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Studies on the effects of light deprivation on the formation of adenosine 3’, 5’ -cyclic monophosphate Nagy, Jim 1976

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STUDIES ON THE EFFECTS OF LIGHT DEPRIVATION ON THE FORMATION OF ADENOSINE 3', 5'-CYCLIC MONOPHOSPHATE by JIM NAGY B.Sc, Un i v e r s i t y of B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE DEPARTMENT OF BIOCHEMISTRY \ FACULTY OF MEDICINE We accept t h i s thesis as conforming to the required standard: THE UNIVERSITY OF BRITISH COLUMBIA March, 1976 (c ) J i m Nagy, 1976 In p re sent ing t h i s t he s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha t permiss ion fo r ex ten s i ve copying o f t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen ta t i ve s . It i s understood that copying or p u b l i c a t i o n o f t h i s t he s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n permi s s ion . c B i o c h e m i s t r y Department of _ The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date M a r c h 2 2 , 1976. i i ABSTRACT Morpho l o g i c a l , e l e c t r o p h y s i o l o g i c a l and biochemical changes have been shown to occur i n the r e t i n a , l a t e r a l g e n i c u l a t e nucleus, and v i s u a l c o r t e x of l i g h t deprived animals. We attempted to determine whether the da r k - r e a r i n g of r a t s from b i r t h to 1 5 , 3 0 and 6 0 days of age a l t e r s the a b i l i t y of noradrenaline (NA) 3 0 u M, potassium c h l o r i d e (KCI) 5 0 u M, adenosine 3 0 yM and combinations of NA and KCI w i t h adenosine to s t i m u l a t e the _in v i t r o formation of c y c l i c AMP (cAMP) i n v i s u a l c o r t i c a l s l i c e s and, as an i n t e r n a l c o n t r o l , i n f r o n t a l c o r t i c a l s l i c e s . At 1 5 and 3 0 days of age there was an 1 1 % and 2 1 7 0 r e d u c t i o n , r e s p e c t i v e l y , compared to normally reared c o n t r o l s , i n the s t i m u l a t i o n of cAMP formation i n a 5 minute i n -cubation w i t h NA i n both f r o n t a l and v i s u a l c o r t i c a l s l i c e s . A f t e r 6 0 days of da r k - r e a r i n g , however, t h i s was reversed i n that the NA s t i m u l a -t i o n of cAMP formation was 2 3 % and 3 5 7 » higher than c o n t r o l s i n f r o n t a l and v i s u a l c o r t i c a l s l i c e s . I n f r o n t a l c o r t i c a l s l i c e s of r a t s dark-reared f o r 1 5 and 3 0 days there was a s i g n i f i c a n t r e d u c t i o n i n the s t i m u l a -t i o n of cAMP formation i n a 2 0 minute i n c u b a t i o n w i t h NA. No d i f f e r e n c e s were observed between 3 0 day o l d experimental and c o n t r o l animals i n studi e s of the accumulation of cAMP i n f r o n t a l and v i s u a l c o r t i c a l s l i c e s incubated f o r various times w i t h KCI. The s t i m u l a t i o n of cAMP formation induced by KCI and adenosine i n a 5 minute i n c u b a t i o n was 5 7 7 0 and 397o higher, r e s p e c t i v e l y , i n f r o n t a l c o r t i c a l s l i c e s of 6 0 day o l d experimental animals than c o n t r o l s w h i l e the response i n v i s u a l c o r t i c a l s l i c e s was un-a f f e c t e d . The d i f f e r e n c e s found between 6 0 day o l d experimental and c o n t r o l animals were a b o l i s h e d i n both v i s u a l and f r o n t a l c o r t i c a l s l i c e s when adenosine was used i n combination w i t h NA or KCI. Studies of the i i i accumulation of cAMP i n s l i c e s incubated f o r v a r i o u s times w i t h NA revealed t h a t the e f f e c t observed i n the v i s u a l c o r t e x a f t e r 30 days of l i g h t d e p r i v a t i o n was due to a decrease i n the maximum l e v e l of cAMP reached w i t h i n a 20 minute i n c u b a t i o n p e r i o d , whereas i n the f r o n t a l c o r t e x the maximum l e v e l a t t a i n e d w i t h i n a 20 minute i n c u b a t i o n p e r i o d was unaf f e c t e d . These r e s u l t s are discussed i n terms of our present knowledge concerning s u p e r s e n s i t i v i t y and p l a s t i c i t y i n the c e n t r a l nervous system and the r o l e of cAMP i n nerve. ACKNOWLEDGMENTS I would l i k e to express my s i n c e r e g r a t i t u d e to Dr. S.C. Sung f o r a l l o w i n g me the opportunity to work w i t h him and f o r u n f a i l i n g confidence i n my a b i l i t i e s . I would l i k e to thank W. Popow who spent innumerable hours p r o v i d i n g v a l u a b l e advice and encouragement when they were needed most. Thanks are a l s o given to Drs. H.C. F i b i g e r and V.K. Singh f o r showing i n t e r e s t , g i v i n g encouragement and o f f e r i n g h e l p f u l suggestions during the course of t h i s work. I would a l s o l i k e to o f f e r my deepest a p p r e c i a t i o n to Drs. E. McGeer and V.K. Singh who took time out from t h e i r busy schedules f o r the c r i t i c a l r;eading of t h i s t h e s i s . V DEDICATION I would l i k e to dedicate t h i s t h e s i s to my w i f e , K r i s t y , who has taken me f o r b e t t e r or f o r worse. v i TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGMENTS . . . i v DEDICATION v INTRODUCTION 1 I. Sensory D e p r i v a t i o n as a Tool to I n v e s t i g a t e C e n t r a l Nervous System Development and Function . 1 I I . Anatomy of the V i s u a l System 4 I I I . E f f e c t s of Sensory D e p r i v a t i o n of the V i s u a l System 6 a) C y t o l o g i c a l 6 b) Morphological 7 c) E l e c t r o p h y s i o l o g i c a l 10 d) Biochemical 13 IV. Adenosine 3', 5 ' - c y c l i c Monophosphate (CAMP) and the Nervous System 17 a) The Metabolism and Function of cAMP i n B r a i n . 17 b) The Development of the cAMP System i n B r a i n . . 21 V. Adaptive Mechanisms i n Nerve . 24 VI. The Present I n v e s t i g a t i o n 27 MATERIALS AND METHODS 30 I. Chemicals 30 I I . Maintenance and Treatment of Animals 30 I I I . Techniques Involved i n the Treatment of B r a i n Tissue 32 a) P r e p a r a t i o n of Krebs-Ringer Bicarbonate B u f f e r . 32 b) Tissue P r e p a r a t i o n 32 c) Incubation Procedures 33 v i : Page IV. I s o l a t i o n and Recovery of cAMP from B r a i n S l i c e s . 35 V. The Measurement of cAMP Levels i n B r a i n Tissue . 36 a) General P r i n c i p l e s of the Assay 36 b) M a t e r i a l s f o r the Assay of cAMP 38 c) D e t a i l s of the Procedure f o r the Assay of cAMP . 39 d) R a d i o a c t i v i t y A n a l y s i s 40 e) P r o t e i n Determination 40 VI. Treatment of Data 41 RESULTS 42 I. Assessment of Some Aspects of the I n v e s t i g a t i v e Procedures 42 I I . The E f f e c t of Noradrenaline and KCI on the Rate of Accumulation of cAMP i n B r a i n S l i c e s 49 I I I . The Ontogenetic Development of Responsiveness of B r a i n S l i c e s to Noradrenaline and KCI . . . . 54 IV. The Accumulation of cAMP i n B r a i n S l i c e s i n Response to Adenosine and Combinations of Adenosine w i t h Noradrenaline and KCI 62 DISCUSSION 67 CONCLUSION 79 REFERENCES 80 INTRODUCTION I. Sensory D e p r i v a t i o n as a Tool to I n v e s t i g a t e C e n t r a l Nervous  System Development and Function. Since the middle of t h i s century there has been an enormous e f f o r t made to determine the extent to which the development and f u n c t i o n i n g of the c e n t r a l nervous system (CNS) may be a f f e c t e d by the environment. The term p l a s t i c i t y has been used to describe the a b i l i t y of the CNS to respond and adapt at d i f f e r e n t s t r u c t u r a l l e v e l s to various types of i n s u l t s . Without environmental demands f o r t h e i r use some CNS or g a n i z a t i o n s are l o s t , t h e i r s t r u c t u r a l substrates diminished, and the b r a i n chemistry a l t e r e d . When c e r t a i n sensory requirements are imposed, exaggerated neu r a l growth patterns and r e s t r u c t u r i n g of f u n c t i o n beyond the or d i n a r y occur. Compensation of s t r u c t u r a l and f u n c t i o n a l c a p a c i t i e s r e s u l t from a s h i f t of environmental demands away from one sensory modality and towards others. Although t h i s i n f o r m a t i o n suggests that the CNS i s h i g h l y p l a s t i c , i t has been d i f f i c u l t to draw conclusions p e r t a i n i n g to the c e l l u l a r mechanisms whereby p l a s t i c i t y i s achieved and the impact that p l a s t i c changes have on f u n c t i o n at various l e v e l s of s t r u c t u r a l o r g a n i z a t i o n . For example, i f use promotes growth and f u n c t i o n of the nervous system and disuse retards development or even induces atrophy, when and how are such e f f e c t s occurring? Furthermore, the e f f e c t s of l e a r n i n g , although c e r t a i n l y l e s s pronounced than those of sensory d e p r i v a t i o n , probably i n v o l v e s i m i l a r f e a t u r e s . Therefore, can a thorough understanding of f i n d i n g s obtained from i n v e s t i g a t i o n s i n v o l v i n g sensory d e p r i v a t i o n lead to the d e l i n e a t i o n of the morphological and chemical substrates of learning? I t i s only 2 through an i n t e r d i s c i p l i n a r y approach i n v o l v i n g physiology, anatomy, and biochemistry that we can begin f i r s t to draw c o r r e l a t i o n s and f i n a l l y to answer these questions. The environmental pe r t u r b a t i o n s most used to i n v e s t i g a t e CNS p l a s t i -c i t y have been sensory d e p r i v a t i o n and c o n t r o l l e d s t i m u l a t i o n . Sensory d e p r i v a t i o n r e f e r s to the r e d u c t i o n of the t o t a l number of s t i m u l i d e l i v e r e d to sensory s t r u c t u r e s such as the motor, a u d i t o r y and v i s u a l systems. More g e n e r a l i z e d approaches include impoverished and enriched environments as w e l l as s o c i a l i s o l a t i o n . Studies i n v o l v i n g d e p r i v a t i o n of the v i s u a l system of animals have been by f a r the most numerous. The main advantages of t h i s system are as f o l l o w s : a) The sensory input, which enters mainly v i a the o p t i c f i b e r s , can be e a s i l y modified and q u a n t i f i e d , b) I t i s a system w i t h minimal convergence of a f f e r e n t f i b e r s from other b r a i n centers, which i s very important because the n o n - v i s u a l a f f e r e n t s might reduce the e f f e c t s of a l t e r e d v i s u a l input, c) The v i s u a l i n f l u x i s l a t e r a l i z e d be-cause of almost complete c r o s s i n g of the o p t i c f i b e r s i n the chiasma. From t h i s p o i n t of view a l b i n o r a t s have been shown to be e s p e c i a l l y s u i t a b l e (1,2). A v a r i e t y of procedures have been used to a l t e r the t o t a l amount of input s t i m u l i to the v i s u a l system. These in c l u d e supplying an excess of l i g h t s t i m u l i , f i t t i n g animals w i t h t r a n s l u c e n t occluders, l i m i t i n g the amount of i n c i d e n t l i g h t t o t a l l y or to a few hours per day, and l i m i t i n g the l i g h t to a p a r t i c u l a r frequency band. Most of the present d i s c u s s i o n w i l l be l i m i t e d to i n v e s t i g a t i o n s i n v o l v i n g the t o t a l e x c l u s i o n of l i g h t from experimental animals. The methods whereby t h i s i s achieved includ e r e a r i n g the animals i n t o t a l darkness, e n u c l e a t i o n of the eye or s u t u r i n g the l i d s over the eye. 3 There are few s t u d i e s that show l i t t l e or no e f f e c t on v i s u a l s t r u c -tures f o l l o w i n g e n u c l e a t i o n . Some of the f i n d i n g s obtained by t h i s method are important and are t h e r e f o r e included i n t h i s d i s c u s s i o n even though i t may be argued that the technique invol v e s d e a f f e r e n t a t i o n to produce the d e s i r e d d e p r i v a t i o n and thus introduces the c o m p l i c a t i n g f a c t o r of anterograde transneuronal degeneration. The techniques of eye l i d - s u t u r i n g and d a r k - r e a r i n g animals have advantages and disadvantages. Although d a r k - r e a r i n g i s r e l a t i v e l y easy to accomplish and i s r e a d i l y r e v e r s i b l e , i t may induce compensatory a c t i v a t i o n of other sense m o d a l i t i e s (3). A l t e r n a t i v e l y , l i g h t d e p r i v a t i o n by l i d - s u t u r i n g can be done u n i l a t e r a l l y thus p r o v i d i n g an i n t e r n a l c o n t r o l but i t i s u n c e r t a i n to what extent l i g h t can penetrate the eye l i d . Many review a r t i c l e s have appeared i n the l a s t decade regarding the e f f e c t s of t o t a l v i s u a l d e p r i v a t i o n on various b r a i n centers. Review a r t i c l e s by Riesen (4-6) i n c l u d e neurochemical, n e u r o p h y s i o l o g i c a l and morphological c o r r e l a t e s of sensory d e p r i v a t i o n and concentrate on the requirement of adequate s t i m u l a t i o n f o r growth of neuronal s t r u c t u r e s and maturation of f u n c t i o n . Mendelson and E r v i n (7) and Globus (8,9) focused t h e i r a t t e n t i o n on the r e l a t i o n of p o s t - s y n a p t i c s t r u c t u r e s to presynaptic f u n c t i o n and i n t e g r i t y . S c h e i b e l and S c h e i b e l (10) summarized the r e l a -t i o n of the d e n d r i t i c spine to presynaptic i n t e g r i t y and f u n c t i o n . Kreech e_t a l . (11) and Rosenzweig (12) reviewed the anatomical f i n d i n g s i n the c o r t e x of animals which have undergone d i f f e r e n t environmental exper-iences. Extensive reviews by F i f k o v a (13), Cragg (14) and Raisman and Mathews (15) have appeared on the morphological e f f e c t s of sensory depriva-t i o n . Rose e_t a l . (16) have discussed the above f i n d i n g s w i t h regard to the i m p l i c a t i o n s they have on the processes that may be i n v o l v e d i n l e a r n -ing. The biochemical c o r r e l a t e s of sensory d e p r i v a t i o n have been reviewed by Bondy and Margolis (17), and more r e c e n t l y by Walker et a l . (18). In the d i s c u s s i o n to f o l l o w an attempt i s made to i n t e g r a t e the key f i n d i n g s on the e f f e c t s of sensory d e p r i v a t i o n . The developmental aspects as w e l l as the metabolism and f u n c t i o n of cAMP i n b r a i n are a l s o discussed. The adaptive nature of nerve i s discussed w i t h the i n t e n t of e s t a b l i s h i n g a connection between the e f f e c t s of sensory d e p r i v a t i o n and the r o l e of cAMP i n the CNS. F i n a l l y , w i t h reference to the foregoing d i s c u s s i o n , the purpose and goal of the present i n v e s t i g a t i o n are d e l i n e a t e d . I I . Anatomy of the V i s u a l System. The e f f e c t s of l i g h t d e p r i v a t i o n on the v i s u a l system have been i n -v e s t i g a t e d at four anatomical l e v e l s i n the h i e r a r c h y of sensory informa-t i o n processing. These are the r e t i n a , s u p e r i o r c o l l i c u l u s , l a t e r a l g e n i c u l a t e nucleus (LGN) and the v i s u a l cortex. The r e l a t i o n s h i p of these s t r u c t u r e s to each other i s shown d i a g r a m a t i c a l l y i n F i g . 1. Although the ganglion c e l l s of the r e t i n a send t h e i r axons v i a the o p t i c t r a c t to a number of b r a i n regions, the primary s i t e of te r m i n a t i o n of these f i b e r s i n mammals i s the LGN. In non-mammalian species the primary s i t e of te r m i n a t i o n i s the s u p e r i o r c o l l i c u l u s or as more g e n e r a l l y r e f e r r e d to i n lower v e r t e b r a t e s , the o p t i c tectum. Neurons i n the LGN which have re c e i v e d input from ganglion c e l l s send t h e i r axons v i a the v i s u a l r a d i a -t i o n to the v i s u a l cortex. Since the f i r s t s ynaptic s i t e from f i b e r s of the r e t i n a i s the LGN, changes i n the LGN as a consequence of any type of i n s u l t on the r e t i n a are c a l l e d primary changes, w h i l e changes i n the v i s u a l c o r t e x being two synapses away from the primary sense organ are secondary e f f e c t s . T e r t i a r y changes are those found three or more syn-Optic nerve-*^ Optic tract^ Retina Ganglion cells Lateral geniculate nucleus Optic radiation Visual cortex^* F I G . 1 A s c h e m a t i c r e p r e s e n t a t i o n o f t h e m a j o r v i s u a l p a t h w a y s i n t h e r a t b r a i n . The g a n g l i o n c e l l s o f t h e t e t i n a s e n d t h e i r a x o n s v i a t h e o p t i c n e r v e and o p t i c t r a c t t o t h e l a t e r a l g e n i c u l a t e n u c l e u s and s u p e r i o r c o l l i c u l u s . The l a t e r a l g e n i c u l a t e n e u r o n s s e n d t h e i r a x o n s v i a t h e o p t i c r a d i a t i o n t o t h e v i s u a l c o r t e x . apses away from the i n i t i a l s i t e of i n s u l t . In the r a t u n l i k e higher mammals only about 107o of the o p t i c f i b e r are uncrossed ( 1 9 ) . Anatomical ( 2 ) , e l e c t r o p h y s i o l o g i c a l ( 1 ) and b e h a v i o r a l ( 2 0 ) s t u d i e s have shown that i n the a l b i n o r a t t h i s percentage i s even l e s s . As has been mentioned e a r l i e r t h i s p a u c i t y of uncrossed f i b e r s i s one of the advantages of employing the a l b i n o r a t i n s t u d i e s of the e f f e c t s of v i s u a l d e p r i v a t i o n . I n v e s t i g a t i o n s i n v o l v i n g the e f f e c t s of a host of v i s u a l d e p r i v a t i o n c o n d i t i o n s on a v a r i e t y of animals have been conducted i n each of the main v i s u a l centers described above. The f o l l o w i n g d i s c u s s i o n w i l l d eal mainly w i t h those changes which have been shown to occur i n the v i s u a l cortex. I I I . E f f e c t s of Sensory D e p r i v a t i o n of the V i s u a l System, a) C y t o l o g i c a l . The evidence accumulated to date s t r o n g l y suggests that transneuronal degeneration i s not h a l t e d at the f i r s t p ost-synaptic element, but that damage i n the main a f f e r e n t supply sets up a progressive involvement of successive neuronal l i n k s on the sensory n e u r a l chain. Although t h i s i s true of the t r a n s - s y n a p t i c e f f e c t s of v i s u a l d e p r i v a t i o n i n the o p t i c system the most obvious changes occur i n the r e t i n a , o p t i c nerve, and sub-c o r t i c a l centers and are more d i f f i c u l t to demonstrate i n the v i s u a l cortex. The e f f e c t s of v i s u a l d e p r i v a t i o n are more pronounced the higher the animal's p o s i t i o n i n the phylogenetic s c a l e and adult.mammals are much les s a f f e c t e d than young animals. The dark r e a r i n g of mice from b i r t h to various times up to 4 months causes a r e d u c t i o n i n the thickness of the v i s u a l c o r t e x ( 2 1 , 2 2 ) . At 3 0 days of age there occurred i n the v i s u a l c o r t e x of these animals a reduc-t i o n i n the nuclear volume i n g l i a and neurons, as w e l l as a re d u c t i o n i n the q u a n t i t y of cytoplasmic m a t e r i a l . A f t e r prolonged dark r e a r i n g the percent d i f f e r e n c e i n these parameters between c o n t r o l and experimental animals was shown to f a l l ( 2 2 ) . The gradual recovery observed i n some s t r u c t u r e s of dark-reared animals has been r e f e r r e d to as n o r m a l i z a t i o n ( 3 ) . The nuclear volume of c e l l s and the q u a n t i t y of i n t e r n u c l e a r m a t e r i a l was a l s o found to be decreased i n the a u d i t o r y cortex of experimental animals at 2 months but increased above c o n t r o l s at 4 months ( 3 ) . In the r a t v i s u a l cortex, monocular d e p r i v a t i o n f o r 3 0 , 6 0 and 9 0 days caused a decrease i n the t i s s u e volume together w i t h an increase i n the c e l l den-s i t y of v arious c o r t i c a l l a y e r s ( 2 3 , 2 4 ) . Thus, a f t e r 3 months there was an 8% decrease i n the thickness of layers I I to IV w i t h a concomitant 117o increase i n the c e l l d e n s i t y of l a y e r s I I I and IV ( 2 5 ) . There have been few c y t o l o g i c a l changes found i n the v i s u a l cortex of r a b b i t , c a t , dog, monkey and chimpanzee a f t e r l i g h t d e p r i v a t i o n although other v i s u a l . c e n t e r s have been shown to be a f f e c t e d . Monocular and bino-c u l a r e y e - l i d s u t u r i n g of the k i t t e n , f o r example, r e s u l t s i n a decrease i n c e l l , n u c l e i , and n u c l e o l a r volume i n the LGN by as much as 3 5 7 » ( 2 6 -2 8 ) . V i s u a l d e p r i v a t i o n of the r a b b i t r e s u l t s i n a 7 4 7 0 decrease i n the dry mass of the r e t i n a l ganglion c e l l s ( 2 9 ) . Dark-rearing of the chimp-anzee f o r 6 months r e s u l t s i n a 9 0 7 o decrease i n the number of ganglion c e l l s i n the r e t i n a ( 4 ) . b) Morphological. Morphological changes i n the v i s u a l cortex of animals dark-reared from b i r t h have been repeatedly reported. Most of the studies have con-centr a t e d on q u a n t i t a t i v e and q u a l i t a t i v e changes i n d e n d r i t e s , s y n a p t i c spines and s y n a p t i c s i t e s . A re d u c t i o n of d e n d r i t i c length and branching 8 of s t e l l a t e neurons was reported i n the v i s u a l cortex of cats r a i s e d i n the dark from b i r t h to 100 days (30). In the v i s u a l c o r t e x of the r a b b i t a greater v a r i a n c e of d e n d r i t i c length has been shown to occur as a r e s u l t of d a r k - r e a r i n g (31,32). Employing the techniques of monocular l i d - s u t u r i n g and d a r k - r e a r i n g mice and r a t s , the deprived and non-deprived v i s u a l c o r t e c i e s have been compared w i t h respect to the number of spines on the a p i c a l dendrites of l a y e r V pyramidal c e l l s . Working w i t h dark-reared mice, Valverde (33) concluded that c e r t a i n spines w i l l not develop without the input of l i g h t to the v i s u a l system. He has shown (33-36) a r e d u c t i o n i n the number of spines on these dendrites and has described the d i s t r i b u t i o n of spines on the dendrites as a f u n c t i o n of distance from the soma. Although there i s a r e d u c t i o n i n the t o t a l number of spines i n dark-reared animals, the shape of the curve of t h e i r d i s t r i b u t i o n i n r e l a t i o n to di s t a n c e from the soma remains the same. In the r a t the mean q u a n t i t a t i v e d e f i c i t i n spines f o r the e n t i r e measured length of the a p i c a l d e n d r i t e was 17% and 28% a f t e r l i d - s u t u r i n g f o r 10 and 60 days, r e s p e c t i v e l y (13,37). In the r a b b i t there have been reports of abnormally formed spines under condi-t i o n s of l i g h t d e p r i v a t i o n r a t h e r than a decrease i n t h e i r numbers (31,32). The e l e c t r o n microscope has been employed f o r studying the e f f e c t s of l i g h t d e p r i v a t i o n on c e l l u l a r u l t r a s t r u c t u r e s i n c e the impregnation technique used i n the above st u d i e s v i s u a l i z e s only a p o r t i o n of the syn-a p t i c p o p u l a t i o n . Cragg (38) u s i n g r a t s which had been deprived of l i g h t f o r 3 weeks found an increase i n sy n a p t i c s i z e i n the s u p e r f i c i a l l a y e r s of the co r t e x but a decrease i n deeper l a y e r s . U n i l a t e r a l l i d - s u t u r i n g of r a t s reduced the number of synap t i c s i t e s by 207<>, the upper l a y e r s of the cort e x being most a f f e c t e d (39). The mean s i z e of the axosomatic syna p t i c 9 contacts of the v i s u a l c o r t e x s u p p l i e d by the deprived eye was smaller by 237, through a l l l a y e r s s t u d i e d when compared to the cor t e x s u p p l i e d by the non-deprived eye (40). Axosomatic synapses having round v e s i c l e s showed a 357, and 297° r e d u c t i o n i n l a y e r s I I and IV, r e s p e c t i v e l y , w h i l e synapses w i t h f l a t v e s i c l e s were decreased by 167, and 147, i n layer s I I and IV r e s p e c t i v e l y (40). Vrensen and Groot (41,42) s t u d i e d the e f f e c t s of d a r k - r e a r i n g and monocular l i d - s u t u r i n g on the synapt i c terminals i n the v i s u a l c o r t e x of the r a b b i t and the recovery from these treatments a f t e r exposure of e x p e r i -mental animals to normal l i g h t i n g . Dark-rearing f o r 7 months d i d not a f f e c t the number of synaptic contacts, t h e i r surface area, or t h e i r mean length. However, there was a 407, decrease i n the number of synaptic v e s i c l e s i n the v i s u a l c o r t e x of dark-reared animals compared to c o n t r o l s , w h i l e no such d i f f e r e n c e was found i n the motor cortex. This decrease was found to p e r s i s t a f t e r r a i s i n g experimental animals under normal condi-t i o n s f o r 1 year. A f t e r e y e - l i d s u t u r i n g the decrease i n synap t i c v e s i c l e s i n the v i s u a l c o r t e x was only 167, and there occurred, i n the motor area of the deprived c o r t e x , an increase i n the d e n s i t y of synapses. I t i s noteworthy that the changes i n the v i s u a l c o r t e x a f t e r enuclea-t i o n or l e s i o n s at various points of the v i s u a l system bear marked s i m i -l a r i t i e s w i t h those observed a f t e r l i g h t d e p r i v a t i o n . Thus, a r e d u c t i o n i n the number of spines of l a y e r V pyramidal c e l l s of the v i s u a l c o r t e x has been reported a f t e r neo-natal e n u c l e a t i o n of the mouse (34,43) and r a b b i t (32). The di m i n u t i o n i n number of the d e n d r i t i c spines i n the mouse has been found to be l a r g e r at 24 days than 48 days of age i n d i c a t -ing some degree of n o r m a l i z a t i o n p o s s i b l y due to the involvement of com-pensatory mechanisms. These f i n d i n g s suggest that the morphological i n t e g r i t y of the v i s u a l c o r t e x i s dependent on the s t r u c t u r a l i n t e g r i t y of the a f f e r e n t systems as w e l l as on i t s f u n c t i o n a l i n t e g r i t y . c) E l e c t r o p h y s i o l o g i c a l . The i n f o r m a t i o n a v a i l a b l e i n d i c a t e s that sensory d e p r i v a t i o n does a f f e c t the general e l e c t r i c a l a c t i v i t y of the c o r t e x although these e f f e c t s are not w e l l understood due to d i f f i c u l t i e s encountered i n i n t e r -p r e t a t i o n . V i s u a l evoked p o t e n t i a l s (VEP) and the a b i l i t y of the c o r t e x to f o l l o w various frequencies of l i g h t s t i m u l a t i o n has been s t u d i e d i n normal r a t s and r a t s reared i n the dark f o r up to 45 days of age (44). D i f f e r e n c e s i n the l a t e n c y of the VEP was small and disappeared by 20 days. The a b i l i t y of the c o r t e x to respond to h i g h - f l a s h frequencies was maximal by 30 days and 45 days i n normally reared and dark-reared animals respec-t i v e l y . Monocular d e p r i v a t i o n of r a t s f o r 70 to 170 days from b i r t h was found (45) to cause a 27%, diminution of the e l e c t r i c a l a c t i v i t y and a 41%, decrease i n the amplitude of the VEP i n the deprived compared to the non-deprived v i s u a l cortex. Although the o v e r a l l c o n c l u s i o n of the l a t t e r study was that v i s u a l d e p r i v a t i o n suppresses c o r t i c a l e l e c t r i c a l a c t i v i t y , i t was found that the v i s u a l c o r t e x as w e l l as non-primary v i s u a l a f f e r e n t c o r t i c a l areas of some animals produced a greater response i n the evoked p o t e n t i a l i n the deprived r a t h e r than the non-deprived cortex. S i m i l a r l y , i n the v i s u a l c o r t e x of dark-reared r a b b i t s (46) there was a longer l a t e n c y and lower amplitude of the VEP compared to c o n t r o l s whereas sound and somesthetic s t i m u l a t i o n produced higher responses i n a l l areas of the c o r t e x of dark-reared animals. I t has been suggested that the hypersensi-t i v i t y observed to v i s u a l , a u d i t o r y , and somesthetic s t i m u l a t i o n i n other than s p e c i f i c p r o j e c t i o n areas of the c o r t e x may be due to n o n - s p e c i f i c systems at the s u b - c o r t i c a l or b r a i n stem l e v e l which have become super-s e n s i t i v e as a r e s u l t of v i s u a l d e p r i v a t i o n . E l e c t r o p h y s i o l o g i c a l r e c o r d i n g from s i n g l e c e l l s of the v i s u a l c o r t e x i s another method that has been used to assess the e f f e c t s of dark-r e a r i n g on the e l e c t r i c a l a c t i v i t y of n e u r a l s t r u c t u r e s . This approach has allowed, to some degree, the determination of the f u n c t i o n a l changes that r e s u l t from d e p r i v a t i o n of sensory experience. In t h i s regard r e -cording from s i n g l e c e l l s i s advantageous over biochemical and morphologi-c a l s t u d i e s s i n c e the l a t t e r a l l o w c o r r e l a t i o n s w i t h f u n c t i o n only to the extent that the consequences of the observed changes can be assessed from the known functions of the parameters a f f e c t e d . There are neurons i n the v i s u a l c o r t e x that respond s e l e c t i v e l y to the o r i e n t a t i o n of an object as w e l l as the d i r e c t i o n of movement. A l s o , there are b i n o c u l a r l y s e n s i t i v e c e l l s which respond only when s t i m u l a t i o n i s provided to both eyes. I t appears that these c e l l s have a dramatic dependence upon e a r l y usage f o r the maintenance of and the f u r t h e r develop-ment of s p e c i f i c i t y of responsiveness. The e f f e c t of u n i l a t e r a l eye c l o s u r e i n the k i t t e n i s to reduce d r a s t i c a l l y the number of c e l l s i n the v i s u a l c o r t e x that remain responsive to s t i m u l a t i o n of the occluded eye (26,47,48). B i n o c u l a r eye c l o s u r e r e s u l t s i n the l o s s of c e l l s respon-s i v e to s t i m u l a t i o n of both eyes and a r e d u c t i o n i n o r i e n t a t i o n s e l e c t i v i t y (49). Some c e l l s a l s o become le s s s e l e c t i v e i n t h e i r d e f i n i t i o n of the o r i e n t a t i o n of moving edges (50) and ste r e o s c o p i c c e l l s show no peak r e -sponsiveness (51). I f only c e r t a i n o r i e n t a t i o n s are a v a i l a b l e to the developing v i s u a l system, the c e l l s that are l a t e r found to respond are r e s t r i c t e d to those having s e n s i t i v i t i e s w i t h i n a range of that o r i e n t a -t i o n . Exposure of k i t t e n s to spots of l i g h t i n a v i s u a l environment w i t h -out s t r a i g h t l i n e s (52) r e s u l t s i n many c e l l s that are o p t i m a l l y s t i m u l a t e d by moving spots. The exposure of a d u l t cats to v e r t i c a l s t r i p s has been shown (53) to decrease the number of neurons s e n s i t i v e to o r i e n t a t i o n s around the v e r t i c a l r e l a t i v e to those s e n s i t i v e to h o r i z o n t a l o r i e n t a t i o n s . This i n d i c a t e s that p l a s t i c i t y of f u n c t i o n a l p r o p e r t i e s of the c o r t i c a l neuronal network s t i l l e x i s t s i n a d u l t animals. The c l o s e c o r r e l a t i o n w i t h f u n c t i o n a f f o r d e d by e l e c t r o p h y s i o l o g i c a l studies of s i n g l e c e l l s enables the determination of the degree of p l a s t i -c i t y inherent i n at l e a s t the v i s u a l c o r t e x i f not the b r a i n . This i s important because from the poin t of view of sensory d e p r i v a t i o n experiments and the i n t e r p r e t a t i o n s thereof, i t must be e s t a b l i s h e d whether the changes t a k i n g p l a c e are indeed due t o p l a s t i c and adaptive mechanisms i n t r i n s i c to the c a p a b i l i t i e s of the CNS or whether they are due to degenerative processes r e s u l t i n g from the s u b j e c t i o n of animals to n o n - p h y s i o l o g i c a l c o n d i t i o n s . The evidence to date i s that a t l e a s t a part of the s t r u c t u r a l basis f o r v i s u a l f u n c t i o n i s l a i d down at b i r t h ; but i t s t i l l remains to be determined to what extent i t i s then modified and r e f i n e d by experience. A f u r t h e r method employed to study the e l e c t r i c a l e f f e c t s of sensory d e p r i v a t i o n i s to create s u r g i c a l l e s i o n s at c r i t i c a l l o c a t i o n s i n a sensory system. Although l e s i o n s i n the v i s u a l system are a form of sensory d e p r i v a t i o n , t h i s procedure may i n v o l v e p h y s i o l o g i c a l and b i o -chemical processes other than those o c c u r r i n g during l i d - s u t u r i n g or dark-r e a r i n g . Nevertheless, some of the f i n d i n g s obtained from these s t u d i e s are r e l e v a n t to the present d i s c u s s i o n s i n c e the bas i c mechanisms respon-s i b l e f o r a l t e r e d b r a i n f u n c t i o n during these two experimental paradigms need not be mutually e x c l u s i v e and may even be inseparable. Lesions of the v i s u a l system may be produced at the l e v e l of the eye ( e n u c l e a t i o n ) , l a t e r a l g e n i c u l a t e nucleus, or the cortex. Fentress and Doty (54) u s i n g c h r o n i c a l l y implanted electrodes i n the cat and monkey to s t i m u l a t e the o p t i c t r a c t and o p t i c r a d i a t i o n showed that the e l e c t r i c a l responsiveness of the v i s u a l c o r t e x increases s e v e r a l f o l d a f t e r enuclea-t i o n . L a t e r a l g e n i c u l a t e l e s i o n s i n the cat r e s u l t e d i n increased e x c i t a -b i l i t y to e l e c t r i c a l s t i m u l a t i o n of the v i s u a l c o r t e x and a r e d u c t i o n i n the e l e c t r i c a l t h r e s h o l d f o r producing a f t e r d i s c h a r g e s (55). The i s o l a t e d c e r e b r a l cortex, a p r e p a r a t i o n which i n v o l v e s under-c u t t i n g the c o r t e x but l e a v i n g the s u p e r f i c i a l blood, supply i n t a c t , has been shown to become more s u s c e p t i b l e to agents e l i c i t i n g e p i l e p t i f o r m a c t i v i t y and to e l e c t r i c a l s t i m u l a t i o n (56). I t was f u r t h e r observed that t h i s increased s u s c e p t i b i l i t y could be prevented from o c c u r r i n g by "exer-c i s i n g " the disused n e u r a l elements through e l e c t r i c a l s t i m u l a t i o n (57). In the i s o l a t e d c e r e b r a l c o r t e x of k i t t e n s there i s extensive c o l l a t e r a l growth from i n j u r e d axons (58). I n the i s o l a t e d c o r t e x of cats there i s increased c a p a c i t y to bind "^C-D-tubocurarine, decreased a c e t y l c h o l i n e s -terase a c t i v i t y , decreased a c e t y l c h o l i n e content and a r e d u c t i o n i n the number of d e n d r i t i c spines (59-61). A l l these changes could be prevented by e l e c t r i c a l l y s t i m u l a t i n g the i s o l a t e d c o r t i c a l slabs.and i t i s t h i s f i n d i n g that brings the above st u d i e s i n t o the realm of the present d i s -c u ssion. I t shows that the changes r e s u l t i n g from i s o l a t i o n of the c o r t e x may not be due to degenerative processes but r a t h e r to disuse. These observations support the c o n t e n t i o n that adaptive mechanisms are present and o p e r a t i v e i n the c e n t r a l nervous system. d) Biochemical. Biochemical i n v e s t i g a t i o n s have not reached the degree of s o p h i s t i -c a t i o n that the other d i s c i p l i n e s have w i t h respect to e s t a b l i s h i n g the 14 e f f e c t s of l i g h t d e p r i v a t i o n on the v i s u a l system. One of the reasons f o r t h i s i s that biochemistry being at a more fundamental l e v e l of s t r u c t u r a l and f u n c t i o n a l o r g a n i z a t i o n , i s i n h e r e n t l y more complex. A l s o , the c e l l u l a r complexity and morphological heterogeneity of b r a i n r e s u l t s i n d i f f i c u l t i e s i n i n t e r p r e t a t i o n of biochemical data. Furthermore, the q u a n t i t i e s of t i s s u e i n v o l v e d i n s t r u c t u r e s of the v i s u a l system are very small thus p r e c l u d i n g some types of i n v e s t i g a t i o n s due to l i m i t a t i o n s i n a v a i l a b l e biochemical techniques. Despite these drawbacks, biochemical s t u d i e s have been conducted and the informa t i o n obtained, although sparce,. does c o n t r i -bute to our understanding of the biochemical basis of neural f u n c t i o n . The emphasis w i t h regard to the biochemical e f f e c t s of environmental manipulation has been on p r o t e i n and r i b o n u c l e i c a c i d (RNA) metabolism s i n c e these are the processes that would be expected to lead to r e l a t i v e l y permanent a l t e r a t i o n s i n f u n c t i o n . The l i t e r a t u r e concerning RNA metabolism i s fragmented and confusing. The polysomes i n the co r t e x of r a t s kept i n darkness f o r 3 days and sub-sequently exposed to l i g h t f o r 15 minutes were c h a r a c t e r i z e d by e l e c t r o n microscopy and sucrose-density g r a d i e n t s . The polysomes i n the v i s u a l c o r t e x and other c o r t i c a l areas increased i n number i n experimental animals compared to c o n t r o l s . Moreover, the p r o t e i n s y n t h e s i z i n g c a p a c i t y of ribosomes i s o l a t e d from the co r t e x of dar k - t r e a t e d animals was increased (62). Consistent w i t h t h i s f i n d i n g i s the demonstration (63) that 22 day o l d dark-reared r a t s exposed to l i g h t f o r 2 hours have a higher i n c o r p o r a -t i o n r a t e of ^ C - o r o t i c a c i d i n t o RNA of the v i s u a l c o r t e x than do dark-reared c o n t r o l s . However, i n the l a t t e r study only the v i s u a l c o r t e x was a f f e c t e d . U n l i k e the f r o n t a l c o r t e x the RNA content of the v i s u a l c o r t e x of exposed animals was lower than unexposed dark-reared animals. De Bold et a l . (64) found 'rio e f f e c t s of darkness and other l i g h t i n g c o n d i t i o n s on the t o t a l amount of c o r t i c a l RNA, but d i d o b t a i n evidence suggesting an i n f l u e n c e on the species of RNA produced i n the v i s u a l cortex. In e x p e r i -ments on i m p r i n t i n g i n newly hatched ch i c k s i n which one eye of the chi c k s 3 was covered i t was shown that the i n c o r p o r a t i o n of H - u r a c i l i n t o RNA and the a c t i v i t y of RNA polymerase were 15% and 34%. lower, r e s p e c t i v e l y , i n the f o r e b r a i n connected w i t h the covered eye as compared to the f o r e b r a i n of the uncovered eye (65,66). S i m i l a r r e s u l t s were obtained when the i n -c o r p o r a t i o n of H - l y s i n e i n t o p r o t e i n was measured (67). Rose and h i s co-workers have conducted a number of st u d i e s i n which they i n v e s t i g a t e d the e f f e c t s of d a r k - r e a r i n g and subsequent exposure to l i g h t on p r o t e i n synthesis i n the v i s u a l c o r t e x of r a t s . Rats t h a t had been dark-reared f o r 7 weeks followed by exposure to l a b o r a t o r y i l l u m i n a -3 t i o n f o r various times a f t e r they had been i n j e c t e d w i t h H - l y s i n e showed a t r a n s i e n t increase i n the i n c o r p o r a t i o n of the r a d i o a c t i v e amino a c i d i n t o p r o t e i n (68). The question of the biochemical s p e c i f i c i t y of these changes i n p r o t e i n synthesis was then i n v e s t i g a t e d by d i f f e r e n t i a l l y l a b e l l i n g the p r o t e i n s of the v i s u a l c o r t e x of experimental and c o n t r o l animals w i t h carbon-14 and t r i t i u m l a b e l l e d amino acids and f r a c t i o n a t i n g the s o l u b l e and p a r t i c u l a t e p r o t e i n s on polyacrylamide gels (69,70). I t was found that 2 out of 21 s o l u b l e p r o t e i n bands and 7 out of,20 p a r t i c u -l a t e p r o t e i n bands e x h i b i t e d high d i f f e r e n t i a l i n c o r p o r a t i o n rates between c o n t r o l (dark-reared) and experimental (light-exposed) animals. This suggests that c e r t a i n p r o t e i n f r a c t i o n s are d i s p r o p o r t i o n a t e l y a f f e c t e d by v i s u a l s t i m u l a t i o n a f t e r dark r e a r i n g . A problem w i t h the approach of f r a c t i o n a t i n g p r o t e i n s by g e l e l e c t r o -phoresis i s the d i f f i c u l t y of a s c r i b i n g f u n c t i o n a l r o l e s to those p r o t e i n s which are s p e c i f i c a l l y a f f e c t e d . For t h i s reason an a l t e r n a t e method of f r a c t i o n a t i o n was adopted by these workers which i n v o l v e d the s e p a r a t i o n of the v i s u a l cortex i n t o two c e l l u l a r components: the neuronal and n e u r o p i l ( g l i a , d e n d r i t e s , axons) f r a c t i o n s . I t was found that the e l e v a -3 t i o n i n i n c o r p o r a t i o n of H - l y s i n e i n t o p r o t e i n which occurs during f i r s t exposure to l i g h t , takes place amongst the neuronal p r o t e i n s (71). In a l l areas of the c o r t e x of normally reared animals the neuronal to n e u r o p i l i n c o r p o r a t i o n r a t i o f o r short l a b e l l i n g times was 1.6 but a f t e r 4 hours decreased to 0.5 (72). Although t h i s was the r a t i o obtained f o r the motor co r t e x of dark-reared animals, the r a t i o f o r the v i s u a l c o r t e x even at short i n c o r p o r a t i o n time i n t e r v a l s was 0.7 and increased to normal values only i f the animal was exposed to l i g h t . From these s t u d i e s i t was sug-gested that the synthesis of r a p i d l y l a b e l l e d , r a p i d l y transported p a r t i c u -l a t e neuronal p r o t e i n s i s supressed i n the v i s u a l c o r t e x but not the motor cor t e x of dark-reared r a t s (72,73). The e f f e c t of u n i l a t e r a l e y e - l i d s u t u r i n g on Na +, K + a c t i v a t e d ATPase and N a + and K*" content has been s t u d i e d i n the o p t i c tectum of the a d u l t pigeon (74). There occurred a t r a n s i e n t increase i n t h i s enzyme a c t i v i t y between 4 and 8 weeks of v i s u a l d e p r i v a t i o n and t h i s was accom-panied by an increase i n N a + content and a decrease i n id" content. These changes i n enzyme a c t i v i t y and Na"*" and K*~ i o n content were c o r r e l a t e d to . the c h a r a c t e r i s t i c s e n s i t i v i t y of the b r a i n p r o t e i n s y n t h e s i z i n g system to the i o n i c environment and i t was suggested that the t r a n s i e n t changes were evidence of f u n c t i o n a l adaptation. The two neurotransmitter m e t a b o l i z i n g enzymes c h o l i n e a c e t y l t r a n s -ferase and a c e t y l c h o l i n e s t e r a s e were s t u d i e d i n the v i s u a l centers of 21 day o l d dark-reared r a t s (75,76). There were no changes i n these enzyme a c t i v i t i e s i n the v i s u a l cortex although changes i n other o p t i c centers (eg. LGN) were found. In the v i s u a l c o r t e x of dark-reared r a t s i t has been found that the amino a c i d l e v e l s e s p e c i a l l y that of glutamate were g e n e r a l l y elevated compared to normally reared c o n t r o l s (77). The f a c t that glutamate was higher by 257o i n experimental animals i s p a r t i c u l a r l y i n t e r e s t i n g s i n c e i t i s suspected that glutamate may f u n c t i o n as a neurotransmitter. The biochemical changes observed i n the v i s u a l c o r t e x as a r e s u l t of the p e r t u r b a t i o n of v i s u a l input must c e r t a i n l y form the basis of the morphological and e l e c t r o p h y s i o l o g i c a l changes that have been observed. I t i s c l e a r that more d e t a i l e d work needs to be done to determine what the biochemical s i g n a l i s f o r a change to take place, what cascade of events t h i s s i g n a l induces, and what the b u i l d i n g blocks are that give r i s e to a l t e r e d neural f u n c t i o n . I t i s p a r t l y toward these problems that the present i n v e s t i g a t i o n i s d i r e c t e d . IV. cAMP and the Nervous System. a) The Metabolism and Function of cAMP i n B r a i n . Adenosine 3 ' , 5 ' - c y c l i c monophosphate i s now recognized as an i n t r a -c e l l u l a r messenger mediating the ac t i o n s of a v a r i e t y of hormones i n s p e c i f i c t a r g e t t i s s u e s . As a r e s u l t of work i n the l a s t decade, the i n -volvement of cAMP i n n e u r o b i o l o g i c a l events i s a l s o g a i n i n g acceptance. What fo l l o w s i s a d i s c u s s i o n of the features of the metabolism and p o s s i b l e r o l e s of cAMP i n nerve t i s s u e i n s o f a r as they are p e r t i n e n t to the present i n v e s t i g a t i o n . Adenylate c y c l a s e , the enzyme r e s p o n s i b l e f o r the sy n t h e s i s of cAMP from i t s s u b s t r a t e ATP, has been shown to be present i n higher a c t i v i t y i n the CNS than i n any other mammalian t i s s u e (78). S u b c e l l u l a r f r a c t i o n a -t i o n s tudies revealed that the enzyme i s l o c a l i z e d to the plasma membrane and i n p a r t i c u l a r to those f r a c t i o n s c o n t a i n i n g nerve endings and sy n a p t i c complexes (79,80). The enzyme r e s p o n s i b l e f o r the degradation of cAMP i s n u c l e o t i d e 3', 5 ' - c y c l i c phosphodiesterase which, l i k e adenylate c y c l a s e , i s present i n higher a c t i v i t y i n the CNS than i n any other mammalian t i s s u e (81). S u b c e l l u l a r d i s t r i b u t i o n and c y t o l o g i c a l l o c a l i z a t i o n s t u d i e s have shown that t h i s enzyme re s i d e s almost e x c l u s i v e l y at the post-s y n a p t i c nerve ending and more p r e c i s e l y at the post-synaptic membrane (82-84). Both adenylate c y c l a s e and phosphodiesterase are concentrated i n those f r a c t i o n s c o n t a i n i n g the great e s t q u a n t i t y of the known neurotrans-m i t t e r s (85) as w e l l as cAMP-dependent enzymes such as cAMP-dependent p r o t e i n kinase (86), the p r o t e i n substrates f o r p r o t e i n kinase (87), phosphoprotein phosphatase (88), and N - a c e t y l t r a n s f e r a s e (89). The pres-ence and s t r a t e g i c l o c a t i o n of t h i s enzymatic machinery, henceforth r e -f e r r e d to as the cAMP system, i n d i c a t e s that cAMP may serve an important f u n c t i o n i n the CNS and that t h i s f u n c t i o n may be r e l a t e d to the process of s y n a p t i c t r a n s m i s s i o n . Further s t u d i e s have l e d to the idea that cAMP may f u n c t i o n as a mediator of the neurohormones involved i n synapt i c t r a n s m i s s i o n . This concept has developed from the demonstration that a v a r i e t y of p u t a t i v e neurotransmitter substances s t i m u l a t e the formation of cAMP, (although only s l i g h t l y i n b r a i n homogenates, do so profoundly i n b r a i n s l i c e s ) . Some of the substances that have been found to increase the content of cAMP i n b r a i n s l i c e s i.include noradrenaline (NA) (2,90,91), histamine (1, 90,91), s e r o t o n i n (92), dopamine (93) and adenosine (94,95). The a b i l i t y of some of these agents to elev a t e cAMP content i n b r a i n s l i c e s i s greater 19 i n some animals than others as i n the case of s e r o t o n i n i n the r a b b i t , and i s greater i n some b r a i n regions than others as i n the case of dopamine i n the caudate nucleus. This f i n d i n g i s c o n s i s t e n t w i t h the heterogeneity of the d i s t r i b u t i o n of neurotransmitters i n the CNS. Nervous t i s s u e f u n c t i o n s by i n t e g r a t i n g i n f o r m a t i o n through e x c i t a -t i o n and i n h i b i t i o n . Thus i t i s noteworthy that p e r t u r b a t i o n of the e l e c t r i c a l processes of nerve a l s o a f f e c t s cAMP l e v e l s . I t has been shown that a v a r i e t y of agents such as K +, ouabain, ba t r a c h o t o x i n , and v e r a t r i d i n e which are known to cause membrane d e p o l a r i z a t i o n a l s o cause profound stimu-l a t i o n of cAMP formation i n b r a i n s l i c e s (96-98). Moreover, i t has been demonstrated that a p p l i c a t i o n of e l e c t r i c a l pulses to b r a i n s l i c e s causes la r g e increases i n t h e i r cAMP content (99). An i n t e r e s t i n g f i n d i n g has been that when adenosine or a d e p o l a r i z i n g agent i s incubated together w i t h some of the biogenic amines (histamine, s e r o t o n i n , NA), the s t i m u l a t i o n of the formation of cAMP i n b r a i n s l i c e s i s much more than a d d i t i v e . Since the mode of a c t i o n of d e p o l a r i z i n g agents and adenosine w i t h regard to t h e i r a b i l i t y to s t i m u l a t e cAMP formation i n b r a i n s l i c e s i s not known, the s i g n i f i c a n c e of the s y n e r g i s t i c e f f e c t s between these agents and the amines i s not c l e a r . The strongest support f o r the involvement of neurohormone-sensitive and more s p e c i f i c a l l y c a t e c h o l a m i n e - s e n s i t i v e adenylate cyclases i n synaptic t r a n s m i s s i o n comes from the work of Greengard and h i s colleagues (100-102) on the sympathetic ganglion and Bloom and h i s a s s o c i a t e s (103-105) on the cerebellum. In the i s o l a t e d s u p e r i o r c e r v i c a l sympathetic ganglion of the r a b b i t e l e c t r i c a l s t i m u l a t i o n causes an a c e t y l c h o l i n e mediated d e p o l a r i z a t i o n of g a n g l i o n i c neurons. This e x c i t a t i o n i s followed by a slow and long l a s t i n g h y p e r p o l a r i z a t i o n of g a n g l i o n i c neurons which i s thought to be mediated by dopamine. The i n h i b i t o r y e f f e c t of dopamine i s thought to be mediated by a dopamine-sensitive adenylate c y c l a s e f o r the f o l l o w i n g reasons: (a) S t i m u l a t i o n of the ganglion increases cAMP l e v e l s . (b) cAMP can mimic the h y p e r p o l a r i z i n g e f f e c t of dopamine when a p p l i e d to g a n g l i o n i c neurons. (c) Phosphodiesterase i n h i b i t o r s p o t e n t i a t e both the h y p e r p o l a r i z a t i o n and the increase i n cAMP l e v e l s induced by e l e c t r i c a l s t i m u l a t i o n and a l s o p o t e n t i a t e the h y p e r p o l a r i z a t i o n induced by exogenous dopamine. In the cerebellum P u r k i n j e c e l l s r e c e i v e an i n h i b i t o r y input from a d i f f u s e system of NA-containing nerve t e r m i n a l s . The i o n t o p h o r e t i c a p p l i -c a t i o n of cAMP onto the surface of P u r k i n j e c e l l s was found to mimic the i n h i b i t o r y a c t i o n s of NE on the discharge r a t e s of these neurons. The i n h i b i t o r y e f f e c t s of both NA and cAMP were p o t e n t i a t e d by phosphodiester-ase i n h i b i t o r s . Furthermore, i n t r a c e l l u l a r recordings from P u r k i n j e c e l l s showed that both NA .and'cAMP caused a h y p e r p o l a r i z a t i o n o f the neurons s i m i l a r to that produced by s t i m u l a t i n g the NA pathway i n n e r v a t i n g these c e l l s . Using an immunocytochemical method f o r d e t e c t i n g cAMP, i t was shown that a p p l i c a t i o n of NA or s t i m u l a t i o n of the NA pathway to these c e l l s caused a la r g e increase i n the p r o p o r t i o n of P u r k i n j e c e l l s that reacted p o s i t i v e l y . A f t e r i t s production i n nerve, l i t t l e i s known concerning the sub-sequent biochemical events that cAMP may p a r t i c i p a t e i n or concerning the p o s s i b l e mechanisms by which changes i n cAMP concentrations would i n f l u e n c e e l e c t r i c a l events at the synaptic membrane. Once these processes are de l i n e a t e d i t w i l l become evident whether cAMP i s , i n f a c t , an i n t r a -c e l l u l a r messenger f o r some neurotransmitters. The study of the ta r g e t enzymes of cAMP and t h e i r a c t i o n s may provide the support f o r the i n v o l v e -merit of cAMP i n mediating the f l u c t u a t i o n s i n the i o n i c environment of the membrane and provide some clues as to the mechanism whereby t h i s i s achieved. I t has been suggested, f o r example, that the i o n p e r m e a b i l i t y c h a r a c t e r i s t i c s of the membrane may be modified by the phosphorylation of s p e c i f i c synaptic membrane pr o t e i n s by cAMP-dependent p r o t e i n kinase (100). I n i t i a l c o n d i t i o n s would be r e s t o r e d through the h y d r o l y s i s of cAMP by phosphodiesterase and dephosphorylation of the membrane p r o t e i n by phos-phoprotein phosphatase. The p a r t i c i p a t i o n of cAMP i n metabolic events other than those a t the synapse have not been s y s t e m a t i c a l l y i n v e s t i g a t e d . Therefore, the problem of r e l a t i n g and connecting the events which intervene between neuronal s t i m u l a t i o n and the general metabolic responses which are known to occur i n nerve under these c o n d i t i o n s remain unsolved i n s o f a r as the involvement of cAMP i s concerned. I t i s b e l i e v e d , however, that the synthesis and ca t a b o l i s m of glycogen i n nerve might be a f f e c t e d by hor-mones i n a manner analogous to that i n other t i s s u e s , w i t h cAMP as a mediator. cAMP has a l s o been i m p l i c a t e d i n the process of axonal elonga-t i o n s i n c e i t i s known that axonal e l o n g a t i o n i s d i r e c t l y dependent on the assembly of microtubules and that the c y c l i c n u c l e o t i d e can s t i m u l a t e t h i s assembly process (106). b) Development of the cAMP System i n B r a i n . Few i n v e s t i g a t i o n s have been conducted on the development of the cAMP system i n animals and as a r e s u l t even fewer studies have been con-ducted where normal development has been perturbed and the e f f e c t s c o r r e -l a t e d w i t h the p o s s i b l e functions of t h i s system. I t i s u n l i k e l y that the pa u c i t y of inf o r m a t i o n i n t h i s area i s due to the idea that such i n v e s t i g a -t i o n s might be f r u i t l e s s s i n c e these studies w i l l s u r e l y be of enormous value i n f i n a l l y determining the f u n c t i o n a l r o l e of cAMP i n b r a i n . I n -stead, i t may be due to the d i v e r s i o n of e f f o r t s towards understanding the enzyme systems and neurohormones inv o l v e d i n the cAMP system. Apart from adding to our understanding of the f u n c t i o n of cAMP i n b r a i n , develop-mental st u d i e s are important because cAMP may play an i n t e g r a l part i n the development and maturation of the nervous system. Moreover, the p a r t i c i p a t i o n of cAMP i n c e r t a i n aspects of ontogenesis of b r a i n may form the basis f o r the f u n c t i o n of cAMP once development i s complete. Thus, the processes that cAMP may r e g u l a t e might overlap the c h a r a c t e r i s t i c processes of development and d i f f e r e n t i a t i o n (107). I f cAMP i s in v o l v e d i n s y n a p t i c t r a n s m i s s i o n a c o r o l l a r y of the above hypothesis i s that syn-a p t i c t r a n s m i s s i o n i t s e l f may be inv o l v e d i n the development of the nervous system. Support f o r t h i s hypothesis i s a v a i l a b l e from work which has been conducted w i t h l i v e r . In t h i s organ, adrenaline and glucagon, both of which s t i m u l a t e adenylate c y c l a s e , s t i m u l a t e the i n d u c t i o n of s e v e r a l hepatic enzymes when i n j e c t e d i n t o the fetus i n utero (108,109) or when a p p l i e d to f e t a l r a t l i v e r c e l l s (110). Exogenously a p p l i e d cAMP, i n a d d i t i o n to mimicking the ac t i o n s of these hormones, has been shown to en-hance t r a n s c r i p t i o n and s t i m u l a t e RNA polymerase i n i s o l a t e d l i v e r n u c l e i (111). In the r a t b r a i n the development of many enzyme systems occurs during the l a t e r h a l f of the second p o s t - n a t a l week (112). Further i n -d i r e c t support f o r the above hypothesis i s a v a i l a b l e from the observation that t h i s increase i n enzyme a c t i v i t y i s preceded by an increase i n b r a i n noradrenaline and an increase i n adenylate c y c l a s e a c t i v i t y (113). More-over, the maximal a c t i v i t y of the e f f e c t o r end of the cAMP system, that of cAMP st i m u l a t e d cAMP-dependent p r o t e i n k i nase, i s f u l l y developed i n the newborn r a t b r a i n (114-116). Neonatal thyroidectomy of r a t s has been shown to impair d r a s t i c a l l y b r a i n development and to lead to anatomical, b e h a v i o r a l and enzymatic d y s f u n c t i o n i n the mature r a t . By t h i s treatment an attempt was made at reducing the a b i l i t y of b r a i n to generate cAMP or to respond to i t , thereby p r o v i d i n g a means to study the p o s s i b l e r o l e of cAMP i n development (116). Although thyroidectomy at b i r t h caused a 16% r e d u c t i o n i n b r a i n weight and a 707. r e d u c t i o n i n body weight by 40 days of age, i t had no e f f e c t on e i t h e r whole b r a i n or c o r t i c a l a c t i v i t y of phosphodiesterase, adenylate c y c l a s e , and the a b i l i t y of NA to s t i m u l a t e the production of cAMP i n b r a i n s l i c e s . I t has been found that i n undernourished r a t s there i s a 25% reduc-t i o n i n b r a i n NA and dopamine and an increase i n t y r o s i n e hydroxylase as compared to adequately fed r a t s (117). This prompted an i n v e s t i g a t i o n to determine the e f f e c t s of m a l n u t r i t i o n on the cAMP system. I t was found that i n undernourished neonatal r a t s the c a p a c i t y of the c e r e b r a l c o r t e x to generate and metabolize cAMP, as shown by adenylate c y c l a s e and phos-phodiesterase a c t i v i t i e s , i s i n s e n s i t i v e to c a l o r i c r e s t r i c t i o n d u r i ng e a r l y p o s t - n a t a l l i f e (118). Studies employing thyroidectomy or m a l n u t r i t i o n appear to cast doubt on the involvement of cAMP, neur o t r a n s m i t t e r s , or synap t i c t r a n s m i s s i o n i n b r a i n ontogenesis. However, the above f i n d i n g s are completely c o n s i s t e n t w i t h the view pointed out e a r l i e r which was that the involvement of these processes i n ne u r a l development may e x i s t only at metabolic and morphologic l e v e l s t h a t i n the f u l l y d i f f e r e n t i a t e d s t a t e succumb to the c o n t r o l of cAMP. A f u r t h e r developmental aspect of the cAMP system i n b r a i n i s the age dependency of the a b i l i t y of various neurotransmitters to s t i m u l a t e cAMP synthesis i n b r a i n s l i c e s . Thus, an important f i n d i n g which has not been, s t r e s s e d i n the l i t e r a t u r e i s that the s t i m u l a t i o n of cAMP formation by some neurotransmitters increases to a maximum at an e a r l y age and t h e r e a f t e r d e c l i n e s to a value observed i n the a d u l t . In r a b b i t c o r t i c a l s l i c e s , f o r example, the histamine-induced formation of cAMP i s highest a t 8 days postpartum and lower a t b i r t h and i n the a d u l t by 75% and 37%., r e s p e c t i v e l y (119). A s i m i l a r d i m i n u t i o n i n the a b i l i t y of NA to s t i m u l a t e cAMP formation occurs by 25 days of age i n the r a b b i t and i s most pro-nounced i n the f r o n t a l c o r t e x and hypothalamus where the decrease from peak s t i m u l a t i o n i s 88%, and 93%. r e s p e c t i v e l y (120). In r a t whole b r a i n s l i c e s , peak responsiveness to NA occurs a t 16 days of age and decreases to 50% of t h i s value by 25 days (113,116). U n l i k e r a b b i t , r a t c o r t i c a l s l i c e s d i d not show a d e c l i n e i n responsiveness to NA w i t h age (121). This could be due to species d i f f e r e n c e s but i s probably due to d i f f e r e n c e s i n b r a i n regions s i n c e i n the r a b b i t f r o n t a l c o r t e x was st u d i e d whereas i n stud i e s w i t h r a t e i t h e r whole b r a i n or whole c o r t e x was employed. An i n t e r p r e t a t i o n of the increased s e n s i t i v i t y of the cAMP system to neurotransmitters i n the developing b r a i n i s that t h i s may be one of the mechanisms whereby cAMP could i n f l u e n c e morphogenesis. More s t r i n g e n t i n v e s t i g a t i o n s are r e q u i r e d to determine the v a l i d i t y of t h i s concept. V. Adaptive Mechanisms i n Nerve. I t has been known f o r some time that e x c i t a b l e t i s s u e s i n c l u d i n g a l l types of muscle, the p i n e a l gland, exocrine organs, and the c e n t r a l nervous system, can e x h i b i t v a r i a b l e s e n s i t i v i t y to neurohormones and chemical agents (122-124). For example, d e p r i v a t i o n of nervous i n f l u e n c e by various methods which b r i n g about disuse causes e f f e c t o r organs to become more e x c i t a b l e w h i l e continuous e x c i t a t i o n causes them to become l e s s s e n s i t i v e . Thus, e x c i t a b l e c e l l s seem to have a feedback system that allows them to compensate f o r chronic changes i n the l e v e l of stimulus they r e c e i v e , be-coming more s e n s i t i v e when the stimulus i s low and l e s s s e n s i t i v e when the stimulus i s high. The terms s u p e r s e n s i t i v i t y and s u b s e n s i t i v i t y have been used to describe these phenomena. The biochemical basis f o r a l t e r a t i o n s i n s e n s i t i v i t y i s not known and, w i t h respect to the CNS, d i f f i c u l t to i n v e s t i g a t e . Therefore, the autonomic n e u r o e f f e c t o r j u n c t i o n and the s k e l e t a l neuromuscular j u n c t i o n have been f r e q u e n t l y used as models of CNS synapses. Some of the p r i n c i -ples that have emerged from i n v e s t i g a t i o n s i n these systems are as f o l l o w s : S u p e r s e n s i t i v i t y i n some e f f e c t o r organs i s n o n - s p e c i f i c , f o r example, the smooth muscle of the n i c t i t a t i n g membrane of the cat which i s normally innervated by adrenergic f i b e r s becomes s e n s i t i z e d a f t e r pro-longed disuse to adrenomimetics, cholinomimetics, s e r o t o n i n , and potassium ions (125). S u p e r s e n s i t i v i t y i n t h i s system i s slow to develop ( r e q u i r i n g s e v e r a l weeks), i s r e v e r s i b l e ( s e n s i t i v i t y r e v e r t i n g to normal when input i s r e s t o r e d (126)), and i s produced by withdrawal of e x c i t a t o r y i n f l u e n c e only and not by pharmacological blockade or denervation of i n h i b i t o r y input (127). To what extent these f i n d i n g s can be e x t r a p o l a t e d to the CNS remains to be e s t a b l i s h e d . At present, however, they are u s e f u l as a poin t of departure. From s t u d i e s of p e r i p h e r a l systems one of the hypothesis put forward f o r the generation of s u p e r s e n s i t i v i t y i s the p r o l i f e r a t i o n of new recep-t o r s f o r neurotransmitters. Although not e n t i r e l y s a t i s f a c t o r y , t h i s e x p l a n a t i o n i s supported by the f i n d i n g that i n normally innervated s t r i -a t a l muscle a c e t y l c h o l i n e (ACh) s e n s i t i v i t y r e s i d e s , and d e p o l a r i z a t i o n can be e l i c i t e d , o n l y w i t h i n a few hundred microns from the neuromuscular j u n c t i o n . A f t e r denervation, however, the e n t i r e muscle becomes s e n s i t i z e d to ACh (128). F e t a l muscle f i b e r s are a l s o s e n s i t i v e along t h e i r e n t i r e length, the ACh - s e n s i t i v e area s h r i n k i n g to"the end-plate r e g i o n only a f t e r a f u n c t i o n a l myoneural connection i s e s t a b l i s h e d (129). Important i n i t s v i n d i c a t i o n of the p e r i p h e r a l system models of CNS f u n c t i o n i s the recent demonstration that a t l e a s t some neurons undergo s i m i l a r changes. A f t e r denervation of the parasynpathetic ganglion c e l l s of the f r o g heart, i t was shown that the ACh s e n s i t i v i t y spread from t h e i r normally confined subsynaptic zones to the e n t i r e surface of the neuron (130,131). A f u r t h e r mechanism that has been proposed f o r the generation of s u p e r s e n s i t i v i t y i n muscle i s an increase i n the e f f i c a c y o f c o u p l i n g between e x c i t a t i o n and c o n t r a c t i o n (54). The analogous process i n nerve would be increased c o u p l i n g between post-synaptic t r a n s m i t t e r - r e c e p t o r i n t e r a c t i o n and the subsequent e l e c t r i c a l a c t i v i t y of the neuronal membrane. The cAMP system i n nerve lends i t s e l f w e l l to a c t i n g as the mediator f o r t h i s c o u p l i n g . This i s supported by the recent demonstration that t r e a t -ments which a l t e r the l e v e l of a c t i v i t y of nervous t i s s u e and thus produce st a t e s of s u p e r s e n s i t i v i t y a l s o a l t e r the e f f i c a c y of neurotransmitter s t i m u l a t i o n of cAMP formation. The i n i t i a l s t u d i e s suggesting a p o s s i b l e involvement of adenylate c y c l a s e and cAMP i n the mechanisms of denervation s u p e r s e n s i t i v i t y were performed by Weiss and Costa (132) and Weiss (133). A b l a t i o n of the su p e r i o r c e r v i c a l g a n glion of the r a t causes denervation of the p i n e a l and increased catecholamine-stimulated adenylate c y c l a s e a c t i v i t y i n v i t r o . Subsequently, i t was shown that i n the c o r t e x the cAMP formation induced by NA was augmented (134,135) a f t e r treatments w i t h r e s e r p i n e or 6-hyd-roxydopamine, both of which reduce the l e v e l of exposure of p o s t - s y n a p t i c s t r u c t u r e s to NA by a f f e c t i n g NA c o n t a i n i n g t e r m i n a l s . That t h i s phenome-non i s not unique to NA was demonstrated by the f i n d i n g that there i s increased dopamine-induced cAMP formation i n homogenates of caudate nucleus a f t e r radiofrequency of 6-hydroxydopamine l e s i o n s of the s u b s t a n t i a n i g r a (136). These procedures r e s u l t i n the degeneration of dopamine c o n t a i n -ing terminals i n the caudate nucleus thus inducing a s t a t e of disuse of post-synaptic s t r u c t u r e s by reducing the exposure of these s t r u c t u r e s to dopamine. The above f i n d i n g s suggest that the cAMP system may be i n t i m a t e l y a s s o c i a t e d w i t h the mechanisms that form the basis f o r the p l a s t i c i t y and a d a p t a b i l i t y demonstrated by the CNS. I t must be pointed out, however, that w i t h regard to s u p e r s e n s i t i v i t y i t i s not known whether the p a r t i c i -p a t i o n of the cAMP system i s the basis f o r or a by-product of t h i s phe-nomenon and t h i s w i l l not be c l e a r u n t i l a b e t t e r understanding i s achieved about the r o l e of cAMP i n nerve. VI. The Present I n v e s t i g a t i o n . In the foregoing d i s c u s s i o n evidence was presented that treatments, p h y s i o l o g i c a l or chemical, which p r e c i p i t a t e disuse of c e r t a i n n e u r a l s t r u c t u r e s r e s u l t i n an increased e x c i t a b i l i t y of these s t r u c t u r e s and, where examined, the s e n s i t i v i t y of the cAMP system to s t i m u l a t i o n by neurotransmitters has been shown to be a l t e r e d . Data have been given which demonstrate that l i g h t d e p r i v a t i o n of animals induces a v a r i e t y of changes i n regions of the b r a i n subservient to t h i s sense modality. I t was f u r t h e r pointed out that l e s i o n s of the anatomical pathway of the v i s u a l system r e s u l t i n changes s i m i l a r i n some respects to those caused by l i g h t d e p r i v a t i o n . These changes i n c l u d e morphological agenesis and/or atrophy and e l e c t r o p h y s i o l o g i c a l s u p e r s e n s i t i v i t y . These f i n d i n g s support the c o n t e n t i o n that l i g h t d e p r i v a t i o n of animals induces i n the v i s u a l cortex a s i m i l a r type of disuse of neu r a l elements as s u r g i c a l and chemical l e s i o n s have been shown to do i n other n e u r a l systems. The involvement of the cAMP system i n synaptic t r a n s m i s s i o n has been discussed. Information was a l s o presented that l i g h t d e p r i v a t i o n of animals a f f e c t s those mor-phologic s t r u c t u r e s of the v i s u a l c o r t e x that the cAMP system i s i n t i m a t e l y a s s o c i a t e d w i t h . I t may be argued, t h e r e f o r e , that s i n c e the a c t i v i t y of the cAMP system i n nerve has been shown to be a l t e r e d as a consequence of disuse or dim i n u t i o n of f u n c t i o n , i t may a l s o be a f f e c t e d i n the v i s u a l c o r t e x of l i g h t - d e p r i v e d animals. The purpose of the present i n v e s t i g a -t i o n i s to t e s t t h i s hypothesis. The experimental approach employed to accomplish t h i s was to measure the a b i l i t y of various agents to s t i m u l a t e the formation of cAMP i n v i s u a l and f r o n t a l c o r t i c a l s l i c e s of dark-reared and normally-reared r a t s at var i o u s p o s t - n a t a l ages. The agents s e l e c t e d were NA, K +, and adenosine, and combinations of NA and IC*" w i t h adenosine. As mentioned e a r l i e r , i n some instances a degree of n o n - s p e c i f i c i t y of e x c i t a t i o n by a v a r i e t y of agents develops i n s u p e r s e n s i t i v e t i s s u e . Although the disuse of neurons imposed by v i s u a l d e p r i v a t i o n and a l t e r e d e x c i t a b i l i t y may i n v o l v e prim-a r i l y neurotransmitters a s s o c i a t e d w i t h the processing of v i s u a l input (and the biochemical i d e n t i t y of these i s unknown) t h i s may g e n e r a l i z e to other substances such as those employed i n t h i s study. The present i n v e s t i g a t i o n represents an i n i t i a l attempt to determine 29 whether the cAMP system with a l l its ramifications can be used as a tool to study what influences environment and experience may have on brain development and subsequent function. MATERIALS AND METHODS I. Chemicals. The sources of the chemicals used i n the assay of cAMP were as f o l l o w s : beef heart cAMP-dependent p r o t e i n kinase and non-radioactive cAMP were obtained from the Sigma Chemical Company; bovine serum albumin (BSA) was obtained from Calbiochem; h y d r o x y l a p a t i t e ( B i o - g e l HTP) was purchased from Bio-Rad L a b o r a t o r i e s ; and t r i t i a t e d cAMP (37.7 Ci/mmole) was purchased from New England Nuclear. The sources of the chemicals to which b r a i n s l i c e s were exposed were as f o l l o w s : 1-noradrenaline (1-arterenol) was obtained from the Sigma Chemical Company; adenosine (grade A) was a product of Calbiochem; and potassium c h l o r i d e (reagent grade) was purchased from the F i s h e r Chemical Company. For l i q u i d s c i n t i l l a t i o n counting T r i t o n X-100 was obtained from the Sigma Chemical Company. Unless otherwise noted, a l l other chemicals were obtained from e i t h e r the F i s h e r Chemical Company or M a l l i n c k r o d t . I I . Maintenance and Treatment of Animals. A l b i n o r a t s of the Wistar s t r a i n , obtained from the v i v a r i u m of the U n i v e r s i t y of B r i t i s h Columbia, were employed throughout t h i s study. Animals were subjected to two d i f f e r e n t environmental c o n d i t i o n s during weaning and subsequent maturation. One of these c o n d i t i o n s involved groups of normally reared or c o n t r o l animals, w h i l e the other involved the r e a r i n g of animals i n complete darkness. For the most p a r t , c o n t r o l r a t s of app r o p r i a t e ages and sex were obtained from the v i v a r i u m on the day they were to be used i n an experiment. However, l i g h t deprived animals were r a i s e d and maintained by us i n animal f a c i l i t i e s i n our l a b o r a t o r y . Since the environment played an important r o l e i n t h i s study i t was necessary to maintain a group of normally reared r a t s i n the l a b o r a t o r y f a c i l i t i e s under our care u n t i l i t could be e s t a b l i s h e d that these animals were not d i f f e r e n t from normally r a i s e d animals of the v i v a r i u m w i t h r e -gard to the biochemical systems under i n v e s t i g a t i o n . I n i t i a l l y t h i s was achieved by t r a n s p o r t i n g from the v i v a r i u m pregnant r a t s that were due to give b i r t h w i t h i n 3 to 5 days. This procedure, however, l e d to a h i g h m o r t a l i t y r a t e among the l i t t e r s and r e s u l t e d i n the death of some of the mothers. I t was found that by t r a n s f e r r i n g the r a t s from the v i v a r i u m roughly 3 days a f t e r the females had given b i r t h , the m o r t a l i t y r a t e could be reduced to v i r t u a l l y zero. C o n t r o l r a t s r a i s e d i n t h i s l a b were subject to s i m i l a r c o n d i t i o n s as those i n the vivarium. This included a l i g h t - d a r k c y c l e of 12 hours, an equal d e n s i t y of r a t s per cage and food (Purina r a t chow) and water ad l i b i t u m . The s e p a r a t i o n of l i t t e r s from mothers was at 21 days of age and the young males were separated from the females a f t e r about 30 days of age. L i g h t d e p r i v a t i o n of animals was achieved by p l a c i n g 3 day o l d l i t t e r s together w i t h the mothers i n t o a l i g h t sealed wooden box. This dark-box was constructed to accommodate 8 such groups of animals and v e n t i l a t e d s u f f i c i e n t l y to maintain the temperature w i t h i n the box, when f i l l e d to c a p a c i t y , equal to that of the surrounding animal room i n which the box was kept. These animals were exposed to a 15 watt red s a f e t y l i g h t f o r a maximum of about 3 minutes each day. During t h i s time i n t e r -v a l the animals were fed and the c l e a n i n g of the cages was accomplished. Due to the l i m i t e d f a c i l i t i e s f o r r a i s i n g animals i n the dark i t was necessary to i n c l u d e both males and females i n a l l experiments i n order to 32 o b t a i n s u f f i c i e n t data. Consequently, a l l data were s c r u t i n i z e d f o r p o s s i b l e sex d i f f e r e n c e s . The l i g h t - d e p r i v e d or experimental animals and the c o n t r o l animals were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n . To avoid.-, exposure of the experimental animals to the f l u o r e s c e n t l i g h t of the l a b o r a t o r y and to all o w the same treatment f o r both the c o n t r o l as w e l l as the experimental r a t s , the s a c r i f i c i n g of a l l animals was c a r r i e d out under the i l l u m i n a -t i o n of a red s a f e t y l i g h t . I I I . Techniques Involved i n the Treatment of B r a i n Tissue. a) P r e p a r a t i o n of Kreb-Ringer Bicarbonate B u f f e r . Krebs-Ringer Bicarbonate b u f f e r (KR-buffer) was used f o r the r i n s i n g of brains during d i s s e c t i o n and i n a l l the incubations of b r a i n s l i c e s . The b u f f e r was prepared (137) by bubbling a mixed gas (95% - 5% CO2) through a 25 mM s o l u t i o n of sodium bicarbonate f o r 40 minutes. The f o l l o w i n g ions, a t the f i n a l concentrations i n the b u f f e r , were then added from 10 times concentrated stock s o l u t i o n s : 118 mM NaCI, 5 mM KGl, 2.5 mM C a C l 2 , 2 mM KH2PO4, 2 mM MgS0 4, and 0.02 mM EDTA. Glucose was added to a f i n a l c o n c e n t r a t i o n of 12 mM. This stock b u f f e r s o l u t i o n was gassed as above f o r an a d d i t i o n a l 10 minutes before use, and then f o r the remainder of the experiment, throughout which i t was kept on i c e . b) Tissue P r e p a r a t i o n . A modified procedure of the method o r i g i n a l l y described by K a k i u c h i and R a i l (1) was used f o r the p r e p a r a t i o n of b r a i n s l i c e s . I n t a c t b r a i n , immediately a f t e r removal from animals, was r i n s e d w i t h i c e - c o l d KR-buffer and placed on wet f i l t e r paper mounted on a glass P e t r i d i s h which was i n contact w i t h i c e . The area of the cortex described by Adams and F o r r e s t e r (138) as that r e c e i v i n g the input from primary v i s u a l a f f e r e n t s was then d i s s e c t e d from both the l e f t and r i g h t s i d e of the b r a i n . The area taken as f r o n t a l c o r t e x was the l e f t and r i g h t s i d e of the most a n t e r i o r pole of the f o r e b r a i n . With t h i s procedure four i n t a c t slabs of b r a i n t i s s u e were obtained from each animal. A f t e r c a r e f u l removal of the white matter from these slabs the t i s s u e was weighed. The y i e l d of t i s s u e from each s i d e of the v i s u a l c o r t e x from animals 30 days and ol d e r was u s u a l l y between 40 and 50 mg. The weight of the samples from the f r o n t a l c o r t e x was of the same magnitude. From animals 15 days o l d and younger, the y i e l d of c o r t i c a l t i s s u e from each s i d e of the b r a i n was u s u a l l y l e s s than 25 mg. The i n d i v i d u a l slabs of t i s s u e were s l i c e d u s i n g a M c l l w a i n t i s s u e chopper (Brinkman Instrument Company) w i t h the blade adjustment set f o r a thickness of 0.3 mm. The dimensions of each t i s s u e s l i c e was about 3 x 1.5 x 0.3 mm. The e n t i r e d i s s e c t i o n and chopping procedure r e q u i r e d about 4 minutes, throughout which time the t i s s u e was c o n s t a n t l y r i n s e d w i t h c o l d KR-buffer. c) Incubation Procedures. A f t e r the completion of s l i c i n g , each s l i c e d s l a b of t i s s u e was t r a n s f e r r e d to a 25 ml Ehrlenmyer f l a s k c o n t a i n i n g 3.0 ml of KRj-buffer. The f l a s k s c o n t a i n i n g t i s s u e and b u f f e r were kept i n i c e u n t i l the begin-ning of the inc u b a t i o n . The f l a s k s were topped w i t h a i r t i g h t rubber stoppers and t r a n s f e r r e d to a 37° water bath o s c i l l a t i n g a t 60 - 70 cycles/min. This marked the beginning of two pr e i n c u b a t i o n steps a t the end of which the t i s s u e was exposed to various i n c u b a t i o n c o n d i t i o n s . The f i r s t p r e i n c u b a t i o n was f o r a p e r i o d of 30 minutes during the f i r s t 10 minutes of which the f l a s k was flushed through the rubber stopper w i t h 34 95% O2~57o CO2. Then the b u f f e r was a s p i r a t e d followed by the a d d i t i o n of 3 ml of f r e s h b u f f e r to the f l a s k , which was again gassed f o r the f i r s t 10 minutes of a 15 minute p r e i n c u b a t i o n . A t o t a l of 45 minutes f o r the p r e i n c u b a t i o n i s r e q u i r e d to a t t a i n a constant l e v e l of cAMP i n the c o r t i c a l s l i c e s . During t h i s time the cAMP content i n the t i s s u e i s f a l l i n g , due to metabolism by phosphodiesterase, from an i n i t i a l l y h i g h l e v e l which i s known to be produced i n b r a i n t i s s u e at the time of s a c r i f i c e of the animal (139,140). The zero time f o r the exposure of b r a i n s l i c e s to agents was the 15th minute of the second p r e i n c u b a t i o n . Once the chemical agents were added, the i n c u b a t i o n l a s t e d u s u a l l y f o r 5 minutes except i n the case of time course studies where the i n c u b a t i o n continued up to a maximum of 20 minutes. B a s e l i n e l e v e l s of cAMP was that measured at time zero of the incubation. In i n i t i a l experiments, v i s u a l and f r o n t a l c o r t i c a l t i s s u e from both the l e f t and r i g h t c e r e b r a l hemispheres were incubated s e p a r a t e l y under s i m i l a r experimental c o n d i t i o n s . I t was thought that a more r e l i a b l e value f o r the cAMP content would be obtained per animal by t a k i n g the average of the two values from separate determinations f o r each of the c o r t i c a l areas s t u d i e d . However, i t soon became apparent t h a t , i r r e s p e c -t i v e of the agent the c o r t e x of any p a r t i c u l a r animal was exposed t o , the l e f t and r i g h t sides were always very s i m i l a r w i t h regard to cAMP content. Thus i t was evident that the v a r i a t i o n w i t h which we and other workers i n the f i e l d are plagued regarding the degree of s t i m u l a t i o n of cAMP produc-t i o n i n b r a i n s l i c e s by c e r t a i n chemical agents, does not a r i s e from tech-n i c a l d i f f e r e n c e s such as the measurement of small q u a n t i t i e s of cAMP but may be due r a t h e r to d i f f e r e n c e s among animals. As a r e s u l t of t h i s f i n d i n g f o r r a t s 30 days and ol d e r , the t i s s u e from the l e f t and r i g h t s i d e s , whether v i s u a l or f r o n t a l cortex, were always incubated s e p a r a t e l y and exposed to d i f f e r e n t agents. A l t e r n a t i v e l y , one s i d e was exposed to an agent w h i l e the other s i d e served as a non-exposed c o n t r o l thus a f f o r d i n g a value of cAMP content r e f e r r e d to as the ba s e l i n e l e v e l . For r a t s 15 days of age and younger t h i s was not p o s s i b l e as the y i e l d of c o r t i c a l t i s s u e from one si d e was i n s u f f i c i e n t f o r an i n -cubation. Therefore, the t i s s u e from both sides of e i t h e r the v i s u a l or f r o n t a l c o r t e x was pooled from animals of these ages. IV. I s o l a t i o n and Recovery of cAMP from B r a i n S l i c e s . At the end of the in c u b a t i o n the contents of the in c u b a t i o n f l a s k s were t r a n s f e r r e d to glass tubes which were used f o r both c e n t r i f u g a t i o n and homogenization. The tubes were c e n t r i f u g e d i n the c o l d room f o r about 30 seconds at 1000 x g and the KR-buffer was decanted o f f i n order to avoid i n t e r f e r e n c e from the ions i n the subsequent assay of cAMP (141). The p e l l e t e d b r a i n s l i c e s were homogenized w i t h a T e f l o n p e s t l e i n 1.0 ml of 5% (w/v) t r i c h l o r a c e t i c a c i d (TCA) and the p e s t l e was washed w i t h 0.5 ml of TCA. The TCA homogenate was t r a n s f e r r e d to S o r v a l l c e n t r i f u g e tubes and the homogenization tubes washed w i t h an a d d i t i o n a l 0.5 ml of 5% TCA. The TCA homogenate, t o t a l volume 2.0 ml, was c e n t r i f u g e d a t 10,000 x g f o r 10 minutes. The supernatant, c o n t a i n i n g the cAMP, was t r a n s f e r r e d to 30 ml t e s t tubes. The TCA p r e c i p i t a t e was washed i n 0.5 ml of TCA by rehomogenization and the p e s t l e again r i n s e d w i t h 0.5 ml of 5% TCA. The homogenate was c e n t r i f u g e d as above and the supernatant was pooled w i t h the previous TCA s o l u b l e m a t e r i a l . The numerous washings of the p e s t l e , homogenization tubes and TCA p r e c i p i t a t e were included to maximize the recovery of cAMP. The TCA p r e c i p i t a t e was stored at -20° and assayed f o r the p r o t e i n content at a l a t e r date. The TCA s o l u b l e f r a c t i o n was e x t r a c t e d 4 times w i t h 2 volumes of ether to remove the TCA. The TCA remaining a f t e r e x t r a c t i o n , as determined by t i t r a t i o n , was n e g l i g i b l e . The TCA s o l u b l e f r a c t i o n was then l y o p h i l i z e d and stored at -20° p r i o r to the cAMP assay. To determine the recovery of cAMP from b r a i n s l i c e s the f o l l o w i n g procedure was employed: Before the homogenization of s l i c e s which had been incubated i n the normal manner, a known amount of r a d i o a c t i v e cAMP was added to the homogenization tubes and the samples were taken through the r o u t i n e i s o l a t i o n procedure f o r cAMP. A f t e r r e c o n s t i t u t i o n of the l y o p h i l i z e d TCA s o l u b l e m a t e r i a l , an a l i q u o t was taken f o r determination of r a d i o a c t i v i t y . The percent recovery was c a l c u l a t e d from the amount of r a d i o a c t i v e cAMP i n the a l i q u o t and the known amount added p r i o r to homo-ge n i z a t i o n . V. The Measurement of cAMP Levels i n B r a i n Tissue, a) General P r i n c i p l e of the Assay of cAMP. The method used f o r the assay of cAMP was a m o d i f i c a t i o n of the method of Brostrom and Kon (141) who used a modified procedure of that o r i g i n a l l y described by Gilman (142). The method can be described essen-t i a l l y as the competition between a f i x e d known amount of t r i t i a t e d cAMP and u n l a b e l l e d cAMP f o r cAMP-dependent p r o t e i n kinase (PK), a p r o t e i n which has both high a f f i n i t y and high s p e c i f i c i t y f o r the b i n d i n g of cAMP. The source of the competing u n l a b e l l e d cAMP i s e i t h e r from stock s o l u t i o n s of known concentrations f o r the purpose of generating a standard curve or from a sample c o n t a i n i n g unknown q u a n t i t i e s of cAMP. The c o n s t r u c t i o n of a standard curve involves the in c u b a t i o n of a s e r i e s of tubes c o n t a i n i n g PK w i t h a constant amount of r a d i o a c t i v e cAMP and i n c r e a s i n g concentrations of u n l a b e l l e d cAMP. The net r e s u l t of t h i s i s to incubate PK w i t h decreasing s p e c i f i c a c t i v i t i e s of r a d i o a c t i v e cAMP. The amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h PK i s then measured and when t h i s i s p l o t t e d on l o g - l o g axes against cAMP con c e n t r a t i o n , a s t r a i g h t l i n e i s obtained. For the determination of cAMP content from an e x p e r i -mental sample, an a l i q u o t i s used which w i l l produce a s p e c i f i c a c t i v i t y of cAMP that i s w i t h i n the l i m i t s of the standard curve. The amount of cAMP that must have been present i n the sample to create the r e s u l t a n t s p e c i f i c a c t i v i t y i s then i n t e r p o l a t e d from the standard curve. The b i n d i n g of cAMP to PK can be increased by i n c l u d i n g p r o t e i n kinase i n h i b i t o r i n the assay (142). We have chosen to in c l u d e BSA i n the assay mixture s i n c e i t has been shown (141) that a number of p r o t e i n s , i n c l u d i n g BSA, are p r o t e i n kinase i n h i b i t o r s . A v a r i e t y of methods have been reported to achieve the s e p a r a t i o n of PK-bound cAMP from free cAMP. These include the bindin g of the PK-cAMP complex to n i t r o c e l l u l o s e membrane f i l t e r s (142) or to h y d r o x y l a p a t i t e (141) which i s added to the assay mixture i n the form of a s l u r r y , or the a d s o r p t i o n of free cAMP on char c o a l (143). I n the present i n v e s t i g a -t i o n the h y d r o x y l a p a t i t e procedure was employed e s s e n t i a l l y because i t i s a r e l a t i v e l y l e s s expensive method. I n i t i a l l y the hydroxylapatite-PK-cAMP complex was separated from f r e e cAMP by c e n t r i f u g a t i o n as suggested by Brostrom and Kon (141). Since t h i s procedure r e s u l t e d i n i n c o n s i s t e n c i e s , the hydroxylapatite-PK-cAMP complex was c o l l e c t e d on Whatman No. 1 f i l t e r paper d i s c s by means of f i l t r a t i o n . This technique was r e l a t i v e l y f a s t e r and gave h i g h l y r e p r o d u c i b l e r e s u l t s . b) M a t e r i a l s f o r the Assay of cAMP. Stock s o l u t i o n s of t r i t i a t e d cAMP contained 50 mM sodium acetate b u f f e r , pH 4.5, 5 mg/ml BSA, and s u f f i c i e n t r a d i o a c t i v i t y to give about 80,000 dpm per 0.1 ml. Stock s o l u t i o n s of PK were made by d i s s o l v i n g l y o p h i l y s e d beef heart PK i n d i s t i l l e d water producing a c o n c e n t r a t i o n of 150 yg per ml. Small a l i q u o t s of the H-cAMP s o l u t i o n and of the PK s o l u t i o n were stored at -20° to avoid excessive f r e e z i n g and thawing. The bin d i n g a c t i v i t y of PK was s t a b l e f o r up to 2 months. The amount of PK used i n the assay can be v a r i e d i n v e r s e l y w i t h the amount of r a d i o a c t i v e cAMP used. Thus, f o r the purpose of s c i n t i l l a t i o n counting, the d e s i r e d q u a n t i t y of r a d i o a c t i v e cAMP bound to PK can be achieved e i t h e r by v a r y i n g the q u a n t i t y of p r o t e i n kinase or H-cAMP employed per assay. I t i s important, however, to use an amount of PK that w i l l be saturated by the cAMP concentrations being measured. Batches of the h y d r o x y l a p a t i t e s l u r r y were made by adding 12 ml of d i s t i l l e d water to 1.5 gm of h y d r o x y l a p a t i t e and were stored at 4°. F i l t e r paper d i s c s of 2.8 cm i n diameter were cut from sheets of Whatman No. 1 chromatography paper. The c o n c e n t r a t i o n of the u n l a b e l l e d cAMP s o l u t i o n used f o r the pro-d u c t i o n of standard curves was checked by measuring "the adsorbance at 256 nm usin g the molar e x t i n c t i o n c o e f f i c i e n t of 14,500 f o r cAMP at pH 2.0. For the f i l t r a t i o n procedure M i l l i p o r e funnels (15 ml capacity) equipped w i t h a 25 mm diameter base and f r i t t e d glass f i l t e r supports, were employed. A Duo-Seal r o t a r y vacuum pump was used to provide the vacuum f o r f i l t r a t i o n . c) D e t a i l s of the Procedure f o r the Assay of cAMP. The l y o p h o l i z e d TCA s o l u b l e f r a c t i o n was r e c o n s t i t u t e d w i t h 2.0 ml of 50 mM sodium acetate b u f f e r , pH 4.5, (NaAc b u f f e r ) and an a l i q u o t of t h i s was assayed f o r cAMP content. The volume of b u f f e r added to the d r i e d samples may vary s i n c e the standard curve f o r cAMP e s t i m a t i o n may be constructed to encompass a wide range of cAMP concentrations. However, ca u t i o n must be taken not to d i l u t e the cAMP to excess r e l a t i v e to the concentrations of other adenine n u c l e o t i d e s . The reason f o r t h i s i s the f i n d i n g that adenine n u c l e o t i d e s can i n t e r f e r e i n the assay of cAMP by competing f o r b i n d i n g to PK. A d i l u t i o n of no greater than 30 of the o r i g i n a l t i s s u e i s recommended by Gilman (142). In the present work, problems were not encountered unless the t i s s u e d i l u t i o n was greater than 70. Thus, the l y o p h i l i z e d TCA s o l u b l e f r a c t i o n of a sample of b r a i n t i s s u e weighing about 0.05 g was not taken up i n more than 2.0 ml of acetate b u f f e r , thereby a f f o r d i n g a t i s s u e d i l u t i o n of about 40. The assay of cAMP was conducted i n a t o t a l volume 0.2 ml. To a sample volume of 80 u l , was added 0.1 ml of r a d i o a c t i v e cAMP s o l u t i o n c o n t a i n i n g u s u a l l y between 3 - 4 pmoles of cAMP. The assay tubes were cooled to 4° and a f t e r the a d d i t i o n of 20 p1 of PK, the tubes were shaken and incubated f o r 1 hour a t 4°. To generate the standard curve the 80 y l of sample was replaced by i n c r e a s i n g amounts of u n l a b e l l e d cAMP ranging from about .7 to 45 pmoles and where r e q u i r e d , the 0.2 ml i n c u b a t i o n volume was achieved by the a d d i t i o n of NaAc b u f f e r . At the end of the 1 hour inc u b a t i o n , 0.2 ml of the h y d r o x y l a p a t i t e s l u r r y (precooled to 4°) was added to the assay tubes and the tubes were incubated f o r an a d d i t i o n a l period of 5 minutes a t 4°. Fo l l o w i n g t h i s , 1.0 ml of i c e c o l d 10 mM potassium phosphate b u f f e r , pH 6.0, (KP buf f e r ) was added and the assay tubes were maintained at 4 U f o r a minimum of 5 minutes. The contents of the tubes were then f i l t e r e d through Whatman No. 1 f i l t e r paper d i s c s u s i n g the apparatus described e a r l i e r . The hydroxylapatite-PK-cAMP complex i s s t a b l e i n KP-buffer a t 4° f o r up to 2.5 hours. I t was necessary to e s t a b l i s h t h i s s t a b i l i t y f a c t o r s i n c e the f i l t r a t i o n of a set of samples u s u a l l y r e q u i r e d 1 . 5 - 2 hours. The assay tubes were r i n s e d 4 times w i t h 1.0 ml of c o l d KR-buffer and the f i l t e r paper d i s c s were f u r t h e r washed w i t h an a d d i t i o n a l 5.0 ml of t h i s b u f f e r . The f i l t e r paper d i s c s were then placed a t the bottom of s c i n t i l l a t i o n v i a l s and prepared f o r counting. A l l assays of cAMP were conducted i n t r i p l i c a t e . d) R a d i o a c t i v i t y A n a l y s i s . The h y d r o x y l a p a t i t e on the f i l t e r paper d i s c s was d i s s o l v e d by the a d d i t i o n of 1.0 ml of 1.5 N HCl to the s c i n t i l l a t i o n v i a l s i n which the di s c s had been placed. A f t e r the a d d i t i o n of 11 ml of a toluene based s c i n t i l l a t i o n f l u i d c o n t a i n i n g 30% (v/v) T r i t o n X-100, 0.02% (w/v) 1, 4-bis(2-(5-phenyloxazolyl))-benzene (POPOP) and 0.3% (w/v) 2,5 dipheny-l o x a z o l e (PPO), the samples were counted i n a Nuclear Chicago l i q u i d s c i n t i l l a t i o n counter at about 36% e f f i c i e n c y . e) P r o t e i n Determination. The p r e c i p i t a t e obtained a f t e r homogenization of b r a i n s l i c e s i n 5% TCA was d i s s o l v e d i n 0.1 N NaOH and assayed f o r p r o t e i n content by the method of Lowry e_t a_l. (144). The cAMP content i n b r a i n s l i c e s was always expressed as pmoles per mg of p r o t e i n so as to f a c i l i t a t e the comparison of the present data w i t h those i n the l i t e r a t u r e . VI. Treatment of Data. As mentioned e a r l i e r chemical agents which s t i m u l a t e the production of cAMP i n b r a i n s l i c e s always do so w i t h a la r g e degree of v a r i a t i o n among d i f f e r e n t animals. As a r e s u l t , a s u f f i c i e n t number of animals have to be included i n an experiment to enable the treatment of data by s t a t i s t i c a l means. The b a s e l i n e cAMP l e v e l s were obtained from 4 animals whereas at l e a s t 6 animals were used i n a l l other experiments to be described i n t h i s t h e s i s . A l l data are presented as the mean value f o r a group of animals plus or minus the standard e r r o r of the mean (S.E.M.). The student t - t e s t was used f o r the a n a l y s i s of s i g n i f i c a n c e , and the d i f f e r e n c e s were considered s i g n i f i c a n t i f p < 0.05. 42 RESULTS I. Assessment of Various Aspects of the I n v e s t i g a t i v e Procedures. I t i s g e n e r a l l y acknowledged that r e a r i n g animals i n the dark may have a m u l t i p l i c i t y of p h y s i o l o g i c a l and biochemical e f f e c t s not only on the v i s u a l system but on various f a c e t s of the animals' b i o l o g i c a l processes. An attempt was made to exclude some of the p o s s i b l e f a c t o r s other than l i g h t d e p r i v a t i o n which may c o n t r i b u t e to a l t e r a t i o n s i n the f u n c t i o n of the v i s u a l systems and indeed i n other CNS s t r u c t u r e s . One of these f a c t o r s i s n u t r i t i o n and i s r e l a t e d to the e f f e c t of d a r k - r e a r i n g on the animals' body weight gain. Since i n f a n t r a t s were fed by the mothers f o r the f i r s t 21 days of l i f e , the n u t r i t i o n a l status i n terms of body weight of mothers of dark-reared animals may a l s o be an important f a c t o r . The body weight g a i n of dark-reared and c o n t r o l r a t s as w e l l as the body weights of mothers of these two groups of animals up to the time of wean-ing i s shown i n F i g . 2. No s i g n i f i c a n t d i f f e r e n c e s e x i s t e d i n the body weights of normally reared r a t s and those reared i n the dark from b i r t h . Furthermore, there were no s i g n i f i c a n t d i f f e r e n c e s i n the body weights of n u r s i n g mothers of dark-reared and c o n t r o l animals. The procedure f o r the e s t i m a t i o n of cAMP i n b r a i n s l i c e s has been discussed i n d e t a i l i n the m a t e r i a l s and methods s e c t i o n . A t y p i c a l standard curve f o r the cAMP assay i s shown i n F i g . 3. That the technique employed i n t h i s study to measure cAMP concentrations i s v a l i d and r e f l e c t s the true cAMP content of b r a i n t i s s u e i s assumed from the f a c t that p r o t e i n kinase has both very high a f f i n i t y and s p e c i f i c i t y f o r cAMP. Moreover, i n F i g . 3 i t i s shown that when a sample of the TCA s o l u b l e f r a c t i o n of b r a i n t i s s u e which contains cAMP i s used i n the assay at i n c r e a s i n g concentra-43 10 20 30 40 50 60 age (days) F I G . 2 The e f f e c t o f l i g h t d e p r i v a t i o n on t h e body w e i g h t s o f r a t s a t v a r i o u s p o s t n a t a l ages and t h e i r m o t h e r s d u r i n g w e a n i n g . A. The body w e i g h t s o f r a t s i n l i t t e r s o f c o n t r o l (O O ) and d a r k - r e a r e d (©— — — —•) g r o u p s were m e a s u r e d a t t h e ages shown. 15. The body w e i g h t s o f n u r s i n g m o t h e r s o f c o n t r o l ( O O ) and d a r k - r e a r e d (•— — — ••) r a t s w e r e m e a s u r e d 5 d a y s a f t e r t h e y h a d g i v e n b i r t h and a t i n t e r v a l s up t o t h e t i m e o f w e a n i n g . Tn t h e c a s e o f l i g h t d e p r i v e d a n i m a l s t h i s o p e r a t i o n was c o n d u c t e d w i t h t h e a i d of a s a f e t y l i g h t t o a v o i d e x p o s u r e o f a n i m a l s t o t h e i n t e n s i t y o f room l i g h t i n g . E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 1 Q - 1 5 a n i m a l s . F I G . 3 A t y p i c a l standard curve f o r the de t e r m i n a t i o n of the cAMP content of b r a i n s l i c e s . The standard curve ( O" O) i s con s t r u c t e d by i n c u b a t i n g p r o t e i n kinase with a f i x e d c o n c e n t r a t i o n of 3 H - C A M P and i n c r e a s i n g c oncentra-t i o n s of unlabeled cAMP. The t o t a l q u a n t i t y of cAMP per i n c u b a t i o n i s p l o t t e d on the a b s c i s s a and the r a d i o a c t i v e cAMP bound to p r o t e i n kinase i s p l o t t e d on the o r d i n a t e . The broken l i n e (•— — —•) r e p r e s e n t s a separate assay where the sourse of the unl a b l e d cAMP added to the assay tubes was b r a i n t i s s u e . The cAMP content of t h i s t i s s u e was assayed i n a previous d e t e r m i n a t i o n . The degree to which the two l i n e s do not ove r l a p r e p r e s e n t s the e r r o r i n the assay procedure of cAMP which i n t h i s case was about 7%. 45 t i o n s r a t h e r than pure cAMP, thus s i m u l a t i n g the procedure f o r the produc-t i o n of a standard curve, a s t r a i g h t l i n e i s generated that i s p a r a l l e l to the standard curve. Since the m a t e r i a l i n the sample produced a l i n e p a r a l l e l to that of the standard curve and sin c e the a f f i n i t y constant f o r PK of t h i s m a t e r i a l and cAMP i s the same, as c a l c u l a t e d from the two curves, i t i s concluded that the m a t e r i a l i n the sample competing w i t h r a d i o a c t i v e cAMP f o r the bi n d i n g of p r o t e i n kinase must indeed be cAMP o r i g i n a t i n g from b r a i n t i s s u e . The two p a r a l l e l l i n e s i n F i g . 3 should overlap and the extent to which they do not represents the e r r o r i n the assay of cAMP. This e r r o r ranges from 3 to 77.. The recovery of cAMP from b r a i n t i s s u e was 897,. This value i s higher than those reported by others. The reason f o r t h i s may be due to the numerous washings employed i n the present study during the i s o l a t i o n of cAMP. The amount of v i s u a l and f r o n t a l c o r t i c a l t i s s u e employed per in c u -b a t i o n v a r i e s , unavoidably, due to the nature of the d i s s e c t i o n of b r a i n t i s s u e . Thus i t was necessary to show that the b a s e l i n e l e v e l s of cAMP i n b r a i n s l i c e s as w e l l as the s t i m u l a t i o n of cAMP formation by various agents i s l i n e a r w i t h the qu a n t i t y of t i s s u e employed per incubation. This would be an important f a c t o r i f substances were r e l e a s e d from b r a i n s l i c e s during the i n c u b a t i o n which could s t i m u l a t e the accumulation of cAMP sin c e the amount of substance r e l e a s e d would be dependent on the amount of t i s s u e incubated. Moreover, during the i n c u b a t i o n of c o r t i c a l s l i c e s w i t h NA, the NA may be taken up by the t i s s u e or i n a c t i v a t e d by some means thus reducing the q u a n t i t y a v a i l a b l e to s t i m u l a t e cAMP formation. This process would a l s o be dependent on the co n c e n t r a t i o n of t i s s u e employed per incubation. The e f f e c t of t i s s u e weight per in c u b a t i o n as represented by p r o t e i n content on the b a s e l i n e cAMP l e v e l s and the NA-induced accumu-l a t i o n of cAMP i n b r a i n s l i c e s i s shown i n F i g . 4. The b a s e l i n e cAMP content and the NA-induced accumulation of cAMP was l i n e a r f o r t i s s u e -p r o t e i n content per i n c u b a t i o n ranging from 0.5 to 7.5 mg. T y p i c a l l y , the amount of t i s s u e p r o t e i n per i n c u b a t i o n was between 1.5 and 4.0 mg. I t was d e s i r a b l e to determine the concentrations of NA to which b r a i n s l i c e s were to be exposed during incubations w i t h t h i s agent. For t h i s purpose i n v e s t i g a t o r s have used a v a r i e t y of concentrations (134,137, 145) or have adopted the co n c e n t r a t i o n of 100 yM (94,146-148) as produc-i n g maximal e f f e c t s . Employing r a t c e r e b r a l c o r t i c a l s l i c e s P erkins and Moore have shown (121,149) that 30 yM NA induces maximal s t i m u l a t i o n of cAMP formation. In F i g . 5 i s shown the time course of the accumulation of cAMP i n v i s u a l c o r t i c a l s l i c e s incubated w i t h 30 and 100yM NA. There i s no s i g n i f i c a n t d i f f e r e n c e i n the accumulation of cAMP e l i c i t e d by the two concentrations of NA i n 1 arid 5 minute incubations. However, f o r longer i n c u b a t i o n times the cAMP content of b r a i n s l i c e s incubated w i t h 30 and 100 yM NA i s s i g n i f i c a n t l y ' d i f f e r e n t . The cAMP co n c e n t r a t i o n i n t i s s u e incubated w i t h 100 yM NA i s maintained a t higher l e v e l s than that i n t i s s u e incubated w i t h 30 y M NA. This d i f f e r e n c e could be explained i f i n the presence of 100 yM NA the r a t e of s t i m u l a t i o n of cAMP formation by NA keeps pace w i t h the r a t e of degradation of cAMP by phosphodiesterase, whereas i n the presence of 30y M NA the r a t e of degradation p r e v a i l s . An a l t e r n a t i v e e x p l a n a t i o n i s that a f t e r an i n i t i a l s t i m u l a t i o n of cAMP formation by NA no f u r t h e r or continuous s t i m u l a t i o n takes place and f o r an unknown reason the degradation of cAMP i n t i s s u e incubated w i t h 30 yM i s f a s t e r than that i n t i s s u e incubated w i t h 100y M NA. Although t h i s 47 0 ; 1 2 3 4 5 6 7 Tissue Protein (mg) F I G . 4 The e f f e c t o f t i s s u e w e i g h t p e r i n c u b a t i o n on t h e b a s e l i n e cAMP l e v e l s and t h e N A - i n d u c e d a c c u m u l a t i o n o f cAMP i n b r a i n s l i c e s . A r a n g e o f t i s s u e w e i g h t s f r o m t h e c o r t e x o f 15 day o l d r a t s were i n c u b a t e d u n d e r s t a n d -a r d i n c u b a t i o n c o n d i t i o n s . The b a s e l i n e cAMP c o n t e n t (•• — — — •) and t h e N A - i n d u c e d a c c u m u l a t i o n of cAMP ( O O ) w e r e m e a s u r e d . NA was e m p l o y e d a t a c o n c e n t r a -t i o n o f 3 yM and i n c u b a t i o n s were f o r a p e r i o d o f 5 m i n . B a s e l i n e v a l u e s o f cAMP a r e t h o s e d e s c r i b e d i n m a t e r i a l s and m e t h o d s . The amount o f t i s s u e p e r i n c u b a t i o n i s r e p r e s e n t e d on t h e a b s c i s s a i n t e r m s o f p r o t e i n c o n t e n t . F I G . 5 Time c o u r s e o f t h e s t i m u l a t i o n o f cAMP f o r m a t i o n by two c o n c e n t r a t i o n s o f NA. V i s u a l c o r t i c a l s l i c e s f r o m 15 day o l d c o n t r o l r a t s were i n c u b a t e d w i t h 30 pM (O——O) and 100 uM (•- — — • ) NA f o r t h e t i m e p e r i o d s i n d i c a t e d . The cAMP c o n t e n t o f t h e s l i c e s i s e x p r e s s e d i n t e r m s o f p i c o m o l e s / m g p r o t e i n . The p r o t e i n y i e l d f r o m t h e t i s s u e i n t h e i n c u b a t i o n s v a r i e d b e t w e e n 1.5 and 4 mg. The B on t h e a b s i s s c a r e f e r s t o t h e b a s e l i n e cAMP c o n t e n t o f s l i c e s a t z e r o t i m e and p r i o r t o t h e a d d i t i o n o f a g e n t s . The t i m e c o u r s e s t u d i e s b e g i n a t t h e t i m e a g e n t s a r e a d d e d w h i c h i s a t t h e end o f t h e s e c o n d p r e i n c u b a t i o n s t e p as d e s c r i b e d i n m a t e r i a l s and m e t h o d s . E a c h p o i n t and v e r -t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4-6 a n i m a l s . e x p l a n a t i o n appears at f i r s t s i g h t l e s s s a t i s f a c t o r y , i t i s the p r e f e r r e d one s i n c e i t has been shown that there i s a r e f r a c t o r i n e s s to r e p e t i t i v e s t i m u l a t i o n of cAMP formation by such biogenic amines as NA (92,137,146). Since at short i n c u b a t i o n times the maximal accumulation of cAMP e l i c i t e d by the two concentrations of NA st u d i e d correspond, the con c e n t r a t i o n of NA chosen to be used i n incubations of b r a i n s l i c e s was 30 yM. The advantage of employing t h i s c o n c e n t r a t i o n i s that any d i f f e r e n c e s e x i s t i n g between dark-reared and c o n t r o l r a t s may r e s i d e i n the r a t e of cAMP catabolism, thus the lower c o n c e n t r a t i o n of NA would a l l o w inferences to be made i n t h i s regard. Phosphodiesterase i n h i b i t o r s were not used during i n c u b a t i o n of b r a i n s l i c e s f o r s i m i l a r reasons. I I . The E f f e c t of NA and K"1" on the Rate of Accumulation of cAMP i n B r a i n S l i c e s . The e f f e c t of da r k - r e a r i n g animals f o r 15 days on the time course of the NA-induced accumulation of cAMP i n v i s u a l and f r o n t a l c o r t i c a l s l i c e s i s shown i n F i g . 6. Dark r e a r i n g f o r 15 days d i d not a f f e c t the ba s e l i n e l e v e l s of cAMP i n e i t h e r v i s u a l or f r o n t a l c o r t i c a l s l i c e s . Nor were there any d i f f e r e n c e s i n the b a s e l i n e cAMP l e v e l s of v i s u a l compared to f r o n t a l c o r t i c a l s l i c e s of c o n t r o l or dark-reared animals. In a 5 minute in c u b a t i o n w i t h NA there was a s i g n i f i c a n t r e d u c t i o n of 1170 i n the cAMP l e v e l s i n v i s u a l c o r t i c a l s l i c e s of dark-reared r a t s compared to c o n t r o l s . At other i n c u b a t i o n times there was no s i g n i f i c a n t d i f f e r -ence between experimental and c o n t r o l animals i n the NA-induced accumu-lation:!, of cAMP i n v i s u a l c o r t i c a l s l i c e s . In f r o n t a l c o r t i c a l s l i c e s of dark-reared r a t s , although there was a trend toward a r e d u c t i o n i n the NA-induced accumulation of cAMP at a l l i n c u b a t i o n times, the only s i g n i f i c a n t F I G . 6 Time c o u r s e o f t h e s t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n v i s u a l and f r o n t a l c o r t i c a l s l i c e s o f 15 day o l d c o n t r o l and d a r k - r e a r e d r a t s . V i s u a l (- ) and f r o n t a l (-> — — -•) c o r t i c a l s l i c e s f r o m c o n t r o l ( O ) and d a r k - r e a r e d ( • ) r a t s w e r e i n c u b a t e d w i t h 30 yM NA f o r t h e t i m e p e r i o d s i n d i c a t e d . E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 6-15 a n i m a l s . ^f- V a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m t h e same b r a i n r e g i o n o f c o n t r o l s (p < 0 . 0 5 ) . d i f f e r e n c e from c o n t r o l s occurred i n a 20 minute incubation. These r e s u l t s suggest that i n both v i s u a l and f r o n t a l c o r t i c a l s l i c e s of dark-reared animals there i s a r e d u c t i o n i n the maximal.stimulation of NA-induced cAMP formation. Furthermore, the diminution of cAMP l e v e l s i n f r o n t a l c o r t i c a l s l i c e s of dark-reared animals a f t e r 20 minutes of i n c u b a t i o n w i t h NA suggests that there i s a greater r a t e of degradation of cAMP i n t h i s b r a i n region of experimental animals than c o n t r o l s . This i s not evident i n v i s u a l c o r t i c a l s l i c e s of dark-reared animals. A f t e r 30 days of da r k - r e a r i n g , the e f f e c t s on the NA-induced accumu-l a t i o n of cAMP i n v i s u a l c o r t i c a l s l i c e s i s q u a l i t a t i v e l y the same as at 15 days. However, as shown i n F i g . 7, there i s a greater r e d u c t i o n from c o n t r o l values (21%,) i n the s t i m u l a t i o n of cAMP formation by NA i n a 5 minute i n c u b a t i o n and there i s a s i g n i f i c a n t r e d u c t i o n (20%,) i n the cAMP l e v e l i n a 1 minute i n c u b a t i o n . As i n the case of 15 day o l d animals, d a r k - r e a r i n g f o r 30 days had no e f f e c t on the b a s e l i n e cAMP l e v e l s i n v i s u a l c o r t i c a l s l i c e s or on the l e v e l s a f t e r 10 or 20 minute incubations w i t h NA. The e f f e c t s of d a r k - r e a r i n g animals f o r 30 days on the NA-induced accumulation of cAMP i n f r o n t a l c o r t i c a l s l i c e s are somewhat more complex than the e f f e c t s on v i s u a l c o r t i c a l s l i c e s . As shown i n F i g . 8 the base-l i n e cAMP content of f r o n t a l c o r t i c a l s l i c e s was s i g n i f i c a n t l y higher (21%,) fo r dark-reared animals than c o n t r o l s w h i l e the NA-induced accumulation of cAMP was 25% and 21% lower than c o n t r o l s i n 1 and 5 minute incubations, r e s p e c t i v e l y . At 10 minutes of in c u b a t i o n w i t h NA the cAMP l e v e l s i n s l i c e s from experimental and c o n t r o l animals are equal and i n a 20 minute inc u b a t i o n w i t h NA t h i s l e v e l i s maintained i n s l i c e s from c o n t r o l animals but there i s a 13% d e c l i n e i n s l i c e s from experimental animals. 120 n Incubation Time (min.) F I G . 7 Time c o u r s e o f t h e s t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n v i s u a l c o r t i c a l s l i c e s of 30 day o l d c o n t r o l and d a r k - r e a r e d r a t s . B r a i n s l i c e s f r o m c o n t r o l (O o) and d a r k - r e a r e d (•> — —•) r a t s were i n c u b a t e d w i t h 30 pM NA f o r t h e t i m e p e r i o d s i n d i c a t e d . E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4-7 a n i m a l s . • t y V a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s ( p < 0 . 0 5 ) . F I G . 8 Time c o u r s e o f t h e s t i m u l a t i o n o f cAMP f o r m a t i o n by NA i n f r o n t a l c o r t i c a l s l i c e s o f 30 day o l d c o n t r o l and d a r k - r e a r e d r a t s . B r a i n s l i c e s f r o m c o n t r o l (O O) and d a r k - r e a r e d ( • — — ^ ) r a t s w e r e i n c u b a t e d w i t h 30 yM NA f o r t h e t i m e p e r i o d s i n d i c a t e d . E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4-7 a n i m a l s . V a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s (p< 0 . 0 5 ) . Dark-rearing f o r 30 days appears to reduce the a b i l i t y of NA to promote the synthesis of cAMP i n v i s u a l c o r t i c a l s l i c e s . In f r o n t a l c o r t i c a l s l i c e s , d a r k - r e a r i n g f o r t h i s p e r i o d does not a f f e c t the maximal accumula-t i o n of cAMP e l i c i t e d by NA, but there i s a r e d u c t i o n i n the r a t e a t which cAMP i s accumulated i n response to NA, as w e l l as a r e d u c t i o n i n the maintenance of the maximally s t i m u l a t e d l e v e l s of cAMP. The a d d i t i o n of 50 mM KC1 to the in c u b a t i o n medium causes a much l a r g e r s t i m u l a t i o n of cAMP formation i n b r a i n s l i c e s than does NA. Shown i n F i g . 9 i s the time course of t h i s s t i m u l a t i o n i n v i s u a l and f r o n t a l c o r t i c a l s l i c e s of 30 day o l d c o n t r o l and dark-reared r a t s . Dark-rearing d i d not a f f e c t the a b i l i t y of K + to s t i m u l a t e the formation of cAMP i n s l i c e s from e i t h e r b r a i n r e g i o n at any of the in c u b a t i o n times s t u d i e d . In c o n t r o l and experimental animals, the K^"-induced accumulation of cAMP i s greater i n v i s u a l than f r o n t a l c o r t i c a l s l i c e s and there i s a greater d e c r e m e n t i n the cAMP l e v e l s i n f r o n t a l than v i s u a l c o r t i c a l s l i c e s at in c u b a t i o n times of 10 and 20 minutes. The complete reverse i s true of the NA-induced accumulation of cAMP where (see F i g s . 6, 7 and 8) the s t i m u l a t i o n induced by NA i s greater i n f r o n t a l c o r t i c a l s l i c e s and i n normally reared animals the subsequent diminution of cAMP l e v e l s i s greater i n v i s u a l c o r t i c a l s l i c e s . In dark-reared animals the NA-induced accumulation of cAMP i n f r o n t a l c o r t i c a l s l i c e s i s more K + - l i k e i n that the diminution of cAMP l e v e l s at longer i n c u b a t i o n times i s increased. I I I . The Ontogenetic Development of Responsiveness of B r a i n S l i c e s to  NA and K + The c a p a c i t y of NA and K"1" to s t i m u l a t e the formation of cAMP was stu d i e d i n b r a i n s l i c e s from r a t s of various ages. Incubations of 5 360 r B 1; 5 10 15 2 0 Incubation Time (min.) F I G . 9 Time c o u r s e o f t h e s t i m u l a t i o n o f cAMP f o r m a t i o n by K + i n v i s u a l and f r o n t a l c o r t i c a l s l i c e s o f 30 day o l d c o n t r o l and d a r k - r e a r e d r a t s . T i s s u e s l i c e s o f f r o n t a l ( — — •) and v i s u a l ( — ) c o r t e x f r o m c o n t r o l ( O ) and and d a r k - r e a r e d ( • ) r a t s w e r e i n c u b a t e d w i t h 50 mM K + f o r t h e t i m e p e r i o d s shown. E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4-7 a n i m a l s . minutes were chosen s i n c e the time course s t u d i e s i n d i c a t e d that i n b r a i n s l i c e s from normally reared animals the accumulation of cAMP i n response to these agents was maximal or almost maximal at t h i s time. As shown i n Fig s . 10 and 11 s e n s i t i v i t y to NA was present a t 5 days of age i n both v i s u a l f r o n t a l s l i c e s . In v i s u a l c o r t i c a l s l i c e s the responsiveness to NA increases a t 10 days of age and a f t e r 15 days remains r e l a t i v e l y con-s t a n t . In f r o n t a l c o r t i c a l s l i c e s the responsiveness to NA undergoes a d r a s t i c increase at 10 days^and subsequently decreases by 30 days to a constant value. The b a s e l i n e l e v e l s of cAMP i n f r o n t a l and v i s u a l c o r t i c a l s l i c e s g r a d u a l l y decrease w i t h age from an average of 80 pmoles/mg p r o t e i n at 5 days to about 50 pmoles/mg p r o t e i n at 60 days. In instances where the s t i m u l a t i o n of cAMP synthesis by various agents i s low t h i s change i n base l i n e may be important w i t h regard to the i n t e r p r e t a t i o n of r e s u l t s inas-much as the elevated l e v e l s of cAMP caused by these agents i s the sum of newly synthesized cAMP and b a s e l i n e l e v e l s . This i s p a r t i c u l a r l y t r u e i n the v i s u a l c o r t e x ( F i g . 10) where the change i n b a s e l i n e l e v e l s of cAMP w i t h age appear to p a r a l l e l changes i n the NA-induced accumulation of cAMP Thus, the changes i n the response of v i s u a l c o r t i c a l s l i c e s to NA may merely r e f l e c t changes i n b a s e l i n e cAMP l e v e l s . For the most p a r t , how-ever, the changes i n b a s e l i n e l e v e l s are of l i t t l e consequence. As shown i n F i g . 12, the c a p a c i t y of K + to s t i m u l a t e the synth e s i s of cAMP i n v i s u a l c o r t i c a l s l i c e s increases enormously from 5 day to 15 days of age whereupon i t decreases s l i g h t l y at 30 and 60 days. This i s i n marked c o n t r a s t to the K + s e n s i t i v i t y changes observed i n f r o n t a l c o r t i c a l s l i c e s ( F i g . 13). Although a s i m i l a r increase i n K + responsive-ness occurs up to 15 days there i s subsequently a progressive decrease 57 F I G . 10 O n t o g e n e t i c d e v e l o p m e n t o f r e s p o n s i v e n e s s o f v i s u a l c o r t e x t o NA. V i s u a l c o r t i c a l s l i c e s f r o m c o n t r o l ( O — o ) and d a r k - r e a r e d (• •) r a t s o f v a r i o u s a g es w e r e e x p o s e d t o 30 uM NA f o r 5 m i n . T i s s u e cAMP c o n t e n t i n t h e a b s e n c e o f NA ( c o n t r o l O- — O ; d a r k - r e a r e d — •• ) r e p r e s e n t s b a s e l i n e l e v e l s . E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4-15 a n i m a l s . V a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s (p < 0 . 0 5 ) . 40 10 20 30 40 50 60 A g e (Days ) F I G . 11 O n t o g e n e t i c d e v e l o p m e n t o f r e s p o n s i v e n e s s o f f r o n t a l c o r t e x t o NA. F r o n t a l c o r t i c a l s l i c e s f r o m c o n t r o l (o O) and d a r k - r e a r e d (•——•) r a t s o f v a r i o u s ages w ere e x p o s e d t o 30 yM NA f o r 5 min. T i s s u e cAMP c o n t e n t i n t h e a b s e n c e o f NA ( c o n t r o l 0- — —0 : d a r k - r e a r e d •• — - * ) r e p r e s e n t s b a s e l i n e l e v e l s . E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4-15 a n i m a l s . V a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s (p < 0 . 0 5 ) . 59 - J — 1 1 1 1 1 10 20 30 40 50 60 Age (days) F I G . 12 O n t o g e n e t i c d e v e l o p m e n t o f r e s p o n s i v e n e s s o f v i s u a l c o r t e x t o K + . V i s u a l c o r t i c a l s l i c e s f r o m c o n t r o l (O 'O) and d a r k - r e a r e d ( • — — • ) r a t s o f v a r i o u s a g es w ere e x p o s e d t o 50 yM KC1 f o r o f 5 m i n . T i s s u e cAMP c o n t e n t i n t h e a b s e n c e o f K C l ( c o n t r o l o — - o ; d a r k - r e a r e d ) r e p r e s e n t s b a s e l i n e l e v e l s . E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y , o f 4-6 a n i m a l s . 60 F I G . 13 O n t o g e n e t i c d e v e l o p m e n t o f r e s p o n s i v e n e s s o f f r o n t a l c o r t e x t o K +. F r o n t a l c o r t i c a l s l i c e s f r o m c o n t r o l (O O ) and d a r k - r e a r e d (• • ) r a t s o f v a r i o u s ages were e x p o s e d t o 50 mM KC1 f o r 5 m i n . T i s s u e cAMP c o n t e n t i n t h e a b s e n c e o f KC1 ( c o n t r o l O- — o ; d a r k - r e a r e d — —• ) r e p r e s e n t s b a s e l i n e l e v e l s . E a c h p o i n t and v e r t i c a l b a r r e p r e s e n t s t h e mean and S.E.M., r e s p e c t i v e l y o f 4-6 a n i m a l s , -fc V a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s (p< 0 . 0 5 ) . u n t i l a t 60 days the cAMP l e v e l s i n f r o n t a l c o r t i c a l s l i c e s incubated i n the presence of high K + i s equal to that observed at 5 days. The net s t i m u l a t i o n of cAMP synthesis by K + i s s t i l l g reater at 60 days due to a decreased b a s e l i n e at t h i s age. The major d i f f e r e n c e between f r o n t a l and v i s u a l c o r t i c a l s l i c e s w i t h regard to the a b i l i t y to respond to K + and NA by augmenting cAMP l e v e l s i s the t r a n s i e n t nature of the response that occurs w i t h age i n f r o n t a l c o r t i c a l s l i c e s but i s l e s s pronounced i n v i s u a l c o r t i c a l s l i c e s . This t r a n s i e n t response to NA w i t h age has been observed i n s l i c e s of r a t whole b r a i n (113,116) as w e l l as i n s l i c e s of r a b b i t f r o n t a l c ortex, hippocampus and hypothalamus (120). The t r a n s i e n t response of f r o n t a l c o r t i c a l s l i c e s to has not been p r e v i o u s l y reported. The e f f e c t s of d a r k - r e a r i n g animals f o r 15 and 30 days on the NA-and K -induced accumulation of cAMP i n s l i c e s has been discussed. I t might be f u r t h e r pointed out that the responsiveness of b r a i n s l i c e s from dark-reared animals of these ages, whether s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s or not, p a r a l l e l s the developmental p r o f i l e s of c o n t r o l s . At 60 days of age there i s a t u r n of events w i t h regard to the e f f e c t s of dark-r e a r i n g . Whereas, at the former ages, d a r k - r e a r i n g caused a decreased responsiveness to NA and had no e f f e c t on the responsiveness to K +, a f t e r 60 days of d a r k - r e a r i n g the NA-induced accumulation of cAMP was 23%, and 35%, higher than c o n t r o l s i n f r o n t a l and v i s u a l c o r t i c a l s l i c e s , respec-t i v e l y (see F i g s . 10 and 11). The K +-induced accumulation of cAMP at 60 days was 57%, higher i n f r o n t a l c o r t i c a l s l i c e s of experimental animals compared to c o n t r o l s but although the responsiveness to K was higher i n v i s u a l c o r t i c a l s l i c e s of experimental animals by 13%,, t h i s was not s i g n i f i c a n t (p 0.1). IV. The Accumulation of cAMP i n B r a i n S l i c e s i n Response to Adenosine  and Combinations of Adenosine w i t h NA and K +. V i s u a l and f r o n t a l c o r t i c a l s l i c e s from 60 day o l d dark-reared and c o n t r o l r a t s were incubated f o r 5 minutes w i t h adenosine and combinations of adenosine w i t h NA or K +. The r e s u l t s are shown i n Table I together w i t h the p r e v i o u s l y discussed r e s u l t s of incubations of s l i c e s of 60 day o l d animals w i t h NA and K + at comparable i n c u b a t i o n times. Although b r a i n s l i c e s incubated i n the presence of 30 y M adenosine contained l e v e l s of cAMP s i g n i f i c a n t l y greater than b a s e l i n e q u a n t i t i e s , these d i d not approach the l e v e l s reported by others (121,146,148). This could be due i n part to the f a c t that these workers incubated b r a i n s l i c e s f o r periods of 15 to 30 minutes w i t h concentrations of adenosine ranging from 30 to 100 yM. There was no s i g n i f i c a n t d i f f e r e n c e between experimental and c o n t r o l animals i n the adenosine-induced accumulation of cAMP i n v i s u a l c o r t i c a l s l i c e s . However, as i n the case of NA and K +, adenosine caused a s i g n i f i -c a n t l y g r e a t e r accumulation of cAMP i n f r o n t a l c o r t i c a l s l i c e s of dark-reared animals than c o n t r o l s . Incubations of v i s u a l and f r o n t a l c o r t i c a l + s l i c e s i n the presence of adenosine i n combination w i t h NA or K produced no d i f f e r e n c e s between experimental and c o n t r o l animals i n e i t h e r b r a i n r e g i o n . Thus, the d i f f e r e n c e observed i n f r o n t a l c o r t i c a l s l i c e s i n + + incubations w i t h adenosine, NA or K alone was ab o l i s h e d when NA or K was combined w i t h adenosine. The synergism between adenosine and biogenic amines that has been observed by others (96,98) i s a l s o demonstrated i n the present work. For example, the accumulation of cAMP e l i c i t e d by adenosine i n combination w i t h NA was greater than the sum of that e l i c i t e d by NA and adenosine alone. The combined e f f e c t s of d e p o l a r i z i n g agents such as K + and adeno-TABLE I The s t i m u l a t i o n of cAMP formation by adenosine and combinations of adenosine w i t h NA and K + i n v i s u a l and f r o n t a l c o r t i c a l s l i c e s of 60 days o l d c o n t r o l and dark-reared r a t s . cAMP content of s l i c e s ( picomoles/mg ) p r o t e i n Agent (s) V i s u a l c o r t e x F r o n t a l c o r t e x C o n t r o l Dark-reared C o n t r o l Dark-reared B a s e l i n e 47.8 + 4.5 50.5 ± 3.4 46.3 ± 2.2 49.4 ±6.2 Adenosine 83.1 + 8.0 99.0 ± 11 75.2 ±8.1 104 ± 6.4* NA 108 ± 9.1 133 ± 8.8* 125 ± 6.8 169 ±8.4* Adenosine + NA 235 ± 15 275 ± 27 293 ± 25 349 ± 22 K + 235 + 21 268 ± 16 115 ±8.7 180 ± 21* Adenosine + K + 312 + 24 344 ± 53 251± 18 261± 20 T i s s u e s l i c e s w e r e i n c u b a t e d f o r 5 m i n . i n t h e p r e s e n c e o f t h e f o l l o w i n g a g e n t s : 30 AIM a d e n o s i n e ; 30 uM NA; 50 mM KC1; 30 juM a d e n o s i n e + 30 «M NA; 30 uM a d e n o s i n e + 50 mM K C l . V a l u e s a r e e x p r e s s e d as t h e mean jf t h e S.E.M. of 4-7 a n i m a l s . * S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l v a l u e o f t h e same b r a i n r e g i o n . s i n e have been reported not to be s y n e r g i s t i c but only a d d i t i v e (90). This was found to be true when v i s u a l c o r t i c a l s l i c e s were incubated w i t h a combination of adenosine and K +. However, f r o n t a l c o r t i c a l s l i c e s of experimental and c o n t r o l animals gave a d i f f e r e n t i a l response such that i n the former there was a s y n e r g i s t i c response w h i l e an a d d i t i v e response was obtained i n s l i c e s from dark-reared animals. I t i s d i f f i c u l t to e x p l a i n t h i s r e s u l t s i n c e the observed synergism between K + and adenosine i n f r o n t a l c o r t i c a l s l i c e s of c o n t r o l animals was e n t i r e l y unexpected and i s contrary to the r e s u l t s reported by others. I t might be pointed out, however, that many i n v e s t i g a t o r s employ, i n t h e i r s t u d i e s , the e n t i r e c o r t e x which precludes the observation of r e g i o n a l d i f f e r e n c e s . That such d i f f e r e n c e s e x i s t i s not without precedent s i n c e i n the present study numerous d i f f e r e n c e s regarding the time course and degree of cAMP accumulation i n response to NA and K + have been found between f r o n t a l and v i s u a l c o r t i c a l s l i c e s . Assuming that the synergism observed w i t h adenosine and K + i n f r o n t a l c o r t i c a l s l i c e s i s v a l i d then a p o s s i b l e e x p l a n a t i o n of why t h i s was not observed i n these s l i c e s from dark-reared animals may be that i n t h i s b r a i n r e g i o n of experimental animals both adenosine and K + alone produced an accumulation of cAMP greater than that observed i n s l i c e s of c o n t r o l animals. Thus, whatever mechanism i s operative i n the process of synergism may have already been a c t i v a t e d and u t i l i z e d to produce the augmented responses to adenosine and K + alone. There e x i s t s i n the l i t e r a t u r e a wide v a r i a t i o n i n the reported b a s e l i n e values of cAMP i n b r a i n s l i c e s as w e l l as i n the accumulation of cAMP l e v e l s e l i c i t e d by various agents. Numerous b r a i n regions of a v a r i e t y of animals such as mouse, r a t , r a b b i t and guinea p i g have been used i n the study of the cAMP system. Thus, some of the above v a r i a t i o n s may be explained and indeed expected from such a heterogeneous use of animals and b r a i n regions. The problem, however, i s not r e s o l v e d by t h i s explana-t i o n s i n c e d i s c r e p a n c i e s e x i s t i n the reported r e s u l t s of d i f f e r e n t workers u s i n g the same b r a i n r e g i o n of the same animal. For example, i n r a t c o r t i c a l s l i c e s b a s e l i n e values ranging from 12 to 100 pmoles/mg p r o t e i n have been reported (134,146,149,150). The reported values of the NA-induced accumulation of cAMP i n r a t c o r t i c a l s l i c e s incubated w i t h 10 pM NA range from 31 to 400 pmoles/mg p r o t e i n (134,149). Although some of these d i f f e r e n c e s may r e s u l t from the f a c t that a v a r i e t y of i n c u b a t i o n times have been employed i n the study of the NA-induced accumulation of cAMP i n b r a i n s l i c e s , t h i s reason i s not e n t i r e l y s a t i s f a c t o r y s i n c e the maximal accumulation of cAMP i n response to t h i s agent has been reported to occur at a v a r i e t y of i n c u b a t i o n times ranging from 10 to 30 minutes (134,146,149). In s p i t e of the f a c t that the percent increase i n cAMP l e v e l s e l i c i t e d by NA v a r i e s from 200 to 700%, a f a i r l y c o n s i s t e n t f i n d i n g i s that the b a s e l i n e l e v e l s of cAMP i n c o r t i c a l s l i c e s vary p r o p o r t i o n a l l y w i t h the NA-induced accumulation of cAMP. For example, i n the case of a low cAMP b a s e l i n e the increase i n cAMP l e v e l s i n response to NA i s g e n e r a l l y p r o p o r t i o n a l to that i n cases where higher b a s e l i n e s are obtained. In the present study the b a s e l i n e l e v e l s of cAMP of between 45 and 65 pmoles/mg p r o t e i n f o r 30 and 60 day o l d animals agree f a i r l y w e l l w i t h the values reported by some other i n v e s t i g a t o r s (134,151). However, the accumulated l e v e l s of cAMP i n b r a i n s l i c e s incubated i n the presence of NA were g e n e r a l l y lower by 25 to 80% than those reported by others who obatined b a s e l i n e l e v e l s s i m i l a r to that reported here. This may be due to s l i g h t d i f f e r e n c e s i n technique or to the f a c t that descrete b r a i n regions were examined i n the present study whereas others have pooled the e n t i r e c o r t e x i n t h e i r s t u d i e s . The d i s c r e p a n c i e s i n cAMP l e v e l s reported i n the l i t e r a t u r e must c e r t a i n l y r e s u l t from the methodological d i f f i c u l t i e s inherent i n i n v e s t i -gations of such a complex t i s s u e as the b r a i n . I f the study of the cAMP system and the i n t e r a c t i o n s of various agents w i t h t h i s system i s to continue u s i n g the techniques employed i n the present i n v e s t i g a t i o n , i t i s c l e a r that an in"depth a n a l y s i s i s r e q u i r e d to determine and e l i m i n a t e the f a c t o r s i n v o l v e d i n the technique that c o n t r i b u t e to v a r i a t i o n s i n r e s u l t s . DISCUSSION We have found that e f f e c t s of dar k - r e a r i n g on the cAMP system occur i n both the v i s u a l and f r o n t a l c o r t e x and that these e f f e c t s are bimodal w i t h age. Dark-rearing r a t s f o r 1 month or l e s s caused p r i m a r i l y a diminution i n the a b i l i t y of NA to increase cAMP l e v e l s i n b r a i n s l i c e s from these animals, whereas a f t e r 2 months of da r k - r e a r i n g the response to NA and K + was increased. The bimodal nature of the e f f e c t s of l i g h t d e p r i v a t i o n and the f a c t that f r o n t a l c o r t e x which i s not the primary s i t e of t e r m i n a t i o n of v i s u a l input was a f f e c t e d must be r e c o n c i l e d not only w i t h the a v a i l a b l e data on the e f f e c t s of l i g h t d e p r i v a t i o n but w i t h the emerging concepts regarding p l a s t i c i t y and recovery of f u n c t i o n i n the CNS. The const r u c t s i n t o which the f i n d i n g s of the present i n v e s t i g a -t i o n must be placed are not f i r m l y e s t a b l i s h e d . Because t h i s allows a c e r t a i n amount of m a l l e a b i l i t y i n the i n t e r p r e t a t i o n of r e s u l t s i t i s i n d i c a t i v e that perhaps a great deal of conjecture i s unwarranted. However, s p e c u l a t i o n i s d e s i r a b l e to the extent that i t may a i d i n the planning of f u r t h e r experiments. The f i n d i n g that d a r k - r e a r i n g a f f e c t s both the v i s u a l and f r o n t a l c ortex renders suspect the c o n c l u s i o n that these e f f e c t s stem from the e l i m i n a t i o n of v i s u a l s t i m u l a t i o n . For example, an e t i o l o g y i n v o l v i n g the humoral system would be more appr o p r i a t e s i n c e t h i s system would have access to many b r a i n regions. However, the studies a l l u d e d to e a r l i e r regarding the e f f e c t s of m a l n u t r i t i o n and thyroidectomy on the cAMP system tend to r u l e out at l e a s t some extraneous p o s s i b i l i t i e s other than l i g h t d e p r i v a t i o n as the caus a t i v e f a c t o r s f o r the r e s u l t s obtained i n the present study. The redeeming feature of these i n v e s t i g a t i o n s i s that 68 n e i t h e r thyroidectomy nor m a l n u t r i t i o n of r a t s caused changes i n the cAMP system i n the brains of these animals. Both of these experimental approaches undoubtedly lead to gross abnormalities i n the endocrine systems of t r e a t e d animals. Since these humoral imbalances d i d not r e s u l t i n a l t e r a t i o n s i n the b r a i n cAMP system, i t can be assumed that t h i s system would not be a f f e c t e d by the humoral changes (152,153) r e s u l t i n g from a les s traumatic treatment of animals such as l i g h t d e p r i v a t i o n . Despite the t e n t a t i v e c o n c l u s i o n that environment i s r e s p o n s i b l e f o r the modified f u n c t i o n of the cAMP system which we have found i n the co r t e x of dark-reared r a t s , the neu r a l systems that form the basis of these m o d i f i c a t i o n s i s s t i l l u n c e r t a i n . For example, i t i s not c l e a r whether these e f f e c t s are mediated by reduced a f f e r e n t e l e c t r i c a l impulses to the v i s u a l c o r t e x which then i n f l u e n c e the a c t i v i t y of the f r o n t a l c o r t e x through i n t r a c o r t i c a l neuronal a s s o c i a t i o n s or whether d a r k - r e a r i n g a f f e c t s s u b c o r t i c a l s t r u c t u r e s which i n t u r n modulate e l e c t r i c a l a c t i v i t y and thus neurochemical processes i n the cortex. That the e x c l u s i o n of l i g h t stimulus to animals and the r e d u c t i o n of a c t i v i t y i n the v i s u a l system that t h i s a f f o r d s c o n t r i b u t e s d i r e c t l y , although perhaps not s o l e l y to the changes observed i n the v i s u a l cortex, i s suggested by the numerous morphological and biochemical s t u d i e s (see i n t r o d u c t i o n ) where s p e c i f i c e f f e c t s of l i g h t d e p r i v a t i o n on the v i s u a l c o r t e x have been demonstrated. For example, the number of spines on the a p i c a l dendrites of l a y e r V pyramidal neurons have been shown to be reduced s p e c i f i c a l l y i n the v i s u a l cortex and not the temporal c o r t e x of l i g h t deprived mice (36). These are the structures which comprise syna p t i c contacts and to which the cAMP system has been l o c a l i z e d . Thus, the diminished responsiveness of v i s u a l c o r t i c a l s l i c e s of 15 and 30 day o l d dark-reared animals to NA may i n part r e f l e c t reduced numbers of those s t r u c t u r a l e n t i t i e s w i t h which exogenously a p p l i e d neurotransmitters can i n t e r a c t . U n f o r t u n a t e l y , inves-t i g a t o r s studying the e f f e c t s of d a r k - r e a r i n g on b r a i n morphology have chosen as t h e i r c o n t r o l s e i t h e r the motor or temporal cortex, or, i n the case of monocular v i s u a l d e p r i v a t i o n , the v i s u a l c o r t e x of the unoccluded eye. Therefore, i f d a r k - r e a r i n g induces s i m i l a r morphological e f f e c t s i n f r o n t a l c o r t e x as i n v i s u a l c o r t e x then the above e x p l a n a t i o n may apply to the f i n d i n g s obtained f o r f r o n t a l c o r t i c a l s l i c e s of 30 day o l d dark-reared r a t s d e s p i t e the f a c t that only the time course and not the maximal accumulation of cAMP e l i c i t e d by NA was a f f e c t e d . As discussed above, the e f f e c t s of da r k - r e a r i n g which we have observed may r e s i d e i n the simultaneous r e d u c t i o n through decreased neural contacts of a l l those components subservient to the production of cAMP. However, the a l t e r e d c a p a c i t y of b r a i n s l i c e s from dark-reared r a t s to respond to various agents by augmenting cAMP synthesis might a l t e r n a t i v e l y be due s p e c i f i c a l l y to key events i n the s e r i e s of i n t e r a c t i o n s that take place w h i l e a neuron responds to a t r a n s m i t t e r . The e l u c i d a t i o n of the biochemical mechanisms that may be r e s p o n s i b l e f o r a l t e r e d responsiveness must await f u r t h e r i n v e s t i g a t i o n s . This task although not insuperable does pose some d i f f i c u l t i e s . The reason f o r t h i s i s the many parameters that could p o t e n t i a l l y give r i s e to the observed e f f e c t s of l i g h t d e p r iva-t i o n . These e f f e c t s may be a s c r i b e d to a change i n a s i n g l e v a r i a b l e or may be the net outcome of s e v e r a l processes a c t i n g i n unison or o p p o s i t i o n . Moreover, the e f f e c t s of d a r k - r e a r i n g may be b r a i n r e g i o n s p e c i f i c causing d i f f e r e n t sets of events i n b r a i n regions r e c e i v i n g a f f e r e n t supply f o r v i s i o n (e.g. v i s u a l cortex) and areas not r e c e i v i n g v i s u a l input (e.g. f r o n t a l c o r t e x ) . Some of the f a c t o r s that may c o n t r i b u t e to the e f f e c t s of dark-r e a r i n g i n c l u d e those components that are i n v o l v e d i n promoting the syn-t h e s i s and degradation of cAMP. Thus, diminished responsiveness of v i s u a l c o r t i c a l s l i c e s to NA a f t e r 15 and 30 days of d a r k - r e a r i n g may be due t o , (1) decreased e f f i c a c y of the NA-receptor i n t e r a c t i o n which might i n v o l v e cooperative changes i n the receptor, (2) reduced number of receptors f o r NA, (3) decreased adenylate c y c l a s e a c t i v i t y , (4) decreased c o u p l i n g between the NA receptor and adenylate c y c l a s e , or (5) increased phospho-d i e s t e r a s e a c t i v i t y . Methods are a v a i l a b l e to d i s t i n g u i s h between at l e a s t two of these p o s s i b i l i t i e s . Phosphodiesterase i n h i b i t o r s such as t h e o p h y l l i n e , aminophylline or diazepam may be included i n the i n c u b a t i o n of b r a i n s l i c e s to determine whether the e f f e c t s of d a r k - r e a r i n g are p r i m a r i l y on the s y n t h e t i c or degradative processes i n v o l v e d i n the metabolism of cAMP. A l t e r n a t i v e l y , phosphodiesterase a c t i v i t y could be assayed (154,155) i n homogenates of the b r a i n areas i n question. I n s o f a r as adenylate c y c l a s e i s concerned, i t s c a t a l y t i c component could be q u a n t i f i e d without the i n t e r f e r e n c e of other components through the known s t i m u l a t i o n of t h i s a c t i v i t y by f l u o r i d e i o n (133,156), thus a f f o r d i n g a measure of the absolute amount of enzyme p r o t e i n . In the v i s u a l c ortex the time course studies ( F i g s . 6 and 7) tend to exclude the p o s s i b i l i t y that increased phosphodiesterase a c t i v i t y i s mediating the e f f e c t s of d a r k - r e a r i n g s i n c e v i s u a l c o r t i c a l s l i c e s incu-bated f o r 10 and 20 minutes w i t h NA showed no d i f f e r e n c e s i n cAMP l e v e l s between c o n t r o l and experimental animals. However, i n f r o n t a l c o r t i c a l s l i c e s , although the same processes as mentioned above may be o p e r a t i v e to produce the observed changes i n responsiveness to NA a f t e r 15 and 30 days of da r k - r e a r i n g , the p a r t i c i p a t i o n of phosphodiesterase i s more suspect than i n v i s u a l c o r t i c a l s l i c e s . For example, the time course studies ( F i g s . 6 and 8) i n d i c a t e that i n a 20 minute i n c u b a t i o n of f r o n t a l c o r t i c a l s l i c e s w i t h NA there i s greater c a t a b o l i s m of cAMP i n e x p e r i -mental than c o n t r o l animals. I f augmented phosphodiesterase a c t i v i t y i n f r o n t a l c o r t i c a l s l i c e s of experimental animals i s r e s p o n s i b l e f o r the diminished cAMP l e v e l s observed i n s l i c e s a f t e r longer i n c u b a t i o n times, then i t i s reasonable to assume that the increased c a t a b o l i c a c t i v i t y of t h i s enzyme may i n part have caused the changes observed i n these s l i c e s at short i n c u b a t i o n times. The q u a l i t a t i v e d i f f e r e n c e s observed regarding the e f f e c t s of da r k - r e a r i n g on f r o n t a l and v i s u a l cortex may then be ex-p l a i n e d by assuming a d i f f e r e n t i a l e f f e c t on the a c t i v i t y of phosphodi-esterase i n these b r a i n areas. Thus, i n f r o n t a l c o r t i c a l s l i c e s the e f f e c t of da r k - r e a r i n g f o r 30 days on the r a t e of accumulation r a t h e r than the maximal l e v e l s of cAMP e l i c i t e d by NA may r e f l e c t changes i n cAMP degradative c a p a c i t y whereas i n v i s u a l c o r t i c a l s l i c e s the reduced maximal response to NA may inv o l v e other processes more d i r e c t l y r e l a t e d to v i s u a l d e p r i v a t i o n such as the s t r u c t u r a l changes a l l u d e d to e a r l i e r . The c o n t r o l of cAMP l e v e l s by phosphodiesterase may be very s t r i n g e n t such that any attempt to elev a t e these l e v e l s would be immediately countered by degradation. Thus, d a r k - r e a r i n g may have caused a s i t u a t i o n where l a r g e f l u c t u a t i o n s i n cAMP l e v e l s are i n t o l e r a b l e and the mainte-nance of steady s t a t e l e v e l s , achieved i n part by phosphodiesterase, be-comes important. That phosphodiesterase may play a v i t a l r o l e i n the r e g u l a t i o n of cAMP l e v e l s i n the c e l l i s borne out i n studies by Cheung (157,158) and Thompson and Appleman (159). These workers have shown that the enzyme d i s p l a y s a l l the features important f o r a r e g u l a t o r y f u n c t i o n such that i t has high a f f i n i t y f o r i t s s u b s t r a t e , i t e x h i b i t s negative c o o p e r a t i v i t y , and i t s a c t i v i t y i s regulated by a p r o t e i n f a c t o r as w e l l as Oa"*"* ions. There were no d i f f e r e n c e s between 30 day o l d experimental and c o n t r o l animals i n the K +-induced accumulation of cAMP i n f r o n t a l or v i s u a l cor-t i c a l s l i c e s ( F i g . 9 ). This tends to discount phosphodiesterase a c t i v i t y as the f a c t o r that p r e c i p i t a t e s the changes observed i n responsiveness to NA i n b r a i n s l i c e s of dark-reared animals. The reason f o r t h i s i s that a l t e r e d phosphodiesterase as a r e s u l t of da r k - r e a r i n g would presumably be manifested regardless of the circumstances that l e d to elevated cAMP l e v e l s . There are s e v e r a l i n t e r v e n i n g v a r i a b l e s , however, which make t h i s l i n e of reasoning more complex than i t appears. F i r s t of a l l , the s t i m u l a t i o n of cAMP formation i n b r a i n s l i c e s by K"*" i s much greater than that of NA. These l e v e l s of cAMP could be high enough to i n a c t i v a t e phosphodiesterase through the negative c o o p e r a t i v i t y which the enzyme e x h i b i t s and thus o b l i t e r a t e any d i f f e r e n c e s i n i t s a c t i v i t y between b r a i n s l i c e s from c o n t r o l and experimental animals. Secondly, the mechanism whereby IC*" and indeed a l l d e p o l a r i z i n g agents s t i m u l a t e cAMP formation i n b r a i n s l i c e s i s not known. That de-p o l a r i z i n g agents do not exert t h e i r e f f e c t s on cAMP l e v e l s because of the increased r e s p i r a t i o n and g l y c o l y s i s i n b r a i n s l i c e s which they cause i s i n d i c a t e d by the f i n d i n g that the increase i n cAMP l e v e l s i n s l i c e s i n cu-bated w i t h p r o g r e s s i v e l y i n c r e a s i n g K~*~ concentrations roughly p a r a l l e l s the known e f f e c t of K+ concentrations on the e l e c t r o g e n i c membrane poten-t i a l s (160) r a t h e r than the e f f e c t of K on r e s p i r a t i o n and g l y c o l y s i s (161). Furthermore, i t has been shown that under c o n d i t i o n s where malonate i n h i b i t s enhanced metabolic a c t i v i t y by more than 507„ (162) there was no r e d u c t i o n i n the accumulation of cAMP evoked by the depola-r i z i n g agent v e r a t r i d i n e (163). I t i s suspected that d e p o l a r i z i n g agents induce the r e l e a s e of adenosine (98) which then s t i m u l a t e s cAMP formation through an adenosine receptor (96). The problem encountered here i s that phosphodiesterase a c t i v i t y may not be as t i g h t l y coupled to the adenosine receptor as i t i s to the NA receptor or that t h i s c o u p l i n g may e x h i b i t d i f f e r e n t c h a r a c t e r i s t i c s . I t has, i n f a c t , been suggested that biogenic amines a c t i v a t e phosphodiesterase whereas adenosine reverses t h i s a c t i v a -t i o n (148). + F i n a l l y , the p o s s i b i l i t y cannot be excluded that K , i n a d d i t i o n to causing the r e l e a s e of adenosine, causes the r e l e a s e of biogenic amines from nerve t e r m i n a l s . In t h i s event, the i n t e r p r e t a t i o n of the r e s u l t s obtained i n incubations of b r a i n s l i c e s w i t h K + would be very d i f f i c u l t i n view of the a n t a g o n i s t i c e f f e c t of adenosine and biogenic amines on phosphodiesterase a c t i v i t y and the synergism that these substances ex-h i b i t w i t h regard to the promotion of cAMP accumulat ion . \ I f phosphodiesterase plays a g r e a t e r part i n f r o n t a l than v i s u a l c o r t i c a l s l i c e s w i t h regard to the observed d i f f e r e n c e s between e x p e r i -mental and c o n t r o l animals, then some of the f i n d i n g s obtained i n incuba-t i o n s of b r a i n s l i c e s of 60 day o l d animals w i t h K + might be explained s p e c i f i c a l l y i n terms of the d i f f e r e n t i a l e f f e c t of adenosine on phospho-+ d i e s t e r a s e a c t i v i t y i n these b r a i n regions. For example, the study of K induced accumulation of cAMP i n b r a i n s l i c e s of 60 day o l d animals showed ( F i g s . 12 and 13) that cAMP accumulation was higher i n f r o n t a l c o r t i c a l s l i c e s of dark-reared animals than c o n t r o l s whereas there was no d i f f e r -ence between the two groups i n v i s u a l c o r t i c a l s l i c e s . Thus, i t i s p o s s i b l e that the K -induced r e l e a s e of adenosine and the subsequent 74 s t i m u l a t i o n of cAMP formation and the simultaneous i n a c t i v a t i o n of phos-phodiesterase by adenosine was greater i n f r o n t a l than v i s u a l c o r t i c a l s l i c e s of dark-reared r a t s . In support of t h i s i s the demonstration (Table I) that cAMP l e v e l s i n f r o n t a l c o r t i c a l s l i c e s incubated w i t h adenosine were s i g n i f i c a n t l y g reater i n experimental than c o n t r o l animals whereas there was no d i f f e r e n c e between the two groups i n v i s u a l c o r t i c a l s l i c e s . This r e s u l t would be expected i f the K +-induced accumulation of cAMP were mediated by adenosine. Furthermore, t h i s f i n d i n g suggests that the a l t e r e d responsiveness to K + i n b r a i n s l i c e s of 60 day o l d dark-reared animals i s not due to changes i n mechanisms c o n t r o l l i n g r e l e a s e of adenosine from c e l l s but r a t h e r to changes i n events subsequent to r e l e a s e such as those described f o r a l t e r e d responsiveness to NA. The processes that may be involved i n diminished responsiveness to NA i n b r a i n s l i c e s of dark-reared animals have been discussed. The accentuated responsiveness of b r a i n s l i c e s of 60 day o l d dark-reared animals to NA and K may be explained i n terms of the two processes of n o r m a l i z a t i o n and s u p e r s e n s i t i v i t y a c t i n g i n concert. N o r m a l i z a t i o n r e f e r s to the a b i l i t y of the CNS s t r u c t u r e s a f f e c t e d by l i g h t d e p r i v a t i o n to recover p a r t i a l l y from d e f i c i e n c i e s i n morphological (22,25,33) and e l e c t r o -p h y s i o l o g i c a l (44) development a f t e r prolonged durations of d a r k - r e a r i n g . In view of the f i n d i n g s that : '1) the e f f e c t s of l i g h t d e p r i v a t i o n may be l i k e n e d to d e a f f e r e n t a t i o n (32,34,43,59-61); (2) denervated neurons may acquire s u p e r s e n s i t i v i t y to the t r a n s m i t t e r s that normally impinge upon them (164-167); 3) s u p e r s e n s i t i v i t y may g e n e r a l i z e to other t r a n s -m i t t e r s as w e l l as K + i o n (125); and 4) s u p e r s e n s i t i v i t y may lead to a l t e r e d responsiveness of the cAMP system (132-136), i t i s reasonable to assume t h a t i n the co r t e x of 60 day o l d dark-reared animals supersens-i t i v i t y of the cAMP generating system may have developed to or g e n e r a l i z e d to NA and to the agents released from nerve c e l l s during d e p o l a r i z a t i o n . This hypothesis could be t e s t e d by measuring the accumulation of cAMP i n response to v a r y i n g concentrations of NA or K +. I f s u p e r s e n s i t i v i t y i n the c o r t e x of r a t s dark-reared f o r 60 days does occur, there w i l l be observed a s h i f t i n the log dose-response curve from the r i g h t to the l e f t . The recent demonstration of axonal growth i n the CNS of animals a f f o r d s yet another mechanism through which prolonged exposure to complete darkness may cause increased responsiveness of c o r t i c a l s l i c e s to NA and p o s s i b l y K +. Furthermore, axonal growth may e x p l a i n some f i n d i n g s of heightened e l e c t r o p h y s i o l o g i c a l a c t i v i t y (45,46) of b r a i n areas other than v i s u a l c o r t e x a f t e r v i s u a l d e p r i v a t i o n as w e l l as some reports of increased s y n a p t i c d e n s i t i e s of c o r t i c a l l a y e r s which do not i n v o l v e s p e c i f i c a f f e r -ent systems (42). Axonal growth takes two forms, d i r e c t and c o l l a t e r a l . I t has been found (168-170) that ascending noradrenergic f i b e r s begin growing a f t e r i n t e r r u p t i o n by e l e c t r o l y t i c or s u r g i c a l l e s i o n s and invade the area of damage. The phenomena of c o l l a t e r a l s p r o u t i n g i n the CNS involv e s u n i n j u r e d f i b e r s that can form new c o l l a t e r a l s which invade regions deprived of t h e i r normal a f f e r e n t i n f l o w by damage elsewhere (171-173). The new c o l l a t e r a l s make synapt i c contacts w i t h denervated post-synaptic membranes. Although axonal growth has only been demonstrated i n cases where l e s i o n s have been introduced i n the CNS i t may be r e i t e r a t e d that there are numerous s i m i l a r i t i e s between the e f f e c t s of l e s i o n s and v i s u a l d e p r i v a t i o n on v i s u a l c o r t i c a l morphology and e l e c t r o p h y s i o l o g y . During d a r k - r e a r i n g there i s reduced a f f e r e n t i n f l o w to the v i s u a l c o r t e x and p o s s i b l y to other c o r t i c a l areas which may be i n f l u e n c e d d i r e c t l y or 76 i n d i r e c t l y by l i g h t d e p r i v a t i o n . Thus, a f u r t h e r feature that l i g h t d e p r i v a t i o n may have i n common w i t h denervation or d e a f f e r e n t a t i o n i s the increased i n v a s i o n by noradrenergic f i b e r s i n t o those c o r t i c a l areas a f f e c t e d by v i s u a l d e p r i v a t i o n . I t has been suggested that d a r k - r e a r i n g r e s u l t s i n h y p e r a c t i v i t y of the anatomical pathways p r o j e c t i n g from the b r a i n stem to the cor t e x (46). One of these p r o j e c t i o n s i s the noradrenergic f i b e r system. Thus, increased growth of axons and c o l l a t e r a l s as a consequence of d a r k - r e a r i n g may form the anatomical s u b s t r a t e f o r apparent c o r t i c a l h y p e r a c t i v i t y and may be the basis f o r the su s p i c i o n s of i n v e s t i g a t o r s that d a r k - r e a r i n g may have an e f f e c t on s u b c o r t i c a l and b r a i n stem s t r u c t u r e s . Since the NA-fiber system emanating from the b r a i n stem innervates the e n t i r e c o r -tex and s i n c e d a r k - r e a r i n g may perturb whatever f u n c t i o n t h i s system might serve, then the e f f e c t of d a r k - r e a r i n g on both the v i s u a l and f r o n t a l c o r t e x i s explained. That increased responsiveness to NA of cor-t i c a l s l i c e s occurred a f t e r 60 days of d a r k - r e a r i n g a l s o has a ready explanation through axonal growth. Increased a r b o r i z a t i o n of noradrener-g i c f i b e r s would lead to a gr e a t e r number of synapses responsive to NA and thus to a greater c a p a c i t y f o r the production of cAMP. The hypothesis of increased axonal growth could be te s t e d by examining the h i s t o f l u o r -escent p a t t e r n i n the c o r t e x of dark-reard r a t s ; t h i s i s the technique used to demonstrate axonal growth of noradrenergic f i b e r s . The developmental p r o f i l e s of the responsiveness of r a t v i s u a l and f r o n t a l c o r t i c a l s l i c e s to NA were found to be d i f f e r e n t i n that the ca p a c i t y of NA to e l i c i t the formation of cAMP i n f r o n t a l c o r t i c a l s l i c e s passes through a maximum at about 10 - 15 days of age and t h e r e a f t e r d e c l i n e s by 30 and 60 days to values s i m i l a r to that observed i n v i s u a l c o r t i c a l s l i c e s which have changed l i t t l e through the ages 10 to 60 days. This may be due to i n t r i n s i c d i f f e r e n c e s i n the development of NA-sensi-t i v i t y i n d i f f e r e n t c o r t i c a l regions. However, a more p a l a t a b l e explana-t i o n i s o f f e r e d by the demonstration that NA i s capable of s t i m u l a t i n g dopamine ( D A ) - s e n s i t i v e adenylate c y c l a s e , a l b e i t at higher concentrations than DA. I n caudate nucleus of r a t i t has been shown (174) that NA s t i m u l a t e s the maximal accumulation of cAMP as e f f e c t i v e l y as DA although the concentrations needed to produce h a l f maximal s t i m u l a t i o n of cAMP synthesis was 4 yM f o r DA and 28 yM f o r NA. That NA i n t e r a c t s s p e c i f i -c a l l y w i t h the DA receptor i s supported by the f o l l o w i n g : 1) the c l a s s i -c a l -adrenergic agonist L - i s o p r o t e r e n o l d i d not s t i m u l a t e cAMP formation (174); 2) the e f f e c t of combinations of dopamine and NA on adenylate c y c l a s e d i d not exceed that observed w i t h optimal concentrations of the i n d i v i d u a l s t i m u l a t o r y agents (174,175); and 3) the increase i n adenylate c y c l a s e a c t i v i t y caused by NA was reduced by the s p e c i f i c DA antagonist h a l o p e r i d o l . The a b i l i t y of NA to s t i m u l a t e DA receptors takes on g r e a t e r s i g n i f i c a n c e w i t h regard to the present i n v e s t i g a t i o n i n view of recent demonstrations of the existence of dopamine nerve endings i n r a t f r o n t a l c ortex (176), as w e l l as dopamine-sensitive adenylate c y c l a s e i n the a n t e r i o r l i m b i c c o r t e x of the primate (177), i n r a t c e r e b r a l cortex (178) and, s p e c i f i c a l l y , i n the l i m b i c f o r e b r a i n of r a t s (179). Thus, the d i f f e r e n c e s i n responsiveness to NA between v i s u a l and f r o n t a l c o r t e x may be e x p l a i n a b l e i n terms of s p e c i f i c developmental c h a r a c t e r i s t i c s of the dopamine-receptor adenylate c y c l a s e complex i n f r o n t a l c ortex. I f the above i n t e r p r e t a t i o n i s v a l i d then the d i f f e r e n t i a l response of f r o n t a l and v i s u a l c o r t i c a l s l i c e s to NA a f t e r 30 days of d a r k - r e a r i n g may be explained by assuming a d i f f e r e n t i a l e f f e c t of l i g h t d e p r i v a t i o n on the NA and DA systems i n the cortex. Moreover, the increase i n the ca p a c i t y of f r o n t a l c o r t i c a l s l i c e s to generate cAMP seen at 10 and 15 days i n normal r a t pups may be c o r r e l a t e d w i t h the development of be-h a v i o r a l a r o u s a l which a l s o passes through a maximum of about 15 - 20 days (180,181). I n support of t h i s c o r r e l a t i o n i s the f i n d i n g that i n the c o r t e x of r a t s DA l e v e l s peak at 16 days of age, p l a t e a u , and then increase s u b s t a n t i a l l y from 30 day to a d u l t (182). Furthermore, i t has been demonstrated that treatments of r a t pups w i t h 6-OHDA which reduced DA but not. NA l e v e l s i n b r a i n r e s u l t s i n the development of increased b e h a v i o r a l a c t i v i t y e a r l i e r and to a greater degree than untreated c o n t r o l s (180). This may have r e s u l t e d from a s u p e r s e n s i t i v e s t a t e of the DA systems. This c o r r e l a t i o n i s i n agreement w i t h the e a r l i e r suggestion that the cAMP system may p l a y a r o l e i n the development of the nervous system. The hypothesis that i n the present experiments NA was s t i m u l a t i n g DA receptors i n the f r o n t a l c o r t e x might be t e s t e d by employing approp-r i a t e DA and NA agonists and antagonists i n incubations of f r o n t a l c o r t i -c a l s l i c e s . That the developmental c h a r a c t e r i s t i c s of the DA-adenylate c y c l a s e system i n f r o n t a l c o r t e x i s p e c u l i a r i n that i t may undergo a s u p e r s e n s i t i v i t y s t a t e at e a r l i e r ages may be t e s t e d by e s t a b l i s h i n g l o g -dose response curves f o r DA i n f r o n t a l c o r t i c a l s l i c e s of r a t s of appro p r i a t e ages. CONCLUSIONS I n v e s t i g a t i o n s i n t o the morphology, e l e c t r o p h y s i o l o g y , and b i o -chemistry of the brains of l i g h t deprived animals suggested to us that i n the cor t e x of animals so t r e a t e d there may be an a l t e r a t i o n i n the biochemical processes r e s p o n s i b l e f o r the metabolism of cAMP. We have shown that i n the v i s u a l and f r o n t a l c o r t e x of dark-reared r a t s changes i n these processes do occur and that i n some respects these changes are d i f f e r e n t i n the v i s u a l than i n the f r o n t a l cortex. To e x p l a i n our observations on the e f f e c t s of da r k - r e a r i n g on the cAMP system, s e v e r a l p o s s i b i l i t i e s have been o f f e r e d . I t i s evident that our la c k of know-ledge about the r o l e of cAMP i n b r a i n , and a l l the systems i n v o l v e d i n that r o l e , make i t d i f f i c u l t to i n t e r p r e t r e s u l t s showing environmental e f f e c t s on the cAMP system. The question a r i s e s , t h e r e f o r e , whether i t i s deemed worthwile to continue on from these p r e l i m i n a r y s t u d i e s . We b e l i e v e that the p o s s i b l e l i n k between two monumental f i n d i n g s , one demonstrating a second messenger r o l e of cAMP i n c e l l s and the other showing the strong propensity of the CNS to e x h i b i t p l a s t i c i t y i n s t r u c -ture and f u n c t i o n , warrant f u r t h e r i n v e s t i g a t i o n s of the k i n d undertaken here. References C r e e l , D.J., Dustman, R.E., and Beck, E.C., Exp. Neurol. J29, 298 (1970). Lund, R.D., Science 149, 1506 (1965). G y l l e n s t e n , L., Malmfors, T., N o r r l i n , M.L., J . Comp. Neurol. 126, 463 (1966). Riesen, A.H., i n "The B i o s o c i a l Basis of Mental R e t a r d a t i o n " , S.F. Osier and R.E. Cooke (Eds.), Johns Hopkins U n i v e r s i t y Press, Baltimore, 1965, p. 61. Riesen, A.H., i n "Progress i n P h y s i o l o g i c a l Psychology", E. S t e l l a r and J . S t e l l a r (Eds.), Academic Press, New York, 1966, p. 117. Riesen, A.H., (Ed.) "The Developmental Neuropsychology of Sensory D e p r i v a t i o n " , Academic Press, New York, 1975, pp. 1, 153, 277. Mendelson, J.H., and E r v i n , F.R., i n "Neurol Physiopathology", R.G. G r e n e l l (Ed.), Harper, New York, 1963, p. 178. Globus, A., i n " B r a i n Development and Behavior", M.B. Sterman, D.J. McGinty and A.M. A d i n o l f i (Eds.), Academic Press, New York, 1971, p. 253. Globus, A., i n "The Developmental Neuropsychology of Sensory D e p r i v a t i o n " , A.H. Reisen (Ed.), Academic Press, New York, 1975, p. 9. S c h e i b e l , M.E., and S c h e i b e l , A.B., Communications i n Behavior B i o l o g y 1, 231 (1968). Krech, D., Rosenzweig, M.R., and Bennett, E.L., Physiology and Behavior .1, 99 (1966). Rosenzweig, M.R., American P s y c h o l o g i s t 21, 321 (1966). F i f k o v a , E., i n "Advances i n Psychobiology", V o l . 2, G. Newton and A.H. Riesen (Eds.), Wiley and Sons, New York, 1974, p. 59. Cragg, B.G., i n "The S t r u c t u r e and Function of Nervous Tissue", V o l . 4, G.H. Bourne (Ed.), Academic Press, New York, 1972, p. 1. Raisman, G., and Mathews, M.R., i n "The S t r u c t u r e and Function of Nervous Tissue", V o l . 4, G.H. Bourne (Ed.), Academic Press, New York, 1972, p. 61. Rose, S.P.R., Hambley, J . , and Haywood, J . , Asilomar Conference on Neural Mechanisms of Learning and Memory (1974), i n press. 81 17. Bondy, S.C., and M a r g o l i s , F.L., i n "Sensory D e p r i v a t i o n and B r a i n Development: The Avian V i s u a l System as a Model", Jena, F i s c h e r , 1971. 18. Walker, J.P., Walker, J.B., K e l l e y , R.L., and Riesen, A.H., i n "The Developmental Neuropsychology of Sensory D e p r i v a t i o n " , A.H. Riesen (Ed.), Academic Press, New York, 1975, p. 93. 19. Hayhow, W.R., Webb, C , and J e r v i e , A., J . Comp. Neurol. 115, 187 (1960). 20. Sheridan, C.L., J . Comp. P h y s i o l . Psych. 5_9, 292 (1965). 21. G y l l e n s t e n , L., Acta Morphologica Neelando - Scandinavica 2, 331 (1959) . 22. G y l l e n s t e n , L., Malmfors, T., and N o r r l i n , M.L., J . Comp. Neurol. 124, 149 (1965). 23. F i f k o v a , E., B r a i n Res. 6, 763 (1967). 24. F i f k o v a , E., and H a s s l e r , R., J . Comp. Neurol. 135, 167 (1969). 25. F i f k o v a , E., J . Comp. Neurol. 140, 431 (1970). 26. Wiesel, T.N., and Hubel, D.H., J . Neurophysiol. 28, 1029 (1965). 27. Wiesel, T.N., and Hubel, D.H., J . Neurophysiol. 26 978 (1963). 28. Kupfer, C , J . Neuropath, and E x p t l . Neurol. 24, 653 (1965). 29. Gomirato, G., and Baggio, G., J . Neuropath, and E x p t l . Neurol. 21, 634 (1962). 30. Coleman, P.D., and Riesen, A.H., J . Anatomy 102, 362 (1968). 31. Globus, A., and S c h e i b e l , A.B., E x p t l . Neurol. 1_9, 331 (1967). 32. Globus, A., and S c h e i b e l , A.B., E x p t l . Neurol. 18_, 116 (1967). 33. Valverde, F., B r a i n Res. 33, 1 (1971). 34. Valverde, F., E x p t l . B r a i n Res. 5, 274 (1968). 35. Valverde, F., and Esteban, M.E., B r a i n Res. 9, 145 (1968). 36. Valverde, F., E x p t l . B r a i n Res. 3, 337 (1967). 37. F i f k o v a , E., Nature 220, 379 (1968). 38. Cragg, B.G., B r a i n Res. 13, 53 (1969). 39. F i f k o v a , E., J . Neu r o b i o l . 2, 61 (1970). 82 40. F i f k o v a , E., J . N e u r o b i o l . I, 285 (1970). 41. Vrensen, G., and Groot, D. De., B r a i n Res. 7_8, 263 (1974). 42. Vrensen, G., and Groot, D. De., B r a i n Res. 93, 15 (1975). 43. Hess, A., J . Comp. Neurol. 109, 91 (1958). 44. C a l l i s o n , D.A., and Spencer, J.W., Develop. P s y c h o b i o l . 1, 196 (1968) . 45. Yinon, U., and Awerback, E., E x p t l . Neurol. 38, 231 (1973). 46. Scherrer, J . , and Fourment, A., i n "Progress i n B r a i n Research", V o l . 9, W.A. Himwich and H.E. Himwich (Eds.), E l s e v i e r , Amsterdam, 1964, p. 103. 47. Wiesel, T.N., and Hubel, D.H., J . Neurophysiol. 26, 1003 (1963). 48. Hubel, H.D., and Wiesel, T.N., Nature 255, 41 (1970). 49. P e t t i g r e w , J.D., I n v e s t i g a t i v e Optamol. 11, 386 (1972). 50. Wiesel, T.N., and Hubel, D.H., J . Neurophysiol. 28^ 1060 (1965). 51. Barlow, H.B., and P e t t i g r e w , J.D., J . P h y s i o l . 218, 98 (1971). 52. Pettigrew, J.D., and Freeman, R.D., Science 182, 599 (1973). 53. C r e u t z f e l d t , O.D., and Heggelund, P., Science 188, 1025 (1975). 54. Fentress, J.C., and Doty, R.W., E x p t l . Neurol. 30, 535 (1971). 55. Spehlman, R., Chang, CM., and D a n i e l s , J.C., Arch. Neurol. 22, 504 (1970). 56. Sharpless, S., and Halpern, L., EEG and C l i n . Neurophy. 14, 22 (1962). 57. Vazquez, A.S., K r i p , G., and Pinsky, C , E x p t l . Neurol. 23, 318 (1969) . 58. Purpura, D.P., and Housepian, E.M., E x p t l . Neurol. 4, 377 (1961). 59. Chu, N.S., Rutledge, L., and S e l l i n g e r , O.Z., B r a i n Res. 29, 323 (1971). 60. Chu, N.S., Rutledge, L., and S e l l i n g e r , O.Z., B r a i n Res. 29, 331 (1971). 61. Rutledge, L., Rarickj.nJV, and Duncan, J . , EEG and C l i n . Neurophy. 23, 256 (1967). 83 62. Appel, S.H., Davis, W., and S c o t t , S., Science 157, 836 (1967). 63. Dewar, A.J., and Winterburn, A.K., J. Neurol. Sciences 20, 279" (1973). 64. DeBold, R.C., F i r s h e n , W., and C a r r i e r , S.C., Psychonomic Science 7_, 379 (1967). 65. Horn, G., Rose, S.P.R., and Bateson, P.P.G, B r a i n Res. 56, 227 (1973). 66. Haywood, J . , Rose, S.P.R., and Bateson, P.P.G., Nature 228, 373 (1970). 67. Bateson, P.P.G., Horn, G., and Rose, S.P.R., B r a i n Res. 39, 449 (1972). 68. Rose, S.P.R., Nature 215, 253 (1967). 69. Richardson, K., and Rose, S.P.R., B r a i n Res. 44, 299 (1972). 70. Richardson, K., and Rose, S.P.R., J . Neurochem. 21, 521 (1973). 71. Rose, S.P.R., Sinha, A.K., and Broomhead, J . , J . Neurochem. 15, 223 (1974). 72. Rose, S.P.R., and Sinha, A.K., J. Neurochem. 23, 1065 (1974). 73. Rose, S.P.R.,rand Sinha, A.K., L i f e Sciences 15, 223 (1974). 74. C h a k r a b a r t i , T., and Daginawala, H.F., J. Neurochem. 24, 983 (1975). 75. M a l e t t a , G.J., and Timir a s , P.S., J. Neurochem. .15, 787 (1968). 76. M a l e t t a , G.J., and Timiras, P.S., E x p t l . Neurol. 19_, 513 (1967). 77. Rose, S.P.R., B r a i n Res. 38, 171 (1972). 78. Sutherland, E.W., R a i l , T.W., and Menon, T., J. B i o l . Chem. 237, 1220 (1962). 79. De R o b e r t i s , E., A r n a i z , G.R.L., A l b e r i c i , M., Butcher, R.W., and Sutherland, E.W., J. B i o l . Chem. 242, 3487 (1967). 80. Weiss, B., and Costa, E., Biochem. Pharmacol. 17_, 2107 (1968). 81. W i l l i a m s , R.H., L i t t l e , S.A., Beag, A.G., and E n s i r c k , J.W., Metabolism 20, 743 (1971). 82. Gaballah, S., and Popoff, C , B r a i n Res. 25, 220 (1971). 83. Yamamoto, M., and Massey, K., Comp. Biochem. P h y s i o l . 30, 941 (1969). 84 84. Florendo, N.T., B a r r n e t t , R.J., and Greengard, P., Science 173, 745 (1971). 85. Krishna, G., Forn, J . , V o i g t , K., P a u l , M., and Gessa, G.L., Advances i n Biochem. Pharmacol. 3_, 155 (1970). 86. Ueda, T., Maeno, H., Greengard, P., J . B i o l . Chem. 248, 8295 (1973). 87. Johnson, E.M., Ueda, T., Maeno, H., Greengard, P., J . B i o l . Chem. 247, 5650 (1972). 88. Maeno, H., and Greengard, P., J . B i o l . Chem. 247, 2269 (1972). 89. Deguchi, T., Molec. Pharmacol. 9, 184 (1973). 90. Shimizu, H., Daly, J.W., and C r e v e l i n g , C.R., Eur. J . Pharmacol. 17_, 240 (1972). 91. K a k i u c h i , S., and R a i l , T.W., Mol. Pharmacol. 4, 379 (1968). 92. S c h u l t z , J . , and Daly, J.W., J . B i o l . Chem. 248, 860 (1973). 93. Walker, J.B., and Walker, J.P., B r a i n Res. 54, 386 (1973). 94. Shimizu, H., Daly, J.W., and C r e v e l i n g , C.R., J . Neurochem. 16, 1609 (1969)1 95. S a t t i n , A., and R a i l , T.W., Molec.. Pharmacol. 6, 379 (1970). 96. Huang, M., Shimizu, H., and Daly, J . , Molec. Pharmacol. 7_, 155 (1971). 97. Shimizu, H., C r e v e l i n g , C.R., and Daly, J.W., Molec. Pharmacol. 6, 184 (1970). 98. Shimizu, H., C r e v e l i n g , C.R., and Daly, J.W., Proc. Nat. Acad. S c i . U.S.A. 65, 1033 (1970). 99. K a k i u c h i , S., R a i l , T.W., and M c l l w a i n , H., J . Neurochem. 16, 485 (1969). 100. Greengard, P., McAfee, D.A., and Kababian,. J.W., Adv. C y c l i c N u c e l o t i d e Res. I, 337 (1972). 101. McAfee, D.A., and Greengard, P., Science 178, 310 (1972). 102. K a l i x , P., McAfee, D.A., Schorderet, M., and Greengard, P., J . Pharmacol. E x p t l . Ther. 188, 676 (1974). 103. Bloom, F.E., L i f e Sciences 14, 1819 (1974). 104. S i g g i n s , G.R., Battenberg, E.F., Ho f f e r , B.J., Bloom, F.E., and S t e i n e r , A.L., Science 179, 585 (1973). 85 105. Segal, M., P i c k e l , V., and Bloom, F.E., L i f e Sciences 13, 317 (1973). 106. Roisen, F.J., Murphy, R.A., and Braden, W.G., Science 177, 809 (1972). 107. McMahon, D., Science 185, 1012 (1974). 108. Yeung, D., and O l i v e r , I.T., Biochemistry ]_, 3231 (1968). 109. Greengard, P., Science 163, 891 (1969). 110. Wicks, W.D., J . B i o l . Chem. 244, 3941 (1969). 111. J o s t , J.P., and Sahib, K., J . B i o l . Chem. 246, 1623 (1971). 112. P i t t s , F.N., and Quick, C , J . Neurochem. 14, 561 (1967). 113. Schmidt, M.J., Palmer, E.C., Dettbarn, W.D., and Robison, G.A., Develop. P s y c h o b i o l . 3, 53 (1970). 114. Schmidt, M.J., and S o k o l o f f , L., J . Neurochem. 21., 1193 (1973). 115. Gahallah, J . , Popoff, C , and Sooknandan, G., B r a i n Res. 31, 229 (1971). 116. Schmidt, M.J., and Robison, G.A., J . Neurochem. 19^ , 937 (1972). 117. Shoemaker, W.S., and Wartman, R.J., Science 171, 1017 (1971). 118. Kauffman, F.C., Harkoen, M.H.A., and Johnson, E.C. S L i f e Sciences 11, 613 (1972). 119. Palmer, G.C., Schmidt, M.S., and Robison, G.A., J . Neurochem. 1£, 2251 (1972). 120. Schmidt, M.J., and Robison, G.A., L i f e Sciences 10, 459 (1971). 121. P e r k i n s , J.P., and Moore, M.M., Molec. Pharmacol. 9, 774 (1973). 122. B i t o , L.Z., Dawson, M.J., and P e t r i n o v i c , L., Science 172, 583 (1971). 123. Fleming, W.W., M c P h i l l i p s , J . J . , and W e s t f a l l , D.P., Rev. P h y s i o l . Biochem. Exp. Pharmacol. 68, 55 (1973). 124. Sharpless, S., i n "The Developmental Neuropsychology of Sensory D e p r i v a t i o n " , A. Riesen (Ed.), Academic Press, New York, 1975, p. 125. 125. Trendelenburg, TJ., Pharmacol. Rev. 15, 225 (1963). 126. Reas, H.W., and Trendelenburg, TJ., J . . Pharmacol. E x p t l . Thera. 156, 126 (1967). 86 127. Green, R.D., Fleming, W.W., and Schmidt, J.L., J . Pharmacol. 162, 270 (1968). 128. Albuquerque, E.X., and Mclsaac, R.J., E x p t l . Neurol. 26, 183 (1970). 129. Diamond, J . , and M i l e d i , R.A., J . P h y s i o l . 162, 393 (1962). 130. H a r r i s , A.J., K u f f l e r , S., and Dennis, M.J., Proc. Royal Soc. London 177, 541 (1971). 131. K u f f l e r , S., Dennis, M.J., and H a r r i s , A.J., Proc. Royal Soc. London 17_7, 555 (1971). 132. Weiss, B., and Costa, E., Science 156, 1750 (1967). 133. Weiss, B., J . Pharmacol. E x p t l . Thera. 168, 146 (1969). 134. Palmer, G.C, Neuropharmacol. 11, 145 (1972). 135. K a l i s k e r , A., Rutledge, CO., and P e r k i n s , J.P., Molec. Pharmacol. 9, 619 (1973). 136. Mishra, R.K., Gardner, E.L., Katzman, R., and Makman, M.H., Proc. Nat. Acad. S c i . U.S.A. 7_1, 3883 (1974). 137. K a k i u c h i , S., and R a i l , T.W., Molec. Pharmacol. 4, 367 (1968). 138. Adams, A.D., and F o r r e s t e r , J.M., J . E x p t l . P h y s i o l . 53, 327 (1968). 139. Burkard, W.P., J. Neurochem. 19, 2615 (1972). 140. Breckenridge, B.McL., Proc. Nat. Acad. S c i . U.S.A. 52, 1580 (1964). 141. Brostrom, CO., and Kon, C , Anal. Biochem. 58, 459 (1974). 142. Gilman, A.C., Proc. Nat. Acad. S c i . U.S.A. 67, 305 (1970). 143. Brown, B.L., Albano, J.D.M., Ek i n s , R.P., and Sgh e r z i , A.M., Biochem. J . 121, 561 (1971). 144. Lowry, O.H., Rosbrough. N.J., F a r r , A.L., and Ra n d a l l , R.J., J . B i o l . Chem. 193, 265 (1951). 145. F e r r e n d e l l i , J.A., Ki n s c h e r f , D.A., and Chang, M.M., B r a i n Res. 84, 63 (1975). 146. S c h u l t z , J . , and Daly, J.W., J . Neurochem. 21, 1319 (1973). 147. Palmer, G.C, S u l e r , F., and Robison, G.A., Neuropharmacol. 12, 327 (1973). 148. S h u l t z , J . , J . Neurochem. 24, 1237 (1975). 87 149. P e r k i n s , J.P., and Moore, M.M., J. Pharmacol, and E x p t l . Thera. 185, 371 (1973). 150. French, S.W., Reid, P.E., Palmer, D.S., Narod, N.E., and Ramey, C.W., Res. Comm. Chem. Path. Pharmacol. 9_, 575 (1974). 151. S k o l n i c k , P., S c h u l t z , J . , and Daly, J.W., J . Neurochem. 24, 1263 (1975). 152. Riesen, A.H., Amer. J.. Orthopsychiat. 30, 23 (1960). 153. Wase, A.W., and Christensen, J . , Arch. Gen. P s y c h i a t . 2, 171 (1960). 154. Cheung, W.Y., i n "Advances i n Biochemical Psychopharmacology", V o l . 3, P. Greengard and E. Costs (Eds.), Raven Press, New York, 1971, p. 51. 155. Drummond, G.I., and Yamamoto, M., The Enzymes 4, 355 (1971). 156. Weiss, B., and Strada, S.J., Advan. C y c l i c N u c l e o t i d e Res. 1_, 357 (1972). 157. Cheung, W.Y., Biochem. Biophys. Res. Comm. 38, 533 (1970). 158. Cheung, W.Y., J . B i o l . Chem. 246, 2859 (1971). 159. Thompson, W.J., and Appleman, M.M., Biochemistry 10, 311 (1971). 160. Hodgkin, A.L., B i o l o g i c a l Rev. 26, 339 (1951). 161. E l l i o t t , K.A.C., and Wolfe, L.S., i n "Neurochemistry", K.A.C. E l l i o t t , I.H. Page, and J.H. Quastel (Eds.), C.C. Thomas, I l l i n o i s , 1962, p. 192. 162. Quastel, J.H., Proc. 4th I n t e r . Congress of Biochem. 3, 90 (1958). 163. Shimizu, H., and Daly, J.W., Eur. J . Pharmacol. 17, 240 (1972). 164. Schoenfeld, R.I., and Uretsky, N.J., Eur. J . Pharmacol. 20, 357 (1967). 165. Schoenfeld, R.I., and Uretsky, N.J., Eur. J . Pharmacol. 19_, 115 (1972). 166. Ungerstedt, U., Acta P h y s i o l . Scand. Suppl. 367, 69 (1971). 167. Uretsky, N.J., and Schoenfeld, R.I., Nature New B i o l . 234, 157 (1971). 168. B j o r k l u n d , A., and St e n e v i , U., B r a i n Res. 3_1, 1 (1971). 169. Bjorklund, A., and St e n e v i , U., Science 125, 1251 (1972). 170. Katzman, R., Bjorklund, A., Owan, C , St e n e v i , U., and West, K.A., B r a i n Res. 25, 579 (1971). 171. Lynch, G., Deadwyler, S., and Cotman, C , Science 180, 1364 (1973). 172. Moore, R.Y., Bjorklund, A., and S t e n e v i , U., B r a i n Res. 3_3, 13 (1971). 173. Raisman, G., B r a i n Res. 14, 25 (1969). 174. Kebabian, J.W., P e t z o l d , G.L., and Greengard, P., Proc. Nat. Acad. S c i . U.S.A. 69, 2145 (1972). 175. Clement-Cormier, Y.C., Kebabian, J.W., P e t z o l d , G.L., and Greengard, P., Proc. Nat. Acad. S c i . U.S.A. 71, 1113 (1974). 176. L i n d v a l l , 0., and Bj o r k l u n d , A., Acta P h y s i o l . Scand., Suppl. 412, 1 (1974). 177. Mishra, R.K., Demirjian, C., Katman, R., and Makman, M.H., B r a i n Res. 96, 395 (1975). 178. Von Hungen, K., and Roberts, S., Eur. J. Biochem. 36, 391 (1973). 179. M i l l e r , R.J., Horn, A.S., and Iversen, L.L., Mol. Pharmacol. 10, 759 (1974). 180. Shaywitz, B.A., Yager, R.D., and Klopper, J.H., Science 191, 305 (1976). 181. Campbell, A., L y t l e , L.D., and F i b i g e r , H.C., Science 166, 635 (1969). 182. L o i z o u , L.A., and S a l t , P., B r a i n Res. 20, 467 (1970). 9 

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