"Medicine, Faculty of"@en . "Biochemistry and Molecular Biology, Department of"@en . "DSpace"@en . "UBCV"@en . "Nagy, Jim"@en . "2010-02-08T21:04:56Z"@en . "1976"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Morphological, electrophysiological and biochemical changes have been shown to occur in the retina, lateral geniculate nucleus, and visual cortex of light deprived animals. We attempted to determine whether the dark-rearing of rats from birth to 15, 30 and 60 days of age alters the ability of noradrenaline (NA) 30 \u00CE\u00BCM, potassium chloride (KCI) 50 \u00CE\u00BCM, adenosine 30 \u00CE\u00BCM and combinations of NA and KCI with adenosine to stimulate the _in vitro formation of cyclic AMP (cAMP) in visual cortical slices and, as an internal control, in frontal cortical slices. At 15 and 30 days of age there was an 11% and 2170 reduction, respectively, compared to normally reared controls, in the stimulation of cAMP formation in a 5 minute incubation\r\nwith NA in both frontal and visual cortical slices. After 60 days of dark-rearing, however, this was reversed in that the NA stimulation\r\nof cAMP formation was 23% and 357\u00C2\u00BB higher than controls in frontal and visual cortical slices. In frontal cortical slices of rats dark-reared for 15 and 30 days there was a significant reduction in the stimulation\r\nof cAMP formation in a 20 minute incubation with NA. No differences were observed between 30 day old experimental and control animals in studies of the accumulation of cAMP in frontal and visual cortical slices incubated for various times with KCI. The stimulation of cAMP formation induced by KCI and adenosine in a 5 minute incubation was 5770 and 397o higher, respectively, in frontal cortical slices of 60 day old experimental animals than controls while the response in visual cortical slices was unaffected.\r\nThe differences found between 60 day old experimental and control animals were abolished in both visual and frontal cortical slices when adenosine was used in combination with NA or KCI. Studies of the\r\n\r\naccumulation of cAMP in slices incubated for various times with NA revealed that the effect observed in the visual cortex after 30 days of light deprivation was due to a decrease in the maximum level of cAMP reached within a 20 minute incubation period, whereas in the frontal cortex the maximum level attained within a 20 minute incubation period was unaffected. These results are discussed in terms of our present knowledge concerning supersensitivity and plasticity in the central nervous system and the role of cAMP in nerve."@en . "https://circle.library.ubc.ca/rest/handle/2429/19799?expand=metadata"@en . "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 \u00C2\u00BB 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 \u00C2\u00BB ( 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\u00C2\u00B0 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\u00C2\u00B0 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\u00C2\u00B0 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\u00C2\u00B0 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\u00C2\u00B0 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\u00C2\u00B0. 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\u00C2\u00B0 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\u00C2\u00B0. 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\u00C2\u00B0) 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\u00C2\u00B0. 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\u00C2\u00B0 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 (\u00C2\u00A9\u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094\u00E2\u0080\u00A2) 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 (\u00E2\u0080\u00A2\u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u00A2\u00E2\u0080\u00A2) 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 (\u00E2\u0080\u00A2\u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094\u00E2\u0080\u00A2) 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 (\u00E2\u0080\u00A2\u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u00A2) 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\u00E2\u0080\u0094\u00E2\u0080\u0094O) and 100 uM (\u00E2\u0080\u00A2- \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u00A2 ) 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 (-> \u00E2\u0080\u0094 \u00E2\u0080\u0094 -\u00E2\u0080\u00A2) 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 ( \u00E2\u0080\u00A2 ) 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 (\u00E2\u0080\u00A2> \u00E2\u0080\u0094 \u00E2\u0080\u0094\u00E2\u0080\u00A2) 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 . \u00E2\u0080\u00A2 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 ( \u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u0094 ^ ) 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 ( \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u00A2) and v i s u a l ( \u00E2\u0080\u0094 ) 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 ( \u00E2\u0080\u00A2 ) 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 \u00E2\u0080\u0094 o ) and d a r k - r e a r e d (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) 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- \u00E2\u0080\u0094 O ; d a r k - r e a r e d \u00E2\u0080\u0094 \u00E2\u0080\u00A2\u00E2\u0080\u00A2 ) 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 (\u00E2\u0080\u00A2\u00E2\u0080\u0094\u00E2\u0080\u0094\u00E2\u0080\u00A2) 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- \u00E2\u0080\u0094 \u00E2\u0080\u00940 : d a r k - r e a r e d \u00E2\u0080\u00A2\u00E2\u0080\u00A2 \u00E2\u0080\u0094 - * ) 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 \u00E2\u0080\u0094 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 ( \u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u00A2 ) 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 \u00E2\u0080\u0094 - 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 (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2 ) 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- \u00E2\u0080\u0094 o ; d a r k - r e a r e d \u00E2\u0080\u0094 \u00E2\u0080\u0094\u00E2\u0080\u00A2 ) 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 \u00C2\u00B1 3.4 46.3 \u00C2\u00B1 2.2 49.4 \u00C2\u00B16.2 Adenosine 83.1 + 8.0 99.0 \u00C2\u00B1 11 75.2 \u00C2\u00B18.1 104 \u00C2\u00B1 6.4* NA 108 \u00C2\u00B1 9.1 133 \u00C2\u00B1 8.8* 125 \u00C2\u00B1 6.8 169 \u00C2\u00B18.4* Adenosine + NA 235 \u00C2\u00B1 15 275 \u00C2\u00B1 27 293 \u00C2\u00B1 25 349 \u00C2\u00B1 22 K + 235 + 21 268 \u00C2\u00B1 16 115 \u00C2\u00B18.7 180 \u00C2\u00B1 21* Adenosine + K + 312 + 24 344 \u00C2\u00B1 53 251\u00C2\u00B1 18 261\u00C2\u00B1 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 \u00C2\u00ABM 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\u00E2\u0080\u009E (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. 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