 days of l i f e or 1 . 2 5 mg. on day 1 of birth) injections on the a c q u i s i t i o n of both active and passive avoidance was studied. Testing was i n i t i a t e d during both the prepubescent and adult periods of l i f e . Neonatal TP i n j e c t i o n s f a c i l i t a t e d the a c q u i s i t i o n of active avoidance responding p r i o r to puberty and also i n adulthood. Neonatal TP injections had no e f f e c t on passive avoidance responding. A number of physiological (body, gonadal and adrenal weights) and behavioural ( a c t i v i t y , shock s e n s i t i v i t y ) measures were also studied to investigate t h e i r possible role i n influencing our r e s u l t s . The TP injections led to s i g n i f i c a n t l y lower gonadal weights i n the TP-injected group. P r i o r to puberty the TP-injected animals also possessed heavier adrenal glands, and were more active than the control group. The TP-injected group also had lower shock thresholds than the control group. The r e s u l t s suggest that excess neonatal TP i n j e c t i o n s a f f e c t an organism's a b i l i t y to acquire an active avoidance response. The exact mechanism by which TP i n j e c t i o n s have t h e i r e f f e c t i s unknown at t h i s time. Future research would help to c l a r i f y whether the e f f e c t of excess neonatal TP i s d i r e c t l y on the associative process or i n d i r e c t l y through a number of other factors, investigated i n t h i s a r t i c l e . . i i i P a r a d o x i c a l l y , i n c r e a s e d a c t i v i t y w o u l d b e e x p e c t e d t o i n t e r f e r e w i t h t h e a c q u i s i t i o n o f p a s s i v e a v o i d a n c e b e h a v i o u r , b u t t h i s d i d n o t p r o v e t o b e t h e c a s e a s T P i n j e c t e d a n i m a l s a c q u i r e d t h i s r e s p o n s e j u s t a s w e l l o r s l i g h t l y b e t t e r t h a n c o n t r o l s . T h e o b s e r v e d d i f f e r e n c e s b e t w e e n g r o u p s o n s h o c k r e a c t i v i t y m e a s u r e s s u g g e s t a n o t h e r p l a u s i b l e e x p l a n a t i o n o f t h e r e s u l t s . N e o n a t a l T P i n j e c t i o n s c o u l d f a c i l i t a t e a c q u i s i t i o n o f a n a c t i v e a v o i d a n c e r e s p o n s e b y i n c r e a s i n g s e n s i t i v i t y t o t h e m o t i v a t i o n a l s t i m u l u s . T h i s p o s s i b i l i t y b e c o m e s e v e n m o r e t e n a b l e w h e n w e c o n s i d e r t h a t o t h e r i n v e s -t i g a t o r s h a v e f o u n d a p o s i t i v e c o r r e l a t i o n b e t w e e n s h o c k s e n s i t i v i t y a n d l e a r n i n g a n a c t i v e a v o i d a n c e r e s p o n s e ( B e a t t y e t . a l . - 1 9 7 0 , P a r e , 1 9 6 9 ) . S p e c i f i c a l l y , t h e s e i n v e s t i g a t o r s h a v e f o u n d t h a t f e m a l e s a c q u i r e a n a c t i v e a v o i d a n c e r e s p o n s e f a s t e r t h a n m a l e s a n d a l s o h a v e l o w e r r e a c t i v i t y t h r e s h o l d s t o f o o t s h o c k t h a n m a l e s . O u r f i n d i n g o f l o w e r e d s h o c k s e n s i t i v i t y f o l l o w i n g n e o n a t a l T P i n j e c -t i o n s w o u l d a p p e a r t o p a r a l l e l t h e s e o b s e r v a t i o n s a n d s u g g e s t s s h o c k s e n s i t i v i t y m a y b e m a k i n g a n i m p o r t a n t c o n -t r i b u t i o n t o t h e a c t i v e a v o i d a n c e d a t a . i v TABLE OF CONTENTS Page I Introduction 1 (1) E f f e c t s of Adult Hormone Manipulation on Learning. 5 (A) The Pituitary-Adrenal System and Avoidance Learning 5 (B) Vasopressin 13 (C) Melanocyte Stimulating Hormone (MSH) . 15 (D) Gonadal Hormones 16 ( i ) Androgens 17 ( i i ) Estrogens 20 ( i i i ) Progesterone 21 (iv) Conclusion 22 (2) Developmental Neuroendocrinology and Learning 23 (A) Thyroxine and C o r t i s o l 24 (B) Gonadal Hormones * . 2? II Purpose of the Present Investigation 32 III Experiment I - Active and Passive Avoidance. . . 34 (1) Method 3^ (2) Results 36 ( 3 ) Discussion • 41 IV Experiment II - Active and Passive Avoidance Learning hk (1) Method ^ (2) Results ^5 ( 3 ) Discussion ^9 V Page V Experiment III - Open-Field Behaviour, Reactivity to Shock 5 3 ( 1 ) Method 5L ( 2 ) Results 5 5 ( 3 ) Discussion . . . . . 57 VI Experiment IV - Step-Down Passive Avoidance Task 6 0 ( 1 ) Method 6 0 ( 2 ) Results 6 1 ( 3 ) Discussion . . . . . 6 2 VII Experiment V - Active and Passive Training i n Adult Animals . . . . . 7 0 ( 1 ) Method . 7 1 ( 2 ) Results 7 2 ( 3 ) Discussion . . . 7 5 VIII Discussion 80 LIST OF TABLES Page Table 1. Mean Latency t o Cross 39 Table 2. Number of Correct Responses . . . . . . . 40 Table 3. Body, Gonadal and Adrenal Weights . . . . 42 Table 4. Number of Correct Responses 48 Table 5 (a) Body, Gonadal and Adrenal Weights at 42 Days of Age 50 (b) Body, Gonadal and Adrenal Weights at 100 Days of Age 51 Table 6 Body, Gonadal and Adrenal Weights at 30 and 31 Days of Age 56 Table 7 Mean F l i n c h and Jump Thresholds 76 v i i LIST OF FIGURES Page Figure 1. Active Avoidance Training (Experiment I) J8 Figure 2. Active Avoidance Training (Experiment II) 47 Figure J. Amount of Shock Received P r i o r to Remaining on Platform f o r 2 Minute Period 64 Figure 4. Amount of Time on Platform 24 Hours After Training 66 Figure 5» Resistance to Ext i n c t i o n . 68 Figure 6 Active Avoidance Acquisition (Experiment V) 74 ACKNOWLEDGEMENT The writer would l i k e to thank: Dr. A.G. P h i l l i p s f o r both his devoted assistance and c r i t i c a l analysis of t h i s t h e s i s . Dr. R. Wong f o r his analysis of t h i s thesis and f o r his h e l p f u l advice i n regards to future research i n t h i s area. 1 I INTRODUCTION One of the most d i f f i c u l t tasks facing learning re-searchers i s an account of how the central nervous system (CNS) codes the learning process. H i s t o r i c a l l y , models of brain function have r e f l e c t e d advances i n contemporary technology and the emphasis has been placed on hard-wired systems ranging from the telephone exchange to d i g i t a l computers (Milner, 19?1). One of the basic components of these theories has been the concept of a permanent change i n the nervous system brought about by the learning process i t s e l f . The name most commonly associated with t h i s concept i s that of an engram. Semen (1913) was one of the e a r l i e s t to support t h i s concept and Lashley (1950) i n the following years spent a great deal of time searching f o r the neural basis of an engram but never succeeded. Freud also popu-l a r i z e d the term as representing the phys i o l o g i c a l repre-sentation of memory. Over the l a s t twenty years our understanding of brain function has greatly advanced (Milner, 1970). With t h i s came a greater emphasis on molecular models of learning and memory. This was primarily due to: (1) the knowledge that genetic information i s coded into nucleic acid molecules (RNA and DNA), therefore i t may be that learning i s simi-l a r l y coded (Rosenzweig, Krech and Bennett, 1958); (2) the fin d i n g that synaptic transmission i s biochemical and a 2 number of these biochemical substances regulate CNS func-tioning (McGeer, 1971; Barchas, C i a r n e l l o , Stalk, Keth, and Hamburg, 1972; Conner, 1972). These findings led to numerous experiments on the neurochemistry of learning. These have included the role of proteins and l i p i d s (Hyden, 1959? M o r r e l l , 1961; Bennett, Diamond, Krech, and Rosenzweig, 1 9 6 4 ) , s p e c i f i c neurotransmitters (Kety, 1967; Murphy and Henry, 1972), and most important for the present discussion, the role of the neuroendocrine system. The neuroendocrine system operates i n a dynamic and f l e x i b l e i n t e r a c t i o n with the CNS (Harris, 1964; Guxe and Hokfelt, 1967; Sawyer, 1 9 6 8 ) . That i s to say, the CNS controls hormonal release v i a the p i t u i t a r y and at the same time endogenous hormone l e v e l s control CNS functioning v i a po s i t i v e and negative feedback loops. Similar to other neurohumors involved i n CNS functioning, hormones have been found to have a regional d i s t r i b u t i o n i n the CNS (Whalen and Rezek, 1972; P f a f f , 1973? Wade, Harding, and Feder, 1973)» to influence e l e c t r i c a l a c t i v i t y of the CNS (Lissak and Endroczi, 1961; Dufy, Vincent, Bensch, and Faure, 1973; Dufy, Vincent, Dufy-Barbe, and Faure, 1973) and to a l t e r monoamine l e v e l s and synthesis (Ladosky and G a z i r i , 1970; Hardin, 1 9 7 3 ) . P a r a l l e l i n g these developments i n neuroendocrinology have been studies on t h e i r behavioural correlates. These have included the e f f e c t s of hormones on sexual behaviour (Beach, 1 9 4 8 ) , aggression (Endroczi, Lissak, and Gelgedy, 3 1 9 5 8 ; Y o u n g , 1 9 6 1 ; B r o n s o n a n d D e s j a r d i n e s , 1 9 6 8 ) , e m o t i o n -a l i t y ( P h i l l i p s a n d D e o l , 1 9 7 3 ) . a r o u s a l ( W o o d b u r y , 1 9 5 * 0 . a c t i v i t y ( S w a n s o n , 1 9 6 7 ) . T h e f u n c t i o n a l r o l e o f t h e s e h o r m o n e s i n l e a r n i n g h a v e a l s o b e e n e x t e n s i v e l y r e v i e w e d ( D a w s o n , 1 9 7 2 ; D e W i e d , v a n D e l f t , G i s p e n , W e i j e n , a n d v a n W i m e r s m a G r e i d a n u s , 1 9 7 2 ; E n d r o c z i , 1 9 7 2 ; L i s s a k a n d B o h u s , 1 9 7 2 ) , w i t h a p r i m a r y e m p h a s i s o n g o n a d a l a n d a d r e n a l s t e r o i d s ( A p p l e z w e i g a n d B a u d r y , 1 9 5 5 ? L e v i n e a n d J o n e s , 1 9 6 5 ; W e i s e l , M c E w e n , S i l v a , a n d K a l k u t , 1 9 7 0 ? K l a i b e r , B r o v e r m a n , V o g e l , A b r a h a m , a n d C o n e , 1 9 7 1 ? G e l g e d y a n d R o z s a h e g y i , 1 9 7 1 ) a n d a n u m b e r o f p i t u i t a r y h o r m o n e s , i . e . A C T H , v a s o p r e s s i n ( L i s s a k a n d B o h u s , 1 9 7 0 ; B o h u s a n d D e W i e d , 1 9 7 2 ; B o h u s , G i s p e n , a n d D e W i e d , 1 9 7 3 ) . A t t h e s a m e t i m e , t h e s e h o r m o n e s h a v e a l s o b e e n s h o w n t o h a v e p r o f o u n d e f f e c t s o n t h e d e v e l o p m e n t o f t h e C N S , w h i c h m a y d i r e c t l y a f f e c t a n o r g a n i s m ' s a b i l i t y t o l e a r n ( H e i n a n d G i m i a s , 1 9 6 3 ? S c h a p i r o , 1 9 7 1 ) . F o r o u r p u r p o s e s , t h e r o l e o f s e x h o r m o n e s i n t h e d e v e l o p m e n t o f a n o r g a n i s m i s p a r t i c u l a r l y i m p o r t a n t . I n m a m m a l s , a n d r o g e n s a p p e a r t o b e e s s e n t i a l f o r n o r m a l m a s c u l i n i z a t i o n o f t h e C N S s t r u c -t u r e s i n v o l v e d i n r e g u l a t i n g g o n a d o t r o p h i n s e c r e t i o n a n d m a t i n g b e h a v i o u r ( H a r r i s , 1 9 6 4 ; G e r a l l , 1 9 6 6 ; G e r a l l , H e n d r i c k s , J o h n s o n , a n d B o u n d s , 1 9 6 7 ) . N e o n a t a l c a s t r a t i o n i n m a l e s a l l o w s f o r f e m i n i n e b e h a v i o u r t o o c c u r i n a d u l t h o o d a n d a l s o a l l o w s f o r c y c l i c a l g o n a d o t r o p h i n r e l e a s e . T e s t o s -t e r o n e i n j e c t e d i n t o n e o n a t a l f e m a l e s c h a n g e s t h e i r b e h a v i o u r p a t t e r n s t o w a r d s t h e m a l e n o r m a n d p r e v e n t s c y c l i c a l gonadotrophs release. These neonatal e f f e c t s of sex steroids also permanently a l t e r nonsexual behaviour patterns such as aggressive behaviour (Bronson and Desjardins, 1 9 6 8 ) , emotionality ( P h i l l i p s and Deol, 1973)t taste preferences (Wade and Zucker, 1 9 6 9 ) , a c t i v i t y l e v e l s (Gray, Lean, and Keynes, 1 9 6 9 ) . Recently a number of researchers have implicated neo-natal androgens i n the determination of learning a b i l i t i e s l a t e r i n l i f e (Money and Lewis, 1966j Dalton, 1968? Money, 1 9 7 1 l Beatty and Beatty, 1 9 7 0 ) . Consideration of t h i s research led to the hypothesis that neonatal androgen mani-pulation would influence learning a b i l i t i e s . In order to describe the rationale underlying t h i s hypothesis i n greater d e t a i l , the remainder of the introduction w i l l be divided into two parts; Section I w i l l describe the e f f e c t s of hormone manipulation on learning i n mature animals and Section II w i l l discuss evidence f o r t h e i r involvement i n neonatal stages of development. 5 (I) E f f e c t s of Adult Hormone Manipulation on Learning (A) The Pituitary-Adrenal System and Avoidance Learning In attempting to discover the rationale behind studies of adrenal function and learning, one major fi n d i n g appears predominant; namely the high c o r r e l a t i o n between adrenal hyperactivity and s t r e s s f u l situations (Applezweig and Moeller, 1959; Ader, Friedman, and Grota, 196?; Sandman, Kastin, and Schally, 1973). E l e c t r i c shock, which produces hyperactivity i n the pituitary-adrenal system, i s employed i n many animal learning experiments (Anderson, Winn, and Tarn, 1968; Bohus and Endroczi, 1965). Given t h i s r e l a t i o n -ship, i t i s understandable that attempts to delineate the phys i o l o g i c a l correlates of avoidance conditioning have focused on the pituitary-adrenal system (De Wied, van De l f t , Gispen, Weijnan, and van Wimersma Greidanus, 1972). There are of course, many d i f f e r e n t ways of a f f e c t i n g the p i t u i t a r y - a d r e n a l system, some of which can have con-founding e f f e c t s . Therefore, p r i o r to a detailed discussion of studies dealing with the role of adrenocorticotrophic hormone (ACTH) and glucocorticoids i n learning, p o t e n t i a l problems a r i s i n g from the d i f f e r e n t procedures employed should be i d e n t i f i e d . The f i r s t problem involves the intimate r e l a t i o n s h i p between the p i t u i t a r y and the adrenal gland. In response to ACTH secretion from the p i t u i t a r y , the adrenal cortex 6 synthesizes and releases adrenoeorticoids, of which the main steroids are glucocorticoids. In a s i m i l a r fashion there i s regulation of ACTH release by adrenoeorticoids which i s accomplished by adrenoeorticoids having an i n h i -b i t o r y feedback action on the brain and the p i t u i t a r y (Mangili, Mota, and Martini, 1 9 6 6 ) . Therefore removal of either the p i t u i t a r y or the adrenal gland, i n j e c t i o n s of ACTH or glucocorticoids lead to a number of confounding ef f e c t s of which any one (or more) could lead to the behavioural e f f e c t s observed. Removal of the p i t u i t a r y not only abolishes the source of ACTH i n the organism but also eliminates the source of a number of other hormones (Vasopressin, MSH, FSH, LH, oxy-t o c i n ) , many of which have been implicated to be involved i n the learning of aversive tasks (Ader and de Wied, 1 9 7 2 ; Kastin, M i l l e r , Nockton, Sandman, Schally, and Stratton, 1 9 7 3 ) * Secondly, hypophysectomy lowers c o r t i c o s t e r o i d output from the adrenal medulla (Turner and Bagnara, 1 9 7 1 ) . Adrenalectomy eliminates not only the source of c o r t i c o -steroids but leads to increased ACTH release (Cox, Hodges, and Vernikas, 1 9 5 8 ) . This procedure also eliminates the source of adrenalin and noradrenalin (from the adrenal medulla), which have been implicated i n the learning of a number of avoidance tasks (Latane and Schacter, 1 9 6 2 ) . However these findings concerned with NA and A have been d i f f i c u l t to r e p l i c a t e (Singer, 1 9 6 3 ; Stewart and Brook-shire, 1 9 6 8 ) and w i l l not be dealt with i n t h i s review. 7 Injections of either ACTH or corti c o s t e r o i d s lead to s i m i l a r confounding e f f e c t s since ACTH injec t i o n s increase c o r t i c o -s t e r o i d l e v e l s while c o r t i c o s t e r o i d i n j e c t i o n s i n h i b i t ACTH release (De Giusto, Cairncross, and King, 1971). These findings led Giusto, Cairncross, and King (1971) to describe 4 relationships which may account f o r the behavioural e f f e c t s of ACTH and glucocorticoids: (1) low ACTH - low glucocorticoids (2) low ACTH - high glucocorticoids (3) high ACTH - high glucocorticoids (4) high ACTH - low glucocorticoids Unfortunately, most studies do not take these relationships into consideration. Furthermore, a second problem to be i d e n t i f i e d concerns injec t i o n s of excessive amounts of a drug to f a c i l i t a t e the learning process. In these situations i t i s of paramount importance to establish dose-response curves. As an example, Latane and Schachter (1962) showed that a small dose of adrenalin enhanced 2-way shuttlebox avoidance a c q u i s i t i o n but a larger dose retarded i t . This could be due to the fact that adrenalin i n j e c t i o n s lead to motor impairment (Kosman and Gerard, 1955). The e a r l i e s t studies dealing with the pituitary-adrenal system and avoidance learning employed p a r t i a l or t o t a l hypophysectomy. Hypophysectomy, adenohypophysectomy, but not neurohypophysectomy lead to retardation of escape and avoidance a c q u i s i t i o n i n rats (Applezweig and Moeller, 1959). 8 This was reversed with injections of ACTH or a mixture of cortisone, testosterone and thyroxine (De Wied, 1 9 6 4 ; De Wied, 1 9 6 5 ) . Hypophysectomy, adenohypophysectomy and neurohypophysectomy also f a c i l i t a t e d extinction of an active avoidance response which could be reversed with in j e c t i o n s of ACTH i n hypophysectomized rats (Weiss, McEwen, Teresa, S i l v a , and Kalkut, 1 9 6 9 ) t or ACTH or vasopressin plus oxytocin i n neurophypophysectomized rats and by cortisone, testosterone plus thyroxine i n adenohypophysectomized animals (De Wied, 1 9 6 5 ; De Wied, 1 9 6 9 ) . Recently De Wied ( 1 9 6 9 ) has supplied further evidence f o r the role of ACTH i n avoidance a c q u i s i t i o n of hypophysectomized r a t s . Analogs of ACTH (ACTH 1-10 and ACTH 4 - 1 0 ) f a c i l i t a t e d the ac q u i s i -t i o n of an avoidance response i n the same manner as the natural occurring ACTH. Because several factors are affected by hypophysectomy the precise mechanism by which ACTH f a c i l i t a t e s a c q u i s i t i o n of avoidance learning i n the above studies i s unknown. Hypophysectomy i s known to retard the speed of escape i n active avoidance responding but the e l e c t r i c shock threshold i s also lowered. Furthermore there i s weight loss and adrenal and t e s t i c u l a r atrophy. De Wied showed that none of these factors were modified by the ACTH analogs and yet the f a c i l i t a t i o n e f f e c t was s t i l l observed (De Wied, 1 9 6 8 ; De Wied, 1 9 6 9 ; Gispen, Wimersma Greidanus, and De Wied, 1 9 7 0 ) . An alternative hypothesis i s that ACTH may have i t s effects through adrenal a c t i v a t i o n since De Wied ( 1 9 6 9 ) has 9 also shown that the natural peptide, ACTH A, and synthetic ACTH-B 1-24 restored the rate of active avoidance a c q u i s i t i o n to control l e v e l s , hut at the same time restored normal adrenal cortex s i z e . On the other hand, neither ACTH 1-10 nor ACTH 4 - 1 0 stimulate the adrenal cortex but f a c i l i t a t e active avoidance a c q u i s i t i o n (Lebowitz and Engel, 1 9 6 4 ; Levine and Jones, 1 9 6 5 ) . Therefore we may discount the hypothesis that ACTH produces i t s e f f e c t on learning through adrenal a c t i v a t i o n . Recently, a much d i f f e r e n t explanation has been offered as i t has been hypothesized that ACTH may have i t s e f f e c t by acting on the consolidation of memory involved i n acquiring the avoidance task (De Wied, D e l f t , Gispen, Weijnen, and Wimersma Greidanus, 1 9 7 3 ) • In contrast to hypophysectomized subjects, the ef f e c t s of c o r t i c o s t e r o i d i n j e c t i o n s on avoidance a c q u i s i t i o n i n int a c t rats i s quite inconsistent as i l l u s t r a t e d by the following experiments. Cortisone i n j e c t i o n s p r i o r to con-ditioned avoidance responding do not a f f e c t the a c q u i s i t i o n of the response, but lower the i n t e r t r i a l i n t e r v a l (Murphy and M i l l e r , 1 9 5 5 ? Bohus and Lissak, 1968). Injections of hydrocortisone or dexamethazone enhance shuttlebox avoidance (Levine and Brush, 1 9 6 7 ) and bar-press avoidance (Wertheim, Conner, and Levine, 1 9 6 ? ) a f t e r the learning task has been p a r t i a l l y or t o t a l l y acquired. Endroczi and Lissak ( 1 9 6 2 ) found that excess amounts of corti c o s t e r o i d s administered 1 0 minutes p r i o r to the experimental s i t u a t i o n resulted i n a suppression of active avoidance responding whereas 10 administration f o r a week p r i o r to t r a i n i n g had no e f f e c t . The r e s u l t s of experiments employing ACTH injections are equally ambivalent. For example, d a i l y ACTH in j e c t i o n s have been found to have no e f f e c t on the a c q u i s i t i o n of a shuttle-box response (Levine and Brush, 1 9 6 7 ) . Beatty, Beatty, Bowman and G i l c h r i s t ( 1 9 7 0 ) on the other hand showed a f a c i l a t o r y e f f e c t using a higher shock l e v e l and longer t r a i n i n g sessions. To make matters even more confusing ACTH has been shown to enhance a c q u i s i t i o n of a pole-jumping avoidance response and shuttle-box avoidance (Bohus, Nyakas, and Endroczi, 1 9 6 8 ; Levine and Brush, I 9 6 8 ) and Sidman avoidance (Wertheim, Connor, and Levine, 1 9 6 9 ) i f the i n -jec t i o n i s administered when the task has been p a r t i a l l y acquired. Why increased g l u c o c o r t i c o i d and ACTH l e v e l s are p o s i t i v e l y correlated with enhanced active avoidance per-formance only when the avoidance response has been at lea s t p a r t i a l l y learned remains unexplained. Only a few studies have been concerned with passive avoidance responding. Presently, the data indicates that glucocorticoids do not influence passive avoidance learning i n rats (Anderson, Win, and Tarn, 1 9 6 8 ) , while ACTH injec t i o n s enhance passive avoidance learning (Levine and Jones, 1 9 6 5 ) . In the l i t e r a t u r e on pituitary-adrenal a c t i v i t y and avoidance learning which i s replete with contradictory findings, one ef f e c t stands out, that being the r e l i a b l e a l t e r a t i o n of the extinction process. Glucocorticoids appear to f a c i l i t a t e the extinction of avoidance responses 1 1 i n a dose-dependent relationship (De Wied, Bohus, and Greven, 1 9 6 9 ; Van Wimersma Greidanus, 1 9 7 0 ) . ACTH inj e c t i o n s on the other hand i n h i b i t the extinction of an avoidance response (Greven and De-Wied, 1 9 6 7 ; De Wied and P i r i e , 1 9 6 8 ) . This ef f e c t i s also doseTrdependent. Since the natural occurring ACTH molecule i s composed of 39 amino acids, the f i r s t 24 which are es s e n t i a l f o r s t e r o i d o g e n e s i s , De Wied ( 1 9 6 6 ) raised the question of whether the behavioural e f f e c t s of ACTH depend on a si m i l a r sequence. Biochemical studies of the f i r s t 24 amino acids composing the ACTH molecule u l t i -mately showed that the amino acid sequence 4-10 could i n -h i b i t extinction, but ACTH 5 - 1 0 possessed a reduced e f f e c t while ACTH 1-3 or ACTH 11-24 had no e f f e c t . F a c i l i t a t i o n of e x t i n c t i o n was also observed by replacing the phenylalanine i n the amino acid sequence, ACTH 1-10 by the d-isomer (Bohus and De Wied, 1 9 6 6 ) . The next question raised by these authors was the s i t e of action of these substances i n the central nervous system. C o r t i s o l implants i n the median eminence of the hypothalamus or i n the mesencephalic r e t i c u l a r formation f a c i l i t a t e d e x t i n c t i o n of an avoidance response (Van Wimersma Greidanus and De Wied, 1 9 6 9 ) . ACTH 1-10 implants i n the nucleus para-fas c i c u l u s , nucleus posteremedianus thalami and the nucleus l a t e r l i s habenulae i n h i b i t e d e x t i n c t i o n of a pole-jumping avoidance response (Van Wimersma Greidanus, 1 9 7 2 ) . There-fore the junction between mesencephalic and diencephalic areas appears to be involved i n f a c i l i t a t i o n and extinction 12 of a conditioned avoidance response. Further evidence f o r t h i s hypothesis appears when lesio n i n g the nucleus para-fasciculus leads to poorer conditioned avoidance and e l e c t r i c a l stimulation improves i t (Bohus and De Wied, 1 9 6 7 ; Cardo, 1 9 6 ? ) . In conclusion we should note that many of the re s u l t s obtained above are not only contradictory but also l i m i t e d to the r a t . In contrast to the ACTH induced f a c i l i t a t i o n of a p a r t i a l l y acquired avoidance response i n ra t s , ACTH inject i o n s i n rabbits and chicks have been shown to suppress avoidance responding (Koranyi and Endroczi, 1 9 6 5 ; De Wied, 1 9 6 6 ) . Furthermore, ACTH administration i n monkeys increased the extinc t i o n rate of a fear-conditioned response (Mirsky, M i l l e r , and Stein, 1 9 5 3 ) • What lim i t e d conclusions that can be drawn from t h i s l i t e r a t u r e could be summarized as follows; ( 1 ) ACTH and glucocorticoids f a c i l i t a t e the a c q u i s i t i o n of active avoidance responding i n hypophysectomized but not int a c t r a t s . (2) ACTH prevents the extinction of an active avoidance response while cor t i c o s t e r o i d s f a c i l i t a t e e x t i n c t i o n . ( 3 ) The exact mechanism by which these hormones exert t h e i r behavioural effects are unknown. A number of hypo-theses have been forwardedt adaptation (De Wied, Bohus, and Greven, 1 9 6 8 ) , arousal (Weiss, McEwen, S i l v a , and Kalkut, 1 9 7 0 ) , memory (De Wied and Bohus, 1 9 6 6 ) , e f f e c t s on motiva-t i o n a l cues (Levine and Brush, 1 9 6 7 ) and i n h i b i t i o n (Levine, 1 3 1 9 6 8 ) , but the exact role of one or more of these factors has yet to be s p e c i f i e d . (B) Vasopressin A number of factors led to the study of vasopressin i n avoidance learning. F i r s t of a l l , hypophysectomy eliminates not only the source of ACTH but also hormones of the posterior p i t u i t a r y (vasopressin and oxytocin). Neurohypophysectomy has also been shown to impair the maintenance of shuttle-box avoidance responses (De Wied, 1 9 6 5 ) . Secondly, vasopressin i s involved i n the release of ACTH (Grindeland, Wherry, and Anderson, 1 9 6 2 ) . Furthermore, vasopressin i s known to be released i n response to s p e c i f i c s timuli and nonspecific stimulation (Nash, 1 9 7 1 ) . Emotional stress also causes the release of vasopressin (Nash, 1 9 7 1 ) . Given these findings, i t i s not surprising that posterior p i t u i t a r y extracts may influence avoidance behaviour; and t h i s prompted a number of researchers to investigate the relationship between vaso-pressin and avoidance behaviour. Recent reports show that the e f f e c t s of vasopressin are s i m i l a r to ACTH (De Wied, 1 9 6 9 ; De Wied, 1 9 7 1 ? Bohus, Gispen, and De Wied, 1 9 7 3 ) . In passive avoidance learning, vasopressin enhances the retention of the task when administered p r i o r to the learning t r i a l s i n hypophysectomized rats (Ader and De Wied, 1 9 7 2 ; Bohus, Ader, and De Wied, 1 9 7 2 ) . I t should be men-tioned that lysine-vasopressin has the desired e f f e c t on passive avoidance learning but phenylalanine-lysine-v a s o p r e s s i n d o e s n o t ( L i s s a k a n d B o h u s , 1972). A l s o t h e i n t e n s i t y o f s h o c k i s a n i m p o r t a n t v a r i a b l e i n t h a t h i g h s h o c k i n t e n s i t i e s (.5 m A o r 1.0 m A ) a p p e a r t o m a s k t h e e f f e c t s o f l y s i n e - v a s o p r e s s i n , w h i l e .3 m A d i d n o t ( L i s s a k a n d B o h u s , 1970). L y s i n e - v a s o p r e s s i n a l s o r e s t o r e s t h e i m p a i r m e n t o f a v o i d a n c e a c q u i s i t i o n i n h y p o p h y s e c t o m i z e d r a t s i n t h e s a m e m a n n e r a s A C T H ( B o h u s , G i s p e n , a n d D e W i e d , 1973)* T h e m a i n d i f f e r e n c e b e t w e e n t h e s e t w o u n r e l a t e d p e p t i d e s a p p e a r s a t t h e t e r m i n a t i o n o f t r e a t m e n t . W i t h l y s i n e - v a s o p r e s s i n t h e r e i s p r e s e r v a t i o n o f t h e r e s p o n s e d e s p i t e c e s s a t i o n o f t r e a t m e n t . W i t h A C T H , a v o i d a n c e r e s p o n d i n g d e t e r i o r a t e s w i t h t r e a t m e n t c e s s a t i o n . I n i n t a c t r a t s , l y s i n e - v a s o p r e s s i n a p p e a r s t o h a v e n o e f f e c t o n t h e a c q u i s i t i o n o f a n a c t i v e a v o i d a n c e r e s p o n s e ( B o h u s , A d e r , a n d D e W i e d , 1972). B o h u s , A d e r a n d D e W i e d (1972) f o u n d t h a t l y s i n e -v a s o p r e s s i n r e s u l t s i n r e s i s t a n c e t o e x t i n c t i o n o f a c t i v e a v o i d a n c e b e h a v i o u r i f i n j e c t e d o n e h o u r p r i o r t o t h e f i n a l a c q u i s i t i o n s e s s i o n , b u t n o t i f a d m i n i s t e r e d 6 h o u r s p r i o r t o t h e s e s s i o n . T h e e f f e c t i s o n e o f l o n g - t e r m p r e s e r v a -t i o n o f t h e c o n d i t i o n e d r e s p o n s e , s i m i l a r t o A C T H b u t o f a l o n g e r d u r a t i o n i . e . t h e e f f e c t s o f A C T H a r e l i m i t e d t o a f e w h o u r s a f t e r a d m i n i s t r a t i o n , w h i l e t h o s e o f v a s o p r e s s i n l a s t f o r d a y s . T h e p r o c e s s o f v a s o p r e s s i n a c t i o n , a s w i t h A C T H , i s u n c l e a r . A v e r y t e n t a t i v e h y p o t h e s i s w h i c h c a n b e a d v a n c e d i s t h a t v a s o p r e s s i n m a y b e i n v o l v e d i n t h e c o n s o l i d a t i o n o f 15 a v e r s i v e l y m o t i v a t e d b e h a v i o u r . L a n d e ( 1 9 7 2 ) h a s p r o v i d e d s o m e e v i d e n c e f o r t h i s i n m i c e b y s h o w i n g t h a t d e s g l y c i n a m i d e l y s i n e - v a s o p r e s s i n s t i m u l a t e s c o n d i t i o n e d a v o i d a n c e a c q u i s i -t i o n i n h y p o p h y s e c t o m i z e d r a t s a n d a l s o p r e v e n t s t h e s u p p r e s -s i o n o f m e m o r y b y p u r o m y c i n . ( G ) M e l a n o c y t e S t i m u l a t i n g H o r m o n e ( M S H ) M S H i s r e l e a s e d f r o m t h e p a r s i n t e r m e d i a ( V a n d e V e e r d o n k , 1 9 6 7 ) i n r a t s a n d i s b e s t k n o w n f o r i t s p i g m e n t a r y e f f e c t s i n a m p h i b i a n s ( T u r n e r a n d B a g n a r a , . 1 9 7 1 ) • S i n c e h y p o p h y -s e c t o m y r e m o v e s t h e s o u r c e o f M S H , i t h a s a l s o b e e n i m p l i c a t e d t o b e i n v o l v e d i n a v o i d a n c e l e a r n i n g . S e c o n d l y t h e c h e m i c a l s t r u c t u r e o f M S H i s s i m i l a r t o A C T H a n d t h e r e f o r e w a s t h o u g h t t o p o s s e s s s i m i l a r b e h a v i o u r a l e f f e c t s . T h e s e s i m i l a r i t i e s h a v e b e e n r e p o r t e d b y d i f f e r e n c e s a l s o e x i s t . ^ — M S H r e s t o r e s t h e r a t e o f a v o i d a n c e a c q u i s i t i o n i n h y p o p h y s e c t o -m i z e d r a t s i n a m a n n e r s i m i l a r t o A C T H ( D e W i e d , 1 9 6 5 ) . B o t h 06- a n d P - M S H i n c r e a s e r e s i s t a n c e t o e x t i n c t i o n i n a s i m i l a r f a s h i o n a s A C T H ( K a s t i n , M i l l e r , N o c k t o n , S a n d m a n , S c h a l l y , a n d S t r a t t o n , 1 9 7 3 ) • S a n d m a n , K a s t i n a n d S c h a l l y ( 1 9 6 9 ) h a v e a l s o s h o w n t h a t M S H l e a d s t o i n c r e a s e d r e s i s t a n c e t o e x t i n c t i o n o f a p p e t i t i v e t a s k s a n d f a s t e r r e v e r s a l i n a s i m p l e r e v e r s a l t a s k . B u t t h e s e e f f e c t s a r e n o t c o n s i s t e n t s i n c e t h e r e i s a l s o e v i d e n c e f o r a l a c k o f a n M S H e f f e c t o n a n u m b e r o f c o m p l e x t a s k s s u c h a s c o m p l e x b r i g h t n e s s d i s c r i -m i n a t i o n ( S a n d m a n , M i l l e r , K a s t i n , a n d S c h a l l y , 1 9 7 2 ) , s p e c i e s d i f f e r e n c e s ( S a n d m a n , A l e x a n d e r , a n d K a s t i n , 1 9 7 3 ) t 16 active and passive avoidance with d i f f e r e n t v i s u a l cues and shock l e v e l s (Nockton, Kastin, Elder, and Schally, 1972) and on a verbal retention test (Kastin, M i l l e r , Gonzalez-Barcena, Hawley, Dyster-Aers, Schally, Parra, and Velasco, 1971). As with ACTH and vasopressin, the nature of MSH a c t i v i t y i s unknown. I t does not appear to influence general a c t i - ' v i t y (Nockton, Kastin, Elder, and Schally, 1972), the memory process ( M i l l e r and Kastin, unpublished) or perseveration i . e . lack of i n h i b i t i o n of a response tendency (Sandman, Kastin, and Schally, 1971), though some sort of i n t e r a c t i o n of these variables cannot be ruled out. There i s some e v i -dence MSH may influence the emotional state of an animal (Kastin et a l . , 1971) which may d i r e c t l y influence the learning of a task. (D) Gonadal Hormones One of the most important sources of hormones within the body i s the gonads. These glands produce androgens i n the male and estrogens and progestins i n the female. There i s an extensive l i t e r a t u r e pertaining to the role of these sex hormones i n a variety of behaviour patterns? sexual (Young, 1961) , emotional (Gray, 1971)» a c t i v i t y l e v e l s (Burke and Broadhurst, 1966; Anderson, 1 9 6 8 ) , aggressive behaviour ( C o l l i a s , 19^4), food and water intake ( T a r t t e l i n and Gorski, 1971), and l i q u i d preferences (Zucker, 1 9 6 9 ) . Despite the attention directed towards establishing 1 7 behavioural correlates of gonadal hormone function, r e l a -t i v e l y l i t t l e i s known about the role of these hormones i n learning. The following section w i l l present a review of those studies that have examined the relationship between learning and each of the major gonadal hormones. ( i ) Androgens There are a number of natural occurring androgens (testosterone, androstenedione, androstenediol, dihydro-testosterone, androsterone and 5 - -androstanediol), and recent evidence has shown that they have d i f f e r e n t e f f e c t s on sexual behaviour. For example, dihydrotestosterone does not i n i t i a t e sexual behaviour i n castrated male rats (Luttge and Nicholas, 1 9 7 3 ) t whereas i t i s highly potent i n stimu-l a t i n g penile and accessory sex organ growth. This example i d e n t i f i e s an important variable that must be controlled for i n a l l studies on hormones and behaviour, namely the s p e c i f i c chemical structure of the substance employed as an independent variable. To avoid confusion on t h i s issue, the following discussion w i l l focus on the role of a s p e c i f i c androgen: testosterone (T) or i t s synthetic analogue testo-sterone propionate (TP). I t might also be added that the s i t u a t i o n becomes even more complex when s t r u c t u r a l simi-l a r i t i e s between each major class of gonadal hormones (androgens, estrogens and progestins) are considered (Ryan, Naf t o l i n , Reddy, Flores, and Petro, 1 9 7 2 ) , e s p e c i a l l y i n view of the s i g n i f i c a n t l e v e l s of each i n both sexes (Bidlingmaier, Wagner-Barnack, Burtenandt, and Knorr, 1 9 7 3 ) . 18 One of the few adult animal studies to examine the role of androgens i n learning situations has employed pigeons, a species that forms a dominance hierarchy which i n turn pre-sumes an organism's a b i l i t y to discriminate among members of the group i n a complex manner. The authors i n f e r that since dominance i n a group i s correlated with T and aggression (Lumba, 1972), the a b i l i t y to learn the complex discrimina-t i o n should somehow involve T. In four t r a i n i n g paradigms (no treatment, conditioning to peck at a target b i r d , T-alone, conditioning plus T) the group provided with con-d i t i o n i n g plus T pecked opponents more than the conditioning group and moved up the dominance hierarchy. The control and T-alone (injected) groups did not d i f f e r s i g n i f i c a n t l y from pre - t r a i n i n g l e v e l s . The authors believe that the group receiving the con-d i t i o n i n g procedure plus testosterone learned the discrimina-t i o n of other pigeons i n the f l o c k to a higher degree than the no treatment, testosterone only and conditioning only groups. There i s ample evidence that T i s involved i n the aggressive behaviour of pigeons and other species (Guhl, 1961), but i t s function i n a discriminatory r o l e has not been shown. This leads to an alternative explanation of the res u l t s obtained, which i s that T may be exerting i t s e f f e c t by changing the pigeons' aggressive q u a l i t i e s while the con-d i t i o n i n g i t s e l f i s influencing the learning process. The finding that the group treated with the conditioning only procedure also moved up the dominance hierarchy (though not to the degree of the conditioning plus testosterone group) supports this hypothesis. On the other hand, the members i n the testosterone only treated group, did not change from pre-treatment l e v e l s , although other studies have shown that T i n j e c t i o n s alone lead to movement of members up the domi-nance hierarchy and castration has led to movement down the dominance hierarchy (Guhl, 1 9 6 1 ) . Another d i f f i c u l t y with the authors' explanation of these findings i s that even i f the conditioning plus testosterone group are performing better due to enhanced discrimination a b i l i t i e s , associa-t i v e processes need not be involved at a l l . Enhanced d i s -crimination could be a function of a number of variables, i . e . arousal, attention. The s i t u a t i o n with respect to androgen in j e c t i o n s and human learning i s equally questionable. In humans, the main reported e f f e c t s of testosterone have been on automa-ti z e d behaviours. These are behaviours that have been highly practised and minimum mental and physical e f f o r t i s required, i . e . balance, walking, reading (Klaiber, Brover-man, and Kobayashi, 1 9 6 7 ) . Infusion of T has a p o s i t i v e e f f e c t on s e r i a l subtraction performance (Klaiber, Brover-man, Vogel, Abraham, and Cone, 1 9 7 1 ) . Again, the e f f e c t s of T i n j e c t i o n s may not be on the memory process, since the tasks T injections f a c i l i t a t e must be r e p e t i t i v e . The e f f e c t s of T could therefore r e f l e c t changes i n the arousal l e v e l , a ttentional cues or the perseverance of a response as much as on the memory component of learning. 20 ( i i ) Estrogens The main evidence f o r the role of estrogens i n learning comes from studies on avoidance conditioning. Again, as f o r the role of androgens, there i s no evidence f o r estrogens having a d i r e c t e f f e c t on the learning process i t s e l f . Males perform more poorly i n a two-way shuttle-box than females and t h i s performance i s a U-shaped function of the shock i n t e n s i t y . Females, on the other hand, have also been shown to have a more rapid escape reaction to a .3 mA of shock (Beatty and Beatty, 1 9 7 0 ) . These differences are thought to be due to the fac t that females have lower f l i n c h and jump thresholds to e l e c t r i c shock than males. Before these differences are sol e l y attributed to d i f f e r -ences i n shock s e n s i t i v i t y , i t should also be mentioned that T i n j e c t i o n s i n adult ovariectomized or in t a c t females raises the f l i n c h and jump thresholds to those of males and yet the females s t i l l acquire the active avoidance response more quickly. This suggests that the lowered s e n s i t i v i t y to shock i n females i s not the only f a c t o r involved i n acquiring active avoidance f a s t e r . These differences may be due to larger adrenal and p i t u i t a r y glands i n females as compared with males and also to higher corticosterone lev e l s (Swanson and van der Werff ten Bosch, 1 9 6 3 ) . Furthermore, estrogens also have excitatory effects on the central nervous system, therefore heightened arousal to the shock may also be involved i n enhanced a c q u i s i t i o n by female rats (Woodbury and Vernadakis, 1 9 6 7 ) . Though Beatty et a l . 21 (1970) found that ovariectomy had no e f f e c t on female a c q u i s i t i o n of avoidance responding, they may not have waited long enough f o r estrogen to have diminished from the system (2 weeks). Again we see the d i f f i c u l t y of tryi n g to correlate a change i n response a c q u i s i t i o n with any one underlying hormonal process. In an appetitive s i t u a t i o n Beatty (1973) has found that females acquire a DRL-20 schedule more rapidl y than males. Beatty (1973) abolished t h i s sex difference by ovariectomy while gonadectomy i n males had no e f f e c t on t h e i r performance. Recently Wong and Wilson ( i n prepara-tion) have observed the same difference between males and females on DRL-5 and DRL-10 schedules. ( i i i ) Progesterone The main e f f e c t of progesterone i n learning situations has been to f a c i l i t a t e the extinction of a conditioned avoidance response i n a manner si m i l a r to adrenocorticoid steroids (Wimersma Greidanus, 1970). Direct implants of progesterone have a f a c i l i t o r y e f f e c t on extinction i f placed i n various medial posterior thalamic areas. These eff e c t s are s i m i l a r to those observed a f t e r glucocorticoid implants i n the brain (Wimersma Greidanus, 1970). The f a c i l i t o r y e f f e c t of progesterone could be due to i t s anesthetic properties (P*an, Gardocki, Hutcheon, Rudel, Kodet, and Lauback, 1955)» even though the two doses used (.2 and 1.0 mg) had no e f f e c t on ambulation or escape speed 22 i n a runway (Wimersma Greidanus, Wijnen, Deurloo, and De Wied, 1973). Banerjee (1971) found that progesterone administration i n females during pregnancy led to a deterioration of conditioned avoidance behaviour. Since endogenous l e v e l s of progesterone are high during pregnancy i t may be that these excessive amounts had an anesthetic e f f e c t on the organism. (i v ) Conclusion The above findings on gonadal hormones and learning are l i m i t e d i n scope and generalizations at t h i s time are there-fore premature. S i m i l a r l y , the evidence and problems d i s -cussed f o r each of the hormones leads to the conclusion that further research i s required before we can state that these hormones d i r e c t l y influence the learning process. At pre-sent i t appears that a number of the hormones f a c i l i t a t e learning i n adult animals perhaps through a va r i e t y of mechanisms such as arousal (Woolley et a l . , I960), attention (Lumla, 1971)» changes i n motivational l e v e l (Beatty et a l . , 1970) and changes i n discriminative cues (Banerjee, 1971). The question remains as to whether a d i r e c t change i n the anatomy or biochemistry of the brain could possibly account for these e f f e c t s , though t h i s cannot be ruled out at t h i s time. 23 ( 2 ) Developmental Neuroendocrinology and Learning Hormones play an important role i n the development of an organism (Schapho, 1 9 7 1 ) . In addition to t h e i r importance i n determining morphological ontogeny these substances also appear to be a major factor i n the development of adaptive behaviour patterns e s s e n t i a l f o r the s u r v i v a l of an organism. Perhaps the best example of t h i s dual relationship can be found i n the experiments on sex hormones (mainly androgens) and reproductive function, i n i t i a t e d by Harris ( 1 9 6 4 ) . These findings showed that androgens have an organizational influence on the central nervous system and behaviour i n the prenatal or neonatal period of l i f e and an a c t i v a t i o n a l role l a t e r i n l i f e . Therefore the presence or absence of androgens at a c r i t i c a l time period determines i f the organism w i l l have a stronger d i s p o s i t i o n towards male or female behaviour patterns. This c r i t i c a l period varies across species due to the length of t h e i r gestational period. In rats, which have a short gestation, the c r i t i c a l time period i s between days 1 to 5 of age. On the other hand, i n guinea pigs with a longer gestational period, the c r i -t i c a l period occurs before b i r t h (Burns, 1 9 6 1 ) . Non-reproductive behaviours such as general locomotor a c t i v i t y (Swanson, 1 9 6 7 ; Gray, Lean, and Keynes, 1 9 6 8 ) , aggression (Edwards, 1 9 6 8 ; Edwards, 1 9 6 9 ) , food and water intake (Swanson and Van der Werff Ten Bosch, 1 9 6 3 ) and l i q u i d preferences (Zucker, 1 9 6 9 ) are also influenced by neonatal 24 l e v e l s of sex steroids. One other area of adaptive behavi-our that i s s t a r t i n g to receive a great deal of attention and appears to be influenced by neonatal hormones i s the animal's a b i l i t y to form new behaviour patterns on the basis of p r i o r experience ( i . e . learning). This section w i l l therefore provide a summary of recent experiments on neo-natal hormone l e v e l s and subsequent learning a b i l i t y . (A) Thyroxine and C o r t i s o l Although most of the research to be described deals with gonadal hormones, some of the i n i t i a l studies on neo-natal hormone manipulation and learning investigated the e f f e c t s of early i n j e c t i o n s of thyroxine and C o r t i s o l . There i s general agreement that both hormones can have a deleterious e f f e c t on physical development when injected between postnatal days 1 -4, and some evidence suggests that both hormones also a f f e c t behavioural development (Schapiro, 1968; Schapiro, Salas, and Vukovich, 1970; Davenport and Gonzalez, 1 9 7 3 ) . Thyroxine injections slowed the growth rate, but on the other hand they advanced the development of the s t a r t l e r e f l e x and swimming behaviour, while accelerating the EEG development and i t s response to novel stimuli (Schapiro, 1 9 6 8 ) . Thyroxine inj e c t i o n s also increase oxygen consump-ti o n , protein synthesis and brain cholesterol i n the infant but not the adult rat (Schapiro, 1 9 6 8 ) . The e f f e c t s of thyroxine on learning a b i l i t i e s are controversial. Excess 25 thyroxine on one hand led to enhanced a c q u i s i t i o n of a con-ditioned avoidance response, at 16 days of age, but between 35-^5 days of age there was a d e f i c i t i n learning a swimming maze and a Lashley III maze (Schapiro, 1 9 6 8 ) . In adult r a t s , Davenport and Gonzalez ( 1973) showed that thyroxine i n j e c t i o n s on postnatal day 2 , 3 and 4 led to i n f e r i o r performance by t h i s group over the control group on c l o s e d - f i e l d maze tasks at day 70 and 150 of age. Eayrs ( 1964) s i m i l a r l y found per-formance d e f i c i t s i n neonatal thyroxine injected rats i n a Hebb-Williams maze series when tested at 100 days of age. In view of these observations of learning decrements i n adolescent and adult subjects, the previous reports of bene-f i c i a l e f f e c t s of thyroxine administration on learned behavi-our at day 16 may be due to an e a r l i e r maturation of loco-motor functions, i . e . f a c i l i t a t e d swimming and r e f l e x i v e behaviour, rather than associative processes per se. Neonatal hypothyroidism has the opposite e f f e c t s of excess thyroxine on a number of p h y s i o l o g i c a l and behavioural patterns such as s t a r t l e r e f l e x (Eayrs and Lishman, 1 9 5 5 )1 avoidance learning (Eayrs and Levine, 1 9 6 3 ) , EEG patterns (Bradley, Eayrs, and Schmalback, i 9 6 0 ) and eye-opening and neurochemical maturation (Balazo, Cocks, Eayrs, and Kovacs, 1971)• On the other hand neonatal hypothyroidism produces impairments i n maze-learning that are s i m i l a r to behavioural d e f i c i t s produced by neonatal thyroxine injections (Daven-port and Dorcey, 1 9 7 2 ) . One possible explanation may be 26 found i n the recent reports of decreased c e l l counts at 35 days of age (Balazs et a l . , 1971). and reduced synapto-genesis i n 3 ° day old rats (Nicholson and Altman, 1972), i n both hypo- and hyperthyroid states. The l i m i t e d findings on C o r t i s o l administration and learning suggest at best a s l i g h t f a c i l i t a t i o n e f f e c t i n r a t s . Neonatal inj e c t i o n s led to s l i g h t l y improved learning on a Lashley III maze and a bar-pressing task at 30 days of age (Schapiro, 1 9 6 8 ) . However t h i s i s incon-s i s t e n t with other detrimental effects of early C o r t i s o l administration, i . e . retarded growth rate, s t a r t l e r e f l e x , swimming a b i l i t y and EEG evoked p o t e n t i a l response along with a delayed RNA and DNA increase (Schapiro, 1968). Therefore the reason f o r a s l i g h t enhancement on the learning tasks used i s unknown. Perhaps an explanation could be found by studying a c t i v i t y l e v e l s , as i t i s generally accepted that immature rats (prepuberal) are more active than older animals (Campbell and Mabry, 1972). Campbell has shown that rats between 15 and 25 days of age are 10 times as active as adult r a t s . I t could therefore be possible that the physiological d e f i c i t s produced by neonatal c o r t i c o s t e r o i d i n j e c t i o n s slowed the maturation process which i n turn resulted i n a higher a c t i v i t y l e v e l . Some further evidence f o r t h i s hypothesis comes from the finding that early hypercorticism i n mice leads to hyper-a c t i v i t y at 14-26 days of age but i s not present a f t e r 2 months of age (Howard and Granoff, 1968). 27 (B) Gonadal Hormones Most of the research concerned with neonatal hormones and learning has focused on gonadal hormones. In p a r t i c u l a r , attention has been directed towards the organizational influence of p e r i n a t a l androgen l e v e l s because, as men-tioned previously, t h i s appears to be the major factor i n determining the development of neural structures c o n t r o l l i n g male and female patterns of behaviour. Since ample evidence exists f o r the role of androgens i n the organization of a variety of behaviour patterns, the question arose as to whether th i s process may underlie sex differences i n learning a b i l i t i e s (Dawson, 1972). In one of the e a r l i e s t tests of t h i s hypothesis, Dawson (1972) i n an unpublished paper reported that neonatal injections of estrogen into castrated males and androgen injections into ovariectomized females reversed the genotypic learning pattern i n adulthood. S p e c i f i c a l l y , males perform better at i n h i b i t o r y perceptual restructuring tasks than females, while females perform better on simple perceptual motor tasks (Broverman, Klaiber, Kobayashi, and Vogel, 1968). From these r e s u l t s , Dawson concluded that early testosterone i n j e c t i o n s i n ovariec-tomized females led to what he referred to as male patterns of learning (higher s p a t i a l maze learning a b i l i t y ) , while estrogen inj e c t i o n s administered to castrated males led to increased female learning patterns (higher wheel running a c t i v i t y ) . These findings are preliminary and have yet to appear i n p r i n t perhaps because of t h e i r questionable nature. 28 One of the more confusing aspects of t h i s data concerns the use of both androgen and estrogen as previous attempts to manipulate dimorphic behaviour have indicated a unique role f o r androgens. Removal of the ovaries i n neonatal females does not change female sexual or nonsexual behaviour to the male pattern (Levine, 1966). But i n the male, removal of the t e s t i s appears to induce female behaviour patterns with-out i n j e c t i o n s of estrogen being necessary. Therefore estro-gens appear unnecessary f o r these organizational influences. There i s also evidence that the ovaries are not functional i n the neonatal female and estrogen therefore i s not c i r c u -l a t i n g i n the system, bringing into question the l o g i c of i n j e c t i n g estrogen into castrated males, i n order to produce a feminized male. Equally important are the findings which suggest the presence of neonatal estrogens may lead to a male pattern of behaviour since they have been shown to disrupt normal female behaviour (Gorski, 1971)• I t should also be pointed out that Dawson's sole evidence of feminine learning patterns was obtained by measuring "wheel running". The adult female rat's wheel running performance fluctuates with the estrous cycle and can be diminished by ovariectomy, therefore wheel running appears to be an index of performance rather than learning. Therefore i t appears unwarranted f o r Dawson to r e f e r to wheel running as a cognitive task. In another approach to the question of hormonal corre-lates of learning, Beatty and co-workers have examined sex differences i n avoidance conditioning. As already mentioned, 29 males perform more poorly on active avoidance tasks than females (Beatty et a l . , 1970) and Beatty hypothesized that th i s difference may he due to the e f f e c t s of neonatal andro-gen i n the male r a t . Therefore female rats were injected with testosterone propionate or o i l at day 3 of age. As adults they were administered eit h e r estrogen or testosterone propionate following ovariectomy. They found that females injected with testosterone propionate as infants and i n adulthood performed more l i k e normal males i n an active avoidance test than did control females. Beatty suggests androgens have an organizing influence on male patterns of avoidance learning which i s sustained by endogenous le v e l s of androgen i n adults. This need f o r the presence of c i r -c u lating androgen i n adults becomes apparent when i t has shown that neonatal testosterone propionate inj e c t i o n s followed by estrogen injections i n adult females were shown to produce a female l e v e l of a c t i v i t y . S i m i l a r l y o i l treat-ment neonatally and testosterone propionate i n j e c t i o n s i n adulthood also led to female a c t i v i t y suggesting that neo-natal androgen l e v e l s are also necessary f o r a male l e v e l of active avoidance performance. I t would be of i n t e r e s t to see i f s i m i l a r r e s u l t s are obtained by neonatal estrogen treatment, since t h i s also disrupts normal female behaviour. It should also be mentioned that the r e s u l t s obtained may be due to the androgen injections r a i s i n g the response threshold to shock. 30 The e f f e c t s of neonatal androgen administration have also been shown to influence behaviour i n male chicks, a l -though the e f f e c t s are not permanent (Andrew and Rogers, 1972). Testosterone injections increased the persistence fo r searching f o r a p a r t i c u l a r type of food i n a p a r t i c u l a r place. This persistence includes both an a b i l i t y to pro-long attention on a l o c a l i z e d stimulus and also a decreased d i s t r a c t i b i l i t y by i r r e l e v a n t s t i m u l i . The researchers con-cluded that the e f f e c t s of testosterone are on the central nervous system mechanisms involved i n the recognition of s p e c i f i c s t i m u l i . Testosterone i s therefore thought to keep these mechanisms i n more persistent use. This perseverance i s thought to have an adaptive role during the breeding season when androgen l e v e l s are high. At t h i s time the males acquire and defend nest s i t e s and mates, therefore the high androgen l e v e l s would allow the males to acquire these objects i n the face of various d i s t r a c t i n g stimuli and also allow f o r enhanced recognition of p a r t i c u l a r s t i m u l i . The p o s s i b i l i t y of testosterone i n j e c t i o n s influencing associa-t i v e processes d i r e c t l y are remote, due to the finding that s i m i l a r i n j e c t i o n s i n female chicks have no e f f e c t on t h e i r search behaviour. Secondly, the f i n d i n g that the testos-terone treated males have lower body weights and that t h i s may be due to an increased persistence of search i n p a r t i c u -l a r areas with neglect of other areas questions the adapta-b i l i t y of testosterone i n j e c t i o n s . 31 Recently a possible c o r r e l a t i o n between early androgen stimulation and learning has been forwarded i n the human c l i n i c a l l i t e r a t u r e (Money and Lewis, 1 9 6 6 ) . These findings suggest that excess androgens or c l o s e l y related substances i . e . progestins, may augment i n t e l l i g e n c e i f present during the prenatal period since Money and others (Money, 1 9 7 1 ) have found a p o s i t i v e c o r r e l a t i o n between the presence of excessive l e v e l s of androgen (or progestins) i n the fetus and subsequent performance on I.Q. t e s t s . Excess androgen stimulation i n humans has been found to be a manifestation of adrenal cortex malfunctioning and secondly, by synthetic progestin i n j e c t i o n s i n pregnant females. Malfunctioning of the adrenal cortex has been referred to as the adreno-genital syndrome. In t h i s disorder the adrenal cortex instead of synthesizing cortisone secretes a hormone which acts l i k e an androgen (Money and Lewis, 1 9 7 1 ) . This disorder i s genetic i n nature and occurs i n both males and females, causing the female to exhibit male sex organs and premature puberty i n both males and females. Of primary i n t e r e s t i s the finding that many individ u a l s i n t h i s group were shown to have high I.Q. scores. Money also found no difference between the groups that were treated f o r the adrenogenital syndrome at b i r t h , i . e . cortisone replace-ment and in d i v i d u a l s which were not treated and therefore possessed high androgen l e v e l s postnatally. Similar corre-l a t i o n s have been obtained when children of mothers treated with synthetic progestin f o r pregnancy disorders were tested (Ehrhardt and Money, 1 9 6 7 ) . 32 II PURPOSE OF THE PRESENT INVESTIGATION Although there i s not a great deal of research concerned with neonatal androgen l e v e l s and learning a b i l i t y , there are indications of a possible f a c i l i t o r y e f f e c t which war-rants further i n v e s t i g a t i o n . This i n d i c a t i o n i s derived p r i -marily from the persistence behaviour of chicks following testosterone i n j e c t i o n s (Andrew and Rogers, 1972) and the c o r r e l a t i v e data on humans described by Money et a l . (1966, 1971). Although highly speculative, both f i e l d s provide an impetus f o r investigating the relationship between elevated p e r i n a t a l testosterone l e v e l s and subsequent learning a b i l i t y . F u l l y aware of the need fo r c a r e f u l l y controlled procedures the following experiments sought to examine the e f f e c t s of neonatal i n j e c t i o n s of testosterone on avoidance conditioning. Avoidance conditioning paradigms were employed because of the greater degree of control offered by these procedures over a v a r i e t y of confounding variables. This point i s p a r t i c u l a r l y s i g n i f i c a n t i n studies dealing with the develop-ing animal. In contrast to avoidance paradigms, appetitive situations r e l y on the a b i l i t y to lower the animal's body weight to a s p e c i f i e d c r i t e r i o n , or to deprive the animal of water, both of which can have profound e f f e c t s on the physiology of a developing organism. Motivation l e v e l s are also hard to specify i n r a p i d l y growing organisms as i t i s impossible to a t t a i n stable body weight l e v e l s p r i o r to i n i t i a t i n g the experimental t e s t i n g . Avoidance paradigms 33 appear to overcome these major d i f f i c u l t i e s while at the same time permitting t e s t i n g over a short period of time. Due to the c r i t i c i s m l e v e l l e d against the previous studies dealing with hormone manipulation and learning, a number of control procedures were incorporated into the pre-sent study. Since motivational l e v e l s are e a s i l y modified by environmental (Beatty et a l . , 1970 ) or chemical (Beatty et a l . , 1970 ) manipulation, two control procedures were used to measure f o r motivational changes due to our treatment, ( i ) open-field a c t i v i t y and ( i i ) r e a c t i v i t y to shock. Different avoidance paradigms were also employed to observe whether task differences would influence the acqui-s i t i o n of the avoidance response. The tasks employed w e r e i ( i ) one-way active avoidance, ( i i ) passive avoidance i n a runway, and ( i i i ) step-down passive avoidance task. Previous reports of neonatal hormone injec t i o n s on learning have found d i f f e r e n t e f fects as a function of age. Consequently, two age l e v e l s were employed i n the present experiment. Hopefully these controls w i l l allow us to specify whether excess neonatal androgen l e v e l s have an influence on sub-sequent learning. 34 III EXPERIMENT I - ACTIVE AND PASSIVE AVOIDANCE The f i r s t experiment was designed to examine the ef f e c t s of neonatal androgen administration on the acqui-s i t i o n of both active and passive avoidance responses. In sel e c t i n g the appropriate dose of testosterone propionate (TP), a dosage with known eff e c t s on the central nervous system (CNS) and behaviour was required. Consequently a dose of 125 ug per day f o r 5 days was selected as t h i s amount has been shown to induce s t e r i l i t y and a tendency towards higher l e v e l s of masculine behaviour when injected into newborn females (Barraclough, 1967). Method Subjects 1 Subjects (Ss) consisted of 20 male albino rats of the Wistar s t r a i n raised from 5 l i t t e r s bred i n our colonies. Ten Ss were injected with 125 ug of testosterone propionate i n vegetable o i l (100 mg/cc) d a i l y f o r the f i r s t 5 postnatal days. The other 10 Ss (controls) were injected with an equal dose of vegetable o i l d a i l y (12.5 u l ) . At 21 days of age the Ss were weaned. They were housed i n i n d i v i d u a l s t a i n l e s s s t e e l cages with food and water available ad libitum. On days 22 and 23» the animals were handled f o r a 5 minute period and at 24 days of age, f a m i l i a r i z e d to the apparatus for a 15 minute period. Apparatus 1 The apparatus consisted of an aluminum shuttlebox (18 i n . x 5.5 i n . x 10 in.)-. Both one-way active avoidance and passive avoidance tasks were run i n t h i s apparatus. A g u i l l o t i n e door divided the shuttlebox into 2 compartments. One side was painted white and the other black. Footshock was delivered v i a a Lafayette shocker (model #5226) to the f l o o r of the shuttlebox, which con-s i s t e d of 1/8 i n . copper rods placed l / 2 i n . apart. Procedure > One-way active avoidance t e s t i n g was begun at 25 days of age. This consisted of 10 t r i a l s a day u n t i l c r i t e r i o n was reached. The c r i t e r i o n used f o r successful a c q u i s i t i o n was 9 correct responses out of 10 on 2 consecu-t i v e days. Each S was placed i n the white compartment of the shuttlebox. Five seconds l a t e r the g u i l l o t i n e door was raised and the animal given 10 seconds to cross to the safe side. I f the S did not cross i n t h i s time, 1 .0 mA of shock was administered v i a the g r i d f l o o r . The S remained on the safe side f o r 30 seconds, and then the next t r i a l was i n i t i a t e d . The passive avoidance task was run i n one day; the day a f t e r the active avoidance c r i t e r i o n had been reached. To overcome any problem of freezing, t e s t i n g began with an i n i t i a l active avoidance t r i a l . The passive avoidance t r i a l s consisted of placing the animal i n the white compartment of the box and r a i s i n g the door 5 seconds l a t e r . I f the animal crossed to the black side, i t received a shock of 1 .0 mA u n t i l i t returned to the white compartment. A correct res-ponse consisted of remaining i n the white compartment f o r 4-5 seconds with the g u i l l o t i n e door raised. The animal was 36 removed from the shuttlebox between each t r i a l f o r an i n t e r -t r i a l i n t e r v a l of 60 seconds. The c r i t e r i o n f o r learning was 9 consecutive correct responses. The retest f o r both tasks was i n i t i a t e d at 100 days of age. The animals had not been disturbed since completing the passive avoidance task. Except f o r two changes, the procedure was i d e n t i c a l to those employed e a r l i e r . The larger, mature animals required a larger shuttlebox (36 i n . x 11 i n . x 20 i n . ) . The CS-UCS i n t e r v a l was reduced from 10 to 5 seconds. After completion of retesting, the animals were weighed and s a c r i f i c e d i n a COg chamber. Gonads and adrenals were excised quickly as possible and weighed on a Mettler scale (model H6Tdig) to the nearest . 0 1 mg. Results Active avoidance 1 The TP-injected group reached c r i t e r i o n s i g n i f i c a n t l y f a s t e r than the o i l - t r e a t e d group (t = 1.81, df = 18j p *c . 0 5 one-tailed). Experimental animals met c r i t e r i o n i n an average of 4-3 t r i a l s while the o i l - t r e a t e d group required 63 t r i a l s (see Figure 1 ) . Latency to cross on the f i r s t 30 t r i a l s d i f f e r e d between the 2 groups. The TP-treated group had a mean latency of 1 .37 seconds over the f i r s t 30 t r i a l s while the o i l - t r e a t e d group had a mean latency of 1 . 9 3 seconds (t = 2 . 6 6 , df = 5 8 ; p C . 0 5 ) (see Table 1 ) . The TP-treated group also made s i g n i f i c a n t l y more correct responses over the f i r s t 30 t r i a l s (t = 1 . 9 0 , df = 5 8 ; p <^ . 0 5 ) (see Table 2 ) . F i g . 1. T r i a l s t o c r i t e r i o n ( M e a n - S t a n d a r d E r r o r o f t h e M e a n ) i n a o n e - w a y a c t i v e a v o i d a n c e t a s k i n i t i a t e d a t 25 d a y s o f a g e a n d r e t e s t e d a t 100 d a y s o f a g e . S u b j e c t s w e r e i n j e c t e d w i t h e i t h e r 100 u g t e s t o s t e r o n e p r o p i o n a t e (T.P.) o r o i l f o r t h e f i r s t 5 d a y s o f l i f e . 38 1 2 0 f 11 O f ioo\ 90\ 80\ 7 0 r T r i a l s t o 6 0 f C r i t e r i o n < X ) 5 0 4 0 T ±3.67 ±8.98 r±3.78 ±4.41 3 0 f 2 0 h 1 0 T. P. O i l 2 5 D a y s o f A g e T. P. O i l 1 0 0 D a y s o f A g e 39 Table 1 Mean Latency to Gross to the "Safe" Side After Raising the G u i l l o t i n e Door; Over the F i r s t 30 T r i a l s T r i a l Group 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 14 1 ? T.P. 5.0 9.0 8.1 6.3 6.8 8.4 7.0 8.7 3.8 1.6 6.6 7.2 6.5 4.6 4.5 O i l 7.1 9.9 8.3 8.6 9.0 7.6 8.0 6.4 5.3 6.9 10.2 8.5 6.9 7.5 6.2 T r i a l Group 16 17 18 19 20 21 22 2 3 24 25 26 27 28 29 30 T.P. 1.8 2.2 1.7 .5 5.4 2.1 1.1 1.4 1.9 1.8 .6 2.0 1.6 3.2 3.3 O i l 3.9 3.8 2.9 3.8 3.2 7.7 5.3 3.0 5.8 2.4 4.2 2.8 3.4 3.4 2.0 Table 2 Number of Correct Responses Over the F i r s t 30 T r i a l s . Ten Subjects per Group T r i a l Group 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 14 15 16 17 18 19 20 2 1 22 2 3 24 25 26 27 28 29 30 T.P. 8 2 5 6 5 5 7 4 8 1 0 4 4 5 8 7 1 0 9 1 0 10 1 0 7 10 1 0 9 9 9 8 9 8 1 0 O i l 7 3 3 3 2 4 4 5 7 5 3 3 6 7 6 6 9 1 0 1 0 5 8 8 7 1 0 9 10 9 8 9 4 Passive avoidance: No s i g n i f i c a n t difference was observed between the experimental and control groups (t = 1 . 5 2 , df = 18; p > . 0 5 ) . Retest : There were no s i g n i f i c a n t differences between the 2 groups on t r i a l s to c r i t e r i o n on eit h e r active or passive avoidance tests when animals were retested at 1 0 0 days of age (t = .14, df = 14; p > .05 active avoidance and t = .34, df = 14} p > ,05 passive avoidance). Body, adrenal and gonadal weights: There were no s i g n i f i c a n t differences between the body weights of the two groups (t = . 6 2 , df = 17? p ? . 0 5 ) or t h e i r adrenal weights (t = .43, df = 1 7 ; p > .05) at 1 0 0 days of age. The gonads of the TP-treated group weighed s i g n i f i c a n t l y less than the control group (t = 6 . 0 1 , df = 1 7 ; p < . 0 5 ) . Table 3 shows the mean values f o r the above measures. Discussion The r e s u l t s suggest a f a c i l i t a t o r y e f f e c t of excessive neonatal TP on active avoidance learning i n prepuberal r a t s . I t should be mentioned that the f i n d i n g of no s i g n i f i c a n t differences on the passive avoidance task suggests the ef f e c t s of neonatal TP may a f f e c t only active avoidance learning. However, before one can conclude that the f a c i -l i t a t e d a c q u i s i t i o n i s s o l e l y attributable to learning, there are a number of experiments which must be undertaken to investigate whether neonatal TP influences other variables than just learning, the most noted of which are: 42 Table 3 E f f e c t of Testosterone Propionate (TP) Administration During Postnatal Days 1-5 on Body Weights and Gonadal and Adrenal Organ Weights of the Adult Male Rat Treatment Body wt., g Organ wt., mg testes adrenals Organ wt., body testes mg/100 g wt. adrenals T.P. 3 9 3 . 5 0 * -15.41 2 2 5 2 . 3 8 5 2 . 7 3 ± 1 6 8 . 0 3 ± 4 . 0 7 5 9 3 . 0 6 ± 3 5 . 0 3 13.81 ± 0 . 7 0 O i l 3 8 0 . 3 3 ±14.57 3 4 5 5 . 5 4 46.82 ± 8 3 . 9 8 ±3.28 8 8 6 . 5 3 ± 3 2 . 5 4 1 1 . 9 3 ± 0 . 7 9 * Mean - Standard Error ( i ) a single neonatal i n j e c t i o n of TP ( i i ) general a c t i v i t y changes ( i i i ) changes i n footshock s e n s i t i v i t y . The decreased gonadal size of the TP-injected group at 100 days of age confirms previous observations (Rubenstein and Kurland, 1 9 4 1 ) and suggests that increases of neonatal androgen l e v e l s can have a permanent e f f e c t on gonadal development either d i r e c t l y on gonadal tissue or i n d i r e c t l y through i t s action on the CNS. LL IV EXPERIMENT II - ACTIVE AND PASSIVE AVOIDANCE LEARNING Experiment I provides a clear i n d i c a t i o n of enhanced active avoidance behaviour i n male rats injected neonatally with TP, but the question remains as to whether the e f f e c t observed i s due to injections of TP over the f i r s t 5 days of l i f e as observed i n Experiment I or whether a single TP i n j e c t i o n at day 1 of b i r t h would have a s i m i l a r e f f e c t . Therefore the following experiment was conducted to examine the e f f e c t s of a single neonatal i n j e c t i o n on subsequent avoidance learning. In Experiment I, both experimental and control animals displayed rapid a c q u i s i t i o n of the active avoidance responses, therefore a lower shock i n t e n s i t y was employed i n the following experiment i n order to increase the number of t r i a l s to c r i -t e r i o n thereby enhancing group differences. Method Subjects: Subjects consisted of 2k male albino rats of the Wistar s t r a i n raised from 5 l i t t e r s i n our colonies. Twelve were injected with 1.25 mg of TP i n vegetable o i l (100 mg/cc) at day of b i r t h . The other Ss were injected with an equal volume of vegetable o i l (12.5 u l ) . At 21 days of age, a l l Ss were weaned. They were housed i n i n d i v i d u a l s t a i n l e s s s t e e l cages with ad libitum food and water. On day 22 and 23, a l l Ss were handled f o r a 5 minute period and f a m i l i a r i z e d to the apparatus on day 2k f o r a 15 minute period. Apparatus: Apparatus consisted of the shuttlebox and shocker used f o r prepuberal animals i n the previous experi-ment. Procedure t Ss were run i n the same manner as Experiment I with a few modifications. The Ss were given 5 seconds to cross to the safe side, once the g u i l l o t i n e door was raised (previous experiment - 10 seconds). There was no pretest at 100 days of age. Body, general and adrenal weights were taken at 42 days of age i n one-half of each group and at 100 days of age i n the other half of each group. The shock in t e n s i t y was also lowered to .80 mA. Results Active avoidance: The TP-injected group reached c r i -t e r i o n s i g n i f i c a n t l y f a s t e r than the control group requiring a mean of 41.77 t r i a l s as compared to 63.33 t r i a l s f o r the control group (t = 2.85i df = 22; p < .01)-. (see Figure 2). The TP group had a s i g n i f i c a n t l y shorter latency to cross to the safe side than the o i l - i n j e c t e d group over the f i r s t 30 t r i a l s (x = 1.01 seconds f o r the TP group; x = 1.31 seconds fo r the o i l - t r e a t e d group, t = 1.94, df = 58; p *C .05). The TP-injected group also made more correct responses over the f i r s t 30 t r i a l s than the control group (t = 3.32, df = 22; p < .01) (see Table 4). Passive avoidance i There was no difference between the TP and o i l - i n j e c t e d groups on the passive avoidance tasks (t = .40, df = 22; p > .05). F i g . 2 T r i a l s to c r i t e r i o n i n a one-way active avoidance task i n i t i a t e d at 25 days of age. Subjects were injected with either 1.25 mg T.P. or o i l at one day of age. 1 2 0 1 1 0 -1 0 0 -9 0 -8 0 -7 0 -T r i a l s t o 6 0 | -C r i t e r i o n <*> 5 0 -4 0 -3 0 2 0 1 0 ±3.66 ±6.67 T . P . Table 4 Number of Correct Responses Over the F i r s t 30 T r i a l s . Twelve Subjects per Group T r i a l Group 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 14 1 5 16 17 18 1 9 20 21 22 23 24 25;<26 27 28 29 30 T.P. 6 0 6 1 6 5 7 6 7 8 7 7 7 9 9 1 2 1 1 1 1 1 1 1 0 4 1 0 1 1 12 1 0 12 1 1 12 1 0 1 2 O i l 9 2 1 1 0 1 2 4 3 7 1 2 8 7 8 1 0 7 9 10 1 1 7 7 8 9 1 0 11 9 1 0 1 0 1 1 4 9 Body, gonadal and adrenal weights? Body weights d i f f e r e d s i g n i f i c a n t l y between the 2 groups at age 42 (t = 4 . 7 5 , df = 9; p < .01) but not at 1 0 0 days of age (t = . 1 3 , df = 9} p > . 0 5 ) . Gonadal weights of the TP injected group were smaller at both 42 days of age (t = 6 . 4 7 , df = 9? p < . 0 1 ) and 1 0 0 days of age (t = 2 . 9 3 . df = 9; p < . 0 1 ) , Adrenal weights did not d i f f e r s i g n i f i c a n t l y between the groups at both ages studied (t = , 5 0 , df = 9; p > .05 at 42 days of age; t = . 7 3 . df = 9; p > .05 at 1 0 0 days of age). Table 5 gives the mean values f o r the various measures. Discussion The r e s u l t s on the active avoidance task r e p l i c a t e the previous findings i n Experiment I. This was accomplished with one i n j e c t i o n of TP at day one of b i r t h . I t can be seen i n Figure 2 that the r e s u l t s are s i m i l a r to those observed i n Experiment I (see Figure 1 ) , Again, differences on the passive avoidance task did not reach s i g n i f i c a n c e . The passive avoidance re s u l t s suggest three possible expla-nations : ( i ) TP injections do not influence passive avoidance learning ( i i ) the task i s too simple to allow differences to appear ( i i i ) the hormone manipulation may influence a c t i v i t y l e v e l s . 5 0 T a b l e 5 ( a ) E f f e c t o f 1.25 m g T P A d m i n i s t r a t i o n a t P o s t n a t a l D a y 1 o n B o d y W e i g h t a n d G o n a d a l a n d A d r e n a l O r g a n W e i g h t s o f t h e 4 2 D a y O l d M a l e R a t B o d y w t . , g O r g a n w t . , m g O r g a n w t . , m g / 1 0 0 g b o d y w t . t e s t e s a d r e n a l s t e s t e s a d r e n a l s T r e a t m e n t T . P . ± 4 . 3 2 1 5 6 . 0 0 1 1 3 5 . 5 5 2 7 . 6 5 7 2 6 . 8 5 ±34.81 ± . 2 7 ±48.79 1 7 . 7 8 1 7 9 . 6 0 2 1 5 3 . 9 9 28.98 1188.19 1 6 . 1 3 ±4.59 ±41.42 ±4.56 ± 5 1 . 8 6 ± 1 . 2 7 51 Table 5 (b) E f f e c t of 1.25 mg TP A d m i n i s t r a t i o n at P o s t n a t a l Day 1 on Body Weight and Gonadal and Adrenal Organ Weights of the 100 Day Old Male Rat Treatment Body wt., g Organ t e s t e s wt., mg adrenals Organ wt., mg/100 g body wt. t e s t e s adrenals T.P. 362.00 2280.54 50.54 638.54 13.98 ±12.03 ±111.07 ± 1 . 6 1 ± 8 4 . 7 ? ± 1 . 3 9 O i l 375.33 ± 2 8 . 1 6 3303.08 ±56.09 4 4 . 7 3 ± 1 . 3 9 892.68 ± 3 7 . 2 1 11.96 ±.86 52 The gonads weighed s i g n i f i c a n t l y less i n the TP-injected group at both kZ and 100 days of age, while there were no differences between the adrenal glands at both ages. These findings also r e p l i c a t e d those observed i n the previous experiment. At kZ days of age, the TP-treated animals weighed s i g n i f i c a n t l y l e s s than controls. This difference was not observed at 100 days of age suggesting the p o s s i b i l i t y that TP i n j e c t i o n s may retard the weight gaining e f f e c t s of TP at puberty. 5 3 V EXPERIMENT III - OPEN-FIELD BEHAVIOUR, REACTIVITY TO SHOCK Before enhanced a c q u i s i t i o n of active avoidance be-haviour observed i n Experiments I and II can be att r i b u t e d to learning, i t w i l l be necessary to determine whether the neonatal androgen treatment produces any changes i n reac-t i v i t y to footshock or i n general a c t i v i t y l e v e l s . The f a c i l i t a t i o n of active avoidance but not passive avoidance already suggests we are not dealing with a general e f f e c t on learning, and perhaps the differences are larg e l y due to a c t i v i t y . The maturation process i s p a r a l l e l e d by marked changes i n a c t i v i t y , with young animals ( 1 0 - 2 5 days) being much more active than older animals (Campbell and Marby, 1 9 7 2 ) . Any changes i n the maturation process induced by TP injec t i o n s could conceivably a f f e c t a c t i v i t y l e v e l s , since a number of other hormones have been shown to either retard or enhance the maturation process (Radouco-Thomas and Martin, 1 9 6 1 ; Heim, 1 9 6 6 ) . Furthermore, steroid hormones have been shown to influence r e a c t i v i t y to shock (Beatty et a l . , 1 9 7 2 ) , requiring the measurement of the variable i n the present series of experiments. Also i n the following experiment, an attempt was made to provide a more accurate estimate of the ef f e c t s of TP on body, gonadal and adrenal weights, by taking these measures between 26 and 3 1 days of age; the actual time of avoidance t r a i n i n g i n the previous experiments. 54 Method Subjects: Subjects consisted of 24 male albino rats obtained from 5 l i t t e r s i n our colonies. Treatment of Ss was i d e n t i c a l to those i n Experiment II u n t i l the 2 5 t h day of b i r t h . A l l Ss were run i n both the a c t i v i t y and reac-t i v i t y to shock t e s t s . After t h i s , a l l animals were weighed and then s a c r i f i c e d . Adrenal and gonadal weights were then recorded. Apparatus: Apparatus consisted of a black box ( 1 8 i n . x 18 i n . x 18 in.) i n which general a c t i v i t y was measured. The f l o o r was marked off into 16 equal squares. Reactivity to shock was examined i n a pl e x i g l a s s chamber ( 1 0 i n . x 10 i n . x 18 i n . ) . The f l o o r consisted of l / 8 i n . copper rods> 1/2 i n . apart. Shock was delivered v i a a constant current AC shock source. Procedure t On day 26 or 27 of age each S was placed i n one corner of the black box and the number of squares entered over a 5 minute period was recorded. On day 28 or 29 Ss were tested f o r differences i n shock r e a c t i v i t y . A l l animals were subjected to 6 series of e l e c t r i c shock. Each series con-s i s t e d of 6 shock i n t e n s i t i e s ( . 1 , . 2 , .4, . 6 , . 8 , 1 . 0 mA) presented i n an ascending-descending order. Each shock administration was followed by a 20 second i n t e r - t r i a l i n t e r v a l . After completion of a series, a 60 second i n t e r -series i n t e r v a l was i n i t i a t e d . Shock duration was . 2 sec. Following t h i s , body, adrenal, and gonadal weights were recorded on day 3° and 31, 55 Results A c t i v i t y measure: The TP-injected group crossed into s i g n i f i c a n t l y more squares than the o i l - t r e a t e d group (x = 9 6 . 1 ? f o r the TP group; x = 8 2 . 6 7 f o r the o i l group; t = 2 7 . 9 8 , df = 2 2 , p < . 0 1 ) . Reactivity to shock: I n i t i a l l y jump thresholds ( a l l k feet leave the ground) were to be observed. The Ss used i n t h i s study did not jump, but ran instead. Therefore the thresholds observed were redefined as "run" thresholds. Shock i n t e n s i t i e s below the "run" threshold normally lead to the ex h i b i t i o n of a f l i n c h response ( s t a r t l e or crouch). F l i n c h thresholds could not be determined as the shocker did not have a s u f f i c i e n t l y low i n t e n s i t y range. The TP-injected group had a mean "run" threshold of ,L9 mA, while the o i l - i n j e c t e d groups mean run threshold was .60 mA. This difference was s t a t i s t i c a l l y s i g n i f i c a n t (t = 6 . 1 1 , df = 2 2 ; p < . 0 1 ) . Body, gonad, and adrenal weights: There were no d i f f e r -ences between the two groups i n terms of body weight (t = . 7 0 , df = 2 2 ; p > . 0 5 ) . The adrenals of the TP-injected group weighed more than the o i l - t r e a t e d group (t = 2 . 2 3 , df = 2 2 ; p < . 0 1 ) . The gonads of the o i l - t r e a t e d group weighed s i g n i f i c a n t l y more than the TP-injected group (t = 5 2 . 7 5 » df = 2 2 ; p 3* . 0 1 ) . Table 6 provides the mean values f o r these measures. 56 Table 6 E f f e c t of 1 . 2 5 mg TP Administration at Postnatal Day 1 on Body Weight and Gonadal and Adrenal Organ Weights Between Day 30 and JI of Age Treatment Body wt., g Organ testes wt., mg adrenals Organ wt., body testes mg/100 g wt. adrenals T.P. 79.17 ±3.15 290.54 -43 .25 20.95 -.64 178 .26 -16.79 13.^1 ±.56 O i l 75.92 ±3 . 3 9 468.54 ±48.20 18.10 -.80 300.11 -22.11 11.98 ±.32 57 D i s c u s s i o n T h e s e r e s u l t s p o i n t t o a v a r i e t y o f f a c t o r s w h i c h m a y i n f l u e n c e t h e f a c i l i t a t e d a c q u i s i t i o n o f a c t i v e a v o i d a n c e r e s p o n s e s i n T P - i n j e c t e d a n i m a l s . F i r s t o f a l l , a c t i v i t y i n t h e o p e n - f i e l d w a s s i g n i f i c a n t l y i n c r e a s e d i n t h e T P t r e a t e d g r o u p a s c o m p a r e d t o t h e o i l - i n j e c t e d g r o u p . M o o r c r a f t , L y t l e a n d C a m p b e l l ( 1 9 7 1 ) h a v e f o u n d t h a t l o c o m o t o r a c t i v i t y o f n e o n a t e r a t s i n c r e a s e s b e t w e e n 15 a n d 2 0 d a y s o f a g e a n d d e c r e a s e s t o a d u l t l e v e l s b y d a y 2 8 . T h e p e a k b e t w e e n 15 a n d 20 d a y s o f a g e w a s n e a r l y 1 0 t i m e s t h a t a t 1 0 o r 2 8 d a y s . T h i s i s t h o u g h t t o b e d u e t o t h e e a r l i e r m a t u r a t i o n o f e x c i t a t o r y c a u d a l b r a i n s t r u c t u r e s o v e r i n h i b i t o r y r o s t r a l f o r e b r a i n s t r u c t u r e s s i n c e t h e b r a i n g r o u p i n a r o s t r a l -c a u d a l d i r e c t i o n u p t o 25 d a y s o f a g e ( M o o r c r a f t , 1 9 7 1 ) . I t i s t h e r e f o r e p o s s i b l e t h a t T P i n j e c t i o n s m a y h a v e a n i n f l u e n c e o n t h e m a t u r a t i o n o f t h e C N S a n d t h e r e b y d i r e c t l y i n f l u e n c i n g b e h a v i o u r a l r e s p o n s e s o f t h e o r g a n i s m , i . e . h i g h a n d r o g e n l e v e l s m a y r e t a r d t h e r o s t r a l - c a u d a l g r o w t h o f t h e C N S . O t h e r h o r m o n e s , s u c h a s t h y r o x i n e ( S c h a p i r o , 1 9 6 6 ) , C o r t i s o l ( S c h a p i r o , 1 9 6 8 ) , e s t r o g e n ( H e l m , 1 9 6 6 ) a n d A C T H ( R a d o u c o - T h o m a s e t a l . , 1 9 & 1 ) h a v e b e e n s h o w n t o h a v e m a t u r a -t i o n a l e f f e c t s o n t h e C N S , e i t h e r e n h a n c i n g o r r e t a r d i n g C N S d e v e l o p m e n t . S i n c e 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 r e q u i r e s a m o t o r r e s p o n s e , i n i t i a l i n c r e a s e d a c t i v i t y c o u l d e n h a n c e a c q u i s i t i o n o f t h e t a s k . The f i n d i n g of an increased r e a c t i v i t y to shock by the TP-treated group at t h i s age i s of i n t e r e s t , as i t suggests the TP animals are more sensitive to footshock. Although the shock i n t e n s i t i e s employed i n the previous experiments were well above the threshold determined i n the present experiment, the possible contribution of d i f f e r e n t s e n s i t i -v i t i e s to footshock cannot be discounted i n explaining differences i n active avoidance behaviour. The larger adrenal glands i n the TP-injected group point to an e f f e c t of TP on t h i s system at t h i s age; since the pre-vious experiments point to no difference at age 42 or 1 0 0 days. I t has been suggested that increased adrenal a c t i v i t y or size may a f f e c t avoidance behaviour (Beatty, Beatty and Bowman, 197 3» Paul and Havlena, 1 9 6 2 ) and therefore the ob-servation of larger adrenals i n the TP-injected animals i s worthy of note. However, before a t t r i b u t i n g observed d i f f e r -ences i n a c t i v i t y , r e a c t i v i t y to footshock, and active avoidance behaviour to these changes, we should point out that many researchers have questioned the relationship be-tween adrenal size and behaviour (Ader, 1 9 6 9 ; Pare and Cullen, 1 9 6 5 ) . Nor has i t been conclusively demonstrated that increased adrenal size i s correlated with increased adrenocorticoid or ACTH secretion. It i s d i f f i c u l t to explain the present observations of larger adrenals at 3 0 - 3 1 days of age, but not at 42 and 1 0 0 days as seen i n the e a r l i e r experiments. One possible expla-nation may be a complex i n t e r a c t i o n between the adrenal and 5 9 gonadal systems. Kitay ( 1 9 6 3 ) showed that i n adult male rats, c astration increased adrenal weights and increased p i t u i t a r y ACTH secretion. Testosterone replacement lowered ACTH content and adrenal weight to control l e v e l s . At the same time, corticosterone secretion declines a f t e r orchiec-tomy, despite an increase i n ACTH secretion and testosterone replacement increases corticosterone production (Kitay, Coyne, Swygert, and Gaines, 1 9 7 1 ) . The res u l t s of increased adrenal size at 30 days of age but not 42 nor 1 0 0 days of age may be due to a s i m i l a r i n t e r a c t i o n of the pituitary-adrenal-gonadal system. At 30 days of age, the gonads have not yet begun to secrete optimal amounts of testosterone (Resko, Feder and Goy, 1 9 6 8 ) which would i n i t i a t e the process of puberty. The TP treated group may also be thought of as being p a r t i a l l y castrated since smaller gonads are correlated with decreased androgen production (Hunt, 1 9 6 9 ) . This could therefore a f f e c t the adrenals i n a si m i l a r fashion as shown by Kitay but to a smaller degree, at 30 days of age. On the other hand, at 42 and 1 0 0 days of age, i n t e r n a l secretions of testosterone are being secreted. Therefore p r i o r to puberty the smaller t e s t i s may be increasing adrenal weight. 60 VI EXPERIMENT IV - STEP DOWN PASSIVE AVOIDANCE TASK Our previous attempts to demonstrate enhanced passive avoidance behaviour proved unsuccessful. The procedure employed was i n h i b i t i o n of a previously acquired approach response i n which the animals learned to stay on one side of the shuttlebox. T r i a l s to c r i t e r i o n indicated t h i s to be a r e l a t i v e l y easy task. In the following experiment a step-down passive avoidance task was employed i n a f i n a l attempt to appraise the effects of neonatal TP injec t i o n s on passive avoidance behaviour. An added advantage i s gained by using the step-down task, as i t allows us to appraise the con-founding e f f e c t s of a c t i v i t y on the avoidance behaviour. Since Experiment III showed an a c t i v i t y difference between the two groups, th i s increased a c t i v i t y could therefore account f o r the better performance i n the active avoidance s i t u a t i o n by the TP group. At the same time, the TP group should perform poorly on a d i f f i c u l t passive avoidance task where i n h i b i t i o n of a response i s required. Consequently, a "step-down" passive avoidance task was chosen to observe whether neonatal androgen influences the learning of a d i f f i c u l t passive avoidance task and whether the increased a c t i v i t y of the TP group retards learning of t h i s task. Method Subjects> (Ss) Ss consisted of 30 male albino rats bred from 7 l i t t e r s and raised i n our colonies. F i f t e e n Ss 61 were injected with 1.25 mg TP i n vegetable o i l (12.5 ul) at day 1 of b i r t h . The other 15 Ss were injected with an equal dose of vegetable o i l (12.5 u l ) . Ss were weaned at 21 days of age and handled on day 22 and 2k f o r a 5 minute period. Apparatus: The apparatus consisted of a 10 i n . x 10 i n . x 18 i n . pl e x i g l a s s chamber. Placed i n one corner of the chamber was a 2.5 i n . x 2.5 i n . x 2.5 i n . wooden block. The f l o o r of the chamber consisted of l / l 6 i n . copper rods placed l / 2 i n . apart. Shock was provided through a shock scrambler constructed l o c a l l y . Procedure: At 25 days of age each S was placed on the wooden platform and given 30 seconds to descend. Once an animal stepped down from the platform a .50 mA shock was administered v i a the g r i d f l o o r . The shock remained on u n t i l the S stepped back on the platform. I f an animal did not i n i t i a l l y descend i n 30 seconds, i t was dropped from the experiment. The learning c r i t e r i o n was defined as remaining on the platform f o r a 2 minute period a f t e r receiving the shock. Twenty-four hours l a t e r , the S was placed on the wooden platform and had to remain on i t f o r a 3 minute period, which was the c r i t e r i o n used f o r retention. For the next 5 days, Ss were again placed on the platform to observe whether the groups d i f f e r e d during e x t i n c t i o n . Results The two groups did not d i f f e r s i g n i f i c a n t l y from each other i n terms of the amount of shock Ss received p r i o r to 62 remaining on the platform f o r a two minute period (t = 1.28, df = 28; p >^ . 0 5 ) . There was also no s i g n i f i c a n t difference between the groups on the retention variable (t = 1 . 1 0 , df = 28; p y> , 0 5 ) . Nor did the groups d i f f e r s i g n i f i c a n t l y during e x t i n c t i o n testing (P = .36; df = 5 / 1 0 0 ; p > . 0 5 ) . Figures 3» 4 and 5 provide the mean data f o r these three varia b l e s . Discussion The findings that the TP-injected group did not d i f f e r s i g n i f i c a n t l y from the o i l - i n j e c t e d male group on both the a c q u i s i t i o n and retention of the step-down task implies that the treatment had no e f f e c t . Therefore neither our hypo-thesis that neonatal TP injections may f a c i l i t a t e learning by a f f e c t i n g the learning process nor our hypothesis that TP treatment may retard learning due to the f a c t that these animals are more active was confirmed. S t i l l , the f i n d i n g that the TP group was not retarded on t h i s learning task rules out the a c t i v i t y variable as being involved i n the a c q u i s i t i o n of active avoidance i n Experiments I and I I . Although the r e s u l t s were not s i g n i f i c a n t , Figure 6 shows that the TP-injected group had a tendency to acquire the task e a r l i e r and also to have a better retention score 24- hours l a t e r . I t may possibly be that the task did not provide the d i f f i c u l t y to allow the differences to reach s i g n i f i c a n t l e v e l s . One reason f o r t h i s explanation may be due to the f a c t that the wooden platform was placed i n one F i g . 3» Mean d u r a t i o n of shock (seconds) a d m i n i s t e r e d p r i o r t o a t t a i n i n g 2 minute c r i t e r i o n p e r i o d on the p l a t f o r m , i n a p a s s i v e avoidance t a s k . 64 2 0 r 18 16 14 12 A m o u n t o f 10 S h o c k (X) 8 ±2.63 ±3.16 T. P. O i l F i g . k. Mean amount of time on platform i n 2k hour retention test of passive avoidance behaviour. Mean Amount of Time on Platform (Seconds) 170 160 150 140 130 120| 110 100 9 0 8 0 70| 6 0 5 0 4 0 3 0 2 0 10 ±18.84 ±18.86 T. P. Oil F i g , 5» Resistance to extinction a f t e r a c q u i s i t i o n the step-down passive avoidance task. corner of the chamber. Once the Ss stepped off the platform and were shocked, they ran around the sides of the chamber and consequently ran d i r e c t l y into the wooden platform and jumped on i t . S i g n i f i c a n t differences may possibly have been observed i f the platform had been placed i n the center of the chamber, 7 0 VII EXPERIMENT V - ACTIVE AVOIDANCE TRAINING IN ADULT ANIMALS Schapiro ( 1 9 6 8 ) while in v e s t i g a t i n g the role of thyroxine i n learning, showed that although neonatal thyroxine administration f a c i l i t a t e d the a b i l i t y to ac-quire a learning task at 1 6 days of age, the same animals were retarded at 3 5 days of age on a number of other learning tasks. More recently, Davenport and Gonzalez' ( 1 9 7 3 ) findings that neonatal thyroxine administration led to a performance d e f i c i t i n passive avoidance learning i n adulthood and maze-learning d e f i c i t s i n adolescence and adulthood help to confirm a possible biphasic e f f e c t of thyroxine on learning. A s i m i l a r dual mode of neonatal corticosterone action has also been implicated. The findings of s i g n i f i c a n t r e s u l t s i n active avoid-ance a c q u i s i t i o n p r i o r to puberty i n Experiment I and II raises the question of whether the f a c i l i t o r y e f f e c t s of neonatal TP administration would also be apparent i n adult-hood. The following experiment was therefore designed to investigate whether neonatal TP manipulation would a f f e c t a c q u i s i t i o n of one-way active avoidance responding i n adult r a t s . Secondly, f l i n c h and jump thresholds were examined to investigate whether group differences i n avoidance learning could be attributable to t h i s f a c t o r . Although not an i n t e g r a l part of the experimental design, a female group was incorporated into t h i s experiment 7 1 t o i n v e s t i g a t e w h e t h e r n e o n a t a l T P m a n i p u l a t i o n e n h a n c e s t h e a c q u i s i t i o n o f t h i s g r o u p o v e r t h a t o f n o r m a l m a l e s a n d f e m a l e s . M e t h o d S u b j e c t s : S s c o n s i s t e d o f 33 a l b i n o r a t s ( 2 2 m a l e s a n d 1 1 f e m a l e s ) b r e d a n d r a i s e d i n o u r c o l o n i e s . E l e v e n m a l e s w e r e i n j e c t e d w i t h 1.25 m g T P i n v e g e t a b l e o i l ( 1 2 . 5 u l ) . T h e o t h e r 1 1 m a l e s a n d 1 1 f e m a l e s w e r e i n j e c t e d w i t h a n e q u a l d o s e o f v e g e t a b l e o i l ( 1 2 . 5 u l ) . S s w e r e w e a n e d a t d a y 21 o f a g e a n d h o u s e d i n g r o u p s o f 5 o r 6 u n t i l 65 d a y s o f a g e . T h e y w e r e g r o u p e d b y s e x a n d t r e a t m e n t . A p p a r a t u s » T h e a p p a r a t u s c o n s i s t e d o f a w o o d e n s h u t t l e -b o x ( 9 0 c m x 2 3 c m x 5 2 c m ) d i v i d e d i n t o 2 e q u a l c o m p a r t m e n t s b y a p l e x i g l a s s g u i l l o t i n e d o o r . O n e s i d e o f t h e s h u t t l e b o x w a s p a i n t e d w h i t e a n d t h e o t h e r s i d e b l a c k . T h e f l o o r o f t h e s h u t t l e b o x w a s c o m p o s e d o f l / l 6 i n . c o p p e r r o d s p l a c e d 1/2 i n . a p a r t . S h o c k w a s a d m i n i s t e r e d v i a a n A C s h o c k e r c o n s t r u c t e d i n o u r l a b o r a t o r y a n d c o n n e c t e d t o t h e g r i d f l o o r . F l i n c h a n d j u m p t h r e s h o l d s w e r e d e t e r m i n e d i n a p l e x i -g l a s s c h a m b e r ( 2 5 c m x 25 c m x 46 c m ) w i t h s h o c k d e l i v e r e d t h r o u g h a g r i d f l o o r . P r o c e d u r e » A t 65 d a y s o f a g e , S s w e r e h o u s e d i n i n d i -v i d u a l s t a i n l e s s s t e e l c a g e s w i t h a d l i b i t u m f o o d a n d w a t e r . T h e y w e r e g e n t l e d o n d a y 66 a n d 68 f o r a 5 m i n u t e p e r i o d . O n d a y 69 p a i r s o f S s w e r e p l a c e d i n t h e s h u t t l e b o x f o r a 7 2 15 minute period to f a m i l i a r i z e them to the apparatus. Actual t e s t i n g "began at 7 0 days of age. Ss were placed i n the black compartment of the shuttlebox. Ten seconds l a t e r the g u i l l o -tine door was raised, serving as the CS. Ss were given 5 seconds to cross to the "safe" side. Shock (.50 mA) was administered i f the Ss did not cross before the 5 seconds had elapsed. Ss remained i n the safe compartment f o r JO seconds and then the next t r i a l was i n i t i a t e d . Ten t r i a l s a day were run u n t i l Ss reached c r i t e r i o n . The c r i t e r i o n employed was 9 correct responses on 2 consecutive days. Ss were run either u n t i l they reached c r i t e r i o n or f o r 1 7 0 t r i a l s , whichever was the shortest. Once a l l the Ss had completed avoidance t r a i n i n g t h e i r jump and f l i n c h thresholds were determined using an ascending-descending series of shock presentation. Ss were placed i n the p l e x i g l a s s chamber and administered 6 series of shock (.05, . l i .2, .4, .6, .8, 1.0 mA) i n an alternating ascending-descending order. There was a 20 second i n t e r v a l between each shock i n a series and a 6 0 second i n t e r - s e r i e s i n t e r v a l . Results There was a s i g n i f i c a n t difference between the groups on a c q u i s i t i o n of the one-way active avoidance task (x = 7 2 . 7 7 TP group? x = 1 0 5 . 4 5 o i l - t r e a t e d male group; x = 9 0 . 9 0 female group; F = 3 . 9 8 , df = 2 / 3 0 ; p < . 0 2 5 ) (see Figure 6 ) . Post hoc analysis confirmed that t h i s difference was between the TP injected group and o i l - i n j e c t e d male group (t = 2 . 2 1 , F i g . 6 . T r i a l s to c r i t e r i o n (Mean - Standard Er r o r of the Mean) i n a one-way active avoidance task i n i t i a t e d at 7 0 days of age. Subjects were injected with either 1 . 2 5 mg T.P. or o i l at 1 day of age. T r i a l s t o C r i t e r i o n (X) 1 2 0 r 1 1 0 100 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 ±7.77 ±12.63 ±7.21 T . P . O i l - M a l e O i l - F e m a l e 75 df = 2 0 , p < . 0 5 ) . The o i l - i n j e c t e d female group did not d i f f e r s i g n i f i c a n t l y from either the o i l - i n j e c t e d male group (t = 1 . 7 2 , df = 2 0 , p > . 0 5 ) nor the TP-injected male group (Tukey A = . 4 4 , q > 9 5 (k,.f) = . 5 3 , p > . 0 5 ) . Analysis of variance also showed a s i g n i f i c a n t d i f f e r -ence between the groups on the jump threshold measure (F = 5 . 0 0 , df = 2/30; p < . 0 5 ) . Post hoc analysis showed that the female group had a s i g n i f i c a n t l y lower jump threshold than the o i l - i n j e c t e d male group (t = 2 . 8 6 , df = 2 0 , p < . 0 5 ) . while the TP-injected male group did not d i f f e r s i g n i f i c a n t l y from eith e r the female group (t = 1 . 2 5 . df = 2 0 , p > . 0 5 ) or o i l - i n j e c t e d male group (t = 1 . 4 3 , df = 2 0 , p > . 0 5 ) , a l -though there was a trend f o r the TP-injected group to have a lower jump threshold than the o i l - i n j e c t e d male group (x = . 3 9 mA TP groups x = . 4 5 mA o i l - i n j e c t e d male group; x = . 3 5 mA female group). F l i n c h thresholds d i f f e r e d s i g n i f i c a n t l y between the groups (F = 1 9 3 . 3 3 . df = 2 / 3 0 ; p < . 0 1 ) . The TP and female groups did not d i f f e r s i g n i f i c a n t l y from each other (Tukey A = . 4 4 , q g 5 (k,f) = . 5 3 , p > . 0 5 ) but both had lower f l i n c h thresholds than the o i l - i n j e c t e d male group (t = 2 . 5 0 , df = 2 0 , p < . 0 5 , T P - o i l groups, t = 3 . 0 , df = 2 0 , p < . 0 5 . oil-female groups). Table 7 provides the mean data on f l i n c h and jump thresholds. Discussion The re s u l t s confirm the major conclusions drawn from Experiment I and I I , namely that neonatal TP manipulation Table 7 Mean F l i n c h and Jump Thresholds to E l e c t r i c Shock i n Adulthood x F l i n c h x Jump Groups Threshold Threshold. TP-Male .18 . 3 9 Oil-Male . 2 3 . 4 5 Oil-Female .14 . 3 5 77 influences the a c q u i s i t i o n of an active avoidance response. These r e s u l t s point to a possible long-term ef f e c t of neo-natal TP e f f e c t s since adult Ss showed the same f a c i l i t a t e d active avoidance as a prepuberal Ss used i n the e a r l i e r experiments. The difference i n a c q u i s i t i o n of the avoidance response was s i g n i f i c a n t only between the TP-treated and o i l -treated male groups. The female group, as seen i n Figure 8 reached c r i t e r i o n on the average i n 90.90 t r i a l s as opposed to 72.73 t r i a l s f o r the TP group and 105.4-5 t r i a l s f o r the o i l - i n j e c t e d group. Although the difference between the female group and the other two groups was not s i g n i f i c a n t , the trend i s towards the female group acquiring the task fa s t e r than the o i l - i n j e c t e d male group. This sex difference has been observed by a number of other researchers (Beatty and Beatty, 1970; Scouten and Beatty, 1971) and i t may be possible that our difference was not s t a t i s t i c a l l y d i f f e r e n t due to d i s s i m i l a r t r a i n i n g procedures. The jump and f l i n c h thresholds also d i f f e r e d between the groups. The TP and female groups had lov/er f l i n c h thresholds than the o i l - i n j e c t e d male group, though they 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 other. In terms of the jump threshold, only the female group was s i g n i f i c a n t l y d i f f e r e n t from the TP and o i l - i n j e c t e d male groups. Although the jump threshold of the TP group (x = .39 mA) did not d i f f e r from the o i l - i n j e c t e d male group (x = .45 mA) or the female group (.35 mA) the trend i s towards a lower jump threshold than the o i l - i n j e c t e d male group as observed i n Experiment I I I . One 78 reason why the jump threshold values may not have d i f f e r e d s i g n i f i c a n t l y "between the o i l - and TP-treated males i s that testosterone l e v e l s increase at the onset of puberty. Since testosterone injections are known to increase f l i n c h , f l i n c h -s h u f f l e , and jump thresholds i n female rats (Beatty et a l . , 1971)» the i n t e r n a l increase i n testosterone secretion may raise the jump threshold i n a s i m i l a r fashion. The differences i n jump and f l i n c h thresholds between normal males and females have been observed by a number of other investigators (Beatty et a l . , 1970; Pare, 1 9 6 9 ) . Beatty et a l . have shown that females have a lower f l i n c h , f l i n c h -shuffle, and jump thresholds than males, which are s i m i l a r to our findings. These differences appear to be hormone dependent (Beatty et a l . , 1970) as testosterone i n j e c t i o n s i n neonate females or adult females raises the values of these three measures to male l e v e l s . Ovariectomy combined with these procedures augment the e f f e c t . As yet, neo-n a t a l castration of males and subsequent ef f e c t s on f l i n c h and jump thresholds or active avoidance responding have not been investigated. Although Beatty et a l . have shown that adult castration of males has no e f f e c t on avoidance behaviour or shock r e a c t i v i t y , t h i s may possibly have been due to the short time i n t e r v a l between gonadectomy and avoidance learning (2 weeks), since i n t e r n a l testosterone levels may not have diminished i n th i s time period (Phoenix, Goy and Young, 1 9 6 7 ) . 79 Our findings of lowered f l i n c h and jump thresholds by the TP group over control males does provide evidence of excessive neonatal TP on shock s e n s i t i v i t y . Shock s e n s i t i v i t y appears to play a role i n active avoidance a c q u i s i t i o n . I t therefore appears that excessive neonatal TP i n male rats lowers t h e i r shock s e n s i t i v i t y towards the female l e v e l , which may p a r t i a l l y account f o r the f a c i l i t a t e d a c q u i s i t i o n of active avoidance responding. GENERAL DISCUSSION On the basis of the data described above, we may conclude that TP administration to in t a c t neonatal male rats led to enhanced a c q u i s i t i o n of one-way active avoid-ance responding i n both prepubescent and adult animals. The i n a b i l i t y to demonstrate f a c i l i t a t e d a c q u i s i t i o n of passive avoidance behaviour i s equally s i g n i f i c a n t as i t emphasizes the s p e c i f i t y of the f a c i l i t a t i o n e f f e c t . On the basis of these data, i t appears unlikel y that the enhancement of active avoidance i s due to associative processes per se. The observation of increased a c t i v i t y and decreased shock thresholds suggest these factors may be playing an important role i n the observed f a c i l i t a t i o n of active avoidance responding. Increased locomotor a c t i v i t y a t t r i b u t a b l e to neonatal androgen excess has been noticed i n adolescent monkeys and humans (Rose, Money and Alexander, 1971). These authors observed i n -creased motor a c t i v i t y i n both monkeys and humans. Given the changes i n a c t i v i t y which follow neonatal TP i n j e c -tions, i t would seem l o g i c a l to assume that such changes could lead to f a c i l i t a t e d active avoidance learning and also to decreased escape latencies during avoidance. The question s t i l l remains as to how neonatal TP inj e c t i o n s may a f f e c t r e a c t i v i t y to footshock and one l i n e of research which may o f f e r an explanation concerns the ef f e c t of neonatal stero i d l e v e l s on synaptic transmitter 81 l e v e l s i n the brain. S p e c i f i c a l l y , l e v e l s of serotonin have been shown to be important correlates of shock thresholds. Depletion of serotonin by either lesions of the medial forebrain bundle, septum or midbrain tegmentum i s correlated with increased s e n s i t i v i t y to footshock i n male rats (Evans, 1 9 6 l | Lints and Harvey, 1 9 6 9 ) . This e f f e c t i s also observed by depleting serotonin l e v e l s i n the brain with i n h i b i t o r s of serotonin synthesis (Tenen, 1 9 6 ? ) . I f i t could be shown that increased l e v e l s of testosterone during neonatal development produce a decrease i n serotonin l e v e l s , t h i s could conceivably account f o r increased s e n s i t i v i t y to shock. Evidence f o r the e f f e c t of neonatal steroids on serotonin l e v e l s i s somewhat ambiguous, but studies with neonatal rats have shown that testosterone may act on serotonin metabolism to lower i t s concentration i n the brain. Castration of neonatal male rats increases sero-tonin l e v e l s at 1 2 days of age, while testosterone i n j e c -tions into neonatal females decreases serotonin concentra-tions (Ladosky and Gazini, 1 9 ? 0 ) . The s i t u a t i o n becomes more complicated however, when sex differences i n absolute l e v e l s of brain serotonin are considered, as females i n both adulthood and infancy have higher serotonin l e v e l s than males (Kats, 1959; Ladosky and Gazini, 1 9 7 0 ; Hardin, 1 9 7 3 ) * The relationship between serotonin l e v e l s and r e a c t i v i t y to footshock i s undoubtedly most complex and i t i s important to remember that the relationship between 8 2 decreased serotonin l e v e l s and increased shock s e n s i t i v i t y has only been established f o r male r a t s . Although one must be most ca r e f u l i n speculating about possible substrates which may account f o r the e f f e c t of elevated testosterone l e v e l s on footshock thresholds, the "serotonin" hypothesis i s d i r e c t l y testable and thereby worthy of further con-sideration. Inherent i n the rationale f o r the current series of experiments i s the hypothesis that TP i n j e c t i o n s may enhance learning by influencing the neural substrate of associative processing. Sands and Chamberlain ( 1 9 5 2 ) found that t r e a t -ment of adolescents with androgens led to increased a c t i v i t y and better school performance. Others have found that androgen treatment i n adolescent retardates with gonadal immaturity improved the mental state of these boys (Berman, Albert-Gaserek, and Reiss, 1 9 5 9 ) . However, i t i s question-able whether the androgen i n j e c t i o n s f a c i l i t a t e d learning as the changes were not permanent. As already indicated prenatal androgen treatment i n humans has also been corre-lated with high I.Q. scores (Dalton, 1 9 6 8 ; Erhardt and Money, 1 9 6 9 ; Money, 1 9 7 1 ) . In c r i t i c a l l y evaluating these ideas, the c o r r e l a t i o n a l nature of the data must be empha-sized. I t i s c l e a r to impartial observers that t h i s r e l a -tionship i s only a c o r r e l a t i o n and therefore need not be a cause-effect r e l a t i o n s h i p . These researchers on the other hand imply that the elevated prenatal androgen l e v e l s j f a c i l i t a t e I.Q. Although t h i s explanation i s both plausible 83 a n d . i n t e r e s t i n g , i t a p p e a r s t o b e p r e m a t u r e . T h i s i s e s p e c i a l l y t r u e i n v i e w o f a r e c e n t r e - e v a l u a t i o n o f t h e s e f i n d i n g s w h i c h h a s s h o w n t h a t t h e y m a y b e d u e t o t h e u s e o f i n a d e q u a t e c o n t r o l g r o u p s , n a m e l y t h e f a i l u r e t o e m p l o y s i b l i n g s a s c o n t r o l s ( V a l e n s t e i n , p e r s o n a l c o m m u n i c a t i o n ) . W h e n t h e a p p r o p r i a t e s i b l i n g c o n t r o l s w e r e e m p l o y e d , n o d i f f e r e n c e s a p p e a r e d b e t w e e n t h e g r o u p w i t h e l e v a t e d p r e -n a t a l a n d r o g e n l e v e l s a n d t h e c o n t r o l g r o u p . T h e r e f o r e t h e h u m a n d a t a d o e s n o t a p p e a r t o p r o v i d e c o n c l u s i v e e v i -d e n c e t h a t p r e n a t a l a n d r o g e n l e v e l s i n f l u e n c e a s s o c i a t i v e p r o c e s s e s a s i n d i c a t e d b y h i g h I . Q . l e v e l s . S U M M A R Y A N D C O N C L U S I O N D e s p i t e t h e c o n t r o v e r s y w h i c h s u r r o u n d s t h e r e l a t i o n -s h i p b e t w e e n e l e v a t e d p r e - n a t a l s t e r o i d l e v e l s a n d i n t e l l i -g e n c e i n h u m a n s , t h e p r e s e n t d a t a c l e a r l y d e m o n s t r a t e d a f a c i l i t a t e d a c q u i s i t i o n o f 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 f o l l o w i n g n e o n a t a l T P i n j e c t i o n s . H o w e v e r i t i s u n l i k e l y t h a t t h i s f a c i l i t a t i o n o f a c t i v e a v o i d a n c e l e a r n i n g i s d u e t o i m p r o v e d a s s o c i a t i v e p r o c e s s i n g . A m o r e p a r s i m o n i o u s e x p l a n a t i o n i s s u g g e s t e d b y t h e o b s e r v e d c h a n g e s i n a c t i v i t y l e v e l s a n d / o r s h o c k s e n s i t i v i t y . F u r t h e r m o r e t h e p r e s e n t s e r i e s o f e x p e r i m e n t s o n l y e m p l o y e d a r e l a t i v e l y s i m p l e l e a r n i n g p a r a d i g m a n d i t w o u l d b e i n a p p r o p r i a t e t o g e n e r a l -i z e f r o m t h e s e f i n d i n g s t o m o r e c o m p l e x l e a r n i n g t a s k s . O n t h e b a s i s o f t h e s e c o n s i d e r a t i o n s , f u t u r e r e s e a r c h s h o u l d 84 employ more complex discrimination tasks and d i f f e r e n t states of motivation. Only when TP in j e c t i o n s have "been shown to a f f e c t other learning tasks such as v i s u a l or s p a t i a l discrimination i n a Skinner box, can we begin to seriously entertain f a c i l i t a t i o n of the learning process by neonatal s t e r o i d manipulation. 85 BIBLIOGRAPHY Ader, R., Friedman, S.B., and Grota, L . J . " E m o t i o n a l i t y and a d r e n o c o r t i c a l f u n c t i o n ! 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