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Pharmacological analysis of EEG "activation" Ling, George McDonald 1960

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PHARMACOLOGICAL ANALYSIS OF EEG "ACTIVATION" by GEORGE MCDONALD L I N G M.A., University of British Columbia, 1957 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in tiie Department of Pharmacology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, I960 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree th a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n permission. Department of PHARMACOLOGY The U n i v e r s i t y of B r i t i s h Columbia, Vancouver Canada. Date 28 September I 960 G R A D U A T E STUDIES Field of Study: Neuropharmacology. Pharmocology of the central nervous system J. G . Foulks Pharmacology of the autonomic nervous system..E. E. Daniel Neuro-anatomy H . Scherrer Neurophysiology .. W . C. Gibson Related Studies: Neurosurgical techniques J. Wada Psychiatry J. S. Tyhurst Biochemistry ~ G . I. Drummond .S. H . Zbarsky ^i}? Pnttierstiy nf ^British Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL E X A M I N A T I O N FOR T H E D E G R E E O F DOCTOR OF PHILOSOPHY of GEORGE LING B.A. McGil l , 1943 M . A . U.B.C. , 1957 IN LECTURE HALL B2, FACULTY OF MEDICINE HUTS MONDAY, AUGUST 29, I960 AT 3:30 P.M. COMMITTEE IN CHARGE D E A N F. H . S O W A R D , Chairman J. G . F O U L K S F. A . PERRY W . C. G I B S O N J. W A D A H . SCHERRER P. M c G E E R G . I. D R U M M O N D H . M c L E N N A N G . E. D O W E R A. R. P. P A T T E R S O N A. M . C R O O K E R External Examiner: DR. A . R O T H B A L L E R , Albert Einstein College of Medicine, New York City. P H A R M A C O L O G I C A L ANALYSIS O F E E G " A C T I V A T I O N " A B S T R A C T The past decade has witnessed an intense interest in the influence of drugs and metabolic substances upon E E G "activation" and arousal mechanisms, mediated by the reticular activating system of the brain stem. Numerous pharmacological agents produce variations of the elec-trical activity of the brain. Analysis of their effects has suggested that the reticular activating system is an area wherein numerous drugs may act, and point to its multineuronal, polysynaptic character as playing a major role in central drug action. A technique has been developed which lends itself to the study of the direct actions of drugs upon the various components of the reticu-lar activating system of the brain stem. The experimental analysis of E E G "activation" requires the presence of a well deactivated back-ground pattern. Observations made in the cat demonstrate that partial trigeminalectomy, cervical dorsalectomy and low cervical transection produce a preparation in which the resting E E G regularly manifests maximal deactivation. A permanent catheter inserted through the right subclavian artery and just into the innominate artery has furnished a means for simul-taneous bilateral distribution of injected drugs to the brain without embarrassing flow in the carotid arteries. The advantages of this tech-nique include (a) an intact brain stem, (b) the maintenance of ade-quate spontaneous respiratory and circulatory states, and (c) the ability to perform various operative procedures without the necessity for ex-traneous pharmacological agents (anaesthetics, muscle relaxants), which may themselves have complicating effects on the E E G . In this preparation adrenergic and cholinergic agents as well as histamine and serotonin all produced prompt, short-lasting and repro-ducible E E G "activation" in low doses following direct ihtra-innomi-nate adminstration. In "equi-activating" doses, isoproterenol is the most potent E E G activating catechol adrenergic amine and norepine-phrine the least potent with epinephrine occupying an intermediate position. Amphetamine and eserine both produce long lasting E E G "activation", with amphetamine having a much shorter latency than eserine. Clear differentiation between the E E G effects resulting from direct drug-induced influences and those which may occur over reflex pathways has been demonstrated in preparations with bilateral cartoid sinus denervation. Complete temporal independence is shown between the onset and termination of the actions of "activating" agents intro-duced directly by intra-innominate administration and the vascular effecs of these agents as reflected in blood pressure alterations.. Partial destruction in the tegmentum rostral to ponto-mesence-phalic junction produces an increase in threshold for adrenergic E E G activation. Unilateral lesions which destroy most or all of the mesence-phalic-tegmentum abolish adrenergic-induced activation in the insi-lateral cortex but do not affect cholinergic activation. Results obtained with various synaptic blocking agents have sug-gested the possible existence in the brain of three types of receptors capable of converging on the final pathway for E E G activation; one responsive to cholinergic compounds and blocked by atropine; one re-sponsive to serotonin and blocked only by chlorpromazine and atropine; and one responsive to histamine and the short acting adren-ergic amines and blocked by phenoxybenzamine as well as chlorpro-mazine and atropine. The responses of the adrenoceptive components in the reticular activating system of the brain stem are not identical with those of any other known adrenergic receptors. This observation emphasizes the difficulty in attempts to classify receptors into, a few clearly defined and discrete categories. PUBLICATIONS 1959—Ling, G . M . , and Foulks, J. G . A suitable preparation for pharmacological analysis of E E G "Activation". Proc. Soc. Exp. Biol. & Med. 101, 429 (1959). - i i -A B S T R A C T The p a s t decade has w i t n e s s e d an i n t e n s e i n t e r e s t i n t h e i n f l u e n c e o f drugs and m e t a b o l i c s u b s t a n c e s upon EEG " a c t i v a t i o n " and a r o u s a l mechanisms, m e d i a t e d by the r e t i c u l a r a c t i v a t i n g s y s t e m o f the b r a i n stem. Numerous p h a r m a c o l o g i c a l a g e n t s produce v a r i a t i o n s o f the e l e c t r i c a l a c t i v i t y o f the b r a i n . A n a l y s i s o f t h e i r e f f e c t s has s u g -g e s t e d t h a t t h e r e t i c u l a r a c t i v a t i n g s y s t e m i s an a r e a w h e r e i n numerous drugs may a c t , and p o i n t t o i t s m u l t i n e u r o n a l , p o l y s y n a p t i c c h a r a c t e r as p l a y i n g a m ajor r o l e i n c e n t r a l d r u g a c t i o n . A t e c h n i q u e has been d e v e l o p e d w h i c h l e n d s i t s e l f w e l l t o t h e s t u d y o f the d i r e c t a c t i o n s o f d rugs upon the v a r i o u s components o f the r e t i c u l a r a c t i v a t i n g s y s t e m o f t h e b r a i n stem. The e x p e r i m e n t a l a n a l y s i s o f EEG " a c t i v a t i o n " r e q u i r e s t h e p r e s e n c e o f a w e l l d e a c t i v a t e d background p a t t e r n . O b s e r v a t i o n s made i n the c a t d e m o n s t r a t e t h a t p a r t i a l t r i g e m i n a l e c t o m y , c e r v i c a l d o r s a l e c t o m y and low c e r v i c a l t r a n -s e c t i o n p r o d u c e a p r e p a r a t i o n i n w h i c h the r e s t i n g EEG r e g u l a r l y mani-f e s t s maximal d e a c t i v a t i o n . A permanent c a t h e t e r i n s e r t e d t h rough the r i g h t s u b c l a v i a n a r t e r y and i n t o the i n n o m i n a t e a r t e r y so t h a t i t s t i p i s p o s i t i o n e d a t the o r i g i n o f t h e two c a r o t i d s , has f u r n i s h e d a means f o r s i m u l t a n e o u s b i l a t e r a l d i s t r i b u t i o n o f i n j e c t e d d rugs to the b r a i n w i t h o u t e m b a r r a s s i n g f l o w i n the c a r o t i d a r t e r i e s . The a dvantages o f t h i s t e c h n i q u e i n c l u d e (a) an i n t a c t b r a i n stem, (b) the m a i n t e n a n c e o f - H I -adequate spontaneous r e s p i r a t o r y and c i r c u l a t o r y s t a t e s , and ( c ) the a b i l i t y t o p e r f o r m v a r i o u s o p e r a t i v e p r o c e d u r e s w i t h o u t the n e c e s s i t y f o r e x t r a n e o u s p h a r m a c o l o g i c a l a g e n t s ( a n a e s t h e t i c s , m u s c l e r e l a x a n t s ) , w h i c h may t h e m s e l v e s have c o m p l i c a t i n g e f f e c t s on the EEG. In t h i s p r e p a r a t i o n a d r e n e r g i c and c h o l i n e r g i c a g e n t s as w e l l as h i s t a m i n e and s e r o t o n i n a l l p r o duced prompt, s h o r t - l a s t i n g and r e p r o d u c i b l e EEG " a c t i v a t i o n " i n low doses f o l l o w i n g d i r e c t i n t r a -i n n o m i n a t e a d m i n i s t r a t i o n . In " e q u i - a c t i v a t i n g " d o s e s , i s o p r o t e r e n o l i s the most p o t e n t EEG a c t i v a t i n g c a t e c h o l a d r e n e r g i c amine and n o r -e p i n e p h r i n e the l e a s t p o t e n t , w i t h e p i n e p h r i n e o c c u p y i n g an i n t e r -m e d i a t e p o s i t i o n . Amphetamine and e s e r i n e b o t h produce l o n g - l a s t i n g EEG " a c t i v a t i o n " , w i t h amphetamine h a v i n g a much s h o r t e r l a t e n c y than e s e r i n e . C l e a r d i f f e r e n t i a t i o n between the EEG e f f e c t s r e s u l t i n g from d i r e c t d r u g - i n d u c e d i n f l u e n c e s and t h o s e w h i c h may o c c u r o v e r r e f l e x pathways has been d e m o n s t r a t e d i n p r e p a r a t i o n s w i t h b i l a t e r a l c a r o t i d s i n u s d e n e r v a t i o n . Complete temporal independence i s shown between the o n s e t and t e r m i n a t i o n o f the a c t i o n s o f " a c t i v a t i n g " a g e n t s i n r o d u c e d d i r e c t l y by i n t r a - i n n o m i n a t e a d m i n i s t r a t i o n and the v a s c u l a r e f f e c t s o f t h e s e a g e n t s as r e f l e c t e d i n b l o o d p r e s s u r e a l t e r a t i o n s . P a r t i a l d e s t r u c t i o n i n the tegmentum r o s t r a l to ponto-mesen-c e p h a l i c j u n c t i o n p r o d u ces an i n c r e a s e i n t h r e s h o l d f o r a d r e n e r g i c EEG a c t i v a t i o n . U n i l a t e r a l l e s i o n s w h i c h d e s t r o y most o r a l l o f t h e m esencephalic-tegmentum a b o l i s h a d r e n e r g i c - i n d u c e d a c t i v a t i o n i n the i p s i - l a t e r a l c o r t e x but do not a f f e c t c h o l i n e r g i c a c t i v a t i o n . R e s u l t s o b t a i n e d w i t h v a r i o u s s y n a p t i c b l o c k i n g a g e n t s have - iv -suggested the possible existence in the brain of three types of receptors capable of converging on the final pathway for EEG "act ivat ion"; one responsive to cholinergic compounds and blocked by atropine; one responsive to serotonin and blocked only by chlorpromazine and atropine; and one responsive to histamine and the short-acting adrenergic amines and blocked by phenoxybenzamine as well as chlorpro-mazine and atropine. The responses of the adrenoceptive components in the reticular activating system of the brain stem are not identical with those of any other known adrenergic receptors. This observation emphasizes the d i f f i cu l ty in attempts to classify receptors into a few clearly defined and discrete categories. - V -TABLE OF CONTENTS Page I. INTRODUCTION I II. METHODS 11 A. S u r g i c a l Procedures 12 1. Anaesthetized Preparations 12 (a) B a r b i t u r a t e s 12 (b) I n h a l a t i o n anaesthetics ( e t h e r ) . . 12 2. The Unanaesthetized C u r a r i z e d Prepara-t i o n v 13 3. Encephale and Cerveau I s o l e Prepara-t i o n 14 4. Mid-Brain Coagulated P r e p a r a t i o n . . . 14 5. Unanaesthetized De-afferented Prepara-t i o n 16 (a) Low t r a n s e c t i o n of the c e r v i c a l s p i n a l cord and c e r v i c a l sensory roots 16 (b) E x t r a - c r a n i a l t r a n s e c t i o n of tr i g e m i n a l roots 17 6. Intra-lnnominate Cannulation f o r D i r e c t C e n t r a l Drug E f f e c t s 22 B. Experimental Procedures 23 III. RESULTS 27 A. Bas i c EEG P a t t e r n 27 B. P r e p a r a t i o n With Intact B r a i n Stem and I n t a c t C a r o t i d Sinuses 29 1. Con t r o l s 29 2. Adrenergic A c t i v a t i o n 29 (a) E f f e c t s of epinephrine, i s o p r o t e r -e n o l , norepinephrine and racemic-amphetamine 29 (b) E f f e c t s of some isomers of i s o -proterenol administered separately and i n combination 41 ( i ) Isomers of i s o p r o t e r e n o l administered s e p a r a t e l y . . . 42 ( i i ) Isomers administered i n com-b i n a t i o n 42 3. C h o l i n e r g i c A c t i v a t i o n 45 (a) E f f e c t s of a c e t y l c h o l i n e and e s e r i n e (physosti gmine) 45 - vi -' J TABLE OF CONTENTS (cont'd.) III. RESULTS (cont'd) Page 4 . Activating Effects of Other Agents . . . 52 (a) Vasopressin and histamine 52 (i) Vasopressin 5*+ ( i i ) Histamine 54 (b) Serotonin 56 5. Effects of Blocking Agents 60 (a) Phenothiazine derivatives (chlorpro-mazine and promazine 60 (i) Chlorpromazine 60 ( i i ) Promazine 76 (b) Phenoxybenzamine (dibenzyline). . . 77 (i) Adrenergic amines: isoproter-enol, epinephrine, and nor-epinephrine 79 ( i i ) Histamine 85 ( i i i ) Acetylcholine . . . . . . . . 85 (iv) Serotonin 88 (c) Dichloroisoproterenol (DC 1) . . . . 94 (i) Isoproterenol 96 ( i i ) Epinephrine and norepineph-rine 96 ( i i i ) Acetylcholine and serotonin . 100 (d) Atropine 109 C. Preparation With Bilateral Carotid Sinus Denervation 110 1. Controls 110 2. Effects of Pressor Agents: Epinephrine, Norepinephrine and serotonin 117 3 . Effects of Depressor Agents: Acetylchol-ine and isoproterenol . . 117 D. Preparation With Brain Stem Lesions 124 1. Effects of Pontile Lesions on the EEG of the Unanaesthetized De-afferented Cat... 124 2. Effects of Unilateral Ponto-Mesencephalic Lesions on EEG "Activation" (Adrenergic and Cholinergic) 128 IV. DISCUSSION 139 V. SUMMARY AND CONCLUSIONS 166 VI . BIBLIOGRAPHY 169 - v i i — LIST OF FIGURES Figure Page 1 E f f e c t of audiogenic a c t i v a t i o n upon the EEG 20 2 E f f e c t on the EEG of feeding v i a stomach tube 21 3 E f f e c t of intra- innominate s a l i n e on the EEG 30 4 E f f e c t of epinephrine 0.5/ug/kg on the EEG 32 5 E f f e c t of norepinephrine 0 . 5 /ug/kg on the EEG 33 6 E f f e c t of i soproterenol 0 . 2 5/Jg / k g on the EEG 3** 7 E f f e c t of amphetamine 50 /Ug/kg on the EEG 35 8 E f f e c t of epinephrine 0 .25/Ug /kg on the EEG 36 3 E f f e c t of epinephrine 2 . 0 / j g /kg on the EEG 37 10 E f f e c t of norepinephrine 0 .5 yug/kg on the EEG 38 11 E f f e c t of norepinephrine 2 . 0 / j g /kg on the EEG 39 12 E f f e c t of i soproterenol 0 .5 /Ug /kg on the EEG 40 13 E f f e c t of d- and I - i soproterenol on the EEG 43 14 E f f e c t of a combination of d- and 1 - i soproterenol 2.0/ug/kg each on the EEG 2 . 0 / j g /kg each on the EEG . . . 44 15 E f f e c t of a c e t y l c h o l i n e 0 .25 /ug /kg on the EEG 46 16 E f f e c t of a ce ty l cho l i ne 0 .5 Xig/kg on the EEG 47 17 E f f e c t of a ce ty l cho l i ne l . 0 ^ g / k g on the EEG 48 18 E f f e c t of a ce ty l cho l i ne 2 .0 /Ug/kg on the EEG 49 19 E f f e c t of eser ine 50/ug/kg on the EEG 50 20 E f f e c t of eser ine 100/Ug/kg 51 21 E f f e c t of chlorpromazine and atropine on eser ine- induced a c t i v a t i o n 53 - v i i i -LIST OF FIGURES (cont'd.) Figure Page 22 E f f e c t of vasopressin 0.5 u n i t s / k g on the EEG 55 23 -Effect of histamine 1.0^g/kg on the EEG 57 24 E f f e c t of histamine 2.0/jg/kg on the EEG 58 25 E f f e c t of histamine 5.0/ig/kg on the EEG 59 26 E f f e c t of se r o t o n i n 5 /jg/kg on the EEG 61 27 E f f e c t of se r o t o n i n 20 /Ug/kg on the EEG 62 28 E f f e c t of CPZ 2.0 mg/kg on the EEG 64 29 E f f e c t of epinephrine 0.5 /ig/kg on the EEG a f t e r CPZ blockade 66 30 E f f e c t of norepinephrine 2.0 Aig/kg on the EEG i n the presence of CPZ blockade 67 31 E f f e c t of s e r o t o n i n 5.0/jg/kg on the EEG In the presence of CPZ blockade 68 32 E f f e c t of histamine 5.0 /ug/kg on the EEG i n the presence of CPZ blockade 69 33 E f f e c t of is o p r o t e r e n o l 0.5Aig/kg on the EEG i n the presence of CPZ blockade 72 34 E f f e c t of iso p r o t e r e n o l 0.5/jg/kg on the EEG i n the presence of CPZ blockade and vasopressin 73 35 E f f e c t of amphetamine on the EEG i n the presence of CPZ blockade 74 36 E f f e c t of phenoxybenzamine (DBZ) 1.0 mg/kg on the EEG . . 78 37 E f f e c t of i s o p r o t e r e n o l 0.5 /ig/kg on the EEG before phenoxybenzamine 80 30 E f f e c t of is o p r o t e r e n o l 0.5/ug/kg on the EEG a f t e r phenoxybenzamine . 81 39 E f f e c t of is o p r o t e r e n o l l.Oyug/kg on the EEG a f t e r phenoxybenzamine 82 - i x -LIST OF FIGURES (cont'd) Figure Page 40 E f f e c t of epinephrine 0.5 >ug/kg on the EEG before phenoxybenzamine 83 41 E f f e c t of epinephrine 0.5 /ig/kg on the EEG a f t e r phenoxybenzamine 84 42 E f f e c t of histamine 2.0 /ug/kg on the EEG before phenoxy-benzami ne 86 43 E f f e c t of histamine 2.0/jg/kg on the EEG a f t e r phenoxy-benzamine 87 44 E f f e c t of a c e t y l c h o l i n e 2.0 yug/kg on the EEG a f t e r phenoxybenzamine 89 45 E f f e c t of s e r o t o n i n 2.5/Jg/kg on the EEG before phenoxy-benzamine . 90 46 E f f e c t of s e r o t o n i n 2.5 ;ug/kg on the EEG a f t e r phenoxy-benzamine 91 47 E f f e c t of s e r o t o n i n 5.0 /jg/kg on the EEG before phenoxy-benzamine 92 48 E f f e c t of s e r o t o n i n 5.0/Ug/kg on the EEG a f t e r phenoxy-benzamine . . . . . . . . 93 49 E f f e c t of DC I 15 mg/kg on the EEG 95 50 E f f e c t of i s o p r o t e r e n o l 0.5A»g/kg on the EEG a f t e r DCI 7.5 mg/kg 97 51 E f f e c t of i s o p r o t e r e n o l 1 .0/jg/kg on the EEG a f t e r DCI 7.5 mg/kg 98 52 E f f e c t of i s o p r o t e r e n o l 1.0/ug/kg on the EEG a f t e r DCI 15 mg/kg 99 53 E f f e c t of epinephrine 0 .5 /Ug/kg on the EEG a f t e r DCI . . 101 54 E f f e c t of norepinephrine 0 . 5 ytig/kg on the EEG a f t e r DCI . 102 55 E f f e c t of epinephrine 2 . 0 / j g / k g on the EEG a f t e r DCI . . 103 56 E f f e c t of norepinephrine 2 .0 /Ug/kg on the EEG a f t e r DCI . 104 57 E f f e c t of a c e t y l c h o l i n e 2 . 0 / u g / k g on the EEG a f t e r DCI . 1 0 5 - x -LIST OF FIGURES (cont'd) Figure Page 58 Effect of acetylcholine 2 .0 /Ug/kg on the EEG after DCI. . 106 59 Effect of serotonin 5 .0 /Ug/kg on the EEG before DCI . . . 107 60 Effect of serotonin 5/Ug/kg on the EEG after DCI . . . . 108 61 Effect of epinephrine 0 , 5/jg / k g and acetylcholine on the EEG after atropine I l l 62 Effect of isoproterenol 2 .0 /Ug/kg and serotonin 5 .0/Ug/kg on the EEG after atropine . , 112 63 Effect of epinephrine 2 .0 /Ug/kg and norepinephrine 2 . 0 /Ug/kg on the EEG after atropine 113 64 Effect of atropine 0 .5 mg/kg on the EEG after eserine . . 114 65 Effect of control injections of saline on the EEG after bi lateral carotid sinus denervation (C.S.D.) 116 66 Effect of epinephrine 0.5/Ug/kg on the EEG after bi lateral C.S.D 118 67 Effect of serotonin 5 .0 /Ug/kg on the EEG after bi lateraj C.S.D 119 68 Effect of acetylacholine 0 .5/Ug/kg on the EEG after bi lateral C.S.D 121 69 Effect of isoproterenol 0 . 5/jg / k g followed by norepineph-rine 0.5/ug/kg after bi lateral C.S.D 122 70 Effect of isoproterenol 0 .5 /Jg/kg in the presence of phenoxybenzamine after C.S.D 123 71 Effect on the EEG of unilateral lesions in the caudal part of the right mesencephalic tegmentum (R.M.T.) . . . 129 72 Effect of epinephrine 2 .0 /Ug/kg on the EEG in the presence of unilateral lesions in the caudal portion of R.M.T. . . 130 73 Effect of acetylcholine 2 .0 pg/kg on the EEG in the presence of unilateral lesions in the caudal portion of the R.M.T. 131 i - x i -LIST OF FIGURES ( c o n t ' d ) F i g u r e Page 7k E f f e c t on the EEG o f f u r t h e r l e s i o n s i n the R.M.T. . . . 133 75 E f f e c t o f e p i n e p h r i n e 2 . 0/Ug/kg on the EEG In the p r e s e n c e o f e x t e n s i v e u n i l a t e r a l l e s i o n s i n the r i g h t v e n t r o - l a t e r -a i p o r t i o n o f the R.M.T 13^ 76 E f f e c t o f i s o p r o t e r e n o l 2 , 0 / j g / k g on the EEG i n t h e p r e s -ence o f u n i l a t e r a l l e s i o n s i n the r i g h t v e n t r o - l a t e r a l p o r t i o n s o f the R.M.T 135 77 E f f e c t o f e p i n e p h r i n e 2 .0 /jg/kg on the EEG a f t e r u n i -l a t e r a l l e s i o n s e x t e n d i n g i n t o the r i g h t d o r s o - l a t e r a l p o r t i o n o f the R.M.T 136 73 E f f e c t o f i s o p r o t e r e n o l 2 .0 /Ug/kg on the EEG a f t e r u n i -l a t e r a l l e s i o n s e x t e n d i n g i n t o the r i g h t d o r s o - l a t e r a l p o r t i o n o f the R.M.T 137 79 E f f e c t o f a c e t y l c h o l i n e 2 . 0 /Og/kg on the EEG a f t e r e x t e n -s i v e l e s i o n s i n the r i g h t d o r s o - l a t e r a l p o r t i o n o f the R.M.T 138 - x i i -LIST OF PLATES P l a t e Page A R e t i c u l a r f o r m a t i o n o f c a t b r a i n k B C r o s s s e c t i o n s o f c o a g u l a t i o n l e s i o n s i n the p o n t o -m e s e n c e p h a l i c a r e a s o f c a t b r a i n . . . . 127 C S c h e m a t i c s a g i t t a l s e c t i o n o f c a t b r a i n stem w i t h s t e r e o -t a x i c c o o r d i n a t e s showing a r e a s o f c o a g u l a t i o n . . i . 126 D P h o t o g r a p h o f the c e r e b e l l a r a p p roach f o r the p r o d u c t i o n o f e l e c t r o l y t i c l e s i o n s i n the p o n t o - m e s e n c e p h a l i c j u n c t i o n w i t h g l a s s i n s u l a t e d e l e c t r o d e . . . . . . . 125 E E f f e c t o f b l o c k i n g a g e n t s on EEG " a c t i v a t i o n " by some a d r e n e r g i c and c h o l i n e r g i c a g e n t s , and by h i s t a m i n e and s e r o t o n i n 159 - x i t i -ACKNOWLEDGEMENTS The writer wishes to express his deep gratitude to Dr. James G. Foulks for his encouragement, expert advice, and stimulus during the preparation of this thesis. He also wishes to express his appreciation to Dr. E.E. Daniel, Dr. G.I. Drummond and to other members of the Department of Pharmacology for advice and helpful cr i t ic i sm during the course of this investigation; to Dr. W.C,Gibson, Dr. J . Wada and Dr. P.L. McGeer of the Department of Neurological Research, and to Dr. H. Scherrer of the Department of Anatomy, who have a l l offered valuable suggestions and have provided stimulating discussions of the problems treated here. The writer also wishes to express his appreciation to Miss S. Calthrop for the expert typing of the thesis, and to Miss M. Nagai and Mrs. P. Hansen for their rel iable assistance during surgical procedures. - 1 -I. INTRODUCTION The f i rst record of the human electroencephalogram was made by Hans Berger (15) In 1929. Shortly after came the observation that during sleep the pattern of the EEG tended to be composed of large, slow wave-like fluctuations of potential, while in contrast a fast frequency low voltage record was characteristic of the wakeful state. Eight years later in 1937, Rhefnberger and Jasper (143) employing the cat as subject, undertook the f i rst combined EEG and behavioural study of wakefulness evoked by afferent stimulation. They showed that several different types of afferent stimulation could produce arousal and low voltage activity in the EEG. They also observed that this activity was not confined to a specific receiving sensory area, but was spread diffusely over the entire mantle. This low voltage fast activity they called EEG "activation" - a state to which the term "desynchronlzation" also has been applied. These investigators demonstrated that this "activation pattern" had a tendency to persist for periods longer than the brief duration of the arousing stimulus. Numerous subsequent studies have confirmed these findings. When the EEG of a normal cat Is followed from wakefulness to sleep, the low voltage fast activity characteristic of the alert and waking state gives way to a slower and more wave-like discharge during drowsiness, and subsequently, when the animal is undoubtedly asleep, large slow wave and - 2 -spindle bursts are characteristic of the recording. If the sleeping animal Is suddenly awakened by an afferent stimulus, such as a "whistle-i blast" or "hand-clap", a return to low voltage fast EEG activity takes place, and, in addition, coincident motor activity (opening of the eyes and movements of the head) suggests that this electro-cortical change is associated with behavioural alertness (113). The possible relationship of the phenomena of EEG "activation" and behavioural arousal following sensory stimulation has continued to attract attention. This transition from sleep to wakefulness, or from the less extreme states of relaxation and drowsiness to alertness and attention, has been attributed to bombardment of the cortex by asynchronous afferent volleys from peripheral receptors. In immobilized animals EEG "activation" has been induced by stimulation of peripheral nerves (58,165), auditory receptors (58,67,165), the olfactory system (28,38,67), sympathetic nerves (58), and the vagi (l8fc). Comparable effects have also been reported following stimulation of active cortical loci (30,55,155), the fastigial nuclei (123), and the caudate nucleus (158). Evidence (123) has pointed to the presence In the mid-brain stem of a system of reticular synaptic relays in the ascending pathway leading to EEG activation and behavioural arousal. Direct stimulation of this mid-brain structure exerts a general effect on the cortex which is mediated in part by the diffuse thalamic projection system, f irst described by Dempsey and Mori son (41,42), and later in more detail by Jasper (87,88,89), and by Jasper and his collaborators (73,90,92), and by other investigators (6,164,166). The central area In the brain stem which is intimately associated with the arousal mechanism, and which evidence indicates may account for electro-cortical and behavioural features characteristic of the waking, - 3 -and/or the alert state, is known as the reticular activating system (area R.F. in Plate A), and includes the reticular formation of the oblongata and pontine tegmentum, extending from bulbar to mid-brain regions. As shown in Plate A, impulses which follow the classical afferent pathways from peripheral sources f i r s t synapse in the spinal cord or dorsal column nucleus, then enter the medial lemniscus in the lateral brain stem, and synapse once again in one of the relay nuclei of the thalamus, before f i na l l y passing by way of the internal capsule to specif ic cort ical areas. In contrast, a secondary extra-lemnlscal ascending pathway receives impulses via col lateral fibres (57) passing from the lemniscus Into the central brain stem, when impulses are dispatched rostral ly to cause wide-spread and generalized EEG "act ivat ion" . The mid-brain ret icular formation also receives projections from several areas of the cerebellar-hemispheres (122,161,162,171), as well.as from certain discrete areas of the cerebral cortex. The places of origin of this cort icifugal projection as found in the monkey, are the frontal eye f ie lds , the sensory motor cortex, the para-occipital cortex, the f i r s t temporal gyrus, the orbital surface of the frontal lobe, the cingulate gyrus, the tip of the temporal lobe and the f i r s t temporal gyrus. Each of these cort ical f ie lds appears to project down Into the reticular formation to very much the same brain stem areas which receive col laterals from a l l the afferent systems of the body. Impulses from many of the active cortical loci described were found to funnel Into a common pathway which in turn made connections with the ret icular activating system (46,71). Alternatively other cort ical loci were demonstrated to exhibit other connecting pathways; for example, neurones from the central and premotor gyrus were found to accompany the pyramidal tract to bulbar levels where they entered the - k -Reticular Formation of the Brain TH: thalamus F I G U R E 7. Diagram of cat brain shows cortical connections (anterior dotted arrow) capable of influencing reticular formation (RF). The dark arrows in-dicate inhibitory influences which the brain stem exerts upon sensory conduc-tion within the brain. The hatched arrow indicates facilitatory or inhibitor) influences known to be exerted upon the nervous system by the same structure. (From Hernandez-Peon.-") PLATE A - 5 -reticular activating system, whereas impulses from the hippocampus and the entorhinal cortex made connections with the reticular brain stem via the s t r i a medullaris (1,3,66). Livingston (108), as well as Hernandez-Peon et al (76), have shown that different modalities of afferent sensory input w i l l interact in the reticular formation with each other, and with each of the projections des-cending from any one of the cortical areas which project into the reticular formation. Thus, i t seems that the reticular formation constitutes a sort of central integrating switch-board for the interaction of impulses generated in remote and varied parts of the nervous system. In addition, not only is there interaction between descending projections from the cortex with sensory input, but each of the cortical f ie lds interacts with each other, and appears to have a rather special pattern of influence within the brain stem reticular formation (2). For example, some of these f ie lds w i l l augment in t r ins ic ac t iv i ty of the brain stem while others w i l l diminish i t , giving rise to a complex sequence of alternating excitement and depression - a sequence •which may last from several tenths of a second to several seconds. Thus, i t seems that the cortex is not simply the victim of demands made on i t by the reticular activating system, but that the cortex i t se l f possesses cort icifugal regulating mechanisms which in turn can influence the level of act ivi ty within the reticular formation (108). This level of a c t i v i t y , which reflects Input and output relations, taken together with the brain stem's own contributions to these relations, is important to the total organization of behaviour (2,128). Using implanted electrodes In unanaesthetized cats, Hernandez-Peon, Scherrer and Jouvet (76), and others (75,83) have shown that potentials evoked in the specif ic sensory areas of the cortex are depressed during attentive behaviour, and the suggestion has - 6 -been raised that reticular discharges may modify or inhibit transmission in the f i r s t synapse of the pathways involved ( 7 6 ) . Similar results have been reported recently by Hugelin et al ( 8 5 ) . In contrast these latter Investiga-tors relate the reduction of the cochlear response to the f ac i l i t a t i on of contraction of tympanic muscles; this, they state, reduces the pressure transmitted to the cochlea and attenuates cochlear potentials. They therefore consider the reticular effects on auditory Input to be the result of an infralamlnal reflex f ac i l i t a t i on belonging to a generalized motor reaction ( 8 5 ) . That these dynamic alterations along the sensory pathways are related to act iv i ty in the brain stem reticular formation is made clear by the effects of brain stem destruction, since, In fact, neither the phenomena of habituation nor focus of attention, nor of conditioning, appear to survive after brain stem lesions ( 5 1 , 5 4 , 5 5 ) • "The influences of metabolic substances, hormonal agents, drugs and circulat ing hormones upon arousal mechanisms mediated by the reticular system have attracted much attention recently" ( 5 4 ) . Numerous pharmacological agents produce variations of the e lectr ica l act iv i ty of the brain. Analysis of their effects has suggested that the reticular activating system is an area wherein numerous drugs may act, and point to its multineuronal poly-synaptic character as playing a major role In central drug action. Bremer ( 2 9 ) , Forbes and associates ( 5 2 ) , and Barany (11) have emphasized the susceptibi l i ty of complex neurone systems to anaesthesia. Larraboe and Posternack (103) have shown that the blocking effect of central anaesthetics on peripheral nerve f ibre conduction proceeds without reference to f ibre diameter or velocity of f ibre conduction, and that synaptic transmission is a l imiting factor, which can be blocked long before f ibre conduction is affected. French et al (57) have observed a d i f ferent ia l block of ascending conduction i n the r e t i c u l a r a c t i v a t i n g system upon a d m i n i s t r a t i o n of p e n t o b a r b i t a l or ether, and have proposed that t h i s may be of importance i n the production of the a n a e s t h e t i c s t a t e ; B r a z i e r (25,26) has discussed the e f f e c t s of th i o p e n t a l on the evoked c o r t i c a l p o t e n t i a l and on the EEG, and s i m i l a r s t u d i e s have been reported f o r c h l o r a l o s o n e by Munroe e t a l (124). In a d d i t i o n , King e t a l (98). u s i n g low and high concentrations of b a r b i t u r a t e s , have confirmed the f u n c t i o n a l block of ascending i n f l u e n c e s on the b r a i n stem r e t i c u l a r formation i n response to low concentrations and, moreover^ have demonstrated a depressant e f f e c t of l a r g e r concentrations d i r e c t l y upon the thalamic r e l a y n u c l e i . Conversely, i t has been demonstrated that conduction i n the b r a i n stem r e t i c u l a r formation can be markedly enhanced by s t r y c h n i n e and metrazol (3,51.137.138), and EEG arousal can be e l i c i t e d by such d i v e r s e agents as cocaine, methamphetamine and phenylephrine (150. p i p r a d r o l , amphetamine, e s e r i n e , mescaline, l y s e r g i c a c i d d i e t h y l a m i d e , phenidylate (21,86), 2-dimethy 1-amino-ethanol (130. ©(-methyl tryptarnine (168), tremorine (10) and apomorphine (43). Of p a r t i c u l a r i n t e r e s t i s the p o s s i b l e r e l a t i o n s h i p of drug-induced changes i n the s t a t e o f EEG " a c t i v a t i o n " to the important c l i n i c a l e f f e c t s on the mood and behaviour o f n e u r o t i c and p s y c h o t i c i, p a t i e n t s d i s p l a y e d by some of these compounds. S Several of the drugs which e l i c i t EEG " a c t i v a t i o n " (e$. amphetamine and eserine) resemble compounds which are known to serve as p e r i p h e r a l t r a n s m i t t e r s . Furthermore, drugs which produce EEG d e a c t i v a t i o n include agents which produce b l o c k i n g e f f e c t s a t various p e r i p h e r a l s y n a p t i c s i t e s (chlorpromazlne, a t r o p i n e ) . These observations r a i s e the question as to whether the mechanism of a c t i o n of these drugs a t c e n t r a l s i t e s may not be analogous to that which they e x e r t a t p e r i p h e r a l synapses. Increasing evidence continues to lend support to the hypothesis that synapses in the central nervous system are chemically mediated. The chemical transmitters of synapses in the peripheral nervous system have been identif ied and characterized in considerable deta i l . However, less definite information is available concerning the identity, the possible role, the l ikely sites and the presumed mechanisms of action of synaptic mediators in the central nervous system. Insofar as is possible, any candidate considered for the role of mediator of a synaptic s i te within the CNS should f u l f i l l the c r i te r i a which have been required to establish the role of acetylcholine and norepinephrine as transmitters of peripheral synapses. Thus, not only must the presence of the candidate be demonstrated in the CNS, but its concentration should be greatest in the locale of its proposed function, and its release follow-ing act iv i ty should be demonstrable at the specified synaptic s i te . Adequate concentrations of enzymes necessary for Its synthesis and its in-activation must exist in that locale. Demonstrable central effects should be reproduced consistently by the direct administration of the l ikely candidate to the proposed s i te , and i t should be possible to duplicate these effects with the use of agents known to exert an inhibitory effect on the In this regard the complex structural organization of the central nervous system imposes obstacles to the rel iable col lection and ident i f ica-tion of substances liberated at central neurone terminals following either autochthonous discharges or exogenous stimulation. However, in some areas of the brain, increased act iv i ty (electr ical stimulation) as well as certain drug-induced effects (metrazol, ethanol, benzoquinolizlne) have been demon-strated to result in the depletion of the content of certain compounds considered as candidates for the role of synaptic mediator. In addition, similar phenomena have been observed to be associated with the administration of effective doses of certain psychotropic agents (reserplne, desmethoxy-reserpine, tetrabenazine). - 3 -deactivating enzyme. A number of workers have .considered the possibility that serotonin (16,32,33,60,100,115,129,160,169), norepinephrine (33,160, 169,170,179) and acetylcholine (47,74) might play the role of a neuro-hormone at various sites in the central nervous system, since each has been shown to meet several of these criteria. Insofar as the reticular activating system is concerned several groups of investigators have shown that the intra-arterlal administration of cholinergic compounds elicits EEG "activation" (22,111,144) and some have contended that the "mesodiencephal?c activating system" is exclusively cholinergic in nature (144). Other studies have demonstrated EEG "activation" following intravenous administration of epinephrine and have suggested that the "reticular activat-ing system" or a component within it is adrenergic (17,149,151). Although the observed EEG changes may well be the result of direct drug effects, concomitant vascular responses have left open the possibility that the observed changes in the electrical activity of the brain may be a consequence of baroreceptor reflexes; One of the objectives of the present investigation was to confirm the existence of both cholinoceptive and adrenoceptive components in the reticular formation of the brain stem and to attempt to evaluate the possible activating capacity of other postulated neurohumours (serotonin, histamine). A second objective was to try to dissociate vascular and electroencephalo-graphic pharmacodynamic effects and to evaluate the possible role of vascular reflexes in eliciting drug-Induced activation. If EEG "activation" should be produced by different classes of compounds, then the thi rd objective would present itself, namely to attempt to characterize further the nature of the respective receptive synapses and to clarify thei r functional - 10 -sequence through the use of blocking agents. It was evident s£ the outset that in order to achieve these objectives it would be necessary to devise a preparation with a functionally Intact brain stem which would be free from extraneous pharmacological influences. The hope was also entertained that such a preparation would be sensitive to the effects of close intra-arterial injection of compounds in quantities reasonably near those that may be presumed to be physiological. - 11 I I. METHODS A. Surgical Procedures. Of fundamental importance to the analysis of drug-induced EEG "act ivat ion" is the establishment of stable and reproducible conditions under which clear-cut and consistent drug effects can be obtained. The basic requirement is that the experimental animal be placed under controlled conditions in which the nervous system can be manipulated in whole or in part in such a way that signs of functional act iv ity in a chosen area may be recorded and measured (95)• The available data bearing on the effects of drugs on EEG "act ivat ion" have exhibited several discrepancies, some of which may be due in part to species variation (some investigators using cats, other rabbits). In addition, there has been wide variation in the type of experimental preparation used to provide a "deactivated" EEG pattern as a baseline for drug-induced "act ivation" studies. A consistent, reliable and uniform baseline seems essential for the analysis of unmistakable "act ivat ion" effects. Rothballer (149) has stated the problem succinctly: "Whether or not any EEG changes could be detected after intravenous adrenaline depends primarily upon the background of act iv i ty , and to a much lesser extent on the dosage. Working against a background of "arousal", i t was quite d i f f i cu l t to detect any effect at a l l . " Personal experience has substant-iated this view, and has revealed certain disadvantages of some preparations - 12 -commonly used in such investigations, and initially utilized during the early stages of this study. These will be outlined in the following section?, 1• Anaesthetized Preparations. (a) Barbi turates. The administration of adequate doses of barbiturates produces a stable "sleeping" EEG pattern. However, this use o f barbiturates to insure a deactivated EEG background upon which drug-indued EEG "activation" may be observed leaves much to be desired. There is general agreement th?t in all species studied, barbiturates decrease the sensitivity of the reticular system and inhibit the arousal syndrome (39,55)-In rabbits the arousal reaction Is markedly reduced after 2 mg/kg o f sodium pentobarbital, and after 10 mg/kg (about one-third of the anaesthetic dose) an activated pattern can no longer be obtained (8). Similar results have been observed for cats, in which the EEG effects of mid-brain reticular excitation are abolished by 8-10 mg/kg (43,97.141). The use of such terminology as "light anaesthesia" or "during recovery from anaesthesia" usually implies that most of these effects of the barbiturate are not operative; certainly, recent evidence tends to show that such is not the case. For example, rhinencephalic seizure responses to stimulation of the limbic system can be eliminated, and thresholds can be raised more than ten-fold by doses of barbiturates below those producing measurable depression of behaviour and much less than those inducing the loss of motor and sensory activity necessary for experimental procedures (95). (b) Inhalation anaesthetics (ether). Inhalation anaesthetics (of which ether is the one most commonly used for animal surgery), share with the barbiturates, the disadvantages of blocking ascending conduction in the reticular activating system and inhibiting EEG arousal (57). In addition, - 13 -ether anaesthesia provokes massive sympathetic discharge (72,93.135.174), which not only alters and modifies the background EEG act iv i ty , but also complicates the effects observed following the administration of other sympathomimetic amines. 2. The Unanaesthetized Curarized Preparation. In the unanaesthetized curarized preparations, there is as yet no general agreement that the muscle relaxants (d-tubocurarine, gallamine tr i -ethiodide, decamethoniurn or succinylcholine) are without central effects (49,69). The most obvious influence of these agents under these conditions is the loss of respiratory act iv i ty . This makes a r t i f i c i a l venti lation necessary and raises the problem of adequate levels of oxygenation without hypo- or hyper-capnia, since changes of blood pH may markedly a lter the act iv i ty of the CNS (95). Furthermore, the relative potency of the curarizing compounds varies markedly from animal to animal, as does the duration of any single dose so that maintaining the animal at fu l l respiratory paralysis, once a r t i f i c i a l respiration Is properly adjusted, becomes an additional problem. Another str iking effect of curarizing agents, particularly of d-tubocurarine is a marked f a l l In blood pressure to shock levels if adequate care in timing the injection Is not observed. In the cat, for example, a f u l l paralyzing dose of d-tubocurarlne (1 mg/kg, I.v.) must be given over a period of 6 to 8 minutes to prevent severe hypotension, which may be further aggravated by histamine release. In addition, external stimulation ( l ight, noise, handling) must be reduced to minimum levels and a rel iable deactivated background is d i f f i c u l t to reproduce consistently. - 14 -3• Encephate and Cerveau I s o l e P r e p a r a t i o n s . The ericephale i s o l e c a t i s a pr e p a r a t i o n i n which the s p i n a l cord i s severed a t the l e v e l of the f i r s t o r second c e r v i c a l vertebrae (27 . 3 0 ) . Here a r t i f i c i a l r e s p i r a t i o n , w i t h i t s attendant problems of adequate oxygena-t i o n , must a l s o be employed. Furthermore, t h i s p r e p a r a t i o n r a r e l y develops a s u f f i c i e n t l y d e a c t i v a t e d background f o r the r e l i a b l e demonstration of drug-induced " a c t i v a t i o n " . These d i f f i c u l t i e s can be l a r g e l y overcome w i t h the cerveau i s o l e (mid-brain t r a n s e c t i o n from the s u p e r i o r c o l l i cuius to the mammillary body), which o f f e r s s t a b l e synchronized patterns of 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 (23,27,29). However, 1 ike other preparations i n which the cranium i s w i d e l y opened, cereb r a l oedema u s u a l l y develops, d e s p i t e the a p p l i c a t i o n of temporary manual compression a t the base of the s k u l l a t the moment of t r a n s e c t i o n (19,104); Moreover, i n a large percentage of cases, i n c r e a s i n g i n s t a b i l i t y of the blood pressure w i t h time and experimental usage (3-5 hours) tended to make t h i s p r e p a r a t i o n u n s u i t a b l e f o r prolonged use. A recent report by Batset (12) Indicates that b e t t e r experimental s t a b i l i t y can be achieved w i t h the cerveau i s o l e p r e p a r a t i o n i n the dog, but s i n c e no blood pressure readings were reported i n t h i s study the question of the adequacy of the c a r d i o v a s c u l a r status remains i n doubt. k. Mid-Brain Coagulated P r e p a r a t i o n . This procedure followed that o r i g i n a l l y developed by Naquet (126) and s t u d i e d by R o t h b a l l e r (149). Anaesthetized cats were placed i n a Johnson S t e r e o t a x i c Instrument Model No. 2)0, which was c a l i b r a t e d to conform to coorindates of "A S t e r e o t a x i c A t l a s of the Diencephalon of the Cat" by H. Jasper and Cosimo Ajmone-Marsan (91). The muscles o v e r l y i n g the s k u l l were r e f l e c t e d from the mi d - s a g g i t a l l i n e . B i l a t e r a l p o ints of entry i n t o - 15 -the bra in were es tab l i shed by the s te reotax i c c a r r i e r from the p rec i se working coordinates which were to be l a t e r used in coagulat ing the mid-bra in r e t i c u l a r substances. Trephined holes were caut ious ly and c a r e f u l l y made to reduce haemorrhage and leave the dura i n t a c t . Some small degree of b leeding does occur (greater with ether than with thiopental in the majority of cases ) , but hemostasis can read i l y be produced by the use of bone wax. Next, the dura was l i f t e d c a r e f u l l y by means of a dural hook, and inc i sed at the point of e lec t rode entry with a sharp ganglion k n i f e . E lectrodes mounted on a s te reo tax i c c a r r i e r were then introduced, using working coordinates s p e c i f i c f o r mid-bra in r e t i c u l a r coagu lat ion. F i r s t one s ide was coagulated. The e lectrodes were removed and cleaned and next inserted into the con t ra l a te ra l s ide f o r coagulat ion of the homologous area. The current necessary f o r coagulat ion was obtained from a high frequency source, using a Heathkit Amateur Transmitter Model DX-20. The output of this instrument was attenuated by means of a v a r i ab l e output c o n t r o l , con s i s t i n g of a se r ie s lamp and potentiometer, c a l i b r a t e d f o r d i f f e r e n t types of e l ec t rodes , and f o r various spacing of e lec t rodes . This c a l i b r a t i o n was achieved by observing the time necessary to produce a d e f i n i t e amount of coagulat ion of egg albumin at a temperature of 20° C. A se r ie s of inves t i ga t ions with the o s c i l l a t o r set at 3.5 megacycles have shown that th i s instrument, with i t s adjustab le output c o n t r o l , is s a t i s f a c t o r y f o r producing coagu lat ive les ions in the r e t i c u l a r formation of the mid-bra in tegmentum, This technique produces a preparat ion which exh ib i t s a good " deac t i v a ted " EEG pat tern as wel l as a r e l i a b l e card iovascu lar s ta tus , but here again the bra in stem is not i n tac t and coagulat ive les ions may e l iminate - 16 -important sites of drug-induced effects. In addition, residual effects of anaesthesia are difficult to exclude in such acute preparations. 5. Unahaesthe t i zed De-af ferehted P reparat i on. It has been demonstrated by Roger et al (147) that a deactivated pattern follows intracranial destruction of the trigeminal nerves in the encephale isole cat. Electrolytic transection of the mid-brain at the rostro-pohtine level (rostral to the trigeminal roots) produces a similar result ( 1 3 ) . Following this lead, a major objective in the present investigation has been to produce a sufficient degree of somatic de-afferentation so that it becomes possible to elicit a consistently high degree of EEG "deactivation" in an unanaesthetized preparation with intact brain stem without the aid of extraneous pharmacological agents. Such a preparation should also exhibit good cardiovascular status and should not require artificial respiration. A preparation which meets the above desiderata can be accomplished by low transection Of the cervical spinal cord and division of the sensory roots of the cervical spinal nerves, followed by extra-cranial transection of the roots of the maxillary and mandibular branches of the trigeminal nerves ( 107) . (a) Low transection of the cervical spinal cord and cervical sensory roots. The surgical technique for cord transection is essentially similar to that described by Bremer (27). but with the following elaboration. The dorso-lateral surface of the atlas and the axis, and the spinous processes and dorsal vertebral arches of to Tj ere exposed. The atlantal foramina and the intervertebral foramina between the axis and the atlas are located bilaterally and the dorsal - 17 -roots of Cj and C 2 cautiously sectioned to avoid bleeding from accompanying blood vessels. The dorsal' arches and spinous processes of the remaining cervical vertebrae as well as those of the f i r s t thoracic vertebrae are removed and their cut ends packed with s ter i lo bone wax. With a dural hook, the dura at the level of Tj is then elevated and a longitudinal incision made rostral ly to C 2 . The dorsal roots of to Cg are gently elevated and sectioned b i latera l ly after high frequency coagulation of the dorsal radicular arteries that accompany these roots. The dorsal spinal artery is then coagulated just rostral to the point chosen for cord transection. The cord is then severed between the last cervical and f i r s t thoracic vertebrae and under direct vision the section is further explored by gentle suction to make sure that a l l connecting strands of cord tissue are separated. Ster i le gelatin sponge is placed between the sectioned ends of the cord, the dura sutured and the incision closed. (b) Extra-cranial transection of trigeminal roots. Spinal cats are anaesthetized with pentobarbital sodium (35 mg/kg) and placed in the inverted position in a stereotaxic holder. The lower jaw is opened maximally to expose the roof of the mouth. An incision 1.5-2.0 cm long is made 2 mm lateral to the edge of the pterygoid process of the sphenoid and the perpendicular plate of the palatine. Adequate exposure is achieved by careful separation and removal of the external and internal pterygoid muscles, and the maxillary and mandibular roots of the trigeminal nerves are sectioned b i latera l ly a short distance from their points of emergence from the foramina ovale and rotundum. Extreme caution is necessary - 18 -to prevent haemorrhage from the e x t e r n a l r e t e , the middle meningeal anastomatic and tensor tympanic a r t e r i e s (37). The fossae created by removal of the p t e r y g o i d muscles are packed w i t h s t e r i l e g e l a t i n sponge and the i n c i s i o n sutured. This p r e p a r a t i o n recovers r e a d i l y from s p i n a l shock and may be main-•ie tained f o r as long as 2 to 3 months i f s u i t a b l e precautions are observed. These include (1) continued a n t i b i o t i c a d m i n i s t r a t i o n , (2) maintenance of a reasonably uniform and p h y s i o l o g i c a l environment temperature, (3) bladder c a t h e t e r i z a t i o n , and (k) feeding by stomach tube. This p r e p a r a t i o n maintains spontaneous diaphragmatic r e s p i r a t i o n and a blood pressure of 70-90 mm Hg. While some degree of motor i n n e r v a t i o n of the muscles of the neck and f o r e -limb remains i n t a c t , the animal g e n e r a l l y remains quies c e n t . The sensory i n f l o w i n t h i s p r e p a r a t i o n i s l i m i t e d to (1) s p e c i a l and v i s c e r a l a f f e r e n t s of the c r a n i a l nerves, and (2) somatic a f f e r e n t s to the o r b i t and upper e y e l i d v i a the ophthalmic branch of the t r i g e m i n a l nerve. Somatic d e - a f f e r e n t a t i o n makes i t p o s s i b l e to c a r r y out a l l of the f o l l o w i n g procedures on the unanaesthetized c a t : (I) To i n s e r t i n d w e l l i n g polyethylene catheters i n t o a r t e r i e s and at-veins f o r the recording of blood pressure f l u c t u a t i o n s and f o r drug a d m i n i s t r a t i o n . By t h i s technique drugs can be i n j e c t e d repeatedly, d i r e c t l y and b i l a t e r a l l y i n t o the b r a i n without compromising the c e r e b r a l c i r c u l a t i o n and without a p p r e c i a b l e latency f o r observing immediate e f f e c t s . In a d d i t i o n , t h i s approach permits a c l o s e r "check" on the c o n c e n t r a t i o n Some degree of wasting of the muscles of the hindpaws i s noted when the p r e p a r a t i o n i s maintained f o r such long p e r i o d s . - 19 -of agents used, since di lut ion and enzymatic inactivation of the injected drugs are minimized. In such preparations, an indwelling arter ia l catheter has been maintained for several days so that the effects of a number of agents, even long-acting compounds, could be observed in the same animal, ( i i ) To place the animal in a stereotaxic instrument, ( i i i ) To expose the cranium and to place screw electrodes through the skull with tips touching the dura for EEG leads. (iv) To trephine the skull and to place electrode tips in the brain stem for the production of e lectro ly t ic lesions. (v) To perform bilateral' denervation of the carotid sinuses, and to evalua te the effects of th i s procedure in an unanaes the t ized, non-curarized anima 1. - • A welcome and; unexpected1 dividend, which has 'iTiade this preparation a part icularly valuable tool for this investigation, is the fact that a highly deactivated EEG can readily be produced even in the absence of 'bra in stem lesions. After a few days of adaptation to its new situation, a Rothballer (149) type "D" pattern (persistent-spindles in "al1 leads) regular iy appeals (.when the animal is placed in a quiet, dimly lighted environment. Marked EEG "act ivat ion" can be accomplished by suitable audi tory or visual st-imuia,ti,on, but prompt reversion to a synchronized pattern follows removal of the stimulus (Fig. 1). The sens i ti vi ty of this preparation is is further i1 lustrated in Figure 2, where both the introduction arid removal of the stomach tube produces EEG "act ivat ion" , in contrast to the deactivated pattern which is observed even during the administration of milk through the stomach tube. In addition, this preparation manifests a consistently high degree of sensit iv ity to direct pharmacological activation by both cholinergic and adrenergic agents (107). - 20 -R F - P Tin fir BR 90 RF.P WtJ ! I I ! I I i i , i i I!  I i , i 11 I I ill Ml m m LF.P t t t Hand Clap Figure 1 Effect of audiogenic activation upon the EEG (whistle blast and hand claps). On a l l leads, abrupt onset of activation from pattern D to A with reversion to pattern D within 25 seconds. No signif icant change is noted in the pressor response. - 21 -' R F - P I LP-0 i|iw jji Ijll^ ^ E**»l L Ijdrotoctew o{ ttohwfc. Tube. J RF-P D u r i n g -fee<i>r»g v i a . S . T . I. TubeJ Figure 2 . Effect on the f EG. of feeding via stomach tube, immediate activation is noted during the introduction and the removal of the stomach, tube. In contrast the pattern observed during feeding Is very similar to the control tracing. 22 Thus, this preparation has f u l f i l l e d a l l of the desired objectives even without the production of brain stem lesions, and, in fact, without opening the cranium. The preparation is as nearly "physiological" as a paralyzed and de-afferented animal can be, and is completely free of extraneous pharmacological influences. Hence, when brain stem lesions are placed uni lateral ly, the intact side can serve as a control, without the necessity for neuromuscular blockade. In fact, within a relatively short period of adaptation, a synchronized EEG pattern can be maintained with the animal in the stereotaxic instrument, and the EEG can be followed throughout the coagulating procedures, while free of anaesthetic effects. 6 . Direct Central Drug Effects. A satisfactory approach for making simultaneous bi lateral intra-carotid injection of drugs presented another procedural problem. Attention was concentrated on obtaining direct central effects of drug action by means of the intra-arter ia l route (153). Unilateral intracarotid injection, though informative, seemed to have certain disadvantages. The cerebral c irculat ion on the side of the injection may be temporarily embarrassed during the cannulation procedure. Furthermore, studies in which dyes have been injected uni lateral ly into the carotid artery have indicated that drugs injected in this manner may not be distributed to the same sites b i l a tera l l y , equally and simultaneously (81). As Marrazzi (114) has pointed out, "a drug injected into the carotid artery would act somewhat l ike a close arter ia l injection and produce i n i t i a l l y a higher concentration in the brain on the ipsi lateral side, while when i t got out into the systemic c irculat ion and got diluted with the overall blood volume i t would then be in a suf f ic ient ly lower concentration to prove - 23 -sub-threshold for the periphery and the contralateral hemisphere. The amounts that get through the Circ le of Wi l l i s ordinari ly are small under these conditions." A satisfactory technique for simultaneous bi lateral drug administration has been developed. By introducing a small bore polyethylene catheter into the right subclavian artery i t is possible to make injections directly into the innominate artery at the point of bifurcation of the two common carotids. The catheter is introduced until the tip touches the caudal aspect of the aortic arch (a distance of approximately 10-10.7 cms for cats weighing approximately 2 . 5 - 3 . 5 kg) and then the catheter is withdrawn approximately 1.5 cms. The location of the catheter tip with reference to its ab i l i ty to distr ibute injected material b i latera l ly and simultaneously may readily be ascertained by a test injection of 0 . 5 cc of 1-epioephrine containing 10/ug/cc (expressed as 1-epinephrine base). The administration of this com-pound produces immediate, equal and bi lateral trans rent d i lat ion of the pupils when the catheter is at the bifurcation of the two common carotids. B. Experimental Procedures. In a l l animals the blood pressure was recorded from the right femoral artery with a mercury manometer. In some animals the right femoral vein was cannulated with a polyethylene catheter whose tip was introduced into the inferior vena cava, for intravenous administration of drugs. The right subclavian artery was cannulated with a small bore polyethylene catheter, with its tips positioned into the innominate artery in such a way as to permit distribution of injected material into both carotid arteries (107) . In the other end of both the venous and the arter ia l catheters, needles were inserted and connected to a two-way stop-cock, so that repeated - 2k -small injections of saline or various drugs could be introduced without disturbing the animal. The muscles of the scalp were reflected and electrodes (brass screws) introduced through the skull to the dura. One pair of screws (electrodes) was positioned anteriorly over the motor cortices. The middle pair was inserted approximately 1 cm behind the coronal suture and 1.5-2.0 cm to each side of the midline. The posterior pair was located just anterior to the origin of the bony tentorium and 1.2-2.0 cm to each side of the mid-line and an earthing electrode was put in the midline over the frontal sinus. The dural leads were connected to an Offner amplifier, Type 140A, and the EEG recorded on a Dynograph ink writer. Bipolar recordings were carried out between "r ight frontal-r ight par ie ta l " , "r ight parietal-r ight o cc i p i t a l " , and between corresponding positions on the contralateral side. Al l records had to be taken at night in a dimly lighted and quiet environment. In some experiments, destruction in whole or In part of the mid-brain reticular formation was produced by a coagulating current at the end of a steel electrode, insulated to 1.0 mm of the t ip. The Heathkit high frequency generator provided the current source. In six experiments bi lateral denervation of the carotid sinus was carried out according to Koella (99) 12 to 2k hours following partial trigeminalectomy. Carotid sinus denervation was performed by dissecting and cutting a l l nerve filaments connected to the carotid body, and by removing cautiously the outermost sheath surrounding the common carotid arteries to distances approximately 0 .5 cm rostral to and 1.5 cm caudal to the origins of the internal carotid, ascending pharyngeal and occipital arter ies. In order to evaluate the effectiveness of bi lateral carotid sinus denervation, the absence of a blood pressure rise over control values, following clamping of both carotids was always tested. The pressor response - 25 -was found to be consistently and effectively abolished by this procedure. Moreover, the usual compensatory cardiovascular reflex adjustments which result from drug-induced pressor and depressor responses were conspicuously absent. Precautions were taken to avoid arousal reactions caused by external environmental influences during the intra-innominate injection of drugs and repeated controls were established by injecting sal ine. Control saline injections did not influence cort ical act iv i ty at any time during the experiments. The drugs studied were I-epinephrine bitartrate, 1-norepinephrine bitartrate, dl-N-isoproterenol hydrochloride, 1-isoproterenol bitartrate dihydrate, d-isoproterenol bitartrate, dichloroisoproterenol hydrochloride (DCI), r-amphetamihe sulphate, chlorpromazine, promazine, phenoxybenzamine hydrochloride (dibenzyline), acetylcholine chloride, physostigmine (eserine) sulphate, atropine sulphate, vasopressin, histamine phosphate, arid 5-hydroxytryptamine creatinine sulphate (serotoriin); Solutions of these drugs in the concentrations in which they were to be used were freshly made up in normal saline or in glucose sal ine. Al l doses are expressed as weight of free bases (except vasopressin and serotonin) and are reported per kg body weight of the animal. it The author is grateful to the following drug houses for their generous gifts of some of the drugs used in this study: to Sterling-Winthrop Research Institute for supplies of 1-eplnephrlne bitartrate, 1-norepinephrine bitartrate, dl-N-isoproterenol hydrochloride, 1-isoproterenol bitartrate dihydrate arid d-isoproterenol bitartrate; to Poulenc Ltd. (Canada) for chlorpromazine hydrochloride; to Mowatt and Moore Ltd. for promazine hydro-chloride; and to Smith Kline and French Inter American Corporation for phenoxybenzamine hydrochloride (dibenzyline). Special acknowledgement is due Dr. I. H. Slater of E l i L i l l y and Co. Ltd. for supplying us with a generous quantity of dichloroisoproterenol hydrochloride (DCI). - 26 -All drugs were Injected slowly via the intra-innominate route, and made up in such a manner that volumes injected never exceeded O.k ccs. The time taken for the administration of agents to be studied was never less than 5 seconds, so as to avoid acute arterial distension. In the case of some agents (phenoxybenzamine, dichloroisoproterenol) the time taken for injection was spread out over a much longer period. In some experiments more than one adrenergic amine was tested in the same animal. However, the interval between such injections was always at least 15 minutes (unless combined or potentiating actions were sought), and only after apparent and complete recovery from the EEG and vascular effects of the previous injection. Moreover, the sequence of injections was varied so that each agent was employed as the first injection in several different experiments. At the end of the experiment the animal was sacrificed and examined for correct intra-innominate catheter placement. The brain was perfused with formol-saline so that lesions and sections could be determined by subsequent histological examination. o - 27 -III. RESULTS Experiments were performed on 56 cats with an average body weight of 2 .5 kg. Animals were prepared in the manner described previously in Part II on Methods of this presentation and reported elsewhere ( 107) . A. Basic EEG Pattern. In the unanaesthetized de-afferented cat fluctuating periods of partial alertness and drowsiness are characteristic of the preparation when i t is undoutedly not asleep. These two states are easily distinguished by the responses of the head and forelimbs and by the e lectr ica l act iv i ty of brain. In the apparently " a le r t " state (following audiogenic, visual or olfactory stimulation) movements of the head, ears, eyes and occasionally the vibrissae and the forepaws may be observed. Under these circumstances the e lectr ica l act iv i ty of the cortex is rapid (25-40 c/s) and of low voltage (less than 75 uV), similar to the "activation pattern" described for the intact animal by Rheinberger and Jasper (143). When the animal is in a drowsy state the pur iIs are usually constricted, movements of the head and forel irribs are minimal and the EEG is dominated by spindle act iv i ty (up to 500 uV in amplitude), wh>ich alternates with short intervals of electro-cortical act iv ity exhibiting intermediate voltage and varying frequency. Depending oh the type and intensity of the stimulus, the electro-cort ical trans i tion from one of these states to the other may either be abrupt or gradual. Under such circumstances and experimental conditions i t was found - 28 -convenient to u t i l i z e four b a s i c types of EEG patterns f o r the purposes of c h a r a c t e r i z i n g and e v a l u a t i n g drug-induced EEG " a c t i v a t i o n " These EEG p a t t e r n s , designated A, B, C and D by R o t h b a l l e r (149) manifest p r o g r e s s i v e degrees of d e a c t i v a t i o n ( i n c r e a s i n g v o l t a g e , decreasing frequency and s y n c h r o n i z a t i o n , f i n a l l y i n c l u d i n g rhythmic s p i n d l e b u r s t s ) . A t y p i c a l d e a c t i v a t e d record ( p a t t e r n D) of the unanaesthetized de-afferented cat i s presented i n Figure 1. In t h i s record high voltage low frequency a c t i v i t y w i t h s p i n d l e s can be seen, followed a b r u p t l y by immediate a c t i v a t i o n ( p a t t e r n A), r e s u l t i n g from audiogenic s t i m u l i ( w h i s t l e b l a s t and hand c l a p s ) . Between these two extremes of electroencephalograph!c " d e a c t i v a t i o n " and " a c t i v a t i o n " , intermediate patterns i n d i c a t i n g v a r y i n g degrees of EEG d e s y n c h r o n i z a t i o n may occur. Such patterns may take place spontaneously during t r a n s i t i o n between s t a t e s of drowsiness or a l e r t n e s s , and sometimes are observed f o l l o w i n g the intra-innominate i n j e c t i o n of l e s s potent a c t i v a t i n g drugs. R o t h b a l l e r (149) has s t a t e d h i s observations cogently; "In i t s s t r i c t e s t sense, the term EEG " a c t i v a t i o n " r e f e r s not to any one s p e c i f i c EEG p a t t e r n , but to the conversion of one p a t t e r n i n t o another -s p e c i f i c a l l y from "D" towards "A" or towards the l e f t i f the patterns are imagined as being arranged i n a l p h a b e t i c a l o r d e r . Conversion i n the opposite d i r e c t i o n i s " d e a c t i v a t i o n " . Thus, although the end r e s u l t would not n e c e s s a r i l y be the EEG of a l e r t wakefulness, a change from p a t t e r n "D" to "C" i s s t i l l " a c t i v a t i o n " . So i s the conversion from "B" to "A", even though both of these l a t t e r patterns might f a l l i n t o what i s g e n e r a l l y considered the " a c t i v a t i o n p a t t e r n " to begin w i t h . " - 29 -B. Preparation With Intact Brain Stem and Intact Carotid Sinuses. 1. Controls. In our de-afferented preparation, the character of the resting deactivated pattern as well as the prompt onset of EEG "activation" which is elicited by auditory stimulation (whistle blast and hand clap) is demonstrated in Figure I. Abrupt reversion to a deactivated pattern occurs within 20-25 seconds. The absence of any significant EEG or blood pressure alterations following the intra-innominate administration of drug-free normal saline is shown in Figure 3. Painful stimulation to the forepaws, the hindpaws, the tail, the occipital part of the head and the body, are equally without effects on both the resting deactivated pattern and the control blood pressure readings. 2. Adrenergic Activation. (a) Effects of epinephrine, isoproterenol, norepinephrine and  racemic-amphetamine. All of the adrenergic agents when administered in adequate doses into the innominate artery, at the point of bifurcation of the two common carotid arteries, produced clear-cut EEG "activation". The fact that our de-afferented preparation would maintain stable patterns of EEG deactivation and remain responsive to repeated drug-induced transitory activation over many hours made it possible to attempt irie to determine the activating threshold for these agents. Typical patterns * In all results the latent period for the onset of "activation" is reported in seconds and is calculated from the mid-point of the length of time taken for the complete injection of the agents to be tested. ** The threshold Is taken as the smallest dose eliciting consistent clear-cut EEG "activation" (pattern D changing to B^A). - 30 -RF-P , , , ilillJl'llLllUli RP-0 LP-LF-P f SoJitve Cowtrol 1 82 1 18 4 RP-0 LP-0 LF-P BP TO 80 85 j F i g u r e 3, Effect of iotra-innomkiate saline on the EEG, Injection bracketed by arrows. No significant EEG alteration can be observed from the normal resting pattern. - 31 -of adrenergic activation at threshold doses are shown for epinephrine (Fig. k), norepinephrine (Fig. 5 ) . isoproterenol (Fig. 6) and dl-amphetamine (Fig. 7 ) . It was noted that the threshold for isoproterenol-induced activation was consistently lower (0.25/ug/kg) than that for either epinephrine, norepinephrine (0.5/ug/kg), or amphetamine (50/ig/kg) . The characteristic patterns of adrenergic activation and blood pressure fluctuations at various dose levels (0.25-2/ug/kg) are shown for epinephrine (Figs. 8 and 9 ) , norepinephrine (Figs. 10 and 11) and iso-proterenol (Fig. 12) . Saline injection produced no such effect. Generally speaking the onset of activation following the intra-innominate administra-tion of the "short-acting" adrenergic amines was very rapid (3-7 seconds) and usually preceded any alteration in the blood pressure. In some preparations, movements of the forepaws and head were noted but these were not as marked as those observed with amphetamine. Activation produced by norepinephrine usually was somewhat more ephemeral than that produced by equivalent doses of epinephrine and isoproterenol, and the latent period for the activating effect was always less prolonged following the administration of isoproterenol. However, adequate doses (2/jg/kg) of all three compounds produced crisp, unquestionable(and near maximal) activating effects (pattern D —> A). In addition, isoproterenol in low doses (0 .25 /ug/kg) produced a more consistently intense activated record than similar amounts of epinephrine and norepinephrine, and at a dose of 0.5/ug/kg shifts from pattern D to A were observed repeatedly with isoproterenol. In contrast, intense activation was not always obtained with epinephrine and norepineprhine at a dose of 0.5-1.0/ug/kg. In approximately 10% of the preparations studied, epinephrine in low doses (0 .25 /ug/kg) produced only fleeting periods of high voltage spindle activity which fluctuated - 32 -LF-P T Ep, o.5>/Ko. I RF-p BRqs 300/*>J 105 LP-0 J 1 HI I 15 115 Figure 4. Effect of epinephrine 0 . 5 /Ug/kg on the EEG. Injection bracketed by arrows. Activation is immediate and precedes any alterations in blood pressure. Reversion to the control pattern occurs after 80 seconds, while the pressor effect is still maxima 1. - 33 -RF-P LP-0 LF7P t N.E. aSr/kg. 1 ppp BR loo n o u o 14-0 y l l i j':' i " I j [j LP-0 135 i3a I3i 130 F i gu re 5. I n t r a - i n n o m i n a t e n o r e p i n e p h r i n e 0.5/jg/kg. I n j e c t i o n b r a c k e t e d by a r r o w s . Immediate a c t i v a t i o n ( t y p e A) o c c u r r i n g b e f o r e the peak o f the p r e s s o r r e sponse and l a s t i n g about 35 s e c o n d s . T h i s p a t t e r n i s nex t f o l l o w e d by p e r i o d s o f d e a c t i v a t i o n and a c t i v a t i o n , w i t h an a b r u p t r e t u r n to the p r e - i n j e c t i o n t r a c i n g s , w h i l e the b l o o d p r e s s u r e i s s t i l l nea r maximal v a l u e s . 34 -RF-P RP-0 tl.N.E. o.asi/M R F .p B R no 85 tS 40 RP-0 LP-0 70 80 8? Figure 6 Intra-innominate isoproterenol 0.25/Ug/kg. Injection bracketed by arrows. Immediate (3 seconds) activation, pattern D to A, occurring 30 seconds before the peak depressor response, and persisting for an additional 30 seconds before recovery to control spindling. 35 -R F P . J » . W . l « M * l M I | » » l W « IB •>li . l> l »fcHH>tl>*l RP.O •"'">' "** ( w4 1 •» . LP.O 50 l/Kg. 3ocy-V BP. no RF.P RP.O 1 LP. 1 Ii l l 1 i " | LF.P 152 152 FI gu re 7. Intra-innominate amphetamine 50/ug/kg. Activation some-what delayed (approximately 12 seconds) but abruptly occurring before the peak pressor response and lasting for almost 2 minutes. Activation pattern is interrupted intermittently by spindle bursts. The activated EEG returns to control values in the presence of a maximal pressor response. - 36 -RF-P 5 RP-0 LP:0 LF-P T Eft. o.25T/Kg- 1 RF-P B.R IOS ice m | | S '1ml : | inil RP-0 ( i_p.p [I 1 F i gu re 8. Intra-innominate epinephrine 0.25A«9/kg. Transient activation occurring 10 seconds after injection and before the peak pressor response. Reversion to D pattern after approximately 25-30 seconds while pressor effect is still maximal. 37 -H Am I, « . < > » r i i * . « i . l . > i « v i i LF.P R F - P 30Cy*V B.P. 90 RP.O L P mm* 140 F i gu re 9. Intra-innominate epinephrine 2.0/ug/kg. Intense and immediate activation (pattern D to A) lasting more than 2 minutes, with gradual return to previous spindling. - 38 -RF-P ( RP-0 LP-0 T N l . E . 0 . 5 n / k 3 . 1 I , . . ' 300/*V 5 set. 7 pp p B . R MS 120 WO ISO i n rm r n RP-0 i 1*5 1 3 0 Figure 10 Intra-innominate norepinephrine 0.5/ug/kg. Activation (approximately k seconds) preceding the peak pressor response (blood pressure 150 mm Hg) by 30 seconds, and lasting approximately 60 seconds with a gradual return to previous "spindling" while the blood pressure is s t i l l elevated. - 39 -RPO LPO LF P t NE. lot/Kg. 1 RF P BP SO RPO LPO LF P 132 135 FIgu re 1 1 . Intra-innominate norepinephrine 2.0/ug/kg. Transient activation followed by alternating periods of deactiva-tion and activation which is independent of the sustained pressor response. - ko -• > ii»»nm»i»ni|iit».#ii RP-0 ' — — ' f 3o°/-v qo 1 —• nr.p RP.O LP.O So sa LF P 5 0 5 4 5 5 Figure 12. Intra-innominate isoproterenol 0.5/ig/kg. Note the prompt and intense activation (pattern D to A) which occurs 40 seconds before the peak depressor response and lasts for more than 90 seconds. Abrupt return to the pre-existing pattern is seen to occur, while the blood pressure is still below control values. a l t e r n a t i n g l y w i t h periods of low voltage f a s t a c t i v i t y . S i m i l a r r e s u l t s have been observed w i t h comparable doses of norepinephrine, but the f l u c t u a t i o n s were less frequent and the degree of high voltage slow a c t i v i t y l e ss intense than those noted w i t h epinephrine. These types of e f f e c t s were observed i n preparations which responded subsequently to s i m i l a r doses of i s o p r o t e r e n o l w i t h d e f i n i t e and unmistakable " a c t i v a t i n g p a t t e r n s " . Reversion to synchronized c o n t r o l patterns occurred w i t h i n 1-2 minutes w i t h a l l three compounds, the d u r a t i o n of the a c t i v a t i n g e f f e c t being longer w i t h l a r g e r doses ( u s u a l l y not more than 2/Ug/kg i n these experiments. For e q u a l l y e f f e c t i v e a c t i v a t i n g doses, the d u r a t i o n of the a c t i v a t i n g e f f e c t of i s o p r o t e r e n o l was somewhat longer than that of epinephrine and norepinephrine. The return to a d e a c t i v a t e d EEG seemed to be more abrupt i n the case of i s o p r o t e r e n o l , but whether gradual or abrupt, EEG r e v e r s i o n preceded blood pressure recovery w i t h a l l three compounds." Amphetamine i n doses of 150-200 /jg/kg e l i c i t e d i n t e n s e , l o n g - l a s t i n g EEG " a c t i v a t i o n " and behavioural signs of a l e r t n e s s , manifested by movements of the forepaws, eyes and searching motions of the head. Racemic dl-amphetamine i n doses as low as 5 0^ig/kg produced d e f i n i t e a c t i v a t i o n ( F i g . 7 ) . However, the a c t i v a t i o n produced by small doses of amphetamine (50/lig/kg) c o n s i s t e d of s h o r t e r r l a s t i n g desynchronized EEG records, i n t e r r u p t e d i n t e r m i t t e n t l y by s p i n d l e bursts but f r e e from any a s s o c i a t e d behavioural changes. Here, too, the pressor response seemed unrelated to the EEG e f f e c t s . (b) E f f e c t s of some isomers of i s o p r o t e r e n o l administered  separately and i n combination. - kl -(i) Isomers of isoproterenol administered separately. The demonstration by Prinzmetal and Alles (136) that in its CNS stimulating effects, the dextro-isomer (d) of amphetamine (dexedrine) is three to four times more potent than the levo-lsomer (1), and one and one-half to two times more potent than the racemic compound, prompted us to investigate whether the EEG effects observed with isoproterenol could be related to the d- or 1- fractions of this compound. In this regard two procedures were adopted. First, the two isomers were given separately and alternately to observe their threshold EEG activating effects; second, the two isomers were given in combination. Administration of the dextro-isomer in doses ranging from I/Ug/kg to 100/ug/kg produced no demonstrable vascular changes and, in addition, failed to elicit any clear-cut EEG "activation1' (Fig. 13. upper record). In contrast, a transient shift from pattern D to B was observed with 1 /Ug/kg of the levo-form of isoproterenol, which, in addition, produced depressor responses comparable to those observed following the administration of racemic isoproterenol (Fig. 13. lower record). (ii) Isomers administered in combination. Injections of mixtures of the d- and 1-isomers of isoproterenol, produce EEG "activation" and depressor responses comparable to those noted with equivalent amounts of the 1-isomer used in the combination. Figure \k was taken from Cat No. 220360, given a combined amount of the d- and 1-isomers and illustrates the prompt EEG "activation", occurring before the peak of the depressor phase, with an abrupt reversion to the control pattern, while the blood pressure is still unquestionably lower than its pre-existing control va1ue. - kl -h. R F - P LP-0 d- L.N.E. ioor/Kg. i • 1 300M.V 5 s e c . [ r RF-P B . P 102. 10S i o i 105 LP-0 T l - I . N E . I .01/K9 J ftp 10 F i gu re 1 3 . Intra-lnnomlnate (a) d-isoproterenol, 10Q^g/kg, and (b) l-lsoproterenol, 1.0/ug/kg. (a) Upper tracing: d-isoproterenol injected slowly over a period of 2 5 seconds. No demonstrable change can be noted either in the blood pressure of the EEG. (b) Lower tracing: I-isoproterenol; produces prompt type A to B pattern which fluctuates towards type D. Note the rapid depressor response which is similar to dl-Isoproterenol (Fig. 1 2 ) . - kk -RF-P RP-o JJj I LP-0 LF-P I! I T'11 IT LP-0 LF-P 5 0 K ^ ' Figure 14. Intra-innominate administration of combined amounts (2.0/Ug/kg each) of I-isoproterenol and d-1soproterenol. Note that the type of EEG activity and the depressor response produced by this combination display characteristics similar to those of 1-isoproterenol alone. - kS -3. C h o l i n e r g i c A c t i v a t i o n . (a) E f f e c t s of a c e t y l c h o l i n e and e s e r i n e (physostlgmine). Patterns of " a c t i v a t i o n " s i m i l a r to those observed w i t h adrenergic amines are r e g u l a r l y produced by the i n t r a - a r t e r i a l a d m i n i s t r a t i o n of a c e t y l c h o l i n e ( 0 . 2 5 - 2 . 0 /ug/kg) and e s e r i n e (50-100 /ig/kg). T r a n s i e n t depressor e f f e c t s , marked w i t h a c e t y l c h o l i n e and s l i g h t w i t h physostigmine, occurred con-s i s t e n t l y w i t h these agents. A c t i v a t i n g doses of a c e t y l c h o l i n e v a r i e d i n d i f f e r e n t animals from 0 .25 to 2/Ug/kg. Whereas i n some preparations low doses (0.25-0.5/Ug/kg) produced t r a n s i e n t EEG d e s y n c h r o n i z a t i o n , i n t e r r u p t e d at i n t e r v a l s by s h ort bursts of s p i n d l e a c t i v i t y ( F i g s . 15 and 16), l a r g e r doses ( 1 -2/jg/kg) always e l i c i t e d more d e f i n i t i v e " a c t i v a t i o n " , w i t h the 2 /Og/kg dose p r e c i p i t a t i n g immediate, intense and long periods of EEG arousal i n a l l instances. Figures 17 and 18 show p a t t e r n A produced by 1 and 2 /ig/kg of a c e t y l c h o l i n e i n our ananaesthetized de-afferented prepara-t i o n . The EEG a c t i v a t i n g response i s produced a f t e r very short l a t e n t periods (ca. 3-7 seconds), and seems unr e l a t e d to the depressor responses, s i n c e the EEG r e v e r t s to i t s p r e - a c t i v a t e d p a t t e r n w h i l e the blood pressure i s s t i l l below i t s c o n t r o l l e v e l . Under the above circumstances, a c e t y l -c h o l i n e - i nduced " a c t i v a t i o n " i s g e n e r a l l y accompanied by tachypnoea and movements of the f r o n t paws and head i n d i c a t i v e of behavioural a r o u s a l . In c o n t r a s t , eserine-induced a c t i v a t i o n d i d not seem to be a s s o c i a t e d w i t h any change i n behaviour. In doses adequate to e l i c i t c o n s i s t e n t changes i n 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 , the marked and f l e e t -ing depressor e f f e c t s noted w i t h a c t i v a t i n g doses of a c e t y l c h o l i n e were conspicuously absent; i n s t e a d , depending on the dose, the blood pressure remained f a i r l y constant around c o n t r o l values (50/ug/kg), o r showed a s l i g h t Increase (100/Ug/kg) ( F i g s . 19 and 2 0 ) . In s i n g l e low doses kS -Ji L P . . LF.P t , ] S ' 5 ~ s « ' *V-V BP * 70 5 5 1 ^ " / RP.O i i 1 1 j [ y LF-P • , T ° 7 5 85 B P 8 7 Figure 15. Intra-lnnominate acetylcholine 0.25/ug/kg. Transient activation occurring within 7 seconds and lasting for 12-15 seconds, precedes the peak depressor response. Note temporal independence between changes in the EEG and blood pressure variations. - kl -WI'ii'M m* ' ' i W r t ^ w N f l W W q o 15 . p 9 0 9 5 B P 8 0 Figure 16. Intra-Innominate acetylcholine 0.5/ig/kg. Immediate activation (D to A) similar to Figure 15, but of longer duration, with gradual recovery (A to C to D). 48 -I UO MO Figure 17. Intra-innominate acetylcholine 1.0/ig/kg. Intense and more prolonged activation, similar to that seen in Figure 16, is observed in this record. Depressor responses are comparable. - 49 -RF-P LP-0 LF-T ACH 2.0v/Kg. t RF-P 5 sec.. B . P . <to 7 0 5 5 « S 300/-V L' I .P . 40 sec. L F - P , . [ii ] ] HO 10 So 90 Figure 18. Intra-innominate acetylcholine 2.0/Ug/kg. The EEG activation which consists of a sh i f t from pattern D to A, is similar in appearance to Figure 17. but is more prolonged. Note temporal independence between the blood pressure variations and EEG activation and deactivation. - 50-il Ii l l * H L P . O ^ E s e r i r * Scor/K^. ^ if RF-P . S 5 80 75" RP.O LP.O E S LF.P 7S 7 0 9 0 Figure 19. Intra-Innominate eserine 50/jg/kg. Intense type A activation occurs only after a long latent period (approximately 47 seconds) and persists for several minutes before returning to control type D spindling. Note the modest depressor response, when compared with that of Figure 18. - 51 -R F - P E. I' 'Willi HI m H i III n »n inimm ^ U U > w J Id »i» ..ii,»wi|i w HH i mt p B P 78 Igl 3oo/*.v P - 0 FP. II f w t n ^ w w ^ ^ i , « n w « i , ihimmmtn iwfi W|»I«WI »I(»|II. <,III.^I,,».I M.»II»I<,.» IIUHII,II^.Bi'^WH. iWMtffi.i ^'jMWWWWSitt^^lW|l|ji., l» lUit,» li«^^^»WIV4WT^| W W 1 1 » » W * » W » ' * « i » i m Wl»u>'il<mV,V*i • i K . w ^ w n ^ i ^ y V . i n ' Jw^ViMW«<».Xwi»t»»*i(v< i y i ^ m w W ^ ^ . i . w I t f r . W i M f r . r t H i j t o w - ^ W W y , ^ >y a n Figure 2 0 . Intra-innominate eserine I00^ig/kg. The EEG pattern (type A) is similar in appearance to that noted in Figure 19, but in this tracing the latent period is shorter and the duration of activation much longer. The pressor response is unchanged. - 52 -(10-20/Ug/kg), e s e r i n e f a i l e d to produce any EEG changes. However, i f these amounts were administered repeatedly at short i n t e r v a l s u n t i l a t o t a l dose of 50-60/Ug/kg was reached, d i s t i n c t a c t i v a t i o n was observed. This occurred a f t e r a r e l a t i v e l y long l a t e n t p e r i o d (75 seconds), but once i n i t i a t e d the degree of a c t i v a t i o n was more intense and the d u r a t i o n more prolonged (more than 20 minutes) than that observed w i t h a c e t y l c h o l i n e . Larger doses, 100-200 /Ug/kg, e l i c i t e d longer l a s t i n g periods of EEG " a c t i v a t i o n " (more than 90 minutes), as w e l l as r e s p i r a t o r y embarrassment, s a l i v a t i o n and incontinence. Recovery of the c o n t r o l e l e c t r o - c o r t i c a l p a t t e r n a f t e r e s e r i n e a d m i n i s t r a t i o n was u s u a l l y complete i n 3 hours f o l l o w i n g a d m i n i s t r a t i o n of the doses employed i n these experiments (50-200 /ig/kg); however, i n a l l cases the recovery of a d e a c t i v a t e d p a t t e r n could be brought about promptly by the a d m i n i s t r a t i o n of a t r o p i n e , 0 . 5 mg/kg, but not by chlorpromazine, 2 . 0 mg/kg ( F i g . 2 1 ) . 4. A c t i v a t i n g E f f e c t s of Other Agents. (a) Vasopressin and histamine. Since the p o s s i b i l i t y e x i s t s that some of the c e n t r a l a c t i o n s of adrenergic amines and of c h o l i n e r g i c agents may be e x p l a i n e d on the basis of t h e i r v a s c u l a r e f f e c t s (Mf), i t seemed important to a s c e r t a i n whether changes i n blood pressure (pressor o r depressor) produced by these agents may not be r e s p o n s i b l e f o r the e l e c t r o - c o r t i c a l events observed w i t h these compounds. The c a r d i o v a s c u l a r e f f e c t s of both vasopressin (pressor) and histamine (depressor) have been w e l l e s t a b l i s h e d . In a d d i t i o n , there i s general agreement that the vasomotor e f f e c t s of these two compounds are the r e s u l t of a d i r e c t a c t i o n on the v a s c u l a r c o n t r a c t i l e mechanism, and 53 RF-P RP-0 LP-0 LF-P 15 Eserine ioox / K g given Co m m . eorlic 8.P 80 R F - P t CPZ. 2.0".yKj. 1 V ««I>HI.»«I»1WIH»<'»«w. .aw* RP-0 A M LP-0 80 7 L../iijj.^M^iiu4Ljli>4Aii.Jiiii1ltu. LF-P t i A t r o p i n e 83 cs^M 75 70 F i g u r e 2 1 . Effect of chlorpromazine (CPZ) 2.0 mg/kg, followed by atropine 0 .5 mg/kg, on eserine-induced activation. Eserine 100/Ug/kg given 60 minutes earlier produces type A activation. This pattern cannot be reversed by CPZ 2 mg/kg, even after a 15 minute latent period but can be overcome by atropine (0.5 mg/kg) within 2-5 minutes. Note the characteristic atropine spindles and slight depressor response. - 54 -are u n r e l a t e d to adrenergic or c h o l i n e r g i c r e c e p t i v e elements ( 6 5 ) . I t was a n t i c i p a t e d that i f EEG " a c t i v a t i o n " could be the r e s u l t of blood pressure f l u c t u a t i o n s above and below c o n t r o l v a l u e s , then the i n t r a -innominate a d m i n i s t r a t i o n of vasopressin i n pressor amounts ( 0 . 5 units/kg) and histamine i n depressor doses (1 /ug/kg), would a s s i s t i n the c l a r i f i c a -t i o n of t h i s r e l a t i o n s h i p . The general procedure f o l l o w e d was to ad m i n i s t e r these agents i n those preparations i n which the a c t i v a t i n g s e n s i t i v i t y to threshold amounts of both adrenergic and c h o l i n e r g i c agents had been e s t a b l i s h e d . ( i ) Vasopressin. In s i x experiments, vasopressin ( 0 . 5 u n i t s / kg) produced a sustained pressor response (more than I hour), averaging a blood pressure increase of more than 25 mm Hg over c o n t r o l values. This blood pressure e l e v a t i o n i s comparable i n degree to that a t t a i n e d w i t h a c t i v a t i n g doses of pressor adrenergic amines. However, no c l e a r - c u t or demonstrable EEG " a c t i v a t i o n " was observed ( F i g . 2 2 ) . Here, a t t e n t i o n should be drawn to the f a c t t h a t i n the same p r e p a r a t i o n , audiogenic s t i m u l a t i o n and epinephrine a d m i n i s t r a t i o n ( 0 . 5 /ug/kg) had a b r u p t l y evoked a type A arousal p a t t e r n . In one p r e p a r a t i o n , a p a t t e r n f l u c t u a t i n g between B C and delayed i n onset was noted. However, i t i s not in c o n c e i v a b l e that t h i s long latency e f f e c t could have been due to a c c i d e n t a l environmental occurrences beyond the c o n t r o l of the experimenter s i n c e o c c a s i o n a l l y such patterns are observed to occur spontaneously ( i . e . , without drug a d m i n i s t r a t i o n ) . ( i i ) Histamine. Histamine ( 1 . 0/ug/kg), i n f i v e experiments, produced a t r a n s i e n t depressor response, e x h i b i t i n g a decrease of 30 mm Hg below c o n t r o l l e v e l s . This depressor e f f e c t which i s comparable i n - 55 -'RF-P RP-o LP-0 L F - P J I , ' 5 MCT ' 1~AV , I qo 1 I OLbopvessin RF-P B R 8 0 LP-o 135 Figure 22. Effect of vasopressin 0 . 5 units/kg on the EEG. The resting deactivated EEG pattern (type D) remains unchanged after the administration of 0 . 5 units of vaso-pressin, despite the increased and persistent pressor response. - 56 -i n t e n s i t y to that noted w i t h small doses of i s o p r o t e r e n o l (0.25/Jg/kg) and a c e t y l c h o l i n e (0.5/jg/kg), was not a s s o c i a t e d w i t h any d e f i n i t i v e e l e c t r o -c o r t i c a l change ( F i g . 23). However, l a r g e r doses (2-5 /ug/kg) which have r e l a t i v e l y s i m i l a r depressor e f f e c t s are a s s o c i a t e d w i t h c o n s i s t e n t changes i n the e l e c t r i c a l a c t i v i t y of the b r a i n (Figures 24 and 25 i l l u s t r a t e such a response). This a c t i v a t e d p a t t e r n p e r s i s t e d f o r periods longer than the b r i e f d u r a t i o n of the depressor response, and was s t i l l present f o r as long as kO seconds a f t e r the depressor phase had reverted to c o n t r o l l e v e l s . (b) S e r o t o n i n . The demonstration by Twarog and Page (167) i n 1953 that 5-hydroxytryptamine (serotonin) was present i n r e l a t i v e l y high concentrations i n the b r a i n , coupled w i t h the o b s e r v a t i o n that enzymes were present f o r the s y n t h e s i s , as w e l l as f o r the i n a c t i v a t i o n of t h i s compound, have s t i m u l a t e d wide and protean i n t e r e s t i n t h i s agent. In a d d i t i o n , there i s c o n s i d e r a b l e lack of agreement on the p o s s i b l e r o l e which s e r o t o n i n may play i n the c e n t r a l nervous system (see D i s c u s s i o n ) . Some have taken the extreme view that s e r o t o n i n does not cross the blood-brain b a r r i e r , and have provided evidence i n support of the hypothesis that i n j e c t e d s e r o t o n i n does not exert i t s c e n t r a l e f f e c t s through a d i r e c t a c t i o n (142). Others have demonstrated that small amounts of i n j e c t e d s e r o t o n i n may be deposited i n b r a i n (36). In a d d i t i o n , c e n t r a l s y n a p t i c i n h i b i t i o n (100, 114) as w e l l as EEG " a c t i v a t i o n " (151) have been observed f o l l o w i n g the a d m i n i s t r a t i o n of t h i s b iogenic amine. In our preparations i n which there are no r e s i d u a l e f f e c t s of anaesthesia, and the b r a i n stem i s completely i n t a c t , s e r o t o n i n has repeatedly produced EEG " a c t i v a t i o n " , even i n the presence of b i l a t e r a l c a r o t i d sinus denervation. The s m a l l e s t dose of s e r o t o n i n necessary to 57 -R F - P R P 0 LF-P , T Histamine LCi/Kji . 3 0 0 / i V R F - P B P 80 t o S 5 - » 50 I 'if n 0 n Pi 1 "' LP-0 S 5« 5 5 Figure 23. Effect of histamine l.OyUg/kg on the EEG* The resting deactivated EEG pattern (type D) remains unchanged after the administration of histamine I.O/ig/kg The depressor response is not associated with any demon-strable EEG changes. - 58 -RF-P f T Histamine e-Olr/Ka J RF-P B R " ° LF-P 100 l IT sec U S Figure 24. Effect of histamine 2.0/Ug/kg on the EEG. EEG activation begins after a latent period of 7 seconds and consists of a change from pattern D to B A. Activation occurs before the peak depressor response, and persists after the blood pressure has returned to control values. 59 -— i T i r w r wt -M LP-0 . L F - P il^lrt'llT^llWilfa^l.lMil^ilJrilll^llifBLftd^l t H i s t a m i m e E.OK/KQ. 1 — r = — • K v RF-p BP 10 20 sec. RP-0 LP-0 15 I 10 Figure 25. Effects of histamine 5.0/jg/kg on the EEG. The pattern of EEG and the depressor response are similar to those seen in Figure Ik. - 60 -produce c o n s i s t e n t EEG " a c t i v a t i o n " was 2 .5/ug/kg, although i n a few preparations f l e e t i n g " a c t i v a t i o n " has been observed w i t h a dose as low as 1 yug/kg. Larger doses (5 ^g/kg and 20 /Ug/kg) produced EEG responses of longer d u r a t i o n , w i t h the 20 ^ ig/kg dose e l i c i t i n g immediate " a c t i v a t i o n " " (ca. 2 seconds), as compared w i t h l a t e n t periods of approximately 5 seconds f o l l o w i n g a d m i n i s t r a t i o n of both the 2.5/Ug/kg and the 5 . 0 /Ug/kg doses. Figures 26 and 27 i l l u s t r a t e these o b s e r v a t i o n s . The " a c t i v a t i o n " noted w i t h s e r o t o n i n resembled i n some respects the " a c t i v a t i o n 1 1 noted w i t h epinephrine. With smaller doses of s e r o t o n i n ( 2 . 5 - 2 . 0/ug/kg), the f i r s t e f f e c t noted was a t r a n s i e n t p e r i o d of a type A a c t i v a t e d p a t t e r n , l a s t i n g approximately 5 seconds, f o l l o w e d by a short p e r i o d of a l e s s a c t i v a t e d o r p a r t i a l l y d e a c t i v a t e d type C t r a c i n g . S i m i l a r c h a r a c t e r i s t i c s i n patterns are obtained w i t h the 20 /Ug/kg doses, but under these circumstances the i n i t i a l type A a c t i v a t e d p a t t e r n i s increased to ap p r o x i -mately 25 seconds. Blood pressure responses were increased w i t h a l l doses used and are comparable f o r both the 5 /jg and 20/ug/kg doses. 5 . E f f e c t s of B l o c k i n g Agents. (a) Phenothiazine d e r i v a t i v e s (chlorpromazine and promazine). The e f f e c t s of chlorpromazine and a r e l a t e d phenothiazine on the EEG and t h e i r blockade of the a c t i v a t i n g response f o l l o w i n g drug a d m i n i s t r a t i o n and that induced by audiogenic or v i s u a l s t i m u l a t i o n were s t u d i e d i n 10 c a t s . ( i ) Chlorpromazine. In the unanaesthetized de-afferented cat the a d m i n i s t r a t i o n of adequate doses of chlorpromazine alone always produced c h a r a c t e r i s t i c a l t e r a t i o n s of e l e c t r o - c o r t i c a l a c t i v i t y which appear to d i f f e r s l i g h t l y from spontaneous patterns of d e a c t i v a t i o n . Figure 28 i l l u s t r a t e s a t y p i c a l 7-12 c/s s p i n d l e a c t i v i t y p a t t e r n - 61 -RF-P LP-0 mm mm t Serotonin s.ar/K&- 1 1 \ l > r-^z—• 1 »r-v pp_p B.R I O S i I O H5 iio RP-0 MM ? LP-0 us m FI gu re 26. Effect of serotonin 5 A*g/kg on tho EEG, Note the intense EEG activation (type A) which occurs after a latent period of about k seconds and persists for more than 1 minute. This effect is observed before the peak of the pressure response, and reversal to a deactivated type 0 pattern is noted while the blood pressure is s t i l l elevated. - 62 -• LP-0 T Serotonin, i ZO.or/Kg B R MS 1 35 sec . LP-0 I LF-P i*o its 132. Figure 27. Effect of serotonin 20 /tig/kg on the EEG. The EEG pattern and the blood pressure response are similar to those seen in Figure 26, but with this dose the duration of the activating response is increased. - 63 -produced by 2 mg/kg of chlorpromazine. This "spindling" can be distin-guished with difficulty when superjmposed on a prior type D pattern, but its development may be seen clearly vyfien elicited agajpst a background of activation, even as slight as that represented by a C type EEG tracing. In approximately 80% of the EEGs recorded, chlorpromazine-induced spindle activity comes on within 1 to 2 minutes following injection and persists for more than 3 hours. EEG "actjvatfon" produced by audiogenic stimula-tion is not blocked by ch 1 orproma,zine• The "chjorpromazine spindles" induced by 2 mg/kg of this agent a^ re converted immediately into an intense type A pattern by a whistle b^ast (Fig. 28) ^. -In contrast to untreated controls (see Figure I) the duration of EEG "activation" produced by audiogenic stimulation does not exceed the length of the whistle blast in the chlorpromazine treated animal. Following chlorpromazine, visual stimulation, produced by an object entering the visual field, also produced similar activating responses. These activating patterns were not accompanied by evidence of behavioural arousal. The use of smaller doses of chlorpromazine (0.1-0.3 mg/kg) initially did not exert any demonstrable effect on the EEG. Slightly larger doses (0.4-0.8 mg/kg) elicited patterns indicating a very transient shift to faster frequencies and lower voltage than the control patterns, while gradually increasing the dose to 2 mg/kg produced progressive electro-cortical deactivation, with the 2 mg/kg dose establishing definite chlorpromazine "spindling". Still larger doses (5-10 mg/kg) of chlorpromazine did not have any additional effect on the EEG patterns over that produced by the 2 mg/kg dose, except that the activating response produced previously by auditory stimulation in the cat treated with 2 mg/kg could not be obtained following administration of doses of - 64 -Figure 28. Effect of CPZ 2.0 mg/kg on the EEG. Mote the "CPZ spindles" produced by 2 mg/kg of CPZ injected 30 minutes previously. Audiogenic stimulus (whistle blast) produces immediate and intense type A activation, whose duration parallels that of the stimulus. - 65 -chlorpromazine as large as 5-10 mg/kg. Chlorpromazine i n doses as low as 2 mg/kg e f f e c t i v e l y blocked the EEG " a c t i v a t i o n " u s u a l l y produced by adequate doses of a l l of the a c t i v a t i n g amines which we have s t u d i e d (epinephrine, norepinephrine, i s o p r o t e r e n o l , s e r o t o n i n , histamine and amphetamine) w i t h the exception of the c h o l i n e r g i c agents. For each a c t i v a t i n g agent, the minimal doses necessary to achieve maximal EEG " a c t i v a t i o n " (type A) was estab-l i s h e d , and the e f f e c t of the same dose of each compound was then observed i n the same p r e p a r a t i o n f o l l o w i n g the a d m i n i s t r a t i o n of chlorpromazine. F i g u r e 23 i l l u s t r a t e s the f a i l u r e of epinephrine (0.5/Ug/kg)to produce i t s usual EEG response i n the presence of chlorpromazine blockade (2 mg/kg). S i m i l a r r e s u l t s w i t h norepinephrine (2 /ug/kg) a l s o were obtained ( F i g . 3 0 ) . While p r i o r treatment w i t h chlorpromazine r e s u l t e d i n suppression of pressor responses to epinephrine, the e l e v a t i o n s in blood pressure produced by norepinephrine and s e r o t o n i n were only p a r t i a l l y blocked. Serotonin (5.0/ug/kg) administered 25 minutes a f t e r the a d m i n i s t r a t i o n of 2 mg/kg of chlorpromazine produced a s l i g h t e l e v a t i o n i n the blood pressure 13 mm Hg) but f a i l e d to evoke any changes i n the d e a c t i v a t e d c o n t r o l EEG p a t t e r n ( F i g . 31 ) . A l a r g e r dose of s e r o t o n i n (20.0/Ug/kg) administered 20 minutes l a t e r produced a comparable pressor response (15 mm Hg) but was e q u a l l y without e f f e c t on the EEG. A f t e r 2 mg/kg of chlorpromazine, the a d m i n i s t r a t i o n of 0.5/Ug/kg of i s o p r o t e r e n o l and 5 .0^ug/kg of histamine a l s o f a i l e d to produce the t y p i c a l immediate EEG " a c t i v a t i o n " u s u a l l y e l i c i t e d by these compounds ( F i g s . 32 and 3 3 ) . However, i n the case of i s o p r o t e r e n o l a delayed apparent EEG " a c t i v a t i o n " of moderate degree developed i n about 22 seconds. This was not seen w i t h histamine. With histamine the depressor - 66 -RF-P L P - 0 T Epi. 0.51/Kq. i / after 'CPZ Z.Omg./Kg. after CrZ i .urng./rg. | | Joou-V & R « 8 0 S M t - 1 75 L F - P i 7S 73 Figure 2S. Effect of epinephrine 0.5 /Ug/kg on the EEG after CPZ blockade (2 mg/kg) . Absence of any activation and continued persistence of CPZ-induced "spindles". Note the transient depressor effect of epinephrine in the presence of CPZ blockade. - 67 -LF-P 1 RF-P BP t NE. 2r/Kq. after 4 C P Z . . 2Tm3./Kg 300/* LP-0 - , 125 IIS 115 Figure 3 0 . Effects of norepinephrine 2/jg/kg on the EEG after CPZ blockade (2 mg/kg). EEG tracing is similar to Figure 29. The blood pressure Is elevated (ca. 35-40 mm) above control values. - b8 -oittr C P Z . . Z n i g . / K g . RF-P B P . q o IOO |OJ , 0 0 F i gu re 31. E f f e c t of s e r o t o n i n 5.0/ug/kg on the EEG a f t e r CPZ blockade (2 mg/kg). Absence of any a c t i v a t i o n , and continued p e r s i s t e n c e of CPZ-induced s p i n d l e s . The pressor response i s much le s s than that noted i n Figure 26. RF-P t H i sW.n» 5 .01 /Kg. 1 T Pfter C P Z . t O r g / K j . , _ _ _ , jj^ V RF-P BP 8 0 70 1 (S TO Figure 32. Effect of histamine 5.0 fig/kg on the EEG after CPZ Wockade (2 mg/kg). Absence of any activation and continued persistence of CPZ-induced spindles. - 70 -e f f e c t s are moderate ( 1 5 mm Hg f a l l i n blood pressure i n 25 seconds) ( F i g . 32), whereas i n the same per i o d a more intense and immediate drop in blood pressure i s observed f o l l o w i n g the a d m i n i s t r a t i o n of 0.5/ug/kg of i s o p r o t e r e n o l ( F i g . 33). The delayed EEG " a c t i v a t i o n " of moderate degree which developed about 22 seconds a f t e r i s o p r o t e r e n o l a d m i n i s t r a t i o n was followed 35 seconds l a t e r by a f l a t t e n i n g of the EEG ( i n t e r r u p t e d by o c c a s i o n a l and i n t e r m i t t e n t s p i n d l i n g ) . During t h i s i n t e r v a l , the f a l l i n blood pressure was profound (from 85 to 38 mm Hg), and c o n s i d e r a b l y greater than that seen f o l l o w i n g i s o p r o t e r e n o l a d m i n i s t r a t i o n i n the absence of chlorpromazine (e.g. 98 to 60 mm Hg). Presumably t h i s d i f f e r e n c e a r i s e s from ch 1orpromazine-induced c e n t r a l and/or p e r i p h e r a l blockade of the usual compensatory r e f l e x v a s c u l a r adjustments. Gastaut et a l (62) have reported depression of 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 d u r i n g acute anoxia, and have shown that b i o - e l e c t r i c rhythms are most depressed at the c o r t i c o t h a l a m i c l e v e l where neuronal depression i s maximal. I t seemed p o s s i b l e that the " f l a t t e n i n g " of the EEG a s s o c i a t e d w i t h i s o p r o t e r e n o l a d m i n i s t r a t i o n i n the presence of c h l o r p r o -mazine may be the r e s u l t of c e r e b r a l anoxia. Indeed, i t i s not inconceiv-able that the observed t r a n s i e n t p e r i o d of a c t i v a t i o n which preceded the depression of 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 could be due to hypotensive ischemia, consequent to the intense and p r e c i p i t o u s depressor e f f e c t s of i s o p r o t e r e n o l i n the presence of chlorpromazine blockade. This deduction was suggested by the o b s e r v a t i o n that at t h i s p o i n t c a r e f u l t i l t i n g of the experimental t a b l e so that the head of the animal i s at a low»r l e v e l than i t s hind end, produces a reversal of t h i s " f l a t t e n e d " p a t t e r n and e l i m i n a t e s the subsequent development of apparent convulsions manifested by j e r k i n g movements of the head and forepaws. - 71 -i h order to explore f u r t h e r t h i s p o s s i b i l i t y , the blood pressure was s t a b i l i z e d a t a higher i n i t i a l l e v e l (115-120 mm Hg) by the a d m i n i s t r a t i o n of 0 . 5 u n i t s / k g of vasopressin i n the same pr e p a r a t i o n and i n the presence Of chlorpromazine. Then a c h a l l e n g i n g dose of i s o p r o t e r -enol ( 0 . 5/jg/kg), i d e n t i c a l to the one which produced the changes i n e l e c t r o - c o r t i c a l a c t i v i t y j u s t d e s c r i b e d , was administered. The r e s u l t s of t h i s c h a l l e n g i n g dose of i s o p r o t e r e n o l are shown i n F i g u r e 34. A r e l a t i v e l y mild depressor e f f e c t i s observed f o l l o w i n g the a d m i n i s t r a t i o n of i s o p r o t e r e n o l i n the presence of both chlorpromazine and vasopressin (compare Figures 33 and 3 4 ) . F i g u r e 34 i l l u s t r a t e s a l s o the lack of any depression of e l e c t r o - c o r t i c a l a c t i v i t y , as w e l l as the absence of any apparent a c t i v a t i o n of the EEG ( i . e . , there was no s h i f t toward low v o l t a g e f a s t a c t i v i t y such as was demonstrated i n Figure 3 3 ) . Hence, when profound depressor e f f e c t s are prevented chlorpromazine blockade of i s o p r o t e r e n o l induced " a c t i v a t i o n " i s r e a d i l y evident. Chlorpromazine blocked the EEG " a c t i v a t i o n " u s u a l l y seen w i t h small (but competent) doses of amphetamine, although l a r g e r doses of amphetamine e l i c i t e d evidence of some degree of break-through of t h i s blockade. In the presence of chlorpromazine (2 mg/kg), amphetamine (racemic) was administered g r a d u a l l y , i n s u c c e s s i v e increments of 50/ug/kg. No e f f e c t on the ch Iorpromazine-induced deactivated p a t t e r n could be observed a f t e r a t o t a l of 150/ig/kg of amphetamine had been given ( F i g . 3 5 ) . However, an a d d i t i o n a l dose of 50 yug/kg of amphetamine, to make a t o t a l of 2 0 0/jg/kg, produced demonstrable changes i n the t r a c i n g . At t h i s p o i n t , an audiogenic stimulus ( w h i s t l e b l a s t ) produced signs of behavioural a l e r t n e s s (movements of the head end forepaws) and prompt type A a c t i v a t i o n , which l a s t e d 70 seconds longer than the d u r a t i o n - 72 -RF-P RP-0 I IJII 1 XN.E . 0.5 1 after CPZ a.Omg/Kg. Rp_p BP 85 80 70 ts Joo/A/ RP-0 LP-0 LF-4o Figure 33. Effect of isoproterenol 0.5/»g/kg on the EEG in the presence of CPZ (2 mg/kg) blockade. The low voltage fast activity noted in the tracing is not a typical activated response, but is believed to be due to anoxemia rather than to any activating effect of iso-proterenol. Note long latency following drug injection and very low level of blood pressure (38-40 mm Hg) coincident with the appearance of the high frequency low voltage pattern. - 73 -Figure 3^. Effect of isoproterenol 0.5/ug/kg on the EEG in the presence of CPZ blockade (2/jg/kg) and vasopressin 0.5 units/kg. Vasopressin injected 60 minutes ear l ier does not produce any change in the CPZ-spindles. Note that the lack of low voltage fast act iv i ty in the presence of CPZ blockade of i soproterenol -induced activation is evident when anoxic effects of the EEG are prevented by prior administration of vasopressin. - 74-L F - P t A d d . t i o n a j 50 r / K g 1 Amphetamine = ZOO total 150 Amphetamine _ M 1.0 mg./Kg C P Z RF-P B P qo J I LP-u »-t Whjstle Blast 1 Figure. 3S* Effect of amphetamine on the EEG in the presence of CPZ blockade, 2.0 mg/kg. Note the absence of any activation following 150^g/kg of amphetamine, whereas an additional 50 /jg/kg produces a change in the EEG from pattern D to B; k minutes later an audio-genic stimulus (whistle blast) elicits type A activation in which the length of activation exceeds the duration of the blast (see Fig. 28). - 75 -of the w h i s t l e b l a s t . This i s i n c o n t r a s t to the s h o r t e r " a c t i v a t i o n " p e r i o d (same d u r a t i o n as the d u r a t i o n of the s t i m u l u s , see Figure 28) e l i c i t e d by audiogenic s t i m u l a t i o n i n the presence of chlorpromazine alone. In c o n t r a s t to the responses to adrenergic amines, s e r o t o n i n and histamine, EEG " a c t i v a t i o n " produced by c h o l i n e r g i c agents ( a c e t y l c h o l i n e and -.serine) i s not a b o l i s h e d o r depressed by chlorpromazine. In the presence of chlorpromazine blockade (2 mg/kg), the i n t r a -innominate a d m i n i s t r a t i o n of a c e t y l c h o l i n e (2/tig/kg) always produces a s h i f t towards f a s t e r frequency low vo l t a g e a c t i v i t y . In view of the int e n s e , p r e c i p i t o u s and prolonged decreases i n blood pressure observed f o l l o w i n g the a d m i n i s t r a t i o n of depressor agents ( i s o p r o t e r e n o l and a c e t y l c h o l i n e ) i n the presence of chlorpromazine blockade, i t seemed necessary to determine whether o r not c e r e b r a l anoxia might be res p o n s i b l e f o r the a c t i v a t i n g e f f e c t of a c e t y l c h o l i n e i n t h i s circumstance as had been observed i n the case of i s o p r o t e r e n o l . Conseq-uentl y , the blood pressure i n t h i s p r e p a r a t i o n was s t a b i l i z e d w i t h 0.5 u n i t s of vasopressin kS minutes before the i n j e c t i o n of 2 /jg/kg of a c e t y l c h o l i n e . EEG " a c t i v a t i o n " e f f e c t s c h a r a c t e r i s t i c of a c e t y l c h o l i n e , as w e l l as the lack of a marked depressor response f o l l o w i n g the a d m i n i s t r a t i o n of t h i s compound, were observed under these circumstances. The i n j e c t i o n of 100 /Cig/kg of e s e r i n e alone over a period of time produces an intense " a c t i v a t i o n " (type A pattern) ( F i g . 21). The a d m i n i s t r a t i o n of chlorpromazine (2.0 mg/kg) f a i l e d to produce any a l t e r a t i o n i n t h i s a c t i v a t e d p a t t e r n even a f t e r a delay of 15 minutes. Increasing doses of chlorpromazine (2-5 mg/kg) were e q u a l l y without - 76 -e f f e c t on the EEG, although the animal appeared drowsy. In c o n t r a s t , 1 minute a f t e r the i n j e c t i o n of a t r o p i n e (0.5 mg/kg), the low voltage f a s t a c t i v i t y was changed to large amplitude slow waves, i n t e r r u p t e d at i r r e g u l a r i n t e r v a l s w i t h bursts of s p i n d l e a c t i v i t y a t 10-15 c/s. This e f f e c t of e s e r i n e coupled w i t h that observed w i t h a c e t y l -c h o l i n e i n d i c a t e s that i n the doses used i n these experiments, the a d m i n i s t r a t i o n of chlorpromazine i s without any " b l o c k i n g e f f e c t " on the e l e c t r o - c o r t i c a l arousal which i s produced by c h o l i n e r g i c agents ( a c e t y l -c h o l i n e and e s e r i n e ) . ( i i ) Promazine. The EEG a l t e r a t i o n s and v a s c u l a r responses produced by promazine are a l s o very s i m i l a r to those reported i n these experiments f o r chlorpromazine. However, a l a r g e r dose of promazine seems necessary to produce the same a l t e r a t i o n s . Whereas 2.0 mg/kg of chlorpromazine was s u f f i c i e n t to e s t a b l i s h blockade of EEG " a c t i v a t i o n " , when challenged w i t h adrenergic amines i n concentrations known to produce c r i s p " a c t i v a t i o n " , k to 5 mg/kg of promazine was needed to produce a s i m i l a r b l o c k i n g e f f e c t . In a d d i t i o n , the d u r a t i o n of the "blockade" produced by promazine appeared s h o r t e r than that observed w i t h chlorpromazine and seemed less e f f e c t i v e a t the end of 2 hours. These q u a n t i t a t i v e d i f f e r e n c e s between the e f f e c t s of chlorpromazine and promazine may be due i n p a r t to the f a c t that the conc e n t r a t i o n s o f promazine s o l u t i o n s used i n these experiments were made up by d i l u t i o n of intravenous ampoules of i n j e c t a b l e s o l u t i o n s f u r n i s h e d by the pharmaceutical manufacturer cf t h i s compound, and amounts used were based on l a b e l concentrations as claimed by the drug house. - 77 -(b) Phenoxybenzamine ( d i b e n z y l i n e ) . The use of adrenergic b l o c k i n g agents of the beta-halo-alkylamine c l a s s (dibenamine, d i b e n z y l i n e ) has c o n t r i b u t e d much to our knowledge of the e f f e c t s and the a c t i o n s of adrer. r g i c amines at p e r i p h e r a l s i t e s . However, remarkably l i t t l e a t t e n t i o n seems to have been paid to the adjuvant r o l e which these agents may play i n e v a l u a t i n g the p o s s i b l e responses of autonomic agents at c e n t r a l s i t e s w i t h i n the c e n t r a l nervous system. Furthermore, the a b i l i t y of adrenergic b l o c k i n g agents to i n h i b i t the CNS responses to adrenergic amines i s q u i t e u n c l e a r . The p o s s i b l e a b i l i t y of phenoxy-benzamine to produce e f f e c t i v e blockade of adrenergic-induced EEG a c t i v a t i o n was i n v e s t i g a t e d i n 8 c a t s . F o l l o w i n g the slow intra-innominate a d m i n i s t r a t i o n (25 seconds) Of phenoxybenzamine, the EEG becomes a c t i v a t e d f o r s e v e r a l minutes (10 minutes or more). The d u r a t i o n of the a c t i v a t i n g response v a r i e s w i t h the dose administered, " a c t i v a t i o n " being longer w i t h l a r g e r doses. Fi g u r e 36 i l l u s t r a t e s EEG " a c t i v a t i o n " which l a s t e d more than 30 minutes f o l l o w i n g the i n j e c t i o n of an e f f e c t i v e b l o c k i n g dose of phenoxybenzamine (I mg/kg). A p e r i o d of 30 to kO minutes i s required f o r phenoxybenzamine blockade to be e s t a b l i s h e d , and c h a l l e n g i n g agents were not tested e a r l i e r than 60 minutes f o l l o w i n g the a d m i n i s t r a t i o n of phenoxybenzamine. As w i t h chlorpromazine, the doses of various com-pounds required to e l i c i t d i s t i n c t a c t i v a t i n g e f f e c t s were f i r s t e s t a b l i s h e d " and then these doses were tested i n the same pr e p a r a t i o n f o l l o w i n g the a d m i n i s t r a t i o n of phenoxybenzamine. The c a p a c i t y of phenoxybenzamine i n a dose of 1 mg/kg to block e f f e c t i v e l y the EEG a c t i v a t i n g e f f e c t s of i s o p r o t e r e n o l , epinephrine, norepinephrine and - 78 -RF - P RP-0 IM LF-P DBZ I.Omg/Ko. J 5~ i V R F - P B.P. IM RP 0 LP-0 l 5 " v " > Figure 36. Effect of phenoxybenzamine (DBZ) i.O mg/kg on the EEG. Activation somewhat delayed and gradual and lasting for more than 30 minutes with an abrupt return to control pattern. A s l ight drop in blood pressure is noted (12 mm Hg) . - 79 -histamine are iI l u s t r a t e d in Figures 38, 41 and 43. ( i j Adrenergi c amines: i s o p r o t e r e n o l . e p i n e p h r i n e and nor- epinephrine. F i g u r e 37 i l l u s t r a t e s the c o n t r o l t r a c i n g f o l l o w i n g the i n j e c t i o n of 6.5/\jg/kg of i s o p r o t e r e n o l . This i s c h a r a c t e r i z e d by prompt and c l e a r - c u t " a c t i v a t i o h " which l a s t s more than 60 seconds. In F i g u r e 38, taken from the same animal a f t e r pre-treatment With phenoxybenzamine (1 mg/kg), the i n j e c t i o n of the 0.5/ug/kg of iso p r o t e r e n o l f a i I s to produce ah a c t i v a t e d EEG p a t t e r n , s i m i l a r to that which was obtained before the a d m i n i s t r a t i o n of phenoxybenzami ne. Furthermore, i n c r e a s i n g the dose of i s o p r o t e r e n o l to 1/ug/kg ( F i g . 39) does not produce an a c t i v a t e d p a t t e r n ; despi te the f a c t that t h i s dose i s approximately 3-4 times that dose which has been demonstrated to produce c o n s i s t e n t EEG ' ' a c t i v a t i o n " (type A). i n a l 1 preparations which have not been treated w i t h adrenergic b l o c k i n g agents. The depressor responses to iso p r o t e r e n o l f o l l o w i n g phenoxybenzamine blockade are comparable i n many respects to those observed in the c o n t r o l t r a c i n g s but d i f f e r from the c o n t r o l record i n the f a c t that the blood pressure has a tendency to remain at low 1evels f o r longer periods of time (more than 90 seconds). the a d m i n i s t r a t i o n of epinephrine and norepinephrine i n "e q u i -a c t i v a t i n g " doses, 2 hours a f t e r I mg/kg of phenoxybenzami ne, l i k e w i s e f a i l s to produce any a l t e r a t i o n in 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 . In F i gure 46 pressor responses as w e l l as EEG " a c t i v a t i o n " produced by 0.5 /Jg/kg of epinephrine i n the absence of phenoxybenzami ne are i l l u s t r a t e d . In c o n t r a s t , no a c t i v a t i n g e f f e c t i s evoked when a s i m i l a r dose of epinephrine Is i n j e c t e d i n t o the same p r e p a r a t i o n 2 hours a f t e r treatment w i t h 1 mg/kg of phenoxybenzamine ( F i g . 41 ) . - 80 -RF-P L P-0 r - u LF-P , j T I . N E . 0 . 5 ^ K g . t . ' F l ^ ' ' pp.p B.R los go 1 M RPto M sec. 70 80 Figure 37. Effect of isoproterenol 0.5/ug/kg on the EEG before phenoxybenzamine blockade. Note the Immediate and intense type A activation of long duration which is unrelated to the vascular responses of this agent. - 81 -t RP-0 LF-P T I .NE O . S » / K g . I after DBZ 1.0 rrg./Kg. , _____ , -__^\/ RF-P B.R _ * 1 W 5C Figure 33. E f f e c t of Isoproterenol 0.5/tig/kg on the EEG a f t e r phenoxy-benzamine blockade 1.0 mg/kg. Absence of any a c t i v a t i o n and continued p e r s i s t e n c e of s p i n d l e a c t i v i t y . Note the depressor response. - 82 -1 1 h i m i RP-0 t I.N.E. ,.or/kg. T , oft-r DBZ. I.Omg/Kg- . . 300*. V R F . p B.R q S 7 S 55 * ~ 1 m RP-o LF-P m Figure 39. Effect of Isoproterenol 1.0/Ug/kg on the EEG after phenoxybenzamine blockade 1.0 mg/kg. Absence of any change in the EEG, even after a larger dose of isoproterenol. - 83 -R F - P L F P i > t Epv o . 5»/kq. 1 , . . 300<-.V 5 s e t I • ' R F p B.P. 115 120 1 |25 RP-0 LP-0 L F - P 135 120 Figure 40. Effect of epinephrine 0.5/ug/kg o n t n e E E G before phenoxybenzamine blockade. Control record for comparison with Figure 41. - 84 -RF-P JA s RP-0 LP-o LF-P( T Epi O-Sly/Kg afteri . DBZ. l.O rng./Ka. . — — , RPf & R 9 0 90 1 « L F - P 75 70 «0 Figure 41. Effects of epinephrine 0.5/ug/kg on the EEG after phenoxy-benzamine blockade 1.0 mg/kg. No change is apparent in the EEG, but typical epinephrine reversal is obvious. - 85 -Moreover, under these circumstances, epinephrine does not produce its characteristic pressor response, and instead typical epinephrine reversal is obtained (Fig. 41). In the same preparation the administra-tion of norepinephrine in a dose of 0.5-1.0/tig/kg failed to elicit any demonstrable EEG "activation" and did not produce its typical pressor response in the presence of this dose of phenoxybenzamine. (ii) Hi stami ne. The injection of histamine in usually effective doses also is equally without EEG activating effect in the phenoxybenzamine-treated preparation (Fig. 43). Blockade of the electro-cortical responses to histamine is obtained within 50 minutes following phenoxybenzamine administration. This blockade persists for more than 24 hours, and appears to be characterized by the same persistence and stability as the blockade of adrenergic amines. Figure 42 illustrates the control activating response of more than 50 seconds duration occurring 15 seconds after the intra-innominate injection of a dose of 2.0/lig/kg of histamine. In contrast, in the same cat pre-treated 60 minutes earlier with phenoxybenzamine ( 1 . 0 mg/kg), the administration of an "equi-activating" dose of histamine does not elicit any change in the already deactivated EEG (Fig. 43). Furthermore, no significant differences are noted between depressor effects observed in the control and the test conditions; in the control, a fall in blood pressure of 25 mm Hg occurs within 25 seconds, whereas in that same time interval a depressor response of 20 mm Hg is observed under the test conditions. (iii) Acetylcholine. The administration of this compound in a dose of 2 /jg/kg in the presence of phenoxybenzamine blockade did not - 86 -RF-P RP-Q L Ll Uiii i U L F - P J t His_mm_ _.oy%i . *" • ' 3<M«V 5 sec. r Rpp B.P. 95 7 5 7 0 > » RP-0 LF-P M * * ^ ^ . « ^ ' w w » ^ , • J . , _ , i , . , ' , , ^ _ . _ - -a.. ' , . , , . T f * y ^ ^ ; | t T t ^ f 1 f t - f ^ ^ Figure 42. Effect of histamine 2.0 /ig/kg on the EEG before phenoxy-benzami ne. Control tracing for comparison with Figure 43. Activation somewhat delayed (10 seconds) but intense (type A) occurring before the peak of the depressor response with a gradual return to previous spindling. - 87 -, RF-P RP LF-P t Histamine 2.0 »/Kq. 1 , , ofUr DBZ l .omg. /Kg. _ p _ _ , pr:p B-P. loo 95 J BO In f LF-P F i g u r e 43. Effect of histamine 2.0 /Ug/kg after phenoxybenzamine i.0 mg/kg. Absence of any EEG activation and continued persistence of spindling. - 88 -e x h i b i t any s i g n i f i c a n t a l t e r a t i o n s i n i t s c h a r a c t e r i s t i c v a s c u l a r responses. Furthermore, t y p i c a l EEG " a c t i v a t i o n " was produced c o n s i s t e n t l y by the i n j e c t i o n of 2/Ug/kg of a c e t y l c h o l i n e i n the presence of 0 .5 mg/kg phenoxybenzamine. Increasing the dose of phenoxybenzamine to 1.0 mg/kg f a i l e d to block the EEG a c t i v a t i n g e f f e c t s of s i m i l a r amounts of a c e t y l c h o l i n e . Figure 44 i l l u s t r a t e s the EEG " a c t i v a t i o n " obtained f o l l o w i n g the a d m i n i s t r a t i o n of 2/Ug/kg of a c e t y l c h o l i n e i n the presence of phenoxybenzamine blockade ( e s t a b l i s h e d by doses of 0 .5 mg/kg and of 1.0 mg/kg r e s p e c t i v e l y ) . ( i v ) S e r o t o n i n . The a d m i n i s t r a t i o n of s e r o t o n i n i n doses of 2.5/ug/kg and 5.0/ug/kg was observed to produce pressor responses and EEG " a c t i v a t i o n " i n the presence of phenoxybenzamine blockade e s t a b l i s h e d by the i n j e c t i o n of I mg/kg Of phenoxybenzamine. In the m a j o r i t y of cases the s m a l l e s t dose of s e r o t o n i n observed to e l i c i t c o n s i s t e n t and d i s t i n c t EEG " a c t i v a t i o n " f o l l o w i n g the administra t i o n of phenoxybenzamine was 2 . 5/ig/kg, but i n a few preparations i t has been p o s s i b l e to produce " a c t i v a t i o n " w i t h as low a dose as 1 /Ug/kg. Comparisons of the e f f e c t s of t h i s compound i n doses of 2 .5/Ug/kg and 5.0 /Jg/kg, both before and a f t e r the a d m i n i s t r a t i o n of phenoxybenzamine (1 mg/kg) i n the same p r e p a r a t i o n , reveal that although " a c t i v a t i o n " of s i m i l a r i n t e n s i t y i s produced i n both circumstances, the d u r a t i o n of " a c t i v a t i o n " i s approximately 20 seconds'shorter f o l l o w i n g the ad m i n i s t r a -t i o n of phenoxybenzamine ( F i g s . 45, 46, 47 and 48) . However, such v a r i a t i o n s i n the d u r a t i o n of " a c t i v a t i o n " are o c c a s i o n a l l y seen i n the absence of b l o c k i n g agents. - 89 -RF-P RP-0 fan*. 2.o*/K3- i , . , 1*5- M M . a f t e r O B Z . O . S m j . / K g . , , u 5 sec. RF-P BP 92. L P - 0 , BP lot t ACH z . o r / K o . J |U> M b a f t e r 0 8 Z l . o m j / K g Figure 44. Effect of acetylcholine 2.0^g/kg after phenoxybenzamine 0.5 and 1.0 mg/kg. In both instances (upper and lower tracings) EEG activation consists of a shift from pattern D to B, occurring before , the peak depressor response. - 90 -RF-P DD r, LF-P t Seretonm. a-s Ij/Kg. I ca n 5 set. B.P. izo 130 RF-P I 300/<-RP-0 LF-P • , ill J|J LP-0 IV) Figure 45. E f f e c t of s e r o t o n i n 2.5/tig/kg on the EEG before phenoxy-benzami ne. Control t r a c i n g f o r comparison w i t h Figure 46. Immediate a c t i v a t i o n (approximately 5 seconds) preceding the peak pressor response w i t h a gradual r e t u r n to the p r e - I n j e c t i o n p a t t e r n ; w h i l e the blood pressure i s s t i l l e l e v a t e d . - 91 -•t R F - P RP-0 t i«rdb»»»v. £.5 if/Kg 1 o H t r 0 6 Z 1.0 ma/Kg. 30O/J.V MB R F - P & P - 1 0 6 RP-0 L P LF-P Figure 46, Effect of serotonin. 2.5/*g/kg on the EEG after phenoxy-benzamine 1 mg/kg. EEG activation consisting of a change in pattern from 0 to A occurs in 5 seconds, and Is similar in Intensity to the control tracing, but appears to be less prolonged (Fig. 45). The pressor response is similar to that seen in Figure 45. - 92 -RF-P R P O S\ 'I I LP-0 LF-P I S e r o W n 5 . 0 ^ X 0 . i RF-P BR H5 3 0 0 / 0 / \m r 111 I'M T RP-0 20 s e t . LP-0 LF-P 1 3 0 Figure 47, Effect of serotonin 5.0 /ug/kg on the EEG before phenoxy-benzamine 1 mg/kg. Control tracing for comparison with Figure 48, - 93 -t SertAorui\ S.O » / K g 4 af te r DBZ. 1.0 m g / k g . r>r _ B .R UO RFP RP-o LP-0 Ficjure kd. Effect of serotonin 5.0/jg/kg on the EEG after phenoxy-benzamine 1 mg/kg. Intense and immediate EEG activation (type A) occurring before the peak of the pressor response, with an abrupt return to the control pattern. The pressor response is similar to that seen in Figure U7. - 94 -(c) D i ch1o ro i sop ro te reno1 (DCI). This compound ( L i l l y 20522) i s the 3,4 dichloro-analogue of i s o p r o t e r e n o l . Observations made during t h i s study have pointed to the prompt and c l e a r - c u t EEG " a c t i v a t i o n " obtained f o l l o w i n g the a d m i n i s t r a t i o n of low doses (0.5/jg/kg) of i s o p r o t e r e n o l . The recent demonstration by Powell and S l a t e r (134) and others (4,59,106,121) that l - ( 3 , 4 - d i c h l o r o p h e n y l ) - 2 -isopropyl amino ethanol (DCI) blocks the hypotension and tach y c a r d i a caused by i s o p r o t e r e n o l a t p e r i p h e r a l receptor s i t e s , prompted our e f f o r t s to a s c e r t a i n whether or not the p r i o r a d m i n i s t r a t i o n of the analogue w i l l b l o c k o r modify is o p r o t e r e n o l - i n d u c e d EEG " a c t i v a t i o n " . F o l l o w i n g the intra-innomlnate a d m i n i s t r a t i o n of DCI i n the unanaesthetized de-afferented c a t , the type D s p i n d l e a c t i v i t y c h a r a c t e r -i s t i c of the pr e p a r a t i o n i s converted w i t h i n a few seconds to a type A a c t i v a t e d p a t t e r n . Depending on the dose administered t h i s a c t i v a t e d p a t t e r n may be t r a n s i e n t o r prolonged. With small doses of 2-5 mg/kg • a c t i v a t i o n " l a s t s f o r several minutes. Larger doses (15 mg/kg) e l i c i t EEG patterns of low voltage f a s t a c t i v i t y which l a s t f o r more than I hour and are sometimes a s s o c i a t e d w i t h movements of the head and forepaws. The p r e l i m i n a r y i soproterenol-1 ike e f f e c t of DCI on the EEG p r i o r to the appearance of i t s b l o c k i n g a c t i o n p a r a l l e l s the e f f e c t s of t h i s compound on the c a r d i o v a s c u l a r system as reported by Dresel (45) and others (50,121). The EEG " a c t i v a t i o n " produced by large doses of DCI, although normally intense and of long d u r a t i o n , can be reversed by the a d m i n i s t r a -t i o n of doses of chlorpromazine as low as 2-4 mg/kg ( F i g . 49). Moreover, the c a r d i o v a s c u l a r responses ( t a c h y c a r d i a and depressor e f f e c t s ) noted f o l l o w i n g the a d m i n i s t r a t i o n of t h i s analogue of is o p r o t e r e n o l are - 95 -RP-0 i > I hour LP-0 , F-P t DC I 15 mj./Kg. J Rpp B P IOO RP-o 95 LP-0 3 * * LF-P T CPZ 3.0mo/K3- '1 O f e r r x 7 l 5 r ^ K g . 120 4_ Figure 49. Effect of DCI 15 mg/kg. on the EEG. Intense activation promptly elicited by DCI. In this dosage, activation lasted well over an hour. Note that activation has persisted long after any evidence of cardiovascular effects (upper tracing). Blockade of DCI induced EEG activation is readily produced by administration of CPZ (3 mg/kg). - 96 -similar to the effects observed with injections of the parent compound. (i) Isoproterenol. In evaluating the possible interaction between isoproterenol and DC! the procedures adopted were similar to thoseuti1Ized during the investigation of the central blocking effects of phenoxybenzamine and chtoropromazine. In every case, proven "activating" doses of Isoproterenol and other "activators" intended for use as challenging agents were tested in the same preparation before and again not less than 30 minutes after the administration of DCI. Under these circumstances the administration of 0.5/ug/kg of isoproterenol failed to produce any EEG "activation" in preparations previously treated with 7 .5 mg/kg of DCI (Fig. 5 0 ) . In addition, the usual depressor responses associated with isoproterenol injections were not observed, and the blood pressure remained near control levels. Increasing the dose of isoproterenol to 1 /Ug/kg produced short-lived "activation" (20 seconds) without any alterations in vascular responses (Fig. 50 . However, when the dose of the blocking agent is increased to 15 mg/kg in the same preparation, a subsequent dose of J flig/kg of isoproterenol fails to produce any change in the cortical electrical activity (Fig. 5 2 ) . (ii) Epinephrine and norepinephrine. No attempt was made to evaluate in detail the activating effects of the other biogenic amines when these agents are administered in the presence of DCI blockade. At present, work is^progress which may shed more light on this problem. However, preliminary and less detailed investigations with epinephrine and norepinephrine in adequate doses of 0.5/ug/kg have revealed that EEG - 37 RF-P MM t I.N.E. 0.5v/Kg. i a f t e y D C I 7.5"mg./Kg. RF-P S 8 5 8 5 S M c 1-A V , Figure 50. Effect *f isoproterenol 0.5Aig/kg on the EEG after DCi 7.5 mg/kg. Note complete blockade of isoproterenol-induced activation, and also greatly reduced depressor response. - 98 -1 I.N.E, I .Ol/Kq t alter DCI I.Smg/Kg. ftp.p BR 105 LP-o LF- P _ Figure 51. Effect of isoproterenol l.O/ug/kg on the EEG after DCI 7.5 mg/kg. In this relatively large dose of isoproterenol, fleeting activation occurred (break through of blockade). Compare with Figure 52. - 99 -Figure 52. Effect of isoproterenol i yug/kg on the EEG after DCI 15 mg/kg. Upper tracing: typical DCI-induced activation lasting for more than 2 hours. Lower tracing: relatively large dose of isoproterenol fa i l s to break through the blockade produced by a larger dose of DCI; compare with Figure 51. - 100 -"activation" may be produced by these agents in the presence of blockade established with doses of 7.5-15 mg/kg. Under these conditions the evoked pattern of EEG "activation" fluctuated between type 8 and C (Figs. 53 and 5 4 ) . In addition, more clear-cut and distinct "activation" (type A) was observed when larger doses (2.0/Ug/kg) of epinephrine and norepinephrine were administered in the same preparation in which 15 mg/kg of DCI had been previously injected (Figs. 55 and 5 6 ) , and whichJ.0/ug/kg of isoproterenol was without EEG effect. (iii) Acetylcholine and serotonin. In contrast, the administration of 2.0/jg/kg of acetylcholine (Fig. 58) and 5.0 /Ug/kg of serotonin (Fig. 60) in the DC I-treated preparation ( 7 . 5 mg/kg) always produced definitive EEG "activation" similar in characteristics to the pattern observed follow-ing the injection of equivalent doses of these compounds in the same preparation before DCI administration (Figs. 57 and 5 9 ) . Furthermore, the duration of the acety1cho1ine-induced response was appreciably shorter (35 seconds) in the preparation pre-treated with DCI, Similar effects were not observed for serotonin. These preliminary observations seem to suggest that the activating effects of serotonin and acetylcholine are not blocked by the administra-tion of DC! In the doses used in these studies. Furthermore, initial evidence gives the impression that EEG activating effects could be obtained with epinephrine and norepinephrine in doses which are equivalent in activating potency to those doses of Isoproterenol which are completely ineffective in the presence of DCI blockade. However, without further work it is difficult to say unequivocally that DCI is more potent in blocking the EEG effects of isoproterenol than it is In blocking - 101 -RF-P in I *'»" ilitiMH'lgW» r 5 RP-0 LP-0 LF-R t E>i. i.ov/Kg. 1 . . oiter rSc.r IS mo ./Kg- | 0 £ ) , , I^ Jy/ 3 .R 15 RF-P L P - 0 R P - o LF-P i a s 120 Figure 53. Effect of epinephrine 0.5 /ig/kg on the EEG after DCI 7.5 mg/kg. Moderate activation fluctuating between patterns B and C i l lustrate incomplete blockade of epinephrine by DCI. Note lack of effect on the pressor response to epinephrine. 102 R F - P RP-0 L P - 0 L F - P RF-P 1,0 W M , RP-0 LP-0 LF-P 140 Figure 54. Effect of norepinephrine 0.5 /ig/kg on the EEG after DCI 7.5 mg/kg. EEG patterns noted here are s imiHr in appearance to those of Figure 53. Fluctuating potential associated with muscular movement (upper tracing) should not be confused with the spindjing of deactivation (lower tracing). Note the marked pressor response in the presence of DCI blockade. « - 103 -RF-P l i f i mam ^ HMwtwmimit mm LF-P t Epi. 0.5 r/kq. I , 0/te.v DCI T S ^ K g . - R F - P B.P. ss RP-C J HII LP-( 120 Figure 55. Effect of epinephrine 2 /tig/kg on the EEG after DCI 15 mg/kg. Intense activation (type A) occurring after 8 seconds with reversion to the control pattern while the blood pressure is s t i l l at maximal levels. Little evidence of blockade by large dose of DCI. - 104 -RF-P I ml! t N . E . i . O l / K g . a f t e r J B.P. 8 ? D t I 1 5 RF-P RP-0 Til II 1 ITT I M I 1 ni iTilI i l l LP-0 LF-P \15 Figure 5&. Effect of norepinephrine 2 A*g/kg on the EEG after DCI 15 mg/kg. Intense activation (type A) occurring after a long latent period of approximately 13 seconds, with reversion to the control pattern while the blood pressure is s t i l l at maximal levels. - 105 -1 ACH 2.0 If/Kg. t , b.for* DCI 300/i.V LP-0 LF-P i I Figure 57. Effect of acetylcholine 2 ug/kg on the EEG before DCJ. Typical and immediate activation (type A, occurring before the peak of the depressor response. - 106 -R F " P 1 I i LF-P *** T ACH 2 . 0 » / K g . J . a f t e r DCI 1.S rr.j/Kg. | JOO-.V B.R qs LP-0 LF-P 8 5 •Figure 5£. Effect of acetylcholine _ vgAg on the EEG after DCI 7.5 mg/kg. Absence of blockade i l lustrated by intense type A activation, which is rapid in onset (6 seconds). 107 -RF-P RP-o I mm 1 Serotolim S.O*/Kg. ) RF-P B.P. « 300>cV LF-P M S Figure 59. Effect of serotonin 5/Jg/kg on the EEG before DCi, 7.5 mg/kg. Prompt and intense activation, fluctuating between type B and A, lasting about 60 seconds and reverting to the control pattern while the pressor response is s t i l l maximal. - 103 -RP-0 LP-0 W . . . . l i ^ . . ^ - . . . t r i | . . - r . . 1 y . | ^ i v . .|..1|T ) r f | l ) l 1 l l T r | n r | t||,.. |..;l|.t||.| 1!^ ,^ ,^ tSeYofem.r, S-oykoi . g_p ait** DCI 7 1 3 r^j/K RF-P RP-0 LP-0 115 Figure 60. Effect of serotonin 5/Ug/kg after DCI 7.5 mg/kg. No evidence of blockade. In the presence of DCI, typical serotonin induced EEG activation and pressor response. Again note termination of activation while pressor response rema i ns max i ma 1. - 109 -similar responses of epinephrine and norepinephrine, although this is the Impression gained. (d) Atropine. The intra-innominate administration of atropine in adequate doses (0.5 mg/kg) alone or following the injection of other drugs, produces mydriasis and a long-lasting (several hours) deactivated EEG pattern, characterized in the majority of preparations by high amplitude slow waves interrupted by regular periodic bursts of spindle activity (12 to \k c/s). This activity, which occurs after a latency of 3-6 minutes, is similar in some respects to the activity seen in the relaxed, drowsy or sleeping state of the unanaesthetized de-afferented cat, but the amplitude is larger and the spindles seem to make their appearance with more regularity than that observed for the control preparation. Further-more, although atropine-induced resting or sleeping-type patterns characterized the EEG, the preparations remained behaviourally awake, as manifested by movements of the head and forepaws. In addition, audio-genic or visual stimulation which ordinarily would alert the preparation and evoke temporary EEG "activation" produced the expected behavioural responses but failed to elicit any change in the electrical patterns. The administration of effective activating doses (determined prior to atropine administration) of acetylcholine (2/jg/kg), histamine (2/ug/kg) , and serotonin (5 A»g/kg) , and of epinephrine, norepinephrine and isoproterenol (0.5/ug/kg of each agent), all failed to produce "activation" of the EEG in the presence of subsequent atropine blockade in the same preparation. Figure 61 iI lust rates these observations for epinephrine (0.5 /Ug/kg) and acatyIcho 1 ine (2.0/Ug/kg). The effects of 5/Ug/kg of serotonin are illustrated in the lower half of Figure 62. - no Similarly, no alterations in the EEG were observed following the administra-tion of very large doses of the adrenergic amines (4 times the dose previously used) in the same animal. These observations are illustrated for isoproterenol (2/Ug/kg) in the upper half of Figure 62, and for epinephrine and norepinephrine (2 ^ ig/kg of each agent) in Figure 63. In contrast, the administration of eserine in doses of 100-200 /ug/ kg can overcome (after a delay of about 25 minutes), the deactivation which results from an injection of 0.5 mg/kg of atropine. Furthermore, adequate doses of eserine can produce EEG "activation" which, though lessened in duration, is similar in some respects to the "activation" observed when this compound alone is given in the same animal before atropine administration. As illustrated in Figure 64 the effect of 0.5 mg/kg of atropine on eserine-induced EEG "activation" Is followed approximately 2 minutes later by the appearance of atropine spindles. At this point, eserine in a dose of 100/Ug/kg was administered. A reduction in the spindle activity is noted 16 minutes later, and the progressive transition to a more activated EEG pattern can be observed. C. Preparation With Bilateral Carotid Sinus Denervation. 1. Controls. There is considerable lack of agreement on the possible influences which receptors in the carotid sinus may exert on the production of EEG "activation" following the intravascular injection of pharmacological agents (see Discussion). It seemed possible that the electrocortical alterations produced by the direct administration of the agents used in this investigation I l l -RF-P 1 C _ D f Epi csr/Kg. l' ~ f — after Atropine aSmj/Ka. | J ^ A / D C D B P 1? 5 s e c . RF.p a P. is s « * • 1 RP-0 T ACH a .o v / k g . 1 B P S O a f t e r A t r o p i n e . O.S m a / K g . A F f gu re 61. Effect of epinephrine 0.5Ajg/kg and acetylcholine 2.0 ug/kg on the EEG after atropine 0.5 mg/kg. Complete blockade of both epinephrine and acetylcholine induced activation is evident. - 112 -LF P t I.N.E. i.O*/K.o. I oiter Atropine O-Smg./Kg. RF-P • L p | | | ) | l j | l | ' ! ; i B.P. So t Serotonin S . 0 / K 9 . 1 after A t r o p i n e a . S m g . / K g . Figure 62. Effect of isoproterenol 2 /ug/kg and serotonin 5 /Jg/kg on the EEG after atropine 0.5 mg/kg. Complete blockade of adrenergic and serotonin-induced activa-tion is evident. - 113 -R F - P t Epi. a.os/Kg. 4 , after Atropine 0 . 5 mjj/Kg. i _  Mi J -1 B.R <)0 t N.E. J.Ol /kg. 4 after Atropine O.S mg./Kg. Figure 6 3 . Effect of epinephrine 2 /ug/kg end norepinephrine 2/Ug/kg on the EEG after atropine 0 . 5 mg/kg. Complete blockade of adrenergic activation. - 114 -2. mm Ccd eseYiruzed RF:P T Atropine 0.5 nwj./Kc). 1 1 I 16 n>io l a t e v 27roir> l a t e r Figure 64. Effect of atropine 0 .5 mg/kg on the EEG after eserine 50 /ug/kg. Atropine abol ished eserine induced activation. Larger dose of eserine (100/ug/kg) gradually overcame atropine blockade. - 115 could be due to reflex effects induced by carotid sinus mechano- or baro-receptor influences, and studies were undertaken to try to ascer-tain whether acetylcholine, serotonin and the adrenergic amines would still elicit EEG "activation" in the unanaesthetized de-afferented preparation in which bilateral denervation of the carotid sinuses had been performed. The technique of carotid sinus denervation and the evaluation of the effectiveness of this procedure has already been treated under section B of Methods. Any preparations so treated which exhibited compensatory vascular adjustments to bilateral clamping of the common carotids were not used in the experiment; nor were preparations employed in these studies which demonstrated continuous ooziness from the "stripped" areas of carotid arteries. The drugs used for study under such conditions were acetylcholine (0.5/ug/kg), serotonin (5.0 /Ug/kg) and the three short-acting adrenergic amines (0.5/Ug/kg). In every case, each drug to be tested was evaluated for its "activating" threshold in the same preparation before carotid sinus denervation. Figure 65 illustrates the control tracing following the injection of 0.3 ccs of normal saline. Close comparison with tracings from preparations with intact carotid sinuses reveals that in preparations in which the carotid sinuses have been denervated, spontaneous deactivation Is associated with an Increase in high amplitude slow wave activity and a decrease in the degree and intensity of spindling. These patterns bear some resemblance to those seen after chlorpromazine admini stration. - 116 -Fi g u r e 65. Effect of control injections of saline on the EEG after bilateral carotid sinus denervation. Control injections of saline are without effect. - 117 -Failure of this preparation to develop an appreciable neurogenic hypertension presumably is due to the elimination of higher central influences on sympathetic outflow below the level of spinal section. In this preparation, al 1 t h a agents studied produce definite EEG "activation" and varying degrees of vascular responses. 2. Effects of Pressor Agents: epinephrine, norepinephrine and  serotonin. Following the administration of 0.5/ug/kg of epinephrine, "activa-tion" occurred after a latent period of 10 seconds and persisted for approximately 2 minutes (Fig. 66). Similar responses were observed with norepinephrine. Serotonin in doses of 5/Ug/kg produced low voltage fast activity (type A pattern in about 6 seconds, with a duration of approximately 60 seconds (Fig. 67)). With both of these agents, pressor responses ranging from 10 to 20 mm Hg were observed following the doses mentioned. 3. Effects of Depressor Agents: acetylcholine and Isoproterenol. EEG "activation", developing 14 seconds after the administration of 0.5^ig/kg of acetylcholine and lasting for 1 minute, is demonstrated in Figure 68, In the absence of compensatory baro-receptor reflexes, the depressor responses observed with this agent (35 mm fall in 15 seconds) were more precipitous than those seen in the intact preparation (15 mm fall in 15 seconds). Larger doses, \-2 flag/kg, also produced EEG "activation", but the depressor effects were so intense that electro-cortical changes suggestive of cerebral anoxia subsequently developed. This condition, if not reversed promptly by adequate supportive measures, - 118 -^ p Carotid Sinosea TJervervoied ^ | R P - 0 I R P - 0 Lp-o [if n ] f f n l i m LF-P I t Ep'l. 0 5 T / k g . i R F . p BP 80 n 5 * " 1 ,oo 9 RP-0 / LF-P 100 10 J5 Figure 66. Effect of epinephrine 0.5/ug/kg on the EEG after bi lateral carotid sinus denervation. Typical epinephrine induced activation (D to B to A) can be observed. - 119 -Carotid Smustt Denervateo lllll'l I I I M I I M I I I II I I I I i I I flTflt mm | Serotcmin. I 1 1 1 "^T, 110 m^*¥^m^m^»>>0> ^ ^ ^ ^ ... LF-P 105 S8 SS q» Figure 67. E f f e c t of s e r o t o n i n 5/ug/kg on the EEG a f t e r b i l a t e r a l c a r o t i d s i n u s d e n e r v a t i o n . Prompt and i n t e n s e a c t i v a t i o n ( t y p e A p a t t e r n w i t h i n 6 seconds) f o l l o w i n g s e r o t o n i n a d m i n i s t r a t i o n . - 120 quickly led to Irreversible cerebral deterioration. In Figure 69 the sequential effect of 0.5/ug/kg each of isoproterenol and norepinephrine are i l lustrated. Approximately 8 seconds following the injection of ispproterenol, type A "act ivat ion" occurs. This is short-l ived (10 seconds) and gives way to a pattern of varying amplitude and frequency which quickly flattens out. This " f lat tening" of the EEG occurs at depressor levels of 40 mm Hg and seems similar to, but may not be identical with the anoxic pattern reported by others (62) and noted above following chlorpromazine administration. At this point the administration of 0.5/tjg/kg of norepinephrine provokes immediate "act ivat ion" as well as rapid restoration of vascular effects. Inquiry into the transient activation observed with isoproterenol in this instance again suggested the poss ib i l i ty that concomitant factors, occurring just previous to the presumed anoxic effect, could have been associated with this electro-cort ical change. In order to rule out this poss ib i l i ty , 1 mg/kg of phenoxybenzamine was administered to the carotid sinus denervated preparation, and 60 minutes later a similar challenging dose (0.5/ug/kg) of isoproterenol was given. Figure 70 i l lustrates these results. No EEG "act ivat ion" can be observed after isoproterenol administration in the presence of phenoxybenzamine blockade, despite the fact that the depression of the vascular responses and the length of time taken to achieve this degree of depression are comparable in this instance with those of the preparation in which phenoxybenzamine was not administered. Furthermore, the " f lattening" of the EEG which is noted at 40 mm Hg is rapidly reversed by the administration of a dose of - 121 -RF-P C a r e t . , , S i . u » e s Dene»v_«4 1 L F - P t A C H . O . 5 T / K 3 . 1 BP. no RF-P qo 300/*V (1 • to Figure (68-E f f e c t of a ce ty l cho l i ne 0.5 ug/kg on the EEG a f t e r b i l a t e r a l c a r o t i d sinus denervat ion. Ac t i v a t i on produced, poss ib ly a f t e r s l i g h t e r longer latency than is usua l ly observed with th is agent. - 122 " RF-P Co-toiid Smuses Innervated I ff [I j)f"[[tfTIf[-f R [iilili |[ i J j L F - P t I . N . E 0.5 *^3 I 7 0 co •4M f N.E. 0.5l/^ * Figure 6 9 . Effect of Isoproterenol 0,5/ug/kg followed by norepinephrine 045yug/kg after bi lateral carotid sinus denervation. Prompt in i t ia l adrenergic activation followed by the devel-opment of an anoxic pattern due to profound uncompensated depressor responses. Restoration of blood pressure with norepinephrine overcomes anoxic pattern and again reveals adrenergic-1 reduced activation. - 123 -R F P Carol «J t I . N.E. 0.5*7 K g . 1 D B Z I. 1 - to Figure 704 E f f e c t o f i s o p r o t e r e n o l 0^5 / i g / kg i n t h e p r e s e n c e o f phenoxy -benzamine I mg/kg a f t e r b i l a t e r a l c a r o t i d s i n u s d e n e r v a t i o n . A n o x i c p a t t e r n d e v e l o p s d u r i n g p r o f o u n d uncompensated d e p r e s s o r re sponse to i s o p r o t e r e n o l and a g a i n i s overcome by r e s t o r a t i o n o f b l o o d p r e s s u i e by n o r e p i n e p h r i n e . However, m a n i f e s t a t i o n s o f a d r e n e r g i c - i n d u c e d a c t i v a t i o n a r e com-p l e t e l y b l o c k e d by phenoxybenzatnLne (a 1 though p r e s s o r r e sponse to n o r e p i n e p h r i n e Is i n c o m p l e t e l y b l o c k e d ) . - 124 -norepinephrine identical to that used in Figure 6 9 . This produces an immediate pressor response which is more delayed and less intense than in the untreated carotid sinus preparation, but, in marked contrast to Figure 6 9 , there is a conspicuous lack of any associated EEG "act ivat ion". D. Preparation With Brain Stem Lesions. 1. Effects of Pontile Lesions on the,EEG of the Unanaesthetized  De-afferented Cat. Since the ophthalmic branch of the trigeminal nerve was intact in our unanaesthetized de-afferent preparation, i t was of interest to deter-mine whether or not residual sensory inflow over this pathway was a s ignif icant source of EEG influences in the "rest ing" state. Consequently, an effort was made to eradicate the sensory nuclei of the 5th nerve by e lectro lyt ic coagulation. Access to the pons by the conventional cerebral route was not attempted, since an intact cranium was necessary for compar-ing drug-induced EEG tracings obtained under these circumstances with those previously recorded following drug administration. However, the electrode tip was placed in the desired area of the pons by t i l t i n g the electrode carr ier to an angle of about 40° to the vert ical and inserting the electrode via the cerebellar approach (Plate D). In this way, three series of e lectro lyt ic lesions (Plate C, lesions A, B and C) destroyed the greater part of the central gray and the tegmentum of the central and right lateral portions of the pons anterior to the root of the 5th cranial nerve and just posterior to the decussation of the branchium conjunctivum (Plate B, sections 16-18, 20 and 28). The resting synchronized pattern, and the drug-induced activation responses were - 125 -P l a t e D Photograph showing the c e r e b e l l a r approach to the production of l e s i o n s i n the ponto-mesencephalic tegmentumof the cat b r a i n by a glass Insulated e l e c t r o d e . - 126 -S C H E M A T I C S A G I T T A L S E C T I O N O F C A T B R A I N S T E M W I T H S T E R E O T A X I C C O O R D I N A T E S S H O W I N G A R E A S O F C O A G U L A T I O N . P L A T E C - 127 -PLATE B IS ID at P l a t e B Photographs of cross sections through lesions in the tegmentum of ponto-mesecnephalic area. Weigert's s tain. - 128 -indistinguishable from those of the preparation with an intact brain stem. 2 . Effects of Unilateral Ponto-mesencephalic Lesions on EEG "Activa-tion" (Adrenergic and Cholinergic). Lesions placed in the mesencephalic tegmentum rostral to the ponto-mesencephalic junction (Plate C, lesions C| and C2, and Plate 6, sections 10, 11 and 13) did not prevent the development of a substantial degree of "activation" when the cat was placed initially in the stereotaxic instrument (Fig. 7 1 ) . However, a well synchronized, deactivated pattern developed in all k leads after a 30 minute period of adaptation, and "activation" in response to auditory stimulation (hand claps) still could be elicited (Fig. 70 . In this preparation, spindle bursts occasionally appeared on the side of the lesion while the simultaneous EEG on the intact side displayed a highly activated pattern. On some occasions, auditory activation was of briefer duration on the side with the lesions. Adrenergic activation still could be elicited but only with doses larger than those which usually produced EEG "activation" in the prepara-tion with an intact brain stem. The duration of response to a larger dose (2/ug/kg) was less than that observed previously, but the pattern was similar on both the coagulated and intact sides (Fig. 7 2 ) . Similar EEG "activation" was obtained with 2 /ug/kg of acetylcholine (Fig. 7 3 ) . Electrolytic lesions were next extended on the right side in a ventro-lateral and a dorso-lateral direction. The rapid recovery of a synchronized EEG pattern following electrolytic coagulation is illustrated in Figure 7k. Following this lesion, which was placed in the right ventro-lateral portion of the mid-brain (Plate B, sections 8-10, and Plate C, 129 -Flora. Cat in Stereotaxic io.05ec. I S O m i o . 85 90 I oatrument". after 0.0 Sec R F . P 300/tV RP.O LP.O F. P L31 w>ir>-a f t e r 0 . 0 S e c . lll^iirtu^i/lU>«tWl>lllliii^Mnll»V'll1i.''»*l<iii^rtii,tilli«rf Mi I i n til s nana Figure 71. E f f e c t of u n i l a t e r a l les ions in the caudal part of the r ight mesencephalic tegmentum (P la te C, les ions C| and C2). Deactivated EEG (type D) developing a f t e r 30 minute period of adaptation in the s te reotax i c instrument. Typ ica l t rans i to ry EEG a c t i v a t i o n in response to audiogenic s t imulat ion (hand c l a p s ) . - 130 -1 300/-V 132. LF.P 135 130 F i g u r e 72. E f f e c t of epinephrine 2 yug/kg on the EEG i n the presence of u n i l a t e r a l l e s i o n s i n the caudal p o r t i o n of the mesencephalic tegmentum. Intense and prompt a c t i v a t i o n (type A) occurs w i t h i n k seconds and l a s t s longer than 40 seconds. - 131-: W l l f ^ 4#»MJjjj^ f l H f f f M l ^ ^ - »/Kq. J B . R 80 LF.P < 6 0 Figure 73. E f f e c t of a c e t y l c h o l i n e 2>ug/kg on the EEG i n the presence of u n i l a t e r a l l e s i o n s i n the caudal p o r t i o n of the mesen-c e p h a l i c tegmentum. Immediate and intense EEG a c t i v a t i o n i s observed l a s t i n g f o r more than 1 minute and i n t e r r u p t e d w i t h occasional and i s o l a t e d s p i n d l e s . - 132 -lesion Cj), auditory activation was of much briefer duration on the side with the lesions (Fig. 74). Under these circumstances, bilateral adrenergic activation with epinephrine (Fig. 75) and isopropyl nor-epinephrine (Fig. 76), while still demonstrable, developed later and was less intense and briefer in duration on the coagulated side. Following a further coagulation in the right dorso-alteral portion of the mesencephalon (Plate B, sections 8 and S, and Plate C, lesion Ch) similar doses of epinephrine (2/Ug/kg) and isoproterenol (2.0/ug/kg) failed to produce discernible activation on the coagulated side, while characteristic desynchronization was produced simultaneously on the intact side (Figs. 77 and 78). In contrast, bilateral and simultaneous EEG "activation" was obtained following the administration of 2/Ug/kg of acetylcholine (Fig. 79). The finding that larger doses of epinephrine and isoproterenol are needed to produce EEG "activation" in the presence of coagulatlve lesions of the mesencephalic tegmentum, together with the observation that the pathways of adrenergic activation are bilaterally discrete, conforms with Rothballer1s data (149), and lends support to the view that the intact side can serve as a valid control for the adrenergic system. - 133 -w LF.P : I S I RFP End of EC-C3 B . P . 10 nil " , B LP 0 LFP < < » r t r f r # ^ i l i ^ * » 4 » , ^ ^ ,*<^^iFw.wr^''|y,'*>v.w*«-*«.*>*i«' - . ' Whistle I Blast Figure 74. Effect of further lesions in the right mesencephalic tegmentum (Plate C, lesion C3) Upper tracing: prompt restoration of deactivated (type D) pattern following the termination of unilateral electrolytic coagulation (lesion placed In the right ventro-lateral portion of the mid-brain). Lower tracing: prompt intense bilateral EEG activation elicited by audiogenic stimulation in the presence of lesion c3-Note prompt reversion to deactivated EEG on the side with the lesion and persistence of activation on the intact side. - 134 -i l l tai In " i ' ilL J<fi^hiiillML^J ,i'-"- j J"- 1 B . P . i o RF.P H'.'mwm Epi. 2.0 Jt/Kq. o « « r EC -C3 . 300*A/ ; sec. [ Mi RP-0 3 1 2 0 10 110 Figure 75. Effect of epinephrine 2.0/jg/kg on the EEG in the presence of extensive unilateral lesions in the right ventro-lateral portion of the mesencephalic tegmentum (lesion C3). Typical prompt and intense EEG activation produced on both sides. Note that on this side of the lesion the activated pattern is interrupted with high voltage low frequency activity which is largely absent on the intact side. - 135 -' I . N . E 1.0 l ^ / K g . 1 a f t e r E C -C"3 10 LS 60 55 50 Figure 76. Effects of isoproterenol 2 /Ug/kg on the EEG in the presence of unilateral lesions in the right ventro-lateral portions of the mesencephalic tegmentum. Mote typical immediate and intense (type A) EEG activation on the intact side. Delayed,less intense, and more transitory activation, frequently interrupted by high voltage low frequency activity, is seen on the side with the lesions. - 130 -Eft. 2.0 >r/K q . a f t e r E f - C 4 ***** 30/.V B.R 8S ")0 R F - P p . L F . P 1 0 5 F i g u r e 77. E f f e c t of epinephrine 2 /Ug/kg on the EEG a f t e r u n i l a t e r a l l e s i o n s extending i n t o the r i g h t d o r s o - l a t e r a l p o r t i o n of the mesencephalic tegmentum ( P l a t e C, l e s i o n C^). Note prompt and intense (type A) a c t i v a t i o n on the i n t a c t s i d e , and lack of app r e c i a b l e adrenergic a c t i v a t i o n on the si d e w i t h l e s i o n s . - 137 -[PHI ^ \ ^ t%H» ^ ^ y \ v » f Figure 73. Effect of isoproterenol 2/jg/kg on the EEG after unilateral lesions extending into the right dorso-1ateral portion of the mesencephalic tegmentum (Plate C, lesion C^). Note prompt and intense (type A) activation on the intact side. Note eventual appearance of f a i r l y intense activation on the side with lesions following this relatively large dose of isoproterenol. Note delay of approximately 30 seconds before the appearance of activation on the side with lesions and its occasional interruption with high voltage low frequency act! vi ty. - 138 -Figure 79. Effect of acetylcholine 2/tig/kg on the EEG after extensive lesions in the right dorsolateral portion of the mesenceph-alic tegmentum (Plate C, lesion Ci*). Prompt and intense type A activation elicited in all leads. - 139 -VI. DISCUSSION A. Advantages of the Preparation. These studies demonstrate that EEG "activation", temporally independent of peripheral cardiovascular responses, can be produced by direct intra-arterial administration of low (physiological) doses of several pharmacological agents. Such drug-induced EEG "activation" can be elicited repeatedly in the same unanaesthetized de-afferented prepara-tion both before and after carotid sinus denervation. These experiments also have shown that certain blocking agents can act selectively to inhibit or modify the usual effects of such activating compounds. These observations indicate that this technique (unanaesthetized de-afferented preparation and intra-innominate administration of drugs) should be a fruitful one for the qualitative study of drugs presumed to exert their influences on pathways leading to EEG "activation". This view is further enhanced by the fact that in this preparation it is still possible to observe some signs indicative of concomitant behavioural alertness (movement of the head and forepaws) following the administration of some activating drugs. This technique also permits the placing of lesions in the mid-brain reticular formation without the complicating effects of anaesthesia or curarizing agents so that subsequent drug-induced electro-cortical activity can be observed without superimposed pharmaco-logical influences. - 140 -B. Requirements for Deactivation. Current hypotheses of the neural mechanism by which mammals achieve and maintain sleep generally are related to decreased activity of the neurones in the reticular activating system, since destruction of the cephalic reticular core results in persistent EEG deactivation and behavioural sleep (56). The results of various techniques for producing deactivated (sleep type) EEG patterns, as well as the converse phenomena of arousal and "activation", have led to emphasis on the role of sensory inflow in initiating and maintaining activated EEG patterns. Deactivated (sleeping) EEG patterns of a persistent character were initially produced by transection of the mid-brain by Bremer (29), who introduced the cerveau isole preparation. In this preparation, sensory afferent inflow to the cortex is restricted to the olfactory and optic nerves. Rossi et al (148) and Battini et al (13) have reported that the most caudal level of brain stem transection which will produce a consistently deactivated EEG pattern is at the cephalic border of the pons (rostral to the roots of the trigeminal nerve). Rothballer (149) has shown that electrolytic coagulation of the reticular formation of the tegmentum at the ponto-mesencephalic junction also will produce such an EEG pattern. The role of various sensory modalities in the maintainance of wakeful-ness has been investigated by Roger, Rossi and Zirondoli (147). In the encephale isole cat they have demonstrated persistent EEG sleeping patterns following bilateral intracranial destruction cf the Gasserlan ganglion but not after the obliteration of olfactory, visual, acoustic, vestibular or vagal afferent inputs, as long as the trigeminal nerve was intact. This observation raised the suggestion that the deactivated - 141 -sleeping-type EEG might be particularly dependent upon reduction in the influences of trigeminal afferent inflow upon the activity of the brain stem reticular formation. In contrast, Randt and Collins (140) have suggested that in the cat, wakefulness is facilitated rather than inhibited by the restriction of afferent sensory inflow. In paralyzed (Flaxedil treated) and blind-folded preparations, some of which (53%) were submerged in water kept at 36-38° C, activated EEG patterns were observed regularly. It Is not Inconceivable that these alien and indeed hostile conditions could have provoked some adrenergic discharge, for, as Randt and Collins have stated, "There appears to be an elaboration of epinephrine and nor-epinephrine, which acts upon the normal or sensitized core of the midbrain", and which in turn could exert arousal influences on the EEG. A comparable report of the release of epinephrine and norepinephrine into the systemic circulation of human males subjected to sensory deprivation in a tank respirator has been made by Mendelson et al (120). Apparent sensory deprivation may be illusory under highly abnormal circumstances and endocrine as well as neuronal influences on the brain stem may be brought into play. Our own observations lend support to the view that reduction in the sensory input to the brain stem is necessary for the development of a deactivated EEG. External activating influences characteristic of routine activities in our laboratory area (intermittent noise and vibra-tion) precluded the day-time investigation of drug-induced EEG "activation". However, deactivation was regularly elicited at night when intermittent noise and vibration were absent, even though low level noise (dynograph - 142 -recorder, quiet conversation) was impossible to eliminate. Apparently, adaptation to continuous, low level sensory input to the auditory nerves permits the development of deactivated EEG patterns. It was anticipated that the exclusion of audiogenic input by the use of cotton ear-plugs and the reduction of vibration with the aid of rubber blocks placed under the experimental table, might minimize extraneous activating influences and permit day-time experimentation in a dimly lighted environment. These measures employed continuously in one preparation over a period of 3 days did not produce the anticipated results. Repeatedly activated EEG patterns, similar in duration to those observed as a result of external environmental influences, were still demonstrable . The possibility that insertion of the cotton ear-plugs established a persistent somatic sensory inflow from the external ear canal appears unlikely since under suitable conditions (at night), deactivated patterns were readily obtained even when the preparation was fixed in the stereotaxic instrument, a procedure which involves inserting the tips of rigid metal rods into the external auditory meati. Although unavoidable manipulation necessary for the precise positioning of the head of the preparation produced intense activation during the initial phase of the latter procedure, deactivated patterns always returned after a 30 minute period of adaptation. Furthermore, no branches to the auditory meatus from the ophthalmic division of the trigeminal are known, although the possible existence of unknown pathways from sensory receptors in the auditory canals should be kept in mind. It is conceivable that longer periods of adaptation to the cotton ear-plugs may have produced the desired results. However, it would appear that even though adaptation - 143 -may be achieved to c e r t a i n s t i m u l i of low i n t e n s i t y , abrupt changes or m o d i f i c a t i o n s i n the environment, even those d i r e c t e d towards reducing sensory i n f l o w , may produce e f f e c t s opposite to those that were being sought. Under such circumstances p e r s i s t e n t a c t ! v a t l o n without adaptation may be due to the complete e x c l u s i o n of i n f l u e n c e s u s u a l l y present and may be r e l a t e d to the strangeness of such experimental s i t u a t i o n s . Recently, Batsel (12) has reported that although the c h r o n i c cerveau i s o l e dog i n i t i a l l y does e x h i b i t a high degree of continuous s p i n d l i n g and slow waves, e v e n t u a l l y (2 weeks or more a f t e r operation) t h i s type of a c t i v i t y i s gradually replaced by low voltage f a s t a c t i v i t y only o c c a s i o n a l l y i n t e r r u p t e d by b r i e f periods of d e a c t i v a t i o n . B a t s e l suggests that these f a c t s o f f e r presumptive evidence that neurones r e l a t e d to the a c t i v a t i n g system remain i n the i s o l a t e d cerebrum and can assume rates of f i r i n g s u f f i c i e n t to a c t i v a t e the EEG. We, too, have observed the s p o r a d i c development of r e l a t i v e l y l o n g - l a s t i n g (£ to I hour) spontaneous EEG " a c t i v a t i o n " patterns i n our preparations as long as 6 weeks a f t e r d e - a f f e r e n t a t i o n , p a r t i c u l a r l y during i n s e r t i o n of the stomach tube p r i o r to feeding and accompanying d e f e c a t i o n and i n the presence of sudden a l t e r a t i o n s im e x t e r n a l environmental f a c t o r s (changes i n the noise l e v e l of the room, v a r y i n g degrees of l i g h t i n g , personnel a l i e n to the p r e p a r a t i o n , e t c . ) . However, these patterns always e v e n t u a l l y subsided and under s u i t a b l e c o n d i t i o n s ( see s e c t i o n 5-b under Methods) c o n s i s t e n t and r e l i a b l e d e a c t i v a t i o n has always been obtained f o r prolonged periods of time ( o c c a s i o n a l l y these preparations were fo l l o w e d f o r as long as 1-3 months). Hence, our - ]kk -observation appears to lend support to the current concensus that the critical prerequisite for the production of persistent deactivation of the EEG is a substantial degree of sensory de-afferentation. Our experience (with extra-cranial partial trigeminal de-afferentation) indicates that consistent and reliable EEG "activation" may be accomplished without brain stem lesions and in the presence of some intact sensory pathways (visceral via vagus, somatic sensory via op! thai mic branch of trigeminal, auditory, vestibular, optic and olfactory) providedthat inflow over these pathways is reduced to low levels. C. A Direct Central Site of Action of Drug-Induced EEG "Activation". For drugs to exert direct effects on neurones within the CNS requires that these compounds be able to cross the so-called blood-brain barrier. It should be recognized that this barrier is not thought of as lying between the brain cells and the fluid surrounding them, since drugs are known to penetrate the cells of the brain as readily as those of other tissues ( 3 1 ) . Rather, the barrier seems to be between the plasma and the extra-cellular fluid of the central nervous system ( 3 1 ) . The anatomical and physiological characteristics of the blood-brain barrier have not been clearly defined. There is remarkably little direct evidence that biogenic amines (serotonin, histamine, short-acting adrenergic agents and cholinergic agents) can or cannot cross the blood-brain barrier easily (31) . The suggestion has been put forward by Krogh (101) that the rapid exchange observed for narcotics between plasma and brain is due to the lipoid solubility of these compounds, since observations made with other lipoid soluble substances (chloroform, ethanol and - 145 -ethy lurethane) have demonstrated that the relative rate of passage of these substances is directly proportional to their lipoid solubility. More recently Brodie and Hogben (31) have supported this view, and have pointed to the comparison between the faster rate of brain penetration by thiopental which exhibits high lipoid solubility, and the slower passage of the less-lipoid soluble analogue barbital. Furthermore, they have suggested that the low fat solubility characteristics of serotonin and norepinephrine makes it difficult for these compounds to cross into the brain in measurable amounts, and have raised the question of whether the central effects observed following the intra-vascul«r administration tf these compounds may not be due to their effect on peri-pheral receptors, rather than to a direct action on central receptors. Indirect evidence indicates that serotonin may penetrate the blood-brain barrier (7,36,61,100,114,153) Efforts to obtain direct evidence that epinephrine can cross the blood-brain barrier generally have been unsuccessful. However, to my knowledge, there is no direct evidence that serotonin and norepinephrine are unable to cross the blood-brain barrier and the observation that other lipoid insoluble substances, such as the sulfonamides, show no correlation between brain penetration and lipoid solubility (9,63) emphasizes the hazards of reasoning by analogy. Failure to obtain any increase in the concentration of epinephrine and norepinephrine in the brain of rats following the intra-venous infusions of these amines ( 139) , coupled with the observation that following subcutaneous administration of labelled epinephrine (157) in the same species, high counts could be obtained only ?n the liver, plasma and kidneys, have led to the view that epinephrine fails to pass - 146 -the blood-brain barrier. In the cat, attempts by Leimdorfer et al (105) to detect adrenaline in the cerebrospinal fluid following intravenous administration of this amine has met with little success, despite the demonstration by Becht (14) that in this species epinephrine is stable in the CSF for prolonged periods (up to 6 hours). The recent report by Mayer (118) that DCI, an analogue of isoproterenol, reaches a high and persistent concentration in the brain following the injection of H3-DCI suggests the possibility that the parent compound (isoproterenol) may have a similar pattern of distribution, following intravascular administration. Recently, Wei 1-Maiherbe et al (175) have infused tritiurn-label led epinephrine into the femoral vein of cats and have concluded that epinephrine can cross the blood-brain barrier in small but significant amounts only in the hypothalamus. They, therefore, interpret the central effects of epinephrine, which occur following peripheral administration, to be the result of the interaction of this amine with hypothalamic or peripheral receptors. However, it should be borne in mind that these observations were made following intravenous administration of large amounts of this compound in anaesthetized preparations. In addition, the analytic methods used cannot be relied upon to detect small and physiologically active concentrations of substances which may have penetrated various parts of the brain. Granting the possibility that biogenic amines and their congeners may be able to cross the blood-brain barrier in physiologically signific-ant amounts, little information exists as to whether or not there are - 147 -neuronal elements within the brain stem which are directly receptive to the action of these agents. The removal of areas believed to be the site of such action provides some relevant evidence. We have found that uni-lateral brain stem lesions at the ponto-mesencephalic junction which destroy most or all of the reticular tegmentum, abolish EEG "activation" Induced by the administration of adrenergic amines only on the coagulated side. However, under the same circumstances acetylcholine still can produce bilateral patterns of cortical electrical activity similar to those observed in the intact preparation. This is in complete agreement with earlier observations of Rothballer (149) who demonstrated that the integrity of the mesencephalic tegmentum was essential for the EEG activating action of adrenaline. Porter (132,133) has shown (by progressive destruction of the brain stem in a rostral direction) that the electrical activity recorded from both the anterior and posterior hypothalamus, following intravenous epinephrine also is dependent upon the integrity of the brain stem. It is not inconceivable that hypothalamic-mid-brain activity may be functionally inter-related, and that the hypo-thalamus may be another site at which adrenergic agents may act to elicit EEG "activation". However, the demonstration by Bradley et al (24) that the intravenous administration of epinephrine and norepinephrine can influence unit activity of the reticular formation in the "isolated mid-brain" preparation (transection of the brain stem at the precol1icular level) indicates adrenergic responsiveness on the part of neuronal elements above the level of section. Similar observations have been made by Bonvallet et al (18) for epinephrine In the isolated "reticular - 148 -slab".* More direct support for this view is provided by the observation that EEG "activation" has been demonstrated following the injection of small amounts (1 /jg) of epineprhine "directly into the brain stem under stereotaxic guidance" (150,152). (Under these circumstances control Injections of comparable amounts of saline were without effect.) The prompt EEG responses seen following the intra-innominate injection of various biogenic amines, indicates that these agents can indeed cross the blood-brain barrier and do find receptive sites within the CNS. It may be argued that the alteration of cortical electrical activity observed following the direct intra-innominate drug administra-tion could be induced reflexly. The onset, duration and termination of drug-induced EEG "activation" usually does not coincide with changes in the general level of blood pressure often produced by these agents. Hence, it is unlikely that the altered electro-cortical activity is the result of reflexes arising from peripheral receptors discharged by these compounds when they reach the general circulation. Further evidence supporting this conclusion derives from the very short latency observed for the initiation of EEG "activation" (approximately 4 seconds) following the intra-innominate administration of these compounds. The possibility that changes in electro-cortical activity could have resulted from mechano-receptor reflexes elicited by direct distension of the vessels into which drug-containing solutions were injected, was tested by giving frequent injections of normal saline, in volumes comparable ic A preparation in which the mesencephalic portion of the reticular formations has been completely isolated from all nervous connections by a transpontine section posteriorly, a pre-mammi1lary section anteriorly, and destruction of the corpora quadrigemina. - 149 -to those of the test agents. Under these circumstances, no change in the resting type EEG pattern was observed. The possibility exists that the injected drugs might directly stimulate baro- and chemo-receptors in the walls of the carotid sinus and thereby initiate EEG "activation" reflexly. Heymans et al (77,73) have reported that the carotid sinus can be excited by the topical application of drugs which modify the distensibi1ity of the wall, but have stated that " i t is unlikely that any drug given systemically exerts any important : action of this kind." However, Bonvallet et al (17) and Dell (39) have reported that distension of carotid sinuses ("prepared as a cul-de-sac by ligature of all vessels save the main carotid which is cannulated") within the normal physiological range produces a deactivated EEG. Nakao et al (125) have reported similar results, and further have emphasized the fact that a lowering of the blood pressure with its resultant decrease in afferent inflow over Herlng's nerve may produce cortical activation. In our experiments, EEG "activation" has been obtained with both pressor (epinephrine) and depressor (isoproterenol) agents. Furthermore, no demonstrable change has been observed in the control EEG following the administration of pressor amounts of vasopressin and small but depressor doses of histamine, even though the changes in blood pressure were as great as those seen with the injection of activating agents. Experiments In preparations whose carotid sinuses have been completely denervated have provided more direct and. -conclusive evidence bearing on this problem, The validity of carotid sinus denervation in these preparations has been confirmed by the repeated demonstration that the usual cardiovascular - 150 -response to bilateral carotid clamping was completely absent. EEG activating effects were elicited by all activating compounds after carotid sinus denervation, the patterns being identical with those elicited by the corresponding agents in the same preparation prior to denervation. This observation rules out the possibility that the EEG activating effects observed following the administration of these agents was in any way dependent upon stimulation of carotid sinus receptors. The observation of serotonin-induced EEG "activation" in our carotid sinus denervated preparation is of particular interest. Some (43, 142) have taken the view that exogenous serotonin does not have any effect on the central nervous system, except indirectly over reflexes arising from peripheral receptors. Others (114) have concluded that intra-carotid serotonin may act directly on the visual cortex to produce transitory inhibition of the ipsilateral transcallosal response. The view that serotonin has a direct action on the visual cortex has in part been confirmed by Koella et al (100), who under more carefully controlled conditions have reported a direct effect of serotonin on the brain stem as well as an indirect cerebral effect mediated by means of the carotid sinuses. Following intra-innominate injection of serotonin we, too, have noted its effects on the EEG of preparations with intact carotid sinuses, in agreement with similar observations of Rothballer (151) and others (61,64). Moreover, our observation that serotonin-induced EEG "activation" also can be elicited in preparations in which the carotid sinuses have been denervated provides additional support in favour of a direct effect for the central actions of this compound. - 151 -It is known that cerebral anoxemia elicits changes in the EEG. Cerebral ischaemia might result from direct vasoconstriction on the part of some vasopressor agents or might be associated with profound hypotension produced by potent vasodepressor compounds. An illustration of this type of phenomenon is furnished by experiments in which isoproterenol was administered to carotid sinus denervated preparations or in the presence of chlorpromazine. In these circumstances, isoproterenol produced precipitous depressor responses, and intense and short-lived EEG "activation". This prompt EEG response is followed directly by an electro-cortical pattern whose amplitude and frequency fluctuates and then declines gradually until, at depressor levels of about 40 mm Hg, It is quickly transformed into a flattened EEG tracing. Others (62,84,172) have reported a similar "flattening" of the EEG and depression of cortical electrical activity during anoxia, also preceded by transient periods of low voltage high frequency electro-cortical activity. It seems possible that the EEG "activation" observed with isoproterenol might be consequent to the preliminary effects of cerebral anoxia. However, the very short latency of the EEG "activation" observed with isoproterenol, together with the fact that this activation occurred at blood pressure levels that were considerably higher than the levels associated with the anoxia-induced alterations of electro-cortical activity, make this very unlikely. Furthermore, the lack of any EEG "activation" in the presence of a more precipitous depressor response after the same dose of isoproterenol was given in the "phenoxybenzamine-treated" denervated carotid sinus preparation, also argues against this possibility; i.e., the prompt intense activating effect is blocked by phenoxybenzamine, whereas the - 152 -anoxic flattening of the EEG is not. It seems unlikely that such vascular changes could have any bear-ing on the EEG effects produced by the other activating compounds used in these experiments, especially since the diverse agents known to elicit consistent EEG "activation" are characterized by such a wide variety of effects on the cerebral circulation (163, also see Rbthballer, 1956). For example, epinephrine produces an Increase in total cerebral blood flow, whereas norepinephrine produces the opposite effects (163). Yet these agents have similar EEG activating effects. Chlorpromazine and histamine have been reported to have no demonstrable effect on cerebral hemodynamics (163), yet these compounds have opposite effects on the EEG; histamine produces activation, while chlorpromazine elicits slow wave spindle activity. Furthermore, doses of amphetamine which cause no perceptible effect on cerebral vascular responses (163) produce intense activation, whereas small doses of the barbiturates which also are without effect on cerebral circulation, block EEG "activation" (18). It therefore seems reasonable to conclude that the EEG "activation" observed following the intra-innominate administration of the compounds we have studied probably is due to the direct effects of these agents on neurones in the CNS, rather than to indirect influences mediated by vascular, reflex or sensory mechanisms. D. Characterization of Central Synapses Receptive to Drug-Induced EEG "Activation". The compounds which we have employed are all active at peripheral - 153 -receptor cells. Some are mediators at autonomic receptor sites, while others are active at other peripheral synapses. The fact that these compounds have direct central effects raise the possibility that some of them may act as mediators or may mimic mediators at central synaptic sites. Several of these compounds are present in the CNS, and some have received consideration as a possible candidate for central synaptic mediation (33,35,48,94,116,130,170,176). The demonstration that these compounds all have direct central actions certainly does not establish them as potential central synaptic mediators. However, their direct effects are compatible with such a role and furnish one of the props that ultimately would be required for the identification of such mediators. Recently the demonstration that epinephrine and norepinephrine are present in certain areas of the CNS ( 1 7 0 ) , coupled with the observa-tion that enzymes necessary for the synthesis and inactivation of these compounds are located in precisely the same areas of the brain,have focused major attention on these amines as possible candidates for the role of central adrenergic mediator. However, epinephrine and norepineph-rine should not be looked upon as the sole candidates for this role. The observation that isoproterenol can produce consistent and crisp EEG "activation" at lower doses than the other adrenergic amines we have studied is of particular interest. In this connection it is of interest that isoproterenol seems to be more potent than epinephrine in eliciting the anxiety which has long been known to be associated with infusions or injections of the latter agent in man ( 8 0 ) . Matthews has demonstrated that isoproterenol in small doses can augment ganglionic transmission - 154 -In the cervical ganglion of decerebrate, cat, during submaximal stimulation (117) . It has been reported that a substance clearly distinguishable from epinephrine and norepinephrine both pharmacologic-ally and chroma tographica 11 y comprises 80-100% of the adrenergic material released into the blood stream during the stimulation of the pulmonary sympathetic nerves in the cat heart-lung preparation ( 110) . This substance could not be differentiated from isoproterenol by the techniques employed and although the evidence strongly suggest that it may be identical with isoproterenol, clear differentiation between the Isopropyl and other high N-alkyl derivatives of norepinephrine has not been established. A similar material also has been obtained in small amounts in the adrenal glands in several species including man, but Its physiological significance has not been clarified ( 1 0 9 ) . It seems possible, on. the basis of its observed EEG effects that isoproterenol may have a more pronounced action than other adrenergic amines on various other central adrenergic mechanisms ( 154) , as well as on those concerned with EEG "activation". Our data suggest the possi-bility that isoproterenol may be acting at central receptor sites which are different from receptors sensitive to epinephrine and norepinephrine. Synaptic blocking agents have been the classical tools for differentiating and classifying various types of synaptic receptors. Adrenergic blocking agents have contributed much to our knowledge concerning the various types of adrenergic receptors which are located in visceral tissues. Ahlquist (4) has classified these receptors into two types, alpha and beta, one of which (alpha or excitatory) is most responsive to norepinephrine, and the'other (beta or inhibitory) is most - 155 -responsive to isoproterenol. Epinephrine occupies an intermediate position, being quite active in eliciting both types of responses. This classification is In part based upon the fact that responses mediated through "alpha receptors" are generally those blocked by classical adrenergic blocking agents (e.g. beta-halo-alkylamines, the ergot alkaloids, etc.), whereas responses mediated through beta receptors by isoproterenol have only recently been found to be blocked by DCI (106). Lands (102) has objected to Ahlquist's classification, and has introduced an additional receptor for the heart which he has classified as an undifferentiated receptor (Acr), since "this organ is stimulated by substances with strong affinity for either" the excitatory receptors (f\c) or the inhibitory receptors (Ar). Furchgott (59) has proposed a modification of Ahlquist's classifica-tion and has suggested that the adrenergic receptors mediating various responses be classified into four groups; namely, alpha receptors for the contraction of smooth muscle; beta receptors for relaxation of smooth muscle, other than that of intestin© and for increase in rate and strength of cardiac contraction; gamma receptors for glycogenolysis; and delta receptors for inhibition of intestinal smooth muscle. Our results show that phenoxybenzamine blocks the EEG activating effect of isoproterenol as well as that induced by epinephrine and norepinephrine. If there are two types of adrenergic receptors in the CNS corresponding to the alpha and beta receptors designated by Ahlquist (5), then it would appear that in the CNS both receptors are blocked by phenoxybenzamine. This is in contrast to the response of these receptors to this blocking agent at peripheral sites. On the - 156 -other hand, it is possible that in the central nervous system there is only one adrenergic receptor, an undifferentiated receptor which, like the receptor of the heart,is responsive to all three of these adrenergic • amines. However, the fact that DCI can block the EEG activating effects \ of Isoproterenol while manifesting very little evidence of blockade of epinephrine and norepinephrine-induced EEG "activation" argues against the identity of these central adrenergic receptors. The peripheral adrenergic blocking actions of DCI also are confined to responses activated by isoproterenol; for example, DCI does not block the cardio-vascular or intestinal relaxing effects of epinephrine and norepinephrine (4,59,106,121).* Thus, the selectivity of DCI for isoproterenol-induced EEG "activation" parallels its behaviour at peripheral adrenergic synapses and limits its value in distinguishing different receptor categories. In their responses to all three adrenergic amines (epinephrine, isoproterenol and norepinephrine), and the selective blockade of isoproterenol by DCI, the adrenoceptiye components of the EEG activating system resemble intestinal smooth muscle. However, the central blockade of all three of these EEG "activators" by phenoxy-benzamine is unique. At present no clear-cut explanation can be offered for the phenoxybenzamine blockade of isoproterenol-induced EEG "activation". Ahlquist et al (4) have reported that intestinal receptors (alpha and beta) which are responsive to epinephrine, occasionally can be blocked * Levy (106) recently has reported that (as with DCI) the dlchloro analogues of epinephrine and norepinephrine produce initial cardiovascular and smooth muscle actions resembling those of their parent catechol amines. However, they also resemble DCI In selectively blocking the intestinal relaxing and vasopressor effects of isoproterenol, and do not permit these actions on the part of epinephrine and norepinephrine. - 157 -by large doses of beta-halo-aIkylamines, and they and others (180) have attributed this property to the fact that these blocking agents are able to block the beta- as well as the alpha-receptive mechanism. Green (68) and others (173) have reported similar observations in isolated canine vascular beds (cutaneous and skeletal muscle) following the intra-arterial administration of high doses of adrenergic blocking agents. A criticism which has been levelled against these observations is the possibility that these large doses produce direct smooth muscle depression (paralytic effects); consequently they themselves probably cause maximal inhibitory responses and, hence, are acting as physiological antagonists rather than as selective blocking agents (127). Thus, clear-cut evidence that phenoxybenzamine can block the action of iso-proterenol at any peripheral site is lacking. Adrenergic blocking agents (beta-halo-aIkylamines and DCI) have uniformly failed to prevent cholinergic-induced EEG "activation"; whereas EEG "activation" elicited by adrenergic compounds (epinephrine, norepinephrine, isoproterenol and amphetamine), and cholinergic agents (acetylcholine and eserine), as well as by histamine and serotonin, all can be blocked by atropine. Furthermore, chlorpromazine in doses which blocks the electro-cortical responses of activating amounts of the adrenergic amines, serotonin and histamine, fails to have any effect on the EEG "activation" produced by acetylcholine and eserine. These observations emphasize the fact that cholinergic receptors are quite distinct from the receptors which are responsive to other compounds. Further support for a differentiation between cholinergic and adrenergic receptors is gained from the fact that whereas adequate - 158 -doses of eserine can reverse both the chlorpromazine and atropine-induced EEG deactivation, r-amphetamine in large doses (200/jg/kg) can overcome the characteristic slow wave and spindle activity only of chlorpromazine, but is without any effect on the atropine-induced EEG deactivation. This is In agreement with the observation of Bradley (21) and others (22,112), but differs somewhat from the findings of White et al (177), who have reported a partial antagonism between atropine and d-amphetamine in the Intact, the post-pontine and the cerveau isole rabbit. The similarities between the blockade of the EEG activating effects of histamine both by chlorpromazine and phenoxybenzamine con-forms with the well-known antihistaminic properties of these two blocking agents at other receptor sites which are excited by histamine (65). However, the fact that phenoxybenzamine blocks the action of histamine but fails to block the effects of serotonin makes it likely that there is a difference between the receptor sites at which these two amines act to elicit EEG "activation". It, therefore, does not seem likely that serotonin is acting at (or only at) sites which are receptive either to histamine or to the adrenergic amines. However, until more direct evidence is available, the possIbi1ity cannot be ruled out that histamine is active at sites which are also adrenoceptive. The effects of various types of activating and blocking agents are summarized in Plate E. They suggest at least three separate and distinct receptor sites which are capable of converging on the final pathway for EEG "activation"; one responsive to cholinergic compounds and blocked only by atropine, one which is responsive to serotonin - 159 -EFFECT OF BLOCKING AGENTS ON DRUG-INDUCED EEG ACTIVATION BLOCKING AGENTS ACTIVATING AGENTS Phenoxy-benzamine Chlor-promazine Dichloro-isoproterenol Atropine Epinephrine Norepinephrine Amphetamine + 4- -4- -t- - + + +® - + Isopropyl-Norepinephrine + + r ® + Histamine Serotonin • + + - 4-- + - + Acetylcholine Eserine - - - 4-— - - +® Key: + = blockade — = no blockade ® = large doses may overcome blockade PLATE E - 160 -and blocked only by chlorpromazine and atropine, and a third which is responsive to histamine and the short-acting adrenergic amines and which is blocked by phenoxybenzamine as well as chlorpromazine and atropine. The poss ib i l i ty of a further differentiat ion within this adrenoceptive category may be indicated by the very selective blocking action of DCI for isoproterenol. Similar observations of the effects of these agents on relaxation of the intestine has prompted Ahlquist (4) to make this inference for this tissue. These observations furnish a basis for some conclusions with regard to the sequence of these links in the chain of neurones which form the pathway for EEG "act ivat ion". The fact that adrenergic, "serotinergic" and "histaminergic" EEG "act ivat ion" a l l are blocked by atropine would seem to suggest that neurones responsive to these agents may feed Into cholinergic-sensitive neurones and that the latter may be the f inal links in the neuronal chain leading to drug-Induced EEG "act ivat ion" . The observation that "serotinergic" activation is not blocked by phenoxybenzamine suggests the poss ib i l i ty that serotonin sensitive neurones may occupy a position intermediate between adreno-ceptive and cholinoceptive components. The effects of blocking compounds on EEG "activation"produced by sensory inflow are of interest. Changes in the e lectr ica l act iv i ty of the brain in response to an arousal stimulus (whistle blast) are blocked by atropine, but not by chlorpromazine or phenoxybenzamine (except in doses far greater than those required to block drug-induced activation). Such observations have been made by other investigators in the case of atropine and chlorpromazine (22,1(^ 0,178). - 16, -Atropine also completely blocks the E€G "activation" produced by direct electrical stimulation of the brain stem reticular formation, whereas chlorpromazine produces a relatively small increase in the threshold for this response without completely abolishing it (23,96,97). However, the lack of selectivity on the part of electrical stimulation has limited the usefulness of observations employing this technique. E. Anatomical Site of Drug Effects on EEG "Activation". Efforts to locate the sites of action of these various compounds have employed the use of brain stem transection or gross destructive lesions. Rothballer (149) has reported that progressive destruction of the mesencephalic tegmentum in a rostral direction leads to loss of responsiveness to EEG "activation" by adrenergic amines. Bradley (21.).'* Bradley and EIkes (22), and others (145) have presented evidence which indicates that cholinergic and anticholinergic agents may act at levels rostral to the ponto-mesencephalic junction, since mesencephalic transection does not eliminate the usual EEG effects observed with these compounds. Our demonstration that similar effects are observed follow-ing the administration of acetylcholine and atropine in preparations with unilateral ponto-mesencephalic lesions, whereas adrenergic activation is completely eliminated on the ipsilateral side (see also Rothballer, 1956), lends additional support to the conceptof more rostrally situated cholinergic-sens?tive areas. However, caution must be observed in the interpretation of experiments designed to locate the site of various receptor elements by - 162 -means of brain stem .transection and gross destructive lesions. While this limitation may still apply, the possibility exists that discrete coagulative lesions and concomitant local recording of unit activity may permit a more critical analysis of the sites of action of agents presumed to produce EEG "activation". The possibility that gross lesions and transections may interrupt an essential link in the pathway rather than a specific receptor site under study must always be kept in mind. F. Correlation Between--Various Aspects of Drug Action of EEG "Activa- tion", Behaviour and Moody In the unanaesthetired de-afferented preparation, behavioural manifestations were confined to vocalization and movements of the head and forepaws. Such effects were observed/vvarying degrees following the administration of most compounds with EEG "activating" effects. These responses are difficult to categorize with respect to mood and alertness, and have not consistently been prevented by the various blocking compounds in doses which selectively abolish EEG "activation" Although EEG "activation" has been observed with both adrenergic and cholinergic agents, the adrenergic phase of EEG "activation" is particularly interesting because this phasehas been reported to be more closely linked to behavioural arousal, and because of the possible relationship of adrenergic activity to the action of drugs which have important clinical effects on the mood and behaviour of neurotic and psychotic patients. Atropine-Induced deactivation seems divorced from behavioural - 163 -alertness. Correlated observations made by Bradley (20) and Bradley and Elkes (22) between the EEG and behaviour have shown, that whereas atropine could induce synchronized slow wave activity similar to that of sleep, the animals were behaviourally awake. This is in agreement with the earlier observation of Wikler (178) who first noted the dissociation between EEG sleep- -patterns -(<ieac-tjva*ion^  and behaviour In atropinized dogs. Various observations point to the possibility that disturbances of central adrenergic function may be involved in disturbances of mood. Drugs which mimic the action of adrenergic agents are well known for their central nervous stimulating effects. S^hallek and Walz (156) have reported both EEG "activation" and increased motor activity in conscious dogs following amphetamine administration; an observation also noted in conscious cats by Bradley and Elkes (22). Drugs which prevent the destruction of local concentrations of biogenic amines in the central nervous system by inhibition of the enzyme monoamine oxidase are used in the treatment of depressed patients (33. 82). A number of hallucinogenic agents are structurally similar to adrenergic amines (e.g. mescaline) Or to adrenergic blocking compounds (e.g. LSD). Agents which allay apprehension, anxiety and agitation (chlorpromazine and reserpine) also have been linked to adrenergic mechanisms in the central nervous system (33,79,159), and direct evidence supports the thesis that chlorpromazine decreases sympathetic activity by a central action. Furthermore, the central effects of reserpine have been widely attributed to the ability of this compound to deplete this as well as other tissue of their store of norepinephrine and serotonin (33). - 164 -If increased activity in an adrenergic pathway is associated with alert behaviour and an elevated mood, then compounds such as phenoxybenzamine, which we have shown are able to block adrenergic mechanisms at central sites, should (like chlorpromazine) have the capacity to alleviate an agitated mood and should be expected to display ataractic properties. This suggestion has been put forward by Rockwell (146) who has described a prolonged tranqui1izing effect in anxiety and tension states, following the administration of dibenamine (a congener of phenoxybenzamine) which he believes exerts its action by central blockade of adrenergic substances, i-'urther, Medinets et al (119) have reported on the "dissolving" actions of dibenamine in catatonic patients who displayed definite improvement in abnormalities of motor behaviour for periods of time lasting 13 to 72 hours. These authors have assumed that the actions of dibenamine are related to its direct cerebral vascular effects. However, evidence Is lacking that dibenamine can exert a direct effect on the cerebral circulation (163), or that changes in cerebral circulation (short of anoxemia) can have psychogenic actions. It therefore seems likely that the effects of dibenamine on mood and behaviour are due to a direct central action which is unrelated to the vascular effects of this compound. In a recent report, Freedman et a I (53) have suggested a relationship between tranquiIizing agents and the ability of these agents to suppress apomorphlne-induced vomiting in dogs, and have indicated that the "apomorphine test" may have utility in selecting tranqui1izlng agents. They have concluded that dibenamine (2 mg/kg) is as effective against apomorphine-induced emesis as chlorpromazine, - 1 6 5 -but dif fers from chlorpromazine in that the latent period for the peak action of dibenamine effects is much longer (2 hours) and the duration much longer (2k hours) than chlorpromazine. Thesa.authors als raise the poss ib i l i ty that dibenamine may have ataractic actions and feel that "the demonstration of prolonged central neuronal alterations does raise the poss ib i l i ty that less toxic agents with greater tranquil izing potency may be found among some of the interesting chemical analogues of dibenamine." - 166 V. SUMMARY AND CONCLUSIONS 1. A technique has been developed which lends itself well to the study of the direct actions of drugs upon the various components of the reticular activating system of the brain stem. The experimental analysis of EEG "activation" requires the presence of a well deactivated background pattern. We have found that partial trigeminalectomy, cervical dorsalectomy and low cervical transection produce a preparation in which the resting EEG regularly manifests maximal deactivation. An indwelling catheter inserted via the right subclavian artery so that its tip lies in the innominate artery has furnished a means for the simultaneous bilateral distribution of injected drugs to the brain without embarrassing the flow in the carotid arteries. The advantages of this technique include (a) an intact brain stem, (b) the malntainence of adequate spontaneous respiratory and circulatory states, and (c) the ability to perform various operative procedures without the necessity for extraneous pharmacological agents (anaesthesia, muscle relaxant) which.may themselves have complicating effects on the EEG. 2. In this preparation adrenergic and cholinergic agents as well as histamine and serotonin all produced prompt, short-lasting and reproducible EEG "activation" in low doses (0.25-2.5 /Ug/kg) following direct intra-innominate administration. In "equi-activating" doses, Isoproterenol is the most potent EEG activating catechol adrenergic - 167 -amine and n o r e p i n e p h r i n e the l e a s t p o t e n t , w i t h e p i n e p h r i n e o c c u p y i n g an i n t e r m e d i a t e p o s i t i o n . Amphetamine and e s e r i n e both produce l o n g -l a s t i n g EEG " a c t i v a t i o n " , w i t h amphetamine h a v i n g a much s h o r t e r l a t e n c y than e s e r i n e . 3 . The d r u g - i n d u c e d EEG " a c t i v a t i o n " w h i c h we have o b s e r v e d i n t h i s p r e p a r a t i o n i s the r e s u l t o f a d i r e c t a c t i o n o f t h e s e compounds on the c e n t r a l nervous s y s t e m and i s n o t m e d i a t e d v i a r e f l e x e s from the c a r d i o v a s c u l a r s y s tem. T h i s v i e w i s based upon s e v e r a l l i n e s o f e v i d e n c e ; (a) temporal independence between t h e o n s e t and t e r m i n a t i o n o f d r u g - i n d u c e d changes i n e l e c t r o - c o r t i c a l a c t i v i t y and i n b l o o d p r e s s u r e f l u c t u a t i o n s , (b) d r u g - i n d u c e d a l t e r a t i o n s i n b l o o d p r e s s u r e a r e not u n i f o r m l y accompanied by EEG " a c t i v a t i o n " ( v a s o p r e s s i n , low doses o f h i s t a m i n e ) , and (c) the d e m o n s t r a t i o n o f t y p i c a l EEG " a c t i v a t i o n " p a t t e r n s f o l l o w i n g d r u g a d m i n i s t r a t i o n i n the p r e p a r a t i o n i n w h i c h both c a r o t i d s i n u s e s a r e c o m p l e t e l y d e n e r v a t e d . k. U n i l a t e r a l l e s i o n s w h i c h d e s t r o y most o r a l l o f the mesen-c e p h a l i c tegmentum a b o l i s h a d r e n e r g i c - i n d u c e d a c t i v a t i o n i n the i p s i -l a t e r a l c o r t e x but do not a f f e c t c h o l i n e r g i c a c t i v a t i o n . 5. R e s u l t s o b t a i n e d w i t h v a r i o u s s y n a p t i c b l o c k i n g a g e n t s have s u g g e s t e d t h e p o s s i b l e e x i s t e n c e i n the b r a i n o f t h r e e t y p e s o f r e c e p t o r s c a p a b l e o f c o n v e r g i n g on the f i n a l pathway f o r EEG " a c t i v a -t i o n " ; one r e s p o n s i v e to c h o l i n e r g i c compounds and b l o c k e d by a t r o p i n e ; one w h i c h f $ r e s p o n s i v e to s e r o t o n i n and b l o c k e d o n l y by c h l o r p r o m a z i n e and a t r o p i n e ; and a t h i r d w h i c h i s r e s p o n s i v e t o h i s t a m i n e and the s h o r t - a c t i n g a d r e n e r g i c amines and w h i c h i s b l o c k e d by phenoxybenzamine as w e l l as c h l o r p r o m a z i n e and a t r o p i n e . The responses o f the adreno-- 168 -ceptive components in the reticular activating system of the brain stem are not identical with those of any other known adrenergic receptors. 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