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

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PHARMACOLOGICAL ANALYSIS OF E E G "ACTIVATION" by GEORGE  MCDONALD  LING  M.A., University of British Columbia, 1957  A THESIS SUBMITTED I N 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 p r e s e n t i n g  this thesis i npartial fulfilment of  the r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t freely  a v a i l a b l e f o r r e f e r e n c e and s t u d y .  agree t h a t p e r m i s s i o n f o r e x t e n s i v e  I further  copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood  that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n  Department o f  PHARMACOLOGY  The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver Canada. Date  28  September  I960  permission.  ^i}? Pnttierstiy nf ^British Columbia FACULTY OF GRADUATE STUDIES GRADUATE  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 Neurophysiology ..  PROGRAMME OF THE  H . Scherrer W . C. Gibson  FINAL O R A L E X A M I N A T I O N  Related Studies: Neurosurgical techniques Psychiatry Biochemistry  FOR T H E D E G R E E  J. Wada J. S. Tyhurst  D O C T O R OF  ~ G . I. Drummond .S. H . Zbarsky  OF  PHILOSOPHY  of GEORGE LING B.A. M c G i l l , M . A . U.B.C.,  1943 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 . P E R R Y  W. H. G. G. A.  J. W A D A P. M c G E E R H. McLENNAN A . R. P. P A T T E R S O N  C. G I B S O N SCHERRER I. D R U M M O N D E. D O W E R M . CROOKER  External Examiner: DR. A . R O T H B A L L E R , Albert Einstein College of Medicine, New York City.  PHARMACOLOGICAL ANALYSIS OF EEG " A C T I V A T I O N " ABSTRACT 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 electrical 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 reticular activating system of the brain stem. The experimental analysis of E E G "activation" requires the presence of a well deactivated background 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 simultaneous bilateral distribution of injected drugs to the brain without embarrassing flow in the carotid arteries. The advantages of this technique include (a) an intact brain stem, (b) the maintenance of adequate spontaneous respiratory and circulatory states, and (c) the ability to perform various operative procedures without the necessity for extraneous 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 reproducible E E G "activation" in low doses following direct ihtra-innominate adminstration. In "equi-activating" doses, isoproterenol is the most potent E E G activating catechol adrenergic amine and norepinephrine 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 introduced 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-mesencephalic junction produces an increase in threshold for adrenergic E E G activation. Unilateral lesions which destroy most or all of the mesencephalic-tegmentum abolish adrenergic-induced activation in the insilateral cortex but do not affect cholinergic activation. Results obtained with various synaptic blocking agents have suggested 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 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 chlorpromazine 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).  -  ii -  ABSTRACT  The influence arousal brain  p a s t d e c a d e has  of  stem.  gested  that  d r u g s may  the  act,  playing A  the  of  the  brain.  point  a major  role  t e c h n i q u e has  in central  the  r e t i c u l a r a c t i v a t i n g system of of  EEG  Analysis  of  direct actions  drug  of  the  lends  brain the  The  presence of cat  produce a preparation  bilateral  the  to of  experimental  a well  low  r e s t i n g EEG  deactivated that  cervical  tran-  regularly  mani-  deactivation.  A permanent c a t h e t e r into  the o r i g i n o f  sug-  character  demonstrate  section  and  the  v a r i o u s components  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  artery  the  numerous  itself well  stem.  O b s e r v a t i o n s made i n t h e  in which  has  area wherein  partial  f e s t s maximal  and  action.  d r u g s upon the  " a c t i v a t i o n " requires  background p a t t e r n .  their effects  i s an  been d e v e l o p e d w h i c h  study of  the  "activation"  i t s multineuronal, polysynaptic  the  analysis  the  to  in  agents produce v a r i a t i o n s of  r e t i c u l a r a c t i v a t i n g system and  interest  r e t i c u l a r a c t i v a t i n g system of  Numerous p h a r m a c o l o g i c a l activity  intense  m e t a b o l i c s u b s t a n c e s upon EEG  m e c h a n i s m s , m e d i a t e d by  electrical  as  d r u g s and  w i t n e s s e d an  the  the  inserted  innominate artery  two  carotids,  d i s t r i b u t i o n of  has  through  the  so  i t s tip is positioned  that  furnished  i n j e c t e d drugs to  embarrassing flow  i n the  carotid arteries.  technique  (a)  intact brain  include  an  right  a means f o r  subclavian  simultaneous  the  brain  The  advantages of  s t e m , (b)  the  at  without this  maintenance  of  -  adequate spontaneous ability  r e s p i r a t o r y and  w h i c h may  agents  histamine  preparation adrenergic  and  "activation"  innominate a d m i n i s t r a t i o n . the most p o t e n t  epinephrine  the  the  (c)  EEG  ( a n a e s t h e t i c s , muscle r e l a x a n t s ) ,  and  the  EEG.  c h o l i n e r g i c a g e n t s as w e l l  i n low d o s e s f o l l o w i n g d i r e c t  and intra-  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  activating  least potent, with  mediate p o s i t i o n .  the  necessity  s e r o t o n i n a l l produced prompt, s h o r t - l a s t i n g  r e p r o d u c i b l e EEG  EEG  s t a t e s , and  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 In t h i s  is  circulatory  to perform v a r i o u s o p e r a t i v e procedures w i t h o u t  f o r extraneous pharmacological  as  H I -  A m p h e t a m i n e and  catechol  adrenergic  epinephrine  amine and  occupying  an  nor-  inter-  e s e r i n e both produce l o n g - l a s t i n g  " 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  latency  than  eserine. Clear differentiation d i r e c t drug-induced p a t h w a y s has  by  of  a g e n t s as  activation.  with  independence  a d m i n i s t r a t i o n and  reflected  i n blood  d e s t r u c t i o n i n the  c e p h a l i c j u n c t i o n p r o d u c e s an EEG  in preparations  Unilateral  pressure  cortex  but  Results obtained  do  reflex  bilateral  carotid  i s shown b e t w e e n agents  to  in threshold for  lesions which destroy  ponto-mesenadrenergic  most o r a l l o f activation  in  the the  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 .  with  various  the  inroduced  alterations.  tegmentum r o s t r a l  increase  over  from  the v a s c u l a r e f f e c t s  mesencephalic-tegmentum a b o l i s h adrenergic-induced ipsi-lateral  occur  the a c t i o n s of " a c t i v a t i n g "  intra-innominate  Partial  effects resulting  t h o s e w h i c h may  Complete temporal  termination of  directly these  i n f l u e n c e s and  been d e m o n s t r a t e d  sinus denervation. o n s e t and  b e t w e e n t h e EEG  s y n a p t i c b l o c k i n g agents  have  -  iv -  suggested the possible existence in the brain of three types of receptors capable of converging on the f i n a l pathway for EEG " a c t i v a t i o n " ; 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 chlorpromazine and atropine.  The responses of the adrenoceptive components in  the r e t i c u l a r activating system of the brain stem are not identical with those of any other known adrenergic receptors. emphasizes the d i f f i c u l t y in attempts to c l a s s i f y few c l e a r l y defined and discrete categories.  This observation  receptors into a  - V -  TABLE OF CONTENTS Page I. II.  INTRODUCTION  I  METHODS A. S u r g i c a l Procedures 1. A n a e s t h e t i z e d P r e p a r a t i o n s (a) B a r b i t u r a t e s (b) Inhalation anaesthetics (ether). . 2 . The U n a n a e s t h e t i z e d C u r a r i z e d P r e p a r a tion 3. E n c e p h a l e and Cerveau I s o l e P r e p a r a tion 4. M i d - B r a i n C o a g u l a t e d P r e p a r a t i o n ... 5. U n a n a e s t h e t i z e d D e - a f f e r e n t e d P r e p a r a tion (a) Low t r a n s e c t i o n o f the c e r v i c a l s p i n a l c o r d and c e r v i c a l sensory roots (b) E x t r a - c r a n i a l t r a n s e c t i o n o f trigeminal roots 6. I n t r a - l n n o m i n a t e C a n n u l a t i o n f o r D i r e c t C e n t r a l Drug E f f e c t s B. E x p e r i m e n t a l Procedures v  III.  RESULTS A. B a s i c EEG P a t t e r n B. P r e p a r a t i o n W i t h I n t a c t B r a i n Stem and I n t a c t C a r o t i d Sinuses 1. C o n t r o l s 2. A d r e n e r g i c A c t i v a t i o n (a) E f f e c t s o f e p i n e p h r i n e , i s o p r o t e r e n o l , n o r e p i n e p h r i n e and racemicamphetamine (b) E f f e c t s o f some isomers o f i s o proterenol administered separately and i n c o m b i n a t i o n (i) Isomers o f i s o p r o t e r e n o l administered separately. . . (ii) Isomers a d m i n i s t e r e d i n combination 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 o f a c e t y l c h o l i n e and e s e r i n e ( p h y s o s t i gmine)  11 12 12 12 12 13 14 14 16  16 17 22 23 27 27 29 29 29 29 41 42 42 45 45  - vi -  '  J  TABLE OF CONTENTS III.  RESULTS (cont'd) Activating Effects of Other Agents . . . (a) Vasopressin and histamine (i) Vasopressin (ii) Histamine (b) Serotonin 5 . Effects of Blocking Agents (a) Phenothiazine derivatives (chlorpromazine and promazine (i) Chlorpromazine (ii) Promazine (b) Phenoxybenzamine (dibenzyline). . . (i) Adrenergic amines: isoproterenol, epinephrine, and norepinephrine (ii) Histamine ( i i i ) Acetylcholine . . . . . . . . (iv) Serotonin (c) Dichloroisoproterenol (DC 1) . . . . (i) Isoproterenol (ii) Epinephrine and norepinephrine ( i i i ) Acetylcholine and serotonin . (d) Atropine Preparation With B i l a t e r a l Carotid Sinus Denervation 1. Controls 2 . Effects of Pressor Agents: Epinephrine, Norepinephrine and serotonin 3 . Effects of Depressor Agents: A c e t y l c h o l ine and isoproterenol . . Preparation With Brain Stem Lesions 1. Effects of Pontile Lesions on the EEG of the Unanaesthetized De-afferented C a t . . . 2. Effects of Unilateral Ponto-Mesencephalic Lesions on EEG " A c t i v a t i o n " (Adrenergic and Cholinergic) 4.  C.  D.  IV. V. VI .  (cont'd.) Page 52 52 5*+ 54 56 60 60 60 76 77 79 85 85 88 94 96 96 100 109 110 110 117 117 124 124 128  DISCUSSION  139  SUMMARY AND CONCLUSIONS  166  BIBLIOGRAPHY  169  -vii  —  LIST OF FIGURES  Figure  Page  1  E f f e c t of a u d i o g e n i c  activation  2  E f f e c t on the EEG of  f e e d i n g v i a stomach tube  3  E f f e c t of  4  E f f e c t of e p i n e p h r i n e 0 . 5 / u g / k g on the EEG  32  5  E f f e c t o f n o r e p i n e p h r i n e 0 . 5 /ug/kg on the EEG  33  6  E f f e c t of  3**  7  E f f e c t o f amphetamine  8  E f f e c t o f e p i n e p h r i n e 0 . 2 5 / U g / k g on the EEG  36  3  E f f e c t of e p i n e p h r i n e 2 . 0 / j g / k g on the EEG  37  10  E f f e c t of  n o r e p i n e p h r i n e 0 . 5 yug/kg on the EEG  38  11  E f f e c t o f n o r e p i n e p h r i n e 2 . 0 / j g / k g on the EEG  39  12  E f f e c t of  13  E f f e c t o f d - and  14  E f f e c t o f a combination of d - and  intra-innominate  isoproterenol  upon the EEG  20 21  s a l i n e on the EEG  30  0 . 2 5 / J g / k g on the EEG  50/Ug/kg on the EEG  isoproterenol  35  0 . 5 / U g / k g on the EEG  I - i soproterenol  40  on the EEG  43  1-isoproterenol  2 . 0 / u g / k g each on the EEG 2 . 0 / j g / k g 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 . 2 5 / u g / k g on the EEG  46  16  E f f e c t of a c e t y l c h o l i n e 0 . 5 Xig/kg on the EEG  47  17  E f f e c t of a c e t y l c h o l i n e  l . 0 ^ g / k g on the EEG  48  18  E f f e c t of a c e t y l c h o l i n e 2 . 0 /Ug/kg on the EEG  49  19  E f f e c t o f e s e r i n e 50/ug/kg on the EEG  50  20  E f f e c t of eserine  51  21  E f f e c t o f c h l o r p r o m a z i n e and a t r o p i n e on e s e r i n e - i n d u c e d activation  100/Ug/kg  53  - vi i i -  LIST OF FIGURES (cont'd.)  Figure  Page  22  E f f e c t o f v a s o p r e s s i n 0.5 u n i t s / k g on the EEG  55  23  - E f f e c t o f h i s t a m i n e 1 . 0 ^ g / k g on the EEG  57  24  E f f e c t o f h i s t a m i n e 2.0/jg/kg on the EEG  58  25  E f f e c t o f h i s t a m i n e 5 . 0 / i g / k g on the EEG  59  26  Effect of serotonin  5 /jg/kg on the EEG  61  27  Effect of serotonin  20 /Ug/kg on the EEG  62  28  E f f e c t o f CPZ 2.0 mg/kg on the EEG  64  29  E f f e c t o f e p i n e p h r i n e 0.5 /ig/kg on the EEG a f t e r CPZ blockade  66  E f f e c t o f n o r e p i n e p h r i n e 2.0 Aig/kg on the EEG i n the p r e s e n c e o f CPZ b l o c k a d e  67  Effect of serotonin of CPZ b l o c k a d e  68  30 31  32  5.0/jg/kg on t h e EEG In t h e p r e s e n c e  E f f e c t o f h i s t a m i n e 5.0 /ug/kg on the EEG i n the p r e s e n c e o f CPZ b l o c k a d e  69  E f f e c t o f i s o p r o t e r e n o l 0.5Aig/kg on the EEG i n the p r e s e n c e o f CPZ b l o c k a d e  72  E f f e c t o f i s o p r o t e r e n o l 0.5/jg/kg on the EEG i n the p r e s e n c e o f CPZ b l o c k a d e and v a s o p r e s s i n  73  E f f e c t o f amphetamine on the EEG i n the p r e s e n c e o f CPZ blockade  74  36  E f f e c t o f phenoxybenzamine (DBZ)  78  37  Effect of isoproterenol phenoxybenzamine  0.5 /ig/kg on the EEG b e f o r e  Effect of isoproterenol phenoxybenzamine .  0.5/ug/kg on the EEG a f t e r  E f f e c t of isoproterenol phenoxybenzamine  l.Oyug/kg on the EEG a f t e r  33 34 35  30 39  1.0 mg/kg on the EEG . .  80 81 82  - ix -  LIST OF FIGURES (cont'd) Figure 40 41 42 43 44 45 46 47 48  Page E f f e c t o f e p i n e p h r i n e 0.5 >ug/kg on the EEG phenoxybenzamine  before  E f f e c t o f e p i n e p h r i n e 0.5 /ig/kg on the EEG a f t e r phenoxybenzamine  84  E f f e c t o f h i s t a m i n e 2.0 /ug/kg on the EEG b e f o r e phenoxybenzami ne  86  E f f e c t o f h i s t a m i n e 2.0/jg/kg on the EEG a f t e r phenoxybenzamine  87  E f f e c t o f 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  Effect of serotonin benzamine  2.5/Jg/kg on the EEG b e f o r e phenoxy-  Effect of serotonin benzamine  2.5 ;ug/kg on the EEG a f t e r phenoxy-  Effect of serotonin benzamine  5.0 /jg/kg on the EEG b e f o r e phenoxy-  E f f e c t of serotonin benzamine  5.0/Ug/kg on the EEG a f t e r phenoxy. . . . . . . .  .  92  E f f e c t o f DC I 15 mg/kg on the EEG  50  E f f e c t of isoproterenol 7.5 mg/kg  0.5A»g/kg on the EEG a f t e r DCI  E f f e c t of isoproterenol 7.5 mg/kg  1.0/jg/kg on the EEG a f t e r DCI  E f f e c t of isoproterenol 15 mg/kg  1.0/ug/kg on the EEG a f t e r DCI  52  90 91  49  51  83  93 95 97 98 99  53  E f f e c t o f e p i n e p h r i n e 0 . 5 /Ug/kg on the EEG a f t e r DCI  54  E f f e c t of norepinephrine 0.5  55  E f f e c t o f e p i n e p h r i n e 2 . 0 / j g / k g on the EEG a f t e r DCI  56  E f f e c t o f n o r e p i n e p h r i n e 2.0  57  E f f e c t o f 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  ytig/kg  /Ug/kg  . . 101  on the EEG a f t e r DCI  . 102  . . 103  on the EEG a f t e r DCI . 104 .105  - x -  LIST OF FIGURES (cont'd) Figure 58  E f f e c t of acetylcholine 2 . 0 /Ug/kg on the EEG a f t e r DCI.  59  E f f e c t of serotonin 5 . 0 /Ug/kg on the EEG before DCI  60  Effect of serotonin 5/Ug/kg on the EEG after DCI  . . . .  108  61  E f f e c t of epinephrine 0 , 5 / j g / k g and acetylcholine on the EEG a f t e r atropine  Ill  E f f e c t of isoproterenol 2 . 0 /Ug/kg and serotonin on the EEG a f t e r atropine . ,  112  62  .  106  . . .  107  5.0/Ug/kg  E f f e c t of epinephrine 2 . 0 /Ug/kg and norepinephrine 2 . 0 /Ug/kg on the EEG after atropine  113  64  E f f e c t of atropine 0 . 5 mg/kg on the EEG a f t e r eserine . .  114  65  E f f e c t of control injections of saline on the EEG a f t e r b i l a t e r a l carotid sinus denervation (C.S.D.)  116  63  66  E f f e c t of epinephrine 0.5/Ug/kg on the EEG after b i l a t e r a l C.S.D 118  67  E f f e c t of serotonin 5 . 0 /Ug/kg on the EEG a f t e r b i l a t e r a j C.S.D  119  E f f e c t of acetylacholine 0 . 5 / U g / k g on the EEG a f t e r b i l a t e r a l C.S.D  121  E f f e c t of isoproterenol 0 . 5 / j g / k g followed by norepinephrine 0.5/ug/kg after b i l a t e r a l C.S.D  122  E f f e c t of isoproterenol 0 . 5 /Jg/kg in the presence of phenoxybenzamine a f t e r C.S.D  123  E f f e c t on the EEG of unilateral lesions in the caudal part of the right mesencephalic tegmentum (R.M.T.) . . .  129  68 69 70 71  i  Page  72  E f f e c t of epinephrine 2 . 0 /Ug/kg on the EEG in the presence of u n i l a t e r a l lesions in the caudal portion of R.M.T. . . 130  73  E f f e c t of acetylcholine 2 . 0 pg/kg on the EEG in the presence of u n i l a t e r a l lesions in the caudal portion of the R.M.T.  131  - xi -  L I S T OF FIGURES  (cont'd)  Figure  Page E f f e c t o n t h e EEG o f f u r t h e r  75  E f f e c t o f e p i n e p h r i n e 2 . 0 / U g / k g o n t h e EEG I n t h e p r e s e n c e of 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 t h e R.M.T  13^  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 o n t h e 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 t h e R.M.T  135  E f f e c t o f e p i n e p h r i n e 2 . 0 / j g / k g o n t h e EEG a f t e r u n i l a t e r a l lesions extending into 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 t h e R.M.T  136  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 o n t h e EEG a f t e r u n i l a t e r a l lesions extending into 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 t h e R.M.T  137  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 o n t h e 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  76  77  73  79  lesions  i n t h e R.M.T.  . . .  133  7k  - xii -  L I S T OF  PLATES  Plate  Page  A  Reticular  B  Cross sections of coagulation lesions mesencephalic areas o f c a t brain  k  formation of cat brain  i n the ponto. . . .  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 stereot a x i c c o o r d i n a t e s showing areas o f c o a g u l a t i o n . . i . 126  D  Photograph of the c e r e b e l l a r approach f o r the p r o d u c t i o n of e l e c t r o l y t i c l e s i o n s i n the ponto-mesencephalic junction with glass insulated electrode . . . . . . .  125  E f f e c t o f b l o c k i n g a g e n t s o n EEG " a c t i v a t i o n " by some a d r e n e r g i c a n d c h o l i n e r g i c a g e n t s , a n d by h i s t a m i n e a n d serotonin  159  E  - xiti  -  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 c r i t i c i s m 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 f o r their r e l i a b l e assistance during surgical procedures.  - 1-  I.  INTRODUCTION  The f i r s t 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 wavelike 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 r s t 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 i r s t 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 a l e r t state, is known as the r e t i c u l a r a c t i v a t i n g system (area R.F.  in Plate A ) , and includes the r e t i c u l a r formation of the oblongata and  pontine tegmentum, extending from bulbar to mid-brain regions. As shown in Plate A, impulses which follow the c l a s s i c a l  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 l a t e r a l brain stem, and synapse once again in one of the relay nuclei of the thalamus, before f i n a l l y passing by way of the internal capsule to s p e c i f i c c o r t i c a l areas. In contrast, a secondary extra-lemnlscal ascending pathway receives impulses via c o l l a t e r a l f i b r e s (57) passing from the lemniscus Into the central brain stem, when impulses are dispatched r o s t r a l l y to cause widespread and generalized EEG " a c t i v a t i o n " . The mid-brain r e t i c u l a r formation also receives projections from several areas of the cerebellar-hemispheres (122,161,162,171), as well.as from c e r t a i n discrete areas of the cerebral cortex.  The places of o r i g i n  of this c o r t i c i f u g a l projection as found in the monkey, are the frontal eye f i e l d s , the sensory motor cortex, the p a r a - o c c i p i t a l cortex, the f i r s t temporal gyrus, the o r b i t a l surface of the frontal lobe, the cingulate gyrus, the t i p of the temporal lobe and the f i r s t temporal gyrus.  Each of  these c o r t i c a l f i e l d s appears to project down Into the r e t i c u l a r formation to very much the same brain stem areas which receive c o l l a t e r a l s from a l l the afferent systems of the body.  Impulses from many of the active c o r t i c a l  loci described were found to funnel Into a common pathway which in turn made connections with the r e t i c u l a r a c t i v a t i n g system (46,71).  Alternatively  other c o r t i c a l loci were demonstrated to e x h i b i t 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 7. Diagram of cat brain shows cortical connections (anterior dotted arrow) capable of influencing reticular formation (RF). The dark arrows indicate inhibitory influences which the brain stem exerts upon sensory conduction 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.-") FIGURE  PLATE A  - 5 -  r e t i c u l a r a c t i v a t i n g system, whereas impulses from the hippocampus and the entorhinal cortex made connections with the r e t i c u l a r brain stem v i a the s t r i a medullaris (1,3,66). Livingston (108), as well as Hernandez-Peon et al (76), have shown that d i f f e r e n t modalities of afferent sensory input w i l l  interact  in the  r e t i c u l a r formation with each other, and with each of the projections descending from any one of the c o r t i c a l areas which project into the r e t i c u l a r formation. of central  Thus, i t seems that the r e t i c u l a r formation constitutes  a sort  integrating switch-board f o r the interaction of impulses generated  in remote and varied parts of the nervous system.  In a d d i t i o n , not only is  there interaction between descending projections from the cortex with sensory input, but each of the c o r t i c a l f i e l d s interacts with each other, and appears to have a rather special pattern of influence within the brain stem r e t i c u l a r formation (2).  For example, some of these f i e l d s w i l l augment i n t r i n s i c  a c t i v i t y of the brain stem while others w i l l  diminish i t , giving rise to a  complex sequence of a l t e r n a t i n g 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 r e t i c u l a r a c t i v a t i n g system, but that the cortex i t s e l f possesses c o r t i c i f u g a l regulating mechanisms which in turn can influence the level of a c t i v i t y within the r e t i c u l a r formation (108). a c t i v i t y , which r e f l e c t s  This level of  Input and output r e l a t i o n s , taken together with  the brain stem's own contributions to these r e l a t i o n s , total organization of behaviour (2,128).  is important to the  Using implanted electrodes In  unanaesthetized cats, Hernandez-Peon, Scherrer and Jouvet (76), and others (75,83) have shown that potentials evoked i n the s p e c i f i c sensory areas of the cortex are depressed during attentive behaviour, and the suggestion has  - 6-  been raised that r e t i c u l a r discharges may modify or i n h i b i t transmission the f i r s t synapse of the pathways  involved ( 7 6 ) .  reported recently by Hugelin et al ( 8 5 ) .  in  Similar results have been  In contrast these l a t t e r Investiga-  tors relate the reduction of the cochlear response to the f a c i l i t a t i o n of contraction of tympanic muscles; t h i s , they state, reduces the pressure transmitted to the cochlea and attenuates cochlear p o t e n t i a l s .  They  therefore consider the r e t i c u l a r effects on auditory Input to be the result of an infralamlnal reaction ( 8 5 ) .  reflex f a c i l i t a t i o n belonging to a generalized motor  That these dynamic alterations along the sensory  pathways  are related to a c t i v i t y in the brain stem r e t i c u l a r formation is made clear by the e f f e c t s of brain stem destruction, since, In f a c t , neither the phenomena of habituation nor focus of a t t e n t i o n , nor of conditioning, appear to survive a f t e r brain stem lesions ( 5 1 , 5 4 , 5 5 ) • "The influences of metabolic substances, hormonal agents, drugs and c i r c u l a t i n g hormones upon arousal mechanisms mediated by the r e t i c u l a r system have attracted much attention recently" ( 5 4 ) .  Numerous pharmacological  agents produce variations of 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 .  Analysis  of their e f f e c t s has suggested that the r e t i c u l a r a c t i v a t i n g system is an area wherein numerous drugs may a c t , and point to its multineuronal polysynaptic character as playing a major role In central drug a c t i o n . ( 2 9 ) , Forbes and associates  ( 5 2 ) , and Barany (11)  Bremer  have emphasized the  s u s c e p t i b i l i t y of complex neurone systems to anaesthesia.  Larraboe and  Posternack (103) have shown that the blocking e f f e c t of central anaesthetics on peripheral nerve f i b r e conduction proceeds without reference to f i b r e diameter or v e l o c i t y of f i b r e conduction, and that synaptic  transmission  is a l i m i t i n g f a c t o r , which can be blocked long before f i b r e conduction is a f f e c t e d .  French et al (57) have observed a d i f f e r e n t i a l block of  ascending  conduction  i n t h e 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  o f p e n t o b a r b i t a l o r e t h e r , and have proposed t h a t t h i s may be o f importance i n the p r o d u c t i o n o f 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 d i s c u s s e d  the e f f e c t s o f t h i o p e n t a l on t h e evoked c o r t i c a l p o t e n t i a l and on t h e EEG, and  s i m i l a r s t u d i e s have been r e p o r t e d f o r c h l o r a l o s o n e by Munroe e t a l  (124).  I n a d d i t i o n , K i n g e t a l (98). u s i n g low and h i g h c o n c e n t r a t i o n s o f  b a r b i t u r a t e s , have c o n f i r m e d  the f u n c t i o n a l block o f ascending  influences  on the b r a i n stem r e t i c u l a r f o r m a t i o n i n response t o low c o n c e n t r a t i o n s and, moreover^ have demonstrated a d e p r e s s a n t  e f f e c t o f larger concentrations  d i r e c t l y upon t h e t h a l a m i c r e l a y n u c l e i . Conversely,  i t has been demonstrated t h a t c o n d u c t i o n  i n the brain  stem r e t i c u l a r f o r m a t i o n c a n be markedly enhanced by s t r y c h n i n e and m e t r a z o l (3,51.137.138), and EEG a r o u s a l c a n be e l i c i t e d by such d i v e r s e agents as c o c a i n e , methamphetamine and p h e n y l e p h r i n e eserine, mescaline,  (150. p i p r a d r o l , amphetamine,  l y s e r g i c a c i d d i e t h y l a m i d e , p h e n i d y l a t e (21,86),  2-dimethy 1-amino-ethanol (130. ©(-methyl t r y p t a r n i n e (168), t r e m o r i n e (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 clinical  changes i n t h e s t a t e o f EEG " a c t i v a t i o n " t o t h e i m p o r t a n t  e f f e c t s on t h e mood and b e h a v i o u r 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 o f these compounds.  S  S e v e r a l o f t h e drugs which e l i c i t EEG " a c t i v a t i o n " (e$. amphetamine and e s e r i n e ) resemble compounds w h i c h a r e known t o s e r v e as p e r i p h e r a l transmitters.  F u r t h e r m o r e , drugs w h i c h produce EEG d e a c t i v a t i o n i n c l u d e  agents w h i c h produce b l o c k i n g e f f e c t s a t v a r i o u s 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 o b s e r v a t i o n s  r a i s e the q u e s t i o n as t o  whether t h e mechanism o f a c t i o n o f these drugs a t c e n t r a l s i t e s may n o t be analogous t o t h a t w h i c h they e x e r t a t p e r i p h e r a l s y n a p s e s .  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 i d e n t i f i e d and characterized in considerable d e t a i l .  However, less d e f i n i t e information  is available concerning the i d e n t i t y , the possible r o l e , the l i k e l y 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 t e within the CNS should f u l f i l l  the c r i t e 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 i t s proposed function, and its release following a c t i v i t y should be demonstrable at the s p e c i f i e d synaptic s i t e . Adequate concentrations of enzymes necessary for Its a c t i v a t i o n must e x i s t in that l o c a l e .  synthesis and its i n -  Demonstrable central effects should  be reproduced consistently by the d i r e c t administration of the l i k e l y candidate to the proposed s i t e , and i t should be possible to duplicate these e f f e c t s with the use of agents known to exert an inhibitory e f f e c t on the  In this regard the complex structural organization of the central nervous system imposes obstacles to the r e l i a b l e c o l l e c t i o n and i d e n t i f i c a tion of substances liberated at central neurone terminals following either autochthonous discharges or exogenous stimulation. However, in some areas of the brain, increased a c t i v i t y ( e l e c t r i c a l stimulation) as well as certain drug-induced effects (metrazol, ethanol, benzoquinolizlne) have been demonstrated to result in the depletion of the content of c e r t a i n compounds considered as candidates for the role of synaptic mediator. In addition, s i m i l a r phenomena have been observed to be associated with the administration of e f f e c t i v e doses of c e r t a i n psychotropic agents (reserplne, desmethoxyreserpine, 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 neurohormone 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 activating 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 electroencephalographic 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.  A.  Surgical  METHODS  Procedures.  Of fundamental  importance to the analysis of drug-induced EEG  " a c t i v a t i o n " 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 a c t i v i t y in a chosen area may be recorded and measured (95)• The available data bearing on the effects of drugs on EEG " a c t i v a t i o n " 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 " a c t i v a t i o n " studies. baseline seems essential effects.  A consistent,  r e l i a b l e and uniform  for the analysis of unmistakable " a c t i v a t i o n "  Rothballer (149) has stated the problem succinctly:  "Whether or  not any EEG changes could be detected a f t e r intravenous adrenaline depends primarily upon the background of a c t i v i t y , and to a much lesser extent on the dosage.  Working against a background of " a r o u s a l " ,  d i f f i c u l t to detect any e f f e c t at a l l . "  i t was quite  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 (a)  Preparations.  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 a l t e r s and modifies the background EEG a c t i v i t y , 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  t r i - e t h i o d i d e , decamethoniurn or succinylcholine) are without central effects (49,69).  The most obvious  influence of these agents under these conditions  is the loss of respiratory a c t i v i t y . necessary and raises  This makes a r t i f i c i a l  ventilation  the problem of adequate levels of oxygenation without  hypo- or hyper-capnia, since changes of blood pH may markedly a l t e r the a c t i v i t y of the CNS (95).  Furthermore, the r e l a t i v e 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 f u l l  respiratory  p a r a l y s i s , once a r t i f i c i a l respiration Is properly adjusted, becomes an additional problem. Another s t r i k i n g e f f e c t of curarizing agents, p a r t i c u l a r l y of d-tubocurarine is a marked f a l l  In blood pressure to shock levels  care in timing the injection Is not observed.  if adequate  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. noise, handling)  In addition, external stimulation  must be reduced to minimum levels and a r e l i a b l e  deactivated background is d i f f i c u l t to reproduce consistently.  (light,  - 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 p r e p a r a t i o n i n w h i c h t h e s p i n a l c o r d i s  severed  a t t h e l e v e l o f t h e f i r s t o r second c e r v i c a l v e r t e b r a e  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  t i o n , must a l s o be employed.  (27.30).  problems o f adequate oxygena-  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 t h e r e l i a b l e d e m o n s t r a t i o n induced " a c t i v a t i o n " .  o f drug-  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 t h e  c e r v e a u i s o l e ( m i d - b r a i n t r a n s e c t i o n from t h e s u p e r i o r c o l l i c u i u s t o t h e mammillary body), w h i c h o f f e r s s t a b l e s y n c h r o n i z e d electrical  activity  (23,27,29).  patterns of c o r t i c a l  However, 1 i k e o t h e r p r e p a r a t i o n s  the c r a n i u m i s w i d e l y opened, c e r e b r a l oedema u s u a l l y d e v e l o p s ,  i n which  d e s p i t e the  a p p l i c a t i o n o f temporary manual c o m p r e s s i o n a t the base o f t h e s k u l l a t t h e moment o f t r a n s e c t i o n (19,104);  Moreover, i n a l a r g e p e r c e n t a g e o f c a s e s ,  i n c r e a s i n g i n s t a b i l i t y o f t h e b l o o d p r e s s u r e w i t h time and e x p e r i m e n t a l usage (3-5 hours) tended t o 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 use.  A r e c e n t r e p o r t by Batset  (12) I n d i c a t e s t h a t b e t t e r  prolonged  experimental  s t a b i l i t y can be a c h i e v e d w i t h t h e c e r v e a u i s o l e p r e p a r a t i o n i n t h e dog, but s i n c e no b l o o d p r e s s u r e  r e a d i n g s were r e p o r t e d  i n t h i s study t h e  q u e s t i o n o f t h e adequacy o f t h e c a r d i o v a s c u l a r s t a t u s remains i n doubt. k.  Mid-Brain Coagulated P r e p a r a t i o n . T h i s p r o c e d u r e f o l l o w e d t h a t o r i g i n a l l y d e v e l o p e d by Naquet (126)  and  s t u d i e d by R o t h b a l l e r ( 1 4 9 ) .  Anaesthetized  c a t s were p l a c e d  ina  Johnson S t e r e o t a x i c Instrument Model No. 2 ) 0 , w h i c h was c a l i b r a t e d t o conform to  c o o r i n d a t e s o f "A S t e r e o t a x i c A t l a s o f t h e D i e n c e p h a l o n o f the C a t " by  H. J a s p e r and Cosimo Ajmone-Marsan ( 9 1 ) . were r e f l e c t e d from t h e m i d - s a g g i t a l  line.  The muscles o v e r l y i n g the s k u l l B i l a t e r a l points of entry  into  - 15 -  the b r a i n were e s t a b l i s h e d by the s t e r e o t a x i c c a r r i e r from the p r e c i s e working c o o r d i n a t e s which were to be l a t e r used i n c o a g u l a t i n g  the m i d - b r a i n  reticular  substances. T r e p h i n e d h o l e s were c a u t i o u s l y 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  o c c u r ( g r e a t e r w i t h e t h e r than w i t h t h i o p e n t a l  degree of b l e e d i n g does  i n the m a j o r i t y of c a s e s ) ,  hemostasis can r e a d i l y be produced by the use o f bone wax. was  Next,  but  the dura  l i f t e d c a r e f u l l y by means of a d u r a l hook, and i n c i s e d a t the p o i n t o f  e l e c t r o d e e n t r y w i t h a sharp g a n g l i o n k n i f e .  E l e c t r o d e s mounted on a  s t e r e o t a x i c c a r r i e r were then i n t r o d u c e d , u s i n g working c o o r d i n a t e s for mid-brain r e t i c u l a r coagulation.  specific  F i r s t one s i d e was c o a g u l a t e d .  The  e l e c t r o d e s were removed and c l e a n e d and next i n s e r t e d i n t o the c o n t r a l a t e r a l side f o r coagulation of  the homologous a r e a .  The c u r r e n t necessary  for  c o a g u l a t i o n was o b t a i n e d from a h i g h frequency s o u r c e , u s i n g a H e a t h k i t Amateur T r a n s m i t t e r Model DX-20.  The output of  this  instrument was  a t t e n u a t e d by means o f a v a r i a b l e o u t p u t c o n t r o l , c o n s i s t i n g o f a s e r i e s  lamp  and p o t e n t i o m e t e r , 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 e c t r o d e s , and f o r various  spacing of e l e c t r o d e s .  the time necessary  to produce a d e f i n i t e amount o f c o a g u l a t i o n of  albumin a t a temperature o f 20° A s e r i e s of  of  This  w i t h the o s c i l l a t o r s e t a t 3.5  instrument, with  f o r producing coagulative  the m i d - b r a i n  egg  C.  investigations  have shown that t h i s satisfactory  T h i s c a l i b r a t i o n was a c h i e v e d by o b s e r v i n g  megacycles  i t s adjustable output c o n t r o l , lesions  is  i n the r e t i c u l a r f o r m a t i o n  tegmentum,  technique produces a p r e p a r a t i o n which e x h i b i t s a good  " d e a c t i v a t e d " EEG p a t t e r n as w e l l as a r e l i a b l e c a r d i o v a s c u l a r s t a t u s , here a g a i n the b r a i n stem is  not i n t a c t and c o a g u l a t i v e  but  l e s i o n s may e l i m i n a t e  - 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 (13).  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 ( 1 0 7 ) .  (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 t e r i l o bone wax.  With a dural hook, the dura at the level of Tj is then  elevated and a longitudinal roots of  i n c i s i o n made r o s t r a l l y to C 2 .  The dorsal  to Cg are gently elevated and sectioned b i l a t e r a l l y  high frequency coagulation of the dorsal accompany these roots. j u s t rostral  radicular a r t e r i e s  after  that  The dorsal spinal artery is then coagulated  to the point chosen for cord transection.  The cord is  then severed between the last c e r v i c a l and f i r s t thoracic vertebrae and under d i r e c t v i s i o n the section is further explored by gentle suction to make sure that a l l connecting strands of cord tissue are separated.  S t e r i l e 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. mouth.  The lower jaw is opened maximally to expose the roof of the An i n c i s i o n 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 l a t e r a l l y a short distance from their points of emergence from the foramina ovale and rotundum.  Extreme caution is necessary  - 18 -  to p r e v e n t  haemorrhage from the e x t e r n a l r e t e , the m i d d l e meningeal  anastomatic  and  t e n s o r tympanic a r t e r i e s ( 3 7 ) .  The  fossae  created  by removal o f the p t e r y g o i d muscles a r e packed w i t h s t e r i l e sponge and  the i n c i s i o n  This p r e p a r a t i o n recovers  gelatin  sutured.  r e a d i l y from s p i n a l shock and may  be main-  •ie t a i n e d f o r as l o n g as 2 to 3 months These i n c l u d e (1) c o n t i n u e d a reasonably  i f s u i t a b l e precautions  are observed.  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 o f  u n i f o r m and p h y s i o l o g i c a l environment t e m p e r a t u r e , (3)  c a t h e t e r i z a t i o n , and  (k)  spontaneous d i a p h r a g m a t i c  f e e d i n g by stomach tube.  This p r e p a r a t i o n maintains  r e s p i r a t i o n and a b l o o d p r e s s u r e of 70-90 mm  W h i l e some degree of motor i n n e r v a t i o n o f the muscles of the neck and l i m b remains i n t a c t , the animal The  sensory  bladder  Hg. fore-  g e n e r a l l y remains q u i e s c e n t .  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 t o (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 n e r v e s , and o r b i t and upper e y e l i d v i a the o p h t h a l m i c  (2) s o m a t i c a f f e r e n t s to the  branch o f the t r i g e m i n a l n e r v e .  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 t o c a r r y o u t a l l o f the f o l l o w i n g p r o c e d u r e s on the u n a n a e s t h e t i z e d (I)  To  cat:  insert indwelling polyethylene catheters into a r t e r i e s  and  atv e i n s f o r the r e c o r d i n g of 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 and administration. d i r e c t l y and  By t h i s t e c h n i q u e  bilaterally  drugs can be  i n t o the b r a i n w i t h o u t  f o r drug  injected repeatedly, compromising the c e r e b r a l  c i r c u l a t i o n and w i t h o u t a p p r e c i a b l e l a t e n c y f o r o b s e r v i n g In a d d i t i o n , t h i s approach p e r m i t s a c l o s e r "check" on the  immediate e f f e c t s . concentration  Some degree o f w a s t i n g o f the muscles o f the hindpaws i s noted when the p r e p a r a t i o n i s m a i n t a i n e d f o r such l o n g p e r i o d s .  - 19 -  of agents used, since d i l u t i o n and enzymatic inactivation of the injected drugs are minimized.  In such preparations, an indwelling a r t e r i a 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, (ii) (iii)  To place the animal  in a stereotaxic instrument,  To expose the cranium and to place screw electrodes through the  skull with tips touching the dura f o r EEG leads. (iv)  To trephine the skull and to place electrode tips  in the brain  stem for the production of e l e c t r o l y t i c lesions. (v)  To perform bilateral' denervation of the carotid sinuses, and to  evalua te the e f f e c t s o f th i s procedure in an unanaes the t i z e d , non-curarized anima 1. - • A welcome and; unexpected dividend, which has 'iTiade this preparation 1  a p a r t i c u l a r l y valuable tool for this investigation,  is the fact that a  highly deactivated EEG can readily be produced even in the absence o f ' b r a i n stem lesions.  A f t e r a few days of adaptation to its new s i t u a t i o n , a  Rothballer (149)  type "D" pattern (persistent-spindles  appeals (.when the animal  in "al1 leads) regular iy  is placed in a quiet, dimly lighted environment.  Marked EEG " a c t i v a t i o n " 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 t i 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 " a c t i v a t i o n " , in contrast to the deactivated pattern which is observed even during the administration of milk through the stomach tube.  In a d d i t i o n , this preparation manifests a consistently high  degree of s e n s i t i v i t y  to d i r e c t pharmacological a c t i v a t i o n by both cholinergic  and adrenergic agents (107).  - 20 -  RF-P  Tin fir  BR  90  RF.P  WtJ  !  I  I !  I  Ii  i, i  i I!! I i  , i 11  I  I  ill Ml  mm  LF.P t  t  t  Hand Clap  Figure 1 Effect of audiogenic activation upon the EEG (whistle blast and hand c l a p s ) . On a l l leads, abrupt onset of activation from pattern D to A with reversion to pattern D within 25 seconds. No s i g n i f i c a n t change is noted in the pressor response.  - 21 -  'RF-P  I  LP-0  i|iw  jji Ijll^^ L  E**»l  Ijdrotoctew  o{  ttohwfc.  Tube.  J  RF-P  During  -fee<i>r»g  via.  S.T.  I.  TubeJ  Figure 2 . Effect on the f EG. of feeding v i a stomach tube, immediate a c t i v a t i o n is noted during the introduction and the removal of the stomach, tube. In contrast the pattern observed during feeding Is very s i m i l a r to the control t r a c i n g .  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 f a c t , without opening the cranium.  The preparation is as nearly " p h y s i o l o g i c a l " as a  paralyzed and de-afferented animal can be, and is completely free of extraneous pharmacological  influences.  Hence, when brain stem lesions are  placed u n i l a t e r a l l y , the intact side can serve as a c o n t r o l , without the necessity for neuromuscular blockade.  In f a c t , within a r e l a t i v e l y  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 e f f e c t s .  6.  Direct Central Drug E f f e c t s . A satisfactory  approach for making simultaneous  bilateral  intra-  carotid injection of drugs presented another procedural problem.  Attention  was concentrated on obtaining d i r e c t central effects of drug action by means of the i n t r a - a r t e r i a l Unilateral  route  (153).  intracarotid i n j e c t i o n , though informative, seemed to  have c e r t a i n disadvantages.  The cerebral c i r c u l a t i o n on the side of the  injection may be temporarily embarrassed during the cannulation procedure. Furthermore, studies  in which dyes have been injected u n i l a t e r a l l y  into the  carotid artery have indicated that drugs injected in this manner may not be d i s t r i b u t e d to the same s i t e s b i l a t e r a l l y , equally and simultaneously As Marrazzi  (81).  (114) has pointed out, " a drug injected into the carotid artery  would act somewhat l i k e a close a r t e r i a l  injection and produce i n i t i a l l y a  higher concentration in the brain on the i p s i l a t e r a l s i d e , while when i t got out into the systemic c i r c u l a t i o n and got diluted with the overall blood volume i t would then be in a s u f f i c i e n t l y lower concentration to prove  - 23 -  sub-threshold for the periphery and the contralateral hemisphere.  The  amounts that get through the C i r c l e of W i l l i s o r d i n a r i l y are small under these conditions." A satisfactory has been developed.  technique for simultaneous  b i l a t e r a l drug administration  By introducing a small bore polyethylene catheter into  the right subclavian artery i t is possible to make injections d i r e c t l y into the innominate artery at the point of bifurcation of the two common carotids. The catheter is  introduced u n t i l the t i p touches the caudal aspect of the  a o r t i c 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 a b i l i t y  to  d i s t r i b u t e injected material b i l a t e r a l l y 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 b i l a t e r a l  trans rent d i l a t i o n of the  pupils when the catheter is at the b i f u r c a t i o n of the two common c a r o t i d s .  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 t i p was introduced into the i n f e r i o r vena cava, for intravenous administration of drugs.  The  right subclavian artery was cannulated with a small bore polyethylene catheter, with i t s  tips positioned into the innominate artery in such a way  as to permit d i s t r i b u t i o n of injected material (107).  into both carotid a r t e r i e s  In the other end of both the venous and the a r t e r i a 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. (brass screws)  The muscles of the scalp were reflected and electrodes  introduced through the skull  to the dura.  One pair of screws  (electrodes) was positioned anteriorly over the motor c o r t i c e s .  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 o r i g i n of the bony tentorium and 1.2-2.0 cm to each side of the midline and an earthing electrode was put in the midline over the frontal  sinus.  The dural leads were connected to an Offner a m p l i f i e r , Type 140A, and the EEG recorded on a Dynograph  ink w r i t e r .  Bipolar recordings were carried out  between " r i g h t f r o n t a l - r i g h t p a r i e t a l " , " r i g h t p a r i e t a l - r i g h t o c c i p i t a l " , and between corresponding positions on the contralateral side.  All  had to be taken at night in a dimly lighted and quiet environment.  records In some  experiments, destruction in whole or In part of the mid-brain r e t i c u l a r formation was produced by a coagulating current at the end of a steel electrode, insulated to 1.0 mm of the t i p .  The Heathkit high frequency generator  provided the current source. In six experiments b i l a t e r a l denervation of the carotid sinus was carried out according to Koella (99) 12 to 2k hours following p a r t i a l 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 c a r o t i d , ascending pharyngeal and o c c i p i t a l arteries.  In order to evaluate the effectiveness of b i l a t e r a l 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 e f f e c t i v e l y 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 i n j e c t i o n of drugs and repeated controls were established by i n j e c t i n g s a l i n e .  Control  saline  injections did not influence c o r t i c a l a c t i v i t y at any time during the experiments. The drugs  studied were I-epinephrine b i t a r t r a t e ,  1-norepinephrine  b i t a r t r a t e , dl-N-isoproterenol hydrochloride, 1-isoproterenol  bitartrate  dihydrate, d-isoproterenol b i t a r t r a t e , dichloroisoproterenol hydrochloride (DCI), r-amphetamihe sulphate, chlorpromazine, promazine, phenoxybenzamine hydrochloride (dibenzyline), acetylcholine c h l o r i d e , 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 s a l i n e or in glucose s a l i n e .  All  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 f o r their generous g i f t s of some of the drugs used in this study: to Sterling-Winthrop Research Institute f o r supplies of 1-eplnephrlne b i t a r t r a t e , 1-norepinephrine b i t a r t r a t e , dl-N-isoproterenol hydrochloride, 1-isoproterenol b i t a r t r a t e dihydrate arid d-isoproterenol b i t a r t r a t e ; to Poulenc Ltd. (Canada) f o r chlorpromazine hydrochloride; to Mowatt and Moore Ltd. f o r promazine hydroc h l o r i d e ; and to Smith Kline and French Inter American Corporation f o r phenoxybenzamine hydrochloride (dibenzyline). Special acknowledgement is due Dr. I. H. Slater of E l i L i l l y and Co. L t d . f o r 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. Part II A.  Animals were prepared in the manner described previously  in  on Methods of this presentation and reported elsewhere ( 1 0 7 ) .  Basic EEG Pattern. In the unanaesthetized de-afferented cat f l u c t u a t i n g periods of  p a r t i a l alertness and drowsiness are c h a r a c t e r i s t i c of the preparation when i t is undoutedly not asleep.  These two states are e a s i l y distinguished by  the responses of the head and forelimbs and by the e l e c t r i c a l a c t i v i t y of brain.  In the apparently " a l e r t " state (following audiogenic, visual or  olfactory stimulation)  movements of the head, ears, eyes and occasionally  the v i b r i s s a e and the forepaws may be observed.  Under these circumstances  the e l e c t r i c a l a c t i v i t y of the cortex is rapid (25-40 c/s)  and of low  voltage (less than 75 uV), s i m i l a r to the " a c t i v a t i o n pattern" described for the intact animal by Rheinberger and Jasper (143). in a drowsy state the pur iIs  When the animal  is  are usually c o n s t r i c t e d , movements of the head  and forel irribs are minimal and the EEG is dominated by spindle a c t i v i t y  (up  to 500 uV in amplitude), wh>ich alternates with short intervals of e l e c t r o c o r t i c a l a c t i v i t y exhibiting intermediate voltage and varying frequency. Depending oh the type and intensity of the stimulus, trans i tion from one of these states gradual.  the e l e c t r o - c o r t i c a l  to the other may either be abrupt or  Under such circumstances and experimental conditions  i t was found  - 28  convenient  -  to u t i l i z e f o u r b a s i c types o f EEG  p a t t e r n s f o r the purposes o f  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 d r u g - i n d u c e d EEG These EEG  patterns, designated  A,  "activation"  B, C and  D by R o t h b a l l e r  m a n i f e s t p r o g r e s s i v e degrees o f 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 , 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  i n F i g u r e 1.  ( p a t t e r n A), claps).  de-afferented  In t h i s r e c o r d h i g h v o l t a g e low  a c t i v i t y w i t h s p i n d l e s can be seen, f o l l o w e d a b r u p t l y by r e s u l t i n g from a u d i o g e n i c  stimuli  frequency  immediate a c t i v a t i o n  ( w h i s t l e b l a s t and  Between these two extremes o f e l e c t r o e n c e p h a l o g r a p h ! c  and " a c t i v a t i o n " , i n t e r m e d i a t e p a t t e r n s d e s y n c h r o n i z a t i o n may  occur.  i n d i c a t i n g v a r y i n g degrees o f  Such p a t t e r n s may  a r e o b s e r v e d f o l l o w i n g the i n t r a - i n n o m i n a t e R o t h b a l l e r (149)  take p l a c e  s p e c i f i c EEG  towards "A"  imagined as b e i n g a r r a n g e d  i n j e c t i o n of l e s s potent cogently;  " a c t i v a t i o n " r e f e r s not t o any  one  o r towards the l e f t i f the p a t t e r n s  in alphabetical order.  opposite d i r e c t i o n is " d e a c t i v a t i o n " . not n e c e s s a r i l y be the EEG is s t i l l  So  Conversion  Thus, a l t h o u g h  of a l e r t wakefulness,  "activation".  though both o f these considered  sometimes  p a t t e r n , but to the c o n v e r s i o n o f one p a t t e r n i n t o a n o t h e r -  s p e c i f i c a l l y from "D"  to "C"  EEG  spontaneously  has s t a t e d h i s o b s e r v a t i o n s  " I n i t s s t r i c t e s t s e n s e , the term EEG  hand  "deactivation"  d u r i n g t r a n s i t i o n between s t a t e s o f d r o w s i n e s s o r a l e r t n e s s , and  a c t i v a t i n g drugs.  decreasing  spindle bursts).  A t y p i c a l d e a c t i v a t e d r e c o r d ( p a t t e r n D) o f the u n a n a e s t h e t i z e d cat i s presented  (149)  the end  i n the r e s u l t would  a change from p a t t e r n  i s the c o n v e r s i o n from "B"  l a t t e r p a t t e r n s might f a l l  are  to "A",  even  i n t o what i s g e n e r a l l y  the " a c t i v a t i o n p a t t e r n " to b e g i n w i t h . "  "D"  - 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 druginduced 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  LPLF-P f  SoJitve Cowtrol  1  82  18  1  4  RP-0  LP-0  LF-P  BP  TO  80  85  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.  j  - 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 isoproterenol (Fig. 1 2 ) . Saline injection produced no such effect. Generally speaking the onset of activation following the intra-innominate administration 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 RF-p  Ep,  o.5>/Ko.  I  300/*>J 105  BRqs  LP-0  J  1 HI 115  I 15  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 s t i l l maxima 1.  - 33 -  RF-P  LP-0  LF P 7  t N.E. aSr/kg. 1  y j':' ppp  BR  loo  l  li  no  u o  j [j  14-0  i  "  I  LP-0  F i gu r e Intra-innominate  b r a c k e t e d by a r r o w s .  occurring  before  35  seconds.  deactivation pre-injection maximal  5.  the peak o f This  Immediate a c t i v a t i o n  the p r e s s o r  pattern  is  next  and a c t i v a t i o n , w i t h a n tracings,  values.  130  n o r e p i n e p h r i n e 0.5/jg/kg.  Injection about  I3i  i3a  135  while  response followed  abrupt  the b l o o d  A)  lasting  by p e r i o d s  return  pressure  (type  and  is  to  of  the  still  near  34 -  RF-P  RP-0  tl.N.E. o.asi/M R .p F  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) a c t i v a t i o n , pattern D to A , occurring 30 seconds before the peak depressor response, and p e r s i s t i n g for an additional 30 seconds before recovery to control spindling.  35 -  RF  P . J » .  RP.O  W  . l « M * l M I | » »  l  W «  IB •>li.l> »fcHH>tl>*l l  • " ' " > ' "** ( w4 1 •»  . LP.O  50 l/Kg.  3ocy-V  1  BP. no  RF.P RP.O 1 LP.  Ii l l  1  i  "  |  LF.P 152  FI gu re 7. Intra-innominate amphetamine 50/ug/kg. Activation somewhat 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.  152  - 36 -  RF-P  RP-0  5  LP:0  LF-P T Eft.  RF-P  B.  R  IOS  o.25T/Kg-  1  ice  m  | | S  '1ml  :|  (  RP-0  [I  i_p.p  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 s t i l l maximal.  inil  37 -  H Am  I, « . < » i i * . « i . . > i « v i i >  r  l  LF.P 30Cy*V B.P. 90 RF-P  RP.O  mm*  LP  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 Nl.E.  0.5n/k . 1  I  3  .  pp  p  B.R  120  MS  5 set.  WO  , .  '  300/*V 7  ISO  i nrm r n  RP-0  i  1*5  130  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 " s p i n d l i n g " while the blood pressure is s t i l l elevated.  -  39  -  RPO  LPO LF P t  RF P  NE.  lot/Kg.  1  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 deactivation and activation which is independent of the sustained pressor response.  -  ko  -  •  > ii»»nm»i»n»ii ti.i|#ii RP-0  ' — — ' qo  f °/3o  —•  1  nr.p  RP.O So sa  LP.O LF P 50  54  v  55  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 p e r i o d s o f low v o l t a g e f a s t a c t i v i t y .  Similar  have been observed w i t h comparable doses o f n o r e p i n e p h r i n e , but f l u c t u a t i o n s were l e s s f r e q u e n t and activity  the  the degree o f h i g h v o l t a g e slow  l e s s i n t e n s e than those noted w i t h e p i n e p h r i n e .  e f f e c t s were o b s e r v e d  results  These types o f  i n p r e p a r a t i o n s w h i c h responded s u b s e q u e n t l y  s i m i l a r doses o f 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 u n m i s t a k a b l e  to  "activating  patterns". R e v e r s i o n to s y n c h r o n i z e d c o n t r o l p a t t e r n s o c c u r r e d w i t h i n minutes w i t h a l l t h r e e compounds, the d u r a t i o n o f the a c t i v a t i n g  1-2  effect  b e i n g l o n g e r 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.  F o r 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,  a c t i v a t i n g e f f e c t o f i s o p r o t e r e n o l was e p i n e p h r i n e and n o r e p i n e p h r i n e . be more a b r u p t EEG  The  the d u r a t i o n o f  the  somewhat l o n g e r than t h a t o f  r e t u r n to a d e a c t i v a t e d EEG  seemed to  i n the case o f i s o p r o t e r e n o l , but whether g r a d u a l o r a b r u p t ,  r e v e r s i o n preceded  b l o o d p r e s s u r e r e c o v e r y w i t h a l l t h r e e compounds."  Amphetamine i n doses o f 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 b e h a v i o u r a l s i g n s o f a l e r t n e s s , m a n i f e s t e d by movements  o f the forepaws,  eyes and s e a r c h i n g motions o f the head.  Racemic  dl-amphetamine i n doses as low as 5 0 ^ i g / k g produced d e f i n i t e (Fig.  7).  activation  However, the a c t i v a t i o n produced by s m a l l doses o f amphetamine  (50/lig/kg) c o n s i s t e d o f s h o r t e r r l a s t i n g d e s y n c h r o n i z e d  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 b u r s t s but f r e e from any a s s o c i a t e d b e h a v i o u r a l changes. the EEG  Here, t o o , the p r e s s o r response  seemed u n r e l a t e d t o  effects. (b)  E f f e c t s o f some isomers o f i s o p r o t e r e n o l a d m i n i s t e r e d  s e p a r a t e l y 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 "activation ' 1  (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 s t i l l unquestionably lower than its pre-existing control va1ue.  - kl -  h. RF-P  LP-0  d- L.N.E. ioor/Kg.  i  • RF-P  B . P 102.  10S  ioi  5  sec.  1  [  300M.V r  105  LP-0  T l-I.NE.  I.01/K9 J  ftp 1 0  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  JJj I  RP-o LP-0  LF-P  I T'11 IT  I!  LP-0 LF-P 50  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.  Cholinergic Activation. (a)  E f f e c t s o f a c e t y l c h o l i n e and e s e r i n e ( p h y s o s t l g m i n e ) .  Patterns  o f " a c t i v a t i o n " s i m i l a r to those o b s e r v e d w i t h a d r e n e r g i c amines a r e 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 / i g / k g ) .  Transient depressor  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 p h y s o s t i g m i n e , s i s t e n t l y w i t h these a g e n t s . d i f f e r e n t animals  from 0.25  occurred  con-  A c t i v a t i n g doses o f a c e t y l c h o l i n e v a r i e d i n to 2/Ug/kg.  Whereas i n some p r e p a r a t i o n s  doses (0.25-0.5/Ug/kg) produced t r a n s i e n t EEG at  effects,  desynchronization, interrupted ( F i g s . 15 and  i n t e r v a l s by s h o r t b u r s t s o f s p i n d l e a c t i v i t y  16), l a r g e r  doses ( 1 - 2 / j g / k g ) always e l i c i t e d more d e f i n i t i v e  "activation", with  2 /Og/kg dose p r e c i p i t a t i n g immediate, i n t e n s e and  long periods of  arousal  in a l l instances.  F i g u r e s 17 and  The  EEG  s i n c e the EEG is s t i l l  EEG  de-afferented  a c t i v a t i n g response i s produced a f t e r v e r y s h o r t  p e r i o d s ( c a . 3-7  prepara-  latent  s e c o n d s ) , and seems u n r e l a t e d to the d e p r e s s o r  responses,  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 b l o o d  below i t s c o n t r o l l e v e l .  the  18 show p a t t e r n A produced by  1 and 2 / i g / k g o f a c e t y l c h o l i n e i n o u r a n a n a e s t h e t i z e d tion.  low  Under the above c i r c u m s t a n c e s ,  pressure acetyl-  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 o f the f r o n t paws and head i n d i c a t i v e o f b e h a v i o u r a l a r o u s a l . In c o n t r a s t , e s e r i n e - i n d u c e d 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 b e h a v i o u r . c o n s i s t e n t changes i n c o r t i c a l  In doses adequate to e l i c i t  e l e c t r i c a l a c t i v i t y , the marked and  fleet-  ing d e p r e s s o r e f f e c t s noted w i t h a c t i v a t i n g doses o f 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 b l o o d  pressure  remained f a i r l y c o n s t a n t around c o n t r o l values (50/ug/kg), o r showed a slight  Increase  (100/Ug/kg) ( F i g s . 19 and 2 0 ) .  In s i n g l e low doses  kS -  Ji LP..  LF.P  t  , ]  S  ' BP *  70  ^  55  "  RP.O  i  i 1 1  • °  '  *V-  V  1  /  j[y  LF-P T  5~s«  , 85  75  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.  BP  87  -  kl  -  15  qo  WI'ii'M  m*  ''iWrt^wNflWW  . p 90 BP  95  80  F i g u r e 16. Intra-Innominate acetylcholine 0.5/ig/kg. Immediate a c t i v a t i o n (D to A) similar to Figure 15, but of longer duration, with gradual recovery (A to C to D).  48 -  I  UO  Figure  17.  Intra-innominate acetylcholine 1.0/ig/kg. Intense and more prolonged a c t i v a t i o n , s i m i l a r to that seen in Figure 16, is observed in this record. Depressor responses are comparable.  MO  - 49 -  RF-P  LP-0  L LF-  RF-P  T ACH 2.0v/Kg.  t 5 sec..  B.P.  70  <to  55  300/-V  «S  ' I  40  .P.  LF-P  HO  sec.  ,  [ii  .  10  ]]  So  Figure 18. Intra-innominate acetylcholine 2.0/Ug/kg. The EEG activation which consists of a s h i f t from pattern D to A, is s i m i l a r in appearance to Figure 17. but is more prolonged. Note temporal independence between the blood pressure variations and EEG a c t i v a t i o n and deactivation.  90  - 50-  if  L  il Ii l l * H  P . O  ^ Eserir*  RF-P  Scor/K^.  ^  80  S5  .  RP.O  ES  LP.O  LF.P 7S  70  90  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.  75"  - 51 -  E.  RF-P I' 'Willi HI  m Hi  III n »n inimm  ^UU>wJ  Id »i» ..ii,»wi|i w HH i  mt  3oo/*.v p  B P 78  II f  Igl  W|»I«WI  »I(»|II. ,III.^I,,».I .»II»I<,.» IUHII,II^.Bi'^WH. iWMtffi.i <  M  I  w t n ^ w w ^ ^ i ,  ihimmmtn  «nw«i,  iwfi  ^'jMWWWWSitt^^lW|l|ji., » Uit,» i«^^^»W V4W ^ l  l  l  I  T  |  P-0  FP. WW  1  1 »»W*»W»'*«i»im  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  an  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  these amounts were a d m i n i s t e r e d t o t a l dose o f 50-60/Ug/kg was  -  EEG  changes.  However, i f  repeatedly at short i n t e r v a l s u n t i l  reached, d i s t i n c t a c t i v a t i o n was  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  observed.  seconds),  but once  initiated  the degree o f a c t i v a t i o n was  prolonged  (more than 20 minutes) than t h a t o b s e r v e d w i t h a c e t y l c h o l i n e .  L a r g e r d o s e s , 100-200 /Ug/kg, e l i c i t e d  more i n t e n s e and  a  longer l a s t i n g periods of  " a c t i v a t i o n " (more than 90 m i n u t e s ) , as w e l l as s a l i v a t i o n and  the d u r a t i o n more  EEG  r e s p i r a t o r y embarrassment,  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 e x p e r i m e n t s (50-200 / i g / k g ) ; however, i n a l l cases the r e c o v e r y o f a d e a c t i v a t e d p a t t e r n c o u l d be brought about promptly by the a d m i n i s t r a t i o n o f a t r o p i n e , 0.5 2.0  mg/kg ( F i g . 4.  mg/kg, but not by  chlorpromazine,  21).  A c t i v a t i n g E f f e c t s o f Other A g e n t s . (a)  Vasopressin  and h i s t a m i n e .  S i n c e the p o s s i b i l i t y e x i s t s  that  some o f the c e n t r a l a c t i o n s o f a d r e n e r g i c amines and o f 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 b a s i s o f t h e i r v a s c u l a r e f f e c t s ( M f ) , i t seemed  important  t o a s c e r t a i n whether changes i n b l o o d p r e s s u r e  depressor)  produced by these agents may  (pressor or  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 e v e n t s o b s e r v e d w i t h these compounds. The histamine general  c a r d i o v a s c u l a r e f f e c t s o f both v a s o p r e s s i n  (depressor)  have been w e l l e s t a b l i s h e d .  (pressor)  and  In a d d i t i o n , t h e r e i s  agreement t h a t the vasomotor e f f e c t s o f these  two compounds a r e  the r e s u l t o f 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  V ««I>HI.»« » IH»<'»« . .aw* I  1W  w  15  LP-0  LF-P Eserine i o o x / K g given Co m m . eorlic  RF-P  7  t CPZ. 2.0".yKj. 1  8.P 80  80  RP-0  L../iijj.^M^ii 4Ljli>4Aii.Jiiii ltu. u  A M  1  LP-0 LF-P t  83  Atropine  cs^M  i  75  70  Figure 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 -  a r e u n r e l a t e d t o a d r e n e r g i c o r c h o l i n e r g i c r e c e p t i v e elements ( 6 5 ) .  It  was a n t i c i p a t e d t h a t i f EEG " a c t i v a t i o n " c o u l d be the r e s u l t o f 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 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 o f v a s o p r e s s i n i n p r e s s o r amounts ( 0 . 5 u n i t s / k g ) i n d e p r e s s o r doses (1 /ug/kg), would a s s i s t i n t h e c l a r i f i c a -  and h i s t a m i n e tion of this The  relationship. g e n e r a l p r o c e d u r e f o l l o w e d was t o a d m i n i s t e r these agents i n  those preparations  i n w h i c h the a c t i v a t i n g s e n s i t i v i t y  t o t h r e s h o l d amounts  o f both a d r e n e r g i c 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 e x p e r i m e n t s ,  vasopressin (0.5  units/  kg) produced a s u s t a i n e d p r e s s o r response (more than I h o u r ) , a v e r a g i n g a blood pressure  i n c r e a s e o f more than 25 mm Hg o v e r c o n t r o l v a l u e s .  This  b l o o d p r e s s u r e e l e v a t i o n i s comparable i n degree t o t h a t a t t a i n e d w i t h a c t i v a t i n g doses o f p r e s s o r a d r e n e r g i c amines. d e m o n s t r a b l e EEG " a c t i v a t i o n " was o b s e r v e d  However, no c l e a r - c u t o r  (Fig. 22).  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 t h e same p r e p a r a t i o n , a u d i o g e n i c s t i m u l a t i o n and e p i n e p h r i n e 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 a r o u s a l 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 delayed  i n o n s e t was noted.  However, i t i s not i n c o n c e i v a b l e t h a t t h i s  l o n g l a t e n c y e f f e c t c o u l d have been due t o a c c i d e n t a l occurrences  beyond the c o n t r o l o f t h e e x p e r i m e n t e r  such p a t t e r n s a r e o b s e r v e d  C and  to occur spontaneously  environmental  since occasionally ( i . e . , without  drug  administration). (ii)  Histamine.  produced a t r a n s i e n t d e p r e s s o r below c o n t r o l l e v e l s .  Histamine response,  (1.0/ug/kg), i n f i v e  experiments,  e x h i b i t i n g a d e c r e a s e o f 30 mm Hg  T h i s d e p r e s s o r e f f e c t w h i c h i s comparable i n  - 55  -  'RF-P  RP-o  LP-0  J I  LF-P  , OLbopvessin  RF-P  ' B R  80  qo  5 MCT  1~A  ' 1  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 vasopressin, despite the increased and persistent pressor response.  V  ,  I I  - 56  -  i n t e n s i t y to t h a t noted w i t h s m a l l doses o f 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  change ( F i g . 23).  cortical  not a s s o c i a t e d w i t h any  definitive electro-  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 associated with changes i n 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 ( F i g u r e s 24 and  i l l u s t r a t e such a r e s p o n s e ) . longer  f o r as  to c o n t r o l  This a c t i v a t e d pattern p e r s i s t e d f o r periods  long as kO seconds a f t e r the d e p r e s s o r phase had  Serotonin.  The  d e m o n s t r a t i o n by Twarog and  t h a t 5 - h y d r o x y t r y p t a m i n e ( s e r o t o n i n ) was  concentrations present  i n the b r a i n , c o u p l e d  present  Page (167)  w i t h the o b s e r v a t i o n  in high  t h a t enzymes were  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 o f t h i s compound,  is considerable  play  In a d d i t i o n ,  l a c k of agreement on the p o s s i b l e r o l e w h i c h s e r o t o n i n  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 t h a t s e r o t o n i n does not c r o s s the b l o o d - b r a i n and  reverted  in r e l a t i v e l y  have s t i m u l a t e d wide and p r o t e a n i n t e r e s t i n t h i s agent.  may  still  levels.  (b)  there  25  than the b r i e f d u r a t i o n o f the d e p r e s s o r response, and was  present  1953  consistent  have p r o v i d e d  e v i d e n c e i n s u p p o r t o f the h y p o t h e s i s  barrier,  that injected  s e r o t o n i n does not e x e r t 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 O t h e r s have demonstrated t h a t s m a l l amounts o f deposited 114)  i n b r a i n (36).  as w e l l as EEG  i n j e c t e d s e r o t o n i n may  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  " a c t i v a t i o n " (151)  (142). be  (100,  have been o b s e r v e d f o l l o w i n g the  a d m i n i s t r a t i o n o f t h i s b i o g e n i c amine. In our p r e p a r a t i o n s anaesthesia, repeatedly  and  i n w h i c h t h e r e a r e no r e s i d u a l e f f e c t s o f  the b r a i n stem i s c o m p l e t e l y  produced EEG  intact, serotonin  " a c t i v a t i o n " , even i n the p r e s e n c e o f  c a r o t i d sinus denervation.  The  has  bilateral  s m a l l e s t dose o f s e r o t o n i n n e c e s s a r y  to  57 -  RF-P  RP  0  ,  LF-P T Histamine LCi/Kji  . 300/iV  RF-P  I 'if  B P  to  80  n 0 n Pi  1  S  555  S  -  5«  »  50  "'  LP-0  Figure 23. E f f e c t of histamine l.OyUg/kg on the EEG* The resting deactivated EEG pattern (type D) remains unchanged a f t e r the administration of histamine I.O/ig/kg The depressor response is not associated with any demonstrable EEG changes.  - 58 -  RF-P  f  T Histamine e-Olr/Ka J  RF-P  B  R  l  "°  IT  sec  LF-P  100  US  Figure 24.  E f f e c t of histamine 2.0/Ug/kg on the EEG. EEG a c t i v a t i o n begins a f t e r 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 a f t e r the blood pressure has returned to control values.  59 -  — i  T i r w r wt  -  M  il^lrt'llT^llWilfa^l.lMil^ilJrilll^llifBLftd^l .  LP-0  LF-P  tHistamime  RF-p  BP  E.OK/KQ.  1  —r=—•  10  K  RP-0  20  LP-0  15  sec.  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.  v  - 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 / u g / k g , a l t h o u g h  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 o b s e r v e d w i t h a dose as low  as  L a r g e r doses ( 5 ^ g / k g  1 yug/kg.  and 20 /Ug/kg) produced EEG responses o f  l o n g e r d u r a t i o n , w i t h the 20 ^ i g / k g dose e l i c i t i n g (ca. 2 seconds),  as compared w i t h  immediate " a c t i v a t i o n " " 5 seconds  l a t e n t periods of approximately  f o l l o w i n g a d m i n i s t r a t i o n o f both t h e 2.5/Ug/kg and the 5 . 0 /Ug/kg doses. F i g u r e s 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 . noted w i t h s e r o t o n i n resembled i n some r e s p e c t s  The " a c t i v a t i o n "  the " a c t i v a t i o n  noted  1 1  With s m a l l e r doses o f s e r o t o n i n ( 2 . 5 - 2 . 0 / u g / k g ) , the  with epinephrine.  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 o f 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 s e c o n d s , f o l l o w e d by a s h o r t p e r i o d o f 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 .  Similar characteristics  i n p a t t e r n s a r e o b t a i n e d w i t h the 20 /Ug/kg d o s e s , b u t under these circumstances  the i n i t i a l  mately 25 seconds.  type A a c t i v a t e d p a t t e r n i s i n c r e a s e d  Blood pressure  to approxi-  responses were i n c r e a s e d w i t h a l l doses  used and a r e comparable f o r both the 5 /jg and 20/ug/kg doses. 5.  E f f e c t s o f B l o c k i n g Agents. (a) P h e n o t h i a z i n e  d e r i v a t i v e s (chlorpromazine  and p r o m a z i n e ) .  The  e f f e c t s of chlorpromazine  and a r e l a t e d p h e n o t h i a z i n e  on the EEG and t h e i r  b l o c k a d e o f 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 t h a t induced  by a u d i o g e n i c (i)  or visual  Chlorpromazine.  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 .  In t h e u n a n a e s t h e t i z e d  c a t the a d m i n i s t r a t i o n o f adequate doses o f c h l o r p r o m a z i n e  de-afferented 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 o f e l e c t r o - c o r t i c a l a c t i v i t y w h i c h appear t o d i f f e r s l i g h t l y from spontaneous p a t t e r n s o f d e a c t i v a t i o n . F i g u r e 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  pattern  - 61 -  RF-P  mm LP-0  1  t Serotonin s.ar/K&- 1 >  pp_p  B.R  H5  i IO  IOS  r-^z—•  1 »r-  v  iio  MM RP-0 LP-0  us  m  FI gu re 26.  E f f e c t of serotonin 5 A*g/kg on tho EEG, Note the intense EEG a c t i v a t i o n (type A) which occurs a f t e r a latent period of about k seconds and persists for more than 1 minute. This e f f e c t 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.  mm \l  ?  - 62 -  •  LP-0  T  B R  Serotonin, ZO.or/Kg  i  1  MS  35  sec.  I  LP-0  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 distinguished 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 stimulation 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. E f f e c t 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 a c t i v a t i o n , whose duration p a r a l l e l s that of the stimulus.  - 65  c h l o r p r o m a z i n e as l a r g e as 5-10  mg/kg.  i n doses as low as 2 mg/kg e f f e c t i v e l y  Chlorpromazine the EEG  -  blocked  " a c t i v a t i o n " u s u a l l y produced by adequate doses o f a l l o f  a c t i v a t i n g amines w h i c h we  the  have s t u d i e d ( e p i n e p h r i n e , n o r e p i n e p h r i n e ,  i s o p r o t e r e n o l , s e r o t o n i n , h i s t a m i n e and amphetamine) w i t h the e x c e p t i o n o f the c h o l i n e r g i c a g e n t s . doses n e c e s s a r y l i s h e d , and  F o r each a c t i v a t i n g a g e n t ,  to a c h i e v e maximal EEG  the  " a c t i v a t i o n " ( t y p e A) was  the e f f e c t o f the same dose o f each compound was  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 o f F i g u r e 23  minimal estab-  then  observed  chlorpromazine.  i l l u s t r a t e s the f a i l u r e o f e p i n e p h r i n e (0.5/Ug/kg)to produce  i t s u s u a l EEG (2 mg/kg).  response  i n the presence o f c h l o r p r o m a z i n e  S i m i l a r results w i t h norepinephrine  obtained ( F i g . 30).  blockade  (2 /ug/kg) a l s o were  While p r i o r treatment w i t h chlorpromazine  i n s u p p r e s s i o n o f p r e s s o r responses  resulted  to e p i n e p h r i n e , the e l e v a t i o n s i n  b l o o d p r e s s u r e produced by n o r e p i n e p h r i n e and s e r o t o n i n were o n l y S e r o t o n i n (5.0/ug/kg) a d m i n i s t e r e d 25 minutes a f t e r  p a r t i a l l y blocked.  the a d m i n i s t r a t i o n o f 2 mg/kg o f c h l o r p r o m a z i n e produced a s l i g h t e l e v a t i o n i n the b l o o d p r e s s u r e 13 mm i n the d e a c t i v a t e d c o n t r o l EEG  Hg)  but f a i l e d  p a t t e r n ( F i g . 31).  to evoke any  changes  A l a r g e r dose o f  s e r o t o n i n (20.0/Ug/kg) a d m i n i s t e r e d 20 minutes l a t e r produced a comparable p r e s s o r response  (15  mm  Hg)  but was  e q u a l l y w i t h o u t e f f e c t on the  A f t e r 2 mg/kg o f c h l o r p r o m a z i n e ,  the a d m i n i s t r a t i o n o f 0.5/Ug/kg  o f i s o p r o t e r e n o l and 5.0^ug/kg o f h i s t a m i n e a l s o f a i l e d typical  immediate EEG  ( F i g s . 32 and 3 3 ) . apparent seconds.  EEG  to produce the  " 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  However, i n the case o f i s o p r o t e r e n o l a d e l a y e d  " a c t i v a t i o n " o f moderate degree developed  T h i s was  EEG.  not seen w i t h h i s t a m i n e .  i n about 22  With h i s t a m i n e the  depressor  - 66 -  RF-P  LP-0  T Epi. 0.51/Kq.  i /  after C r Z Z.Omg./Kg. i.urng./rg. after 'CPZ  &R  LF-P  |  «  80  M  t  -  i  7S  Joou-V  |  S  73  Figure 2S. E f f e c t of epinephrine 0.5 /Ug/kg on the EEG a f t e r CPZ blockade (2 mg/kg) . Absence of any activation and continued persistence of CPZ-induced " s p i n d l e s " . Note the transient depressor e f f e c t of epinephrine in the presence of CPZ blockade.  1  75  - 67 -  LF-P  1  t NE. 2r/Kq. after 4  CPZ.. 2Tm./Kg 3  RF-P  300/*  BP  LP-0  - ,  125  IIS  Figure 3 0 .  Effects of norepinephrine 2/jg/kg on the EEG a f t e r CPZ blockade (2 mg/kg). EEG tracing is s i m i l a r to Figure 29. The blood pressure Is elevated (ca. 35-40 mm) above control values.  115  - b8 -  oittr  RF-P  BP.  qo  |OJ  CPZ.. Z n i g . / K g .  IOO  ,00  F i gu re 31. E f f e c t o f s e r o t o n i n 5.0/ug/kg on the EEG a f t e r CPZ b l o c k a d e (2 mg/kg). Absence o f any a c t i v a t i o n , and c o n t i n u e d p e r s i s t e n c e o f CPZ-induced s p i n d l e s . The p r e s s o r response i s much l e s s than t h a t noted i n F i g u r e 26.  RF-P  t  HisW.n» Pfter  RF-P  BP  5.01/Kg.  1  T ,  CPZ. t O r g / K j .  80  70  _ _ _  ,  jj^V  (S  1  TO  Figure 32.  fig/kg  Effect of histamine 5.0 on the EEG after CPZ Wockade (2 mg/kg). Absence of any activation and continued persistence of CPZinduced spindles.  - 70  e f f e c t s are moderate ( 1 5 mm (Fig.  32),  Hg f a l l  -  p r e s s u r e i n 25  i n blood  whereas i n the same p e r i o d a more i n t e n s e and  seconds)  immediate drop  i n b l o o d p r e s s u r e i s o b s e r v e d 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  isoproterenol  ( F i g . 33).  The  degree w h i c h developed about 22 was  f o l l o w e d 35  occasional  " a c t i v a t i o n " o f moderate  seconds a f t e r i s o p r o t e r e n o l  seconds l a t e r by a f l a t t e n i n g o f the EEG  and  intermittent spindling).  i n b l o o d p r e s s u r e was greater  d e l a y e d EEG  profound (from 85  administration  ( i n t e r r u p t e d by  D u r i n g t h i s i n t e r v a l , the t o 38 mm  Hg),  and  considerably  than t h a t 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  absence of c h l o r p r o m a z i n e ( e . g . 98  to 60 mm  Hg).  fall  Presumably  i n the this  d i f f e r e n c e a r i s e s from c h 1 o r p r o m a z i n e - i n d u c e d c e n t r a l and/or p e r i p h e r a l b l o c k a d e o f the usual  compensatory r e f l e x v a s c u l a r  G a s t a u t e t a l (62)  have r e p o r t e d  a c t i v i t y d u r i n g a c u t e a n o x i a , and  depression  adjustments. of c o r t i c a l  electrical  have shown t h a t b i o - e l e c t r i c rhythms  a r e most d e p r e s s e d a t the c o r t i c o t h a l a m i c l e v e l where neuronal i s maximal.  I t seemed p o s s i b l e t h a t the " f l a t t e n i n g " o f the  associated with mazine may  depression EEG  isoproterenol administration  i n the p r e s e n c e of  be the r e s u l t o f c e r e b r a l a n o x i a .  Indeed, i t i s not  chlorproinconceiv-  a b l e t h a t the o b s e r v e d t r a n s i e n t p e r i o d o f a c t i v a t i o n w h i c h preceded depression ischemia,  o f 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 c o u l d be due consequent to the i n t e n s e and  to h y p o t e n s i v e  p r e c i p i t o u s d e p r e s s o r e f f e c t s of  isoproterenol  i n the p r e s e n c e o f c h l o r p r o m a z i n e b l o c k a d e .  d e d u c t i o n was  s u g g e s t e d by the o b s e r v a t i o n  t i l t i n g of the e x p e r i m e n t a l a low»r l e v e l p a t t e r n and  eliminates  This  that at this point careful  t a b l e so t h a t the head of the animal  than i t s h i n d end,  the  is at  produces a r e v e r s a l o f t h i s " f l a t t e n e d "  the subsequent development of a p p a r e n t  m a n i f e s t e d by j e r k i n g movements o f the head and  forepaws.  convulsions  - 71  -  i h o r d e r to e x p l o r e f u r t h e r t h i s p o s s i b i l i t y , the b l o o d was  s t a b i l i z e d at a higher i n i t i a l  a d m i n i s t r a t i o n of 0.5  (0.5/jg/kg),  (115-120 mm  Hg)  by  the  u n i t s / k g o f v a s o p r e s s i n i n the same p r e p a r a t i o n  i n the presence Of c h l o r p r o m a z i n e . enol  level  pressure  identical  Then a c h a l l e n g i n g dose o f i s o p r o t e r -  to the one w h i c h 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  results  o f t h i s c h a l l e n g i n g dose o f i s o p r o t e r e n o l a r e shown i n F i g u r e 34. r e l a t i v e l y mild d e p r e s s o r e f f e c t i s o b s e r v e d  (compare F i g u r e s 33 and 3 4 ) .  F i g u r e 34  A  f o l l o w i n g the a d m i n i s t r a t i o n  o f i s o p r o t e r e n o l i n the p r e s e n c e o f both c h l o r p r o m a z i n e  and  vasopressin  i l l u s t r a t e s a l s o the l a c k o f  d e p r e s s i o n o f 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 o f a p p a r e n t a c t i v a t i o n o f the EEG  ( i . e . , t h e r e was  v o l t a g e f a s t a c t i v i t y such as was when profound  depressor  small  no s h i f t toward  demonstrated i n F i g u r e 3 3 ) .  e f f e c t s are prevented  chlorpromazine  o f 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 Chlorpromazine  b l o c k e d the EEG  50/ug/kg.  any  low Hence,  blockade  " a c t i v a t i o n " u s u a l l y seen w i t h l a r g e r doses o f  amphetamine e l i c i t e d e v i d e n c e o f some degree o f break-through In the p r e s e n c e o f c h l o r p r o m a z i n e  (racemic) was  any  evident.  ( b u t competent) doses o f amphetamine, a l t h o u g h  blockade.  and  of  this  (2 mg/kg), amphetamine  a d m i n i s t e r e d g r a d u a l l y , i n s u c c e s s i v e increments  of  No e f f e c t on the c h I o r p r o m a z i n e - i n d u c e d d e a c t i v a t e d p a t t e r n  c o u l d be o b s e r v e d  a f t e r a t o t a l o f 150/ig/kg o f amphetamine had  given ( F i g . 35).  However, an a d d i t i o n a l dose o f 50 yug/kg o f amphetamine,  been  to make a t o t a l o f 2 0 0 / j g / k g , produced d e m o n s t r a b l e changes i n the tracing.  A t t h i s p o i n t , an a u d i o g e n i c s t i m u l u s ( w h i s t l e b l a s t ) produced  s i g n s o f b e h a v i o u r a l a l e r t n e s s (movements o f 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 -  I  RF-P  RP-0  IJII 1 Rp_p  BP  85  X N . E . 0.5 1 after C P Z a.Omg/Kg. 80  Joo/A/  70  ts  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 isoproterenol. 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 e a r l i e r does not produce any change in the CPZ-spindles. Note that the lack of low voltage fast a c t i v i t y in the presence of CPZ blockade of i soproterenol induced activation is evident when anoxic effects of the EEG are prevented by p r i o r administration of vasopressin.  - 74-  LF-P  150 _ M  RF-P  Amphetamine 1.0 mg./Kg  B P  t Add.tionaj  50  r/Kg  1  Amphetamine = ZOO  CPZ  total  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 audiogenic stimulus (whistle blast) elicits type A activation in which the length of activation exceeds the duration of the blast (see Fig. 28).  - 75 -  o f the w h i s t l e b l a s t .  T h i s i s i n c o n t r a s t t o 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 o f the s t i m u l u s , see F i g u r e 28) e l i c i t e d by a u d i o g e n i c s t i m u l a t i o n i n t h e p r e s e n c e o f c h l o r p r o m a z i n e alone. In c o n t r a s t t o the responses h i s t a m i n e , EEG " a c t i v a t i o n " produced and  t o a d r e n e r g i c amines, s e r o t o n i n and by c h o l i n e r g i c agents  (acetylcholine  -.serine) i s not a b o l i s h e d o r d e p r e s s e d by c h l o r p r o m a z i n e . In the p r e s e n c e o f c h l o r p r o m a z i n e b l o c k a d e (2 mg/kg), the 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 o f 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 v o l t a g e a c t i v i t y .  In view o f the i n t e n s e , p r e c i p i t o u s and p r o l o n g e d d e c r e a s e s i n b l o o d p r e s s u r e o b s e r v e d f o l l o w i n g the a d m i n i s t r a t i o n o f d e p r e s s o r 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 t h e p r e s e n c e o f c h l o r p r o m a z i n e  b l o c k a d e , i t seemed n e c e s s a r y t o d e t e r m i n e whether o r not c e r e b r a l  anoxia  might be r e s 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 o f a c e t y l c h o l i n e i n t h i s c i r c u m s t a n c e as had been o b s e r v e d  i n the c a s e o f i s o p r o t e r e n o l .  Conseq-  u e n t l y , t h e b l o o d p r e s s u r e 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 o f v a s o p r e s s i n kS minutes acetylcholine.  b e f o r e the i n j e c t i o n o f 2 /jg/kg o f  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 o f a c e t y l c h o l i n e ,  as w e l l as the l a c k o f a marked d e p r e s s o r 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 o f t h i s compound, were o b s e r v e d under these c i r c u m s t a n c e s . The  i n j e c t i o n o f 100 /Cig/kg o f e s e r i n e a l o n e o v e r a p e r i o d o f  time produces  an 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 ) ( F i g . 2 1 ) . The  a d m i n i s t r a t i o n o f c h l o r p r o m a z i n e (2.0 mg/kg) f a i l e d  t o 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 d e l a y o f 15 m i n u t e s . I n c r e a s i n g doses o f c h l o r p r o m a z i n e (2-5 mg/kg) were e q u a l l y w i t h o u t  - 76 -  e f f e c t on the EEG, a l t h o u g h 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 o f a t r o p i n e (0.5 mg/kg), the low v o l t a g e f a s t a c t i v i t y was changed t o l a r g e a m p l i t u d e slow waves, i n t e r r u p t e d a t i r r e g u l a r i n t e r v a l s w i t h b u r s t s o f 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 t h a t 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 t h a t i n the doses used  i n these e x p e r i m e n t s , the  a d m i n i s t r a t i o n o f c h l o r p r o m a z i n e i s w i t h o u t 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 a r o u s a l which  i s produced  by c h o l i n e r g i c agents  (acetyl-  c h o l i n e and e s e r i n e ) .  (ii) produced  Promazine.  The EEG a l t e r a t i o n s and v a s c u l a r responses  by promazine a r e a l s o v e r y s i m i l a r t o those r e p o r t e d i n these  experiments  f o r chlorpromazine.  However, a l a r g e r dose o f promazine  seems n e c e s s a r y t o produce the same a l t e r a t i o n s .  Whereas 2.0 mg/kg o f  c h l o r p r o m a z i n e was s u f f i c i e n t t o e s t a b l i s h b l o c k a d e o f EEG " a c t i v a t i o n " , when c h a l l e n g e d w i t h a d r e n e r g i c amines i n c o n c e n t r a t i o n s known to produce c r i s p " a c t i v a t i o n " , k t o 5 mg/kg o f promazine was needed t o produce a similar blocking effect. produced  In a d d i t i o n , t h e d u r a t i o n o f the " b l o c k a d e "  by promazine appeared  s h o r t e r than t h a t observed w i t h  c h l o r p r o m a z i n e and seemed l e s s e f f e c t i v e a t the end o f 2 h o u r s .  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 t h e e f f e c t s o f c h l o r p r o m a z i n e and promazine may be due i n p a r t t o the f a c t t h a t the c o n c 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 e x p e r i m e n t s were made up by d i l u t i o n  o f i n t r a v e n o u s ampoules o f 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 p h a r m a c e u t i c a l m a n u f a c t u r e r cf t h i s compound, and amounts used were based on l a b e l c o n c e n t r a t i o n s as c l a i m e d by t h e drug house.  - 77  (b)  -  Phenoxybenzamine ( d i b e n z y l i n e ) .  b l o c k i n g agents of the b e t a - h a l o - a l k y l a m i n e has  The  use o f  c l a s s (dibenamine,  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  adrer. r g i c amines a t p e r i p h e r a l s i t e s .  adrenergic dibenzyline)  the a c t i o n s  However, remarkably  of  little  a t t e n t i o n seems to have been p a i d to the a d j u v a n t 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 o f autonomic agents  a t 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. a b i l i t y of adrenergic adrenergic  Furthermore,  b l o c k i n g agents to i n h i b i t the CNS  amines i s q u i t e u n c l e a r .  The  responses to  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 b l o c k a d e 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 was  the  EEG  investigated in 8 cats.  Following  the slow i n t r a - i n n o m i n a t e  Of phenoxybenzamine, the EEG (10 minutes o r more).  The  w i t h the dose a d m i n i s t e r e d , F i g u r e 36  a d m i n i s t r a t i o n (25 seconds)  becomes a c t i v a t e d f o r s e v e r a l  minutes  d u r a t i o n o f the a c t i v a t i n g response v a r i e s " a c t i v a t i o n " being  i l l u s t r a t e s EEG  longer w i t h  l a r g e r doses.  " a c t i v a t i o n " w h i c h 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 o f an e f f e c t i v e b l o c k i n g dose o f phenoxybenzamine (I mg/kg). for  A p e r i o d of 30 to kO minutes i s r e q u i r e d  phenoxybenzamine b l o c k a d e to be e s t a b l i s h e d , and  were not  c h a l l e n g i n g agents  t e s t e d 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 c h l o r p r o m a z i n e , the doses o f v a r i o u s com-  pounds r e q u i r e d  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  e s t a b l i s h e d " and  then these doses were t e s t e d 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 o f phenoxybenzamine.  The  capacity  phenoxybenzamine i n a dose o f 1 mg/kg to b l o c k e f f e c t i v e l y 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,  first  the  norepinephrine  of EEG and  - 78 -  RF-P  RP-0  IM LF-P DBZ RF-P  B.P.  I.Omg/Ko.  IM  J 5  ~  iV  RP 0  LP-0  l5  "v">  Figure 36. E f f e c t 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 i g h t drop in blood pressure is noted (12 mm Hg) .  - 79 -  h i s t a m i n e a r e iI l u s t r a t e d in F i g u r e s 38, 41 and 4 3 . ( i j A d r e n e r g i c amines: epinephrine.  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-  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  i n j e c t i o n o f 6.5/\jg/kg o f i s o p r o t e r e n o l .  t r a c i n g f o l l o w i n g the  T h i s 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 " w h i c h 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 p r e - t r e a t m e n t W i t h phenoxybenzamine (1 mg/kg), t h e i n j e c t i o n o f the 0.5/ug/kg o f i s o p r o t e r e n o l f a i I s t o 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 t o t h a t w h i c h was o b t a i n e d b e f o r e the a d m i n i s t r a t i o n o f phenoxybenzami ne. the dose o f i s o p r o t e r e n o l  Furthermore, i n c r e a s i n g  t o 1/ug/kg ( F i g . 39) does n o t produce an  a c t i v a t e d p a t t e r n ; d e s p i te t h e f a c t t h a t t h i s dose i s a p p r o x i m a t e l y 3-4 times t h a t dose w h i c h has been demonstrated t o produce c o n s i s t e n t EEG ' ' a c t i v a t i o n " ( t y p e A ) . i n a l 1 p r e p a r a t i o n s w h i c h have not been t r e a t e d with adrenergic blocking agents.  The d e p r e s s o r responses t o i s o p r o t e r e n o l  f o l l o w i n g phenoxybenzamine b l o c k a d e a r e comparable those o b s e r v e d in the c o n t r o l  i n many r e s p e c t s t o  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 t h a t t h e b l o o d p r e s s u r e has a tendency t o remain a t low 1 e v e l s f o r l o n g e r p e r i o d s o f time (more than 90 s e c o n d s ) . t h e a d m i n i s t r a t i o n o f e p i n e p h r i n e and n o r e p i n e p h r i n e i n " e q u i a c t i v a t i n g " d o s e s , 2 hours a f t e r I mg/kg o f phenoxybenzami ne, fails  likewise  t o 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 p r e s s o r responses as w e l l as EEG " a c t i v a t i o n " produced by 0.5 /Jg/kg o f e p i n e p h r i n e i n the absence o f phenoxybenzami ne a r e illustrated.  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 o f e p i n e p h r i n e 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 t r e a t m e n t w i t h 1 mg/kg o f phenoxybenzamine ( F i g . 4 1 ) .  - 80 -  RF-P  L r - u LP-0  LF-P  j  ,  T  I.NE.  0.5 ^ K g .  t  . '  pp.p  go  B.R los  F l ^  '  ' 1  RPto  M sec.  80  70  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.  M  - 81 -  t  RP-0  LF-P T I.NE  O.S»/Kg.  after RF-P  DBZ  I  1.0 rrg./Kg.  B.R _  , *  _____  ,  -__^\/ 1  5C  Figure 33. E f f e c t o f I s o p r o t e r e n o l 0.5/tig/kg on t h e EEG a f t e r phenoxybenzamine b l o c k a d e 1.0 mg/kg. Absence o f any a c t i v a t i o n and c o n t i n u e d p e r s i s t e n c e o f s p i n d l e a c t i v i t y . Note the d e p r e s s o r response.  W  - 82 -  11  h  RP-0  i  m  t I.N.E. ,.or/kg.  T  ,  oft-r DBZ. I.Omg/KgR F  .p  B.R q  S  i  . 7  S  55  300*. V  . *  ~  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 -  RF-P  L  F  i  P  >  t  Epv  o.5»/kq.  1  ,  .  . 5  R  F  p  B.P. 115  120  1  RP-0  LP-0  LF-P  135  120  Figure 40. Effect of epinephrine 0.5/ug/kg before phenoxybenzamine blockade. Control record for comparison with Figure 41. o n  300<-.V  I •'  set  t n e  E E G  |25  - 84 -  RF-P  JA  s  RP-0  LP-o LF-P  (  T Epi  RPf  & R  O-Sly/Kg afteri DBZ. l.O  . rng./Ka.  . 90  90  — —  , «  1  LF-P  75  70  Figure 41. Effects of epinephrine 0.5/ug/kg on the EEG after phenoxybenzamine blockade 1.0 mg/kg. No change is apparent in the EEG, but typical epinephrine reversal is obvious.  «0  - 85 -  Moreover, under these circumstances, epinephrine does not produce its characteristic pressor response, and instead typical epinephrine reversal is obtained (Fig. 41). tion of norepinephrine  In the same preparation the administra-  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 2 5 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 LLl Uiii i U  LF-P  J  t His_mm_ _.oy%i  .  *"  •  ' 5  R  pp  B.P. 95  75  sec.  3<M«V r  70  >»  RP-0  LF-P  M * * ^ ^ . « ^ ' w w » ^ ,  • .,_,i,.,',,^_._J  -a..',.,,.  T  f*y^^; tTt^f1ft-f^^ |  Figure 42.  Effect of histamine 2.0 /ig/kg on the EEG before phenoxybenzami 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 . o m g . / K g .  pr:p  B-P.  _  95  loo  p  _  _  ,  , BO  J  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. by  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  the i n j e c t i o n o f 2/Ug/kg o f a c e t y l c h o l i n e i n t h e p r e s e n c e o f 0 . 5 mg/kg  phenoxybenzamine.  Increasing  the dose o f phenoxybenzamine t o 1.0 mg/kg  f a i l e d to block  t h e EEG a c t i v a t i n g e f f e c t s o f s i m i l a r amounts o f  acetylcholine.  F i g u r e 44 i l l u s t r a t e s  t h e EEG " a c t i v a t i o n " o b t a i n e d  f o l l o w i n g the a d m i n i s t r a t i o n o f 2/Ug/kg o f a c e t y l c h o l i n e i n t h e p r e s e n c e of phenoxybenzamine b l o c k a d e ( e s t a b l i s h e d by doses o f 0 . 5 mg/kg and o f 1.0 mg/kg r e s p e c t i v e l y ) . (iv) Serotonin.  The a d m i n i s t r a t i o n o f s e r o t o n i n  2.5/ug/kg and 5.0/ug/kg was o b s e r v e d t o produce p r e s s o r  i n doses o f  responses and EEG  " a c t i v a t i o n " i n the p r e s e n c e o f phenoxybenzamine b l o c k a d e e s t a b l i s h e d by the  i n j e c t i o n o f I mg/kg Of phenoxybenzamine. In the m a j o r i t y o f cases the s m a l l e s t dose o f 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 a d m i n i s t r a t i o n o f phenoxybenzamine was 2 . 5 / i g / k g , but i n a few p r e p a r a t i o n s  i t has  been p o s s i b l e t o produce " a c t i v a t i o n " w i t h as low a dose as 1 /Ug/kg. Comparisons o f t h e e f f e c t s o f t h i s compound i n doses o f 2.5/Ug/kg and 5.0 /Jg/kg, both b e f o r e and a f t e r t h e a d m i n i s t r a t i o n o f phenoxybenzamine (1 mg/kg) i n t h e 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 c i r c u m s t a n c e s , t h e d u r a t i o n o f " a c t i v a t i o n " i s a p p r o x i m a t e l y 20 s e c o n d s ' s h o r t e r f o l l o w i n g the a d m i n i s t r a t i o n o f phenoxybenzamine ( F i g s . 45, variations  46,  47 and 4 8 ) .  However, such  i n t h e d u r a t i o n o f " a c t i v a t i o n " a r e o c c a s i o n a l l y seen i n t h e  absence o f b l o c k i n g  agents.  - 89 -  RF-P  RP-0  fan*. 2.o*/K31*5-  RF-P  LP-0  M M .  i  after  ,  OBZ.O.Smj./Kg.  BP 92.  . ,  5 sec.  , ,  u  ,  t  BP lot  ACH |U>  z.or/Ko. M b  after  J 0 8 Z l.omj/Kg  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  RF-P  ca n  B.P.  Seretonm. a-s  Ij/Kg. I  izo  130  5  set.  I  300/<-  RP-0  LP-0  LF-P  ,  •  ill  J|J  IV)  Figure  45.  E f f e c t o f s e r o t o n i n 2.5/tig/kg on the EEG b e f o r e phenoxybenzami ne. C o n t r o l t r a c i n g f o r comparison w i t h F i g u r e 46. Immediate a c t i v a t i o n ( a p p r o x i m a t e l y 5 seconds) p r e c e d i n g the peak p r e s s o r 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 b l o o d p r e s s u r e i s s t i l l e l e v a t e d .  - 91 -  •t RF-P  RP-0  t i«rdb»»»v. £.5 if/Kg 1 RF-P  & P  -  106  oHtr  06Z  1.0  30O/J.V  ma/Kg.  RP-0  L P  LF-P  Figure 46, Effect of serotonin. 2.5/*g/kg on the EEG after phenoxybenzamine 1 mg/kg. EEG activation consisting of a change i n pattern from 0 to A occurs in 5 seconds, and Is similar i n Intensity to the control tracing, but appears to be less prolonged (Fig. 45). The pressor response is similar to that seen in Figure 45.  MB  - 92 -  RF-P  S  RPO  \ 'I  I  LP-0  LF-P ISeroWn  5.0^X0.  i 300/0/  RF-P  BR H5  r1 11 I'M T  \m RP-0  LP-0  20  set.  LF-P 130  Figure 47, Effect of serotonin 5.0 /ug/kg on the EEG before phenoxybenzamine 1 mg/kg. Control tracing for comparison with Figure 48,  - 93 -  t SertAorui\  r> _ r  RFP  after  B.R  S.O » / K g  4  DBZ.  mg/kg.  1.0  UO  RP-o  LP-0  Ficjure kd. Effect of serotonin 5.0/jg/kg on the EEG after phenoxybenzamine 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 t e reno1 ( D C I ) .  the 3,4 d i c h l o r o - a n a l o g u e Observations  20522) i s  of isoproterenol.  made d u r i n g t h i s study have p o i n t e d t o the prompt and  c l e a r - c u t EEG " a c t i v a t i o n " o b t a i n e d doses ( 0 . 5 / j g / k g ) o f i s o p r o t e r e n o l . and S l a t e r (134)  T h i s compound ( L i l l y  and o t h e r s  i s o p r o p y l amino e t h a n o l  f o l l o w i n g the a d m i n i s t r a t i o n o f low The r e c e n t d e m o n s t r a t i o n  by Powell  (4,59,106,121) t h a t l - ( 3 , 4 - d i c h l o r o p h e n y l ) - 2 -  (DCI) b l o c k s the h y p o t e n s i o n  and t a c h 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 r e c e p t o r s i t e s , prompted o u r e f f o r t s to a s c e r t a i n whether o r not the p r i o r a d m i n i s t r a t i o n o f the analogue w i l l b l o c k o r modify i s 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 i n t r a - i n n o m l n a t e a d m i n i s t r a t i o n o f DCI i n the unanaesthetized  d e - a f f e r e n t e d c a t , the type D s p i n d l e a c t i v i t y  character-  i s t i c o f the p r e p a r a t i o n i s c o n v e r t e d w i t h i n a few seconds t o a type A activated pattern.  Depending on t h e dose a d m i n i s t e r e d  p a t t e r n may be t r a n s i e n t o r p r o l o n g e d . • a c t i v a t i o n " l a s t s f o r several minutes. EEG  this activated  W i t h s m a l l doses o f 2-5 mg/kg L a r g e r doses (15 mg/kg) e l i c i t  p a t t e r n s o f low v o l t a g e f a s t a c t i v i t y w h i c h l a s t f o r more than I hour  and a r e sometimes a s s o c i a t e d w i t h movements o f the head and forepaws. The p r e l i m i n a r y i s o p r o t e r e n o l - 1 i k e e f f e c t o f DCI on the EEG p r i o r to the appearance o f 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 t h e e f f e c t s o f t h i s compound on the c a r d i o v a s c u l a r system as r e p o r t e d by D r e s e l (50,121). normally  (45) and o t h e r s  The EEG " a c t i v a t i o n " produced by l a r g e doses o f DCI, a l t h o u g h i n t e n s e and o f long d u r a t i o n , can be r e v e r s e d by the a d m i n i s t r a -  t i o n o f doses o f c h l o r p r o m a z i n e  as low as 2-4 mg/kg ( F i g . 4 9 ) .  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 d e p r e s s o r  Moreover,  e f f e c t s ) noted  f o l l o w i n g t h e a d m i n i s t r a t i o n o f t h i s analogue o f i s o p r o t e r e n o l a r e  - 95 -  RP-0  i  > I hour ,  LP-0  F-P t  Rpp  BP  DCI  15 mj./Kg.  J  95  IOO  RP-o  LP-0  3  **  LF-P T CPZ 3.0mo/K 3  Ofer  '1  rx7l5 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" ( 2 0 seconds) without any alterations in vascular responses (Fig. 5 0 .  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 . 5 v / K g . i aftey DCI 7.5"mg./Kg.  RF-P  S S  8 5  85  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  alter  ftp.p  t  DCI I.Smg/Kg.  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 a f t e r DCI 15 mg/kg. Upper tracing: typical DCI-induced a c t i v a t i o n lasting for more than 2 hours. Lower tracing: r e l a t i v e l y large dose of isoproterenol f a 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 following 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 administration 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  RP-0  5  LP-0  LF-R t E>i. i.ov/Kg. 3.R  1 .  .  15  RF-P  oiter rSc.r  IS mo ./Kg-  | 0 £ )  ,  ,  I^Jy/  RP-o  LP-0  LF-P  i  as  Figure 53. E f f e c t of epinephrine 0.5 /ig/kg on the EEG a f t e r DCI 7.5 mg/kg. Moderate activation f l u c t u a t i n g between patterns B and C i l l u s t r a t e incomplete blockade of epinephrine by DCI. Note lack of e f f e c t on the pressor response to epinephrine.  120  102  RF-P  RP-0  LP-0  LF-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 a f t e r DCI 7.5 mg/kg. EEG patterns noted here are s i m i H r 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  lifi  mam ^HMwtwmimit  mm  LF-P  t  Epi. 0.5 r / k q .  I ,  0/te.v DCI T S ^ K g .  -  RF-P  B.P. ss  J HII  RP-C  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!  i.Ol/Kg.  t N.E. B.P.  8?  D  t  I  1  after  J  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 a f t e r DCI 15 mg/kg. Intense activation (type A) occurring a f t e r 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.  -  1  LP-0  ACH 2.0 If/Kg. b.for* DCI  105 -  t  , 300/i.V  i  I  LF-P  Figure 5 7 . 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 -  RF  "  P  I i  1  *** LF-P  T  ACH  2.0»/Kg. J . a f t e r D C I 1.S rr.j/Kg.  | JOO-.V  B.R qs  LP-0  LF-P 8 5  •Figure 5 £ .  E f f e c t of acetylcholine _ v g A g on the EEG a f t e r DCI 7.5 mg/kg. Absence of blockade i l l u s t r a t e d by intense type A a c t i v a t i o n , which is rapid in onset (6 seconds).  107  -  I  RF-P  RP-o  mm 1 Serotolim S . O * / K g . )  RF-P  300>cV  B.P. «  LF-P MS  Figure 59. Effect of serotonin 5/Jg/kg on the EEG before DCi, 7.5 mg/kg. Prompt and intense a c t i v a t i o n , f l u c t u a t i n g 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  ....li^..^-...  tri|  . . - . . y . | ^ i . .|.. r  1  v  1|T  ) r f | l ) l 1 l l T r | n r |  t  ||,.. ..; |. | . |  l  t  | |  1!^,^^,  tSeYofem.r, S - o y k o i . ait** DCI 1 3 r^j/K  g_p  7  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. Furthermore, 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 administration 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  Ill -  RF-P  C _ D  1  f Epi  .p  D C RF  B P a P.  D  csr/Kg.  l'  ~ f —  after Atropine aSmj/Ka. 1? is  |  5  s «s *e c•.  1  J^A/  RP-0  T ACH B  P  SO  after  a.o v / k g . Atropine.  1 O.S  A  ma/Kg.  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. oiter  i.O*/K.o.  I  Atropine  O-Smg./Kg.  RF-P  • |||)|lj|l|'!  ;i  Lp  t Serotonin S . 0 / K 9 . 1  B.P. So  after  Atropine  a.Smg./Kg.  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 a c t i v a tion is evident.  -  113  -  RF-P  t  Epi.  a.os/Kg. after  4  ,  Atropine 0 . 5 mjj/Kg.  i __ Mi t N.E.  B.R  <)0  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.  J  -1  - 114 -  2. mm  T Atropine  0.5 nwj./Kc). 1  Ccd eseYiruzed  RF:P  1 I  16 n>io  latev  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 ascertain whether acetylcholine, serotonin and the adrenergic amines would s t i l l 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 -  F i 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, "activation" 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 electrocortical changes suggestive of cerebral anoxia subsequently developed. This condition, if not reversed promptly by adequate supportive measures,  - 118 -  ^  p  Carotid  Sinosea TJervervoied  ^  |  I  RP-0 RP-0  Lp-o  [if n ]  ffnlim  LF-P  I  t  R F  .p  B P 80  Ep'l. 0 5 T / k g .  i  n  5  *"  1  ,oo  /  RP-0  LF-P  100  10  Figure  J5  66.  Effect of epinephrine 0.5/ug/kg on the EEG after b i l a t e r a l carotid sinus denervation. Typical epinephrine induced activation (D to B to A) can be observed.  9  -  119 -  lllll'l  Carotid Smustt Denervateo  I I I M I  I  M  I  II  II I I  I I i I I flTflt  mm  |  Serotcmin.  I  m^*¥^m^m^»>>0>  1  1  110 1  ^ ^ ^ ^  "^T,  ...  LF-P  105  S8  SS  Figure 67. E f f e c t of s e r o t o n i n 5/ug/kg o n the EEG a f t e r b i l a t e r a l carotid sinus denervation. Prompt a n d 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 .  q»  - 120  quickly led to Irreversible cerebral d e t e r i o r a t i o n . In Figure 69 the sequential e f f e c t of 0.5/ug/kg each of isoproterenol and norepinephrine are i l l u s t r a t e d .  Approximately 8  seconds following the i n j e c t i o n of ispproterenol, type A " a c t i v a t i o n " occurs.  This is s h o r t - l i v e d (10 seconds) and gives way to a pattern of  varying amplitude and frequency which quickly flattens out.  This  " f l a t t e n i n g " of the EEG occurs at depressor levels of 40 mm Hg and seems s i m i l a r 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 " a c t i v a t i o n " as well as rapid restoration of vascular e f f e c t s . Inquiry into the transient a c t i v a t i o n observed with isoproterenol in this  instance again suggested the p o s s i b i l i t y  that concomitant factors,  occurring j u s t previous to the presumed anoxic e f f e c t , could have been associated with this e l e c t r o - c o r t i c a l change. possibility,  In order to rule out this  1 mg/kg of phenoxybenzamine was administered to the carotid  sinus denervated preparation, and 60 minutes later a s i m i l a r dose (0.5/ug/kg) of isoproterenol was given. results.  challenging  Figure 70 i l l u s t r a t e s  these  No EEG " a c t i v a t i o n " can be observed a f t e r isoproterenol  administration in the presence of phenoxybenzamine blockade, despite the f a c t 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 l a t t e n i n g " of the EEG which is noted at 40 mm Hg is rapidly reversed by the administration of a dose of  - 121 -  Caret.,,  Si.u»es  1  Dene»v_«4  RF-P  (1 LF-P  t  ACH. O.5T/K3.  1 300/*V  BP.  no  qo  RF-P  •  to  Figure (68E f f e c t of a c e t y l c h o l i n e 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 denervation. A c t i v a t i o n produced, p o s s i b l y a f t e r s l i g h t e r longer l a t e n c y than is u s u a l l y observed w i t h t h i s agent.  - 122 "  Co-toiid Smuses Innervated  [iilili |[ i J j  RF-P  R  I ff [I j)f"[[tfTIf[-f  LF-P  t I . N . E 0.5^*3  I 70  co  •4M  f N.E. 0.5l/^ *  Figure 69. E f f e c t of Isoproterenol 0,5/ug/kg followed by norepinephrine 0 5yug/kg after b i l a t e r a l carotid sinus denervation. Prompt i n i t i a l adrenergic a c t i v a t i o n followed by the development 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 a c t i v a t i o n . 4  - 123 -  RFP  Carol «J  t  I.N.E.  DBZ  1  1  0.5*7 K g . I.  to  -  Figure 70  4  Effect of  isoproterenol  0^5 / i g / k g  in  benzamine  I mg/kg a f t e r  bilateral  carotid  Anoxic  pattern  depressor by  restoration  manifestations pletely to  develops  response  to  during  profound  isoproterenol  and  of  blood  of  adrenergic-induced  p r e s s u i e by  Is  incompletely  sinus  phenoxy-  denervation.  uncompensated again  is  overcome  norepinephrine.  b l o c k e d by phenoxybenzatnLne  norepinephrine  the presence of  activation  are  However, com-  (a 1 though p r e s s o r  blocked).  response  - 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  D.  lack of any associated EEG " a c t i v a t i o n " .  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 determine whether or not residual sensory inflow over this pathway was a s i g n i f i c a n t source of EEG influences in the " r e s t i n g " state.  Consequently,  an e f f o r t was made to eradicate the sensory nuclei of the 5th nerve by e l e c t r o l y t i c coagulation.  Access to the pons by the conventional cerebral  route was not attempted, since an intact cranium was necessary for comparing 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 c a r r i e r to an angle of about 40° to the v e r t i c a l and inserting the electrode v i a the cerebellar approach (Plate D). series of e l e c t r o l y t i c lesions  In this way, three  (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 j u s t posterior to the decussation of the branchium conjunctivum (Plate B, sections  16-18, 20 and 28).  The resting  synchronized pattern, and the drug-induced a c t i v a t i o n responses were  -  125  -  Plate D Photograph showing the c e r e b e l l a r approach to the p r o d u c t i o n o f 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 tegmentumof the c a t b r a i n by a g l a s s I n s u l a t e d e l e c t r o d e .  - 126 -  S C H E M A T I C  STEM  S A G I T T A L  W I T H  S H O W I N G  S E C T I O N  STEREOTAXIC  A R E A S  O F  O F  C A T  COORDINATES  C O A G U L A T I O N  PLATE  B R A I N  C  .  - 127 -  PLATE  IS  B  ID  at  Plate B Photographs of cross sections through lesions in the tegmentum of ponto-mesecnephalic area. Weigert's s t a i n .  -  128 -  indistinguishable from those of the preparation with an intact brain stem. 2.  Effects of Unilateral Ponto-mesencephalic Lesions on EEG "Activation" (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) s t i l l could be elicited (Fig. 7 0 .  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 s t i l l could be elicited but only with doses larger than those which usually produced EEG "activation" in the preparation 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 ventrolateral portion of the mid-brain (Plate B, sections 8-10, and Plate C,  129 -  Flora. Cat in Stereotaxic  io.05ec.  I oatrument".  85  I SOmio.  90  after 0.0 Sec 300/tV  RF.P  RP.O  LP.O  F. P LF. lll^iirtu^i/lU>«tWl>lllliii^Mnll»V' l1i.''»*l<iii^rtii tilli«rf Mi I i n til 31 w>ir>after  0.0Sec.  s  l  ,  nana  F i g u r e 71.  E f f e c t o f u n i l a t e r a l l e s i o n s i n the caudal p a r t o f the r i g h t mesencephalic tegmentum ( P l a t e C, l e s i o n s C| and C2). D e a c t i v a t e d EEG (type D) d e v e l o p i n g a f t e r 30 minute p e r i o d o f a d a p t a t i o n i n the s t e r e o t a x i c i n s t r u m e n t . Typical t r a n s i t o r y EEG a c t i v a t i o n i n response to a u d i o g e n i c s t i m u l a t i o n (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 o f e p i n e p h r i n e 2 yug/kg on the EEG i n the presence o f 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 o f the m e s e n c e p h a l i c tegmentum. I n t e n s e and prompt a c t i v a t i o n ( t y p e A) o c c u r s w i t h i n k seconds and l a s t s l o n g e r than 40 seconds.  - 131-  :  4#»MJjjj^  Wllf^  f l H f f f M l ^ ^  -  »/Kq.  B.R  J  80  LF.P <  60  F i g u r e 73.  E f f e c t o f a c e t y l c h o l i n e 2>ug/kg on the EEG i n the p r e s e n c e 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 o f the mesenc e p h a l i c tegmentum. Immediate and i n t e n s e 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 o c c a s i o n a l 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 norepinephrine (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 Rothballer s data (149), and lends support to the view that the intact 1  side can serve as a valid control for the adrenergic system.  - 133 -  w  LF.P :ISI  End of EC-C3 B.P.  RFP  10  nil  "  ,  B  LP 0 LFP <<»rtrfr#^ili^*»4»,^^  -  .  ' Whistle Blast  ,* ^^i w.wr^'' y,'*>v.w*«-*«.*>*i«' <  I  F  |  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 3Note prompt reversion to deactivated EEG on the side with the lesion and persistence of activation on the intact side. c  -  illtai  In "i' ilL <fi^hiiillML^J i'-"- "J  ,  jJ  134 -  1  E p i . 2.0 Jt/Kq. o««r EC-C3  .  ; sec.  B.P. io RF.P H'.'mwm  300*A/  [ Mi  RP-0  3 120  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.  -  '  I.N.E after  1.0  l^/Kg.  EC  135 -  1  -C"3  10  60  55  LS  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  after  E f - C 4 30/.V  B.R  ")0  8S  RF-P  p. LF.P 105  F i g u r e 77. E f f e c t o f e p i n e p h r i n e 2 /Ug/kg on t h e 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 m e s e n c e p h a l i c tegmentum ( P l a t e C, l e s i o n C^). Note prompt and i n t e n s e ( t y p e A) a c t i v a t i o n on t h e i n t a c t s i d e , and l a c k o f a p p r e c i a b l e a d r e n e r g i c a c t i v a t i o n on t h e side with lesions.  - 137 -  [PHI ^\  ^  t%H» ^ ^ y \ v » f  Figure 73. E f f e c t of isoproterenol 2/jg/kg on the EEG a f t e r u n i l a t e r a l lesions extending into the right dorso-1ateral portion of the mesencephalic tegmentum (Plate C, lesion C^). Note prompt and intense (type A) a c t i v a t i o n on the intact side. Note eventual appearance of f a i r l y intense a c t i v a t i o n on the side with lesions following this r e l a t i v e l y large dose of isoproterenol. Note delay of approximately 30 seconds before the appearance of a c t i v a t i o n 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 mesencephalic tegmentum (Plate C, lesion Ci*). Prompt and intense type A activation elicited in all leads.  -  VI.  A.  139  -  DISCUSSION  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 preparation 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 s t i l l 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 pharmacological 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 behavioural sleep (56).  results in persistent EEG deactivation and  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 wakefulness 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 norepinephrine, 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 vibration) 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 s t i l l 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 a c h i e v e d  -  to c e r t a i n s t i m u l i o f low  i n t e n s i t y , a b r u p t changes o r  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 sensory  i n f l o w , may  sought.  produce e f f e c t s o p p o s i t e to those t h a t were b e i n g  Under such c i r c u m s t a n c e s  a d a p t a t i o n may p r e s e n t and may  reducing  be due  p e r s i s t e n t act!vatlon without  to the complete e x c l u s i o n o f i n f l u e n c e s u s u a l l y  be r e l a t e d to the s t r a n g e n e s s  o f such  experimental  situations. R e c e n t l y , B a t s e l (12) has cerveau i s o l e dog  reported  that although  the c h r o n i c  i n i t i a l l y does e x h i b i t a h i g h degree o f  continuous  s p i n d l i n g and s l o w waves, e v e n t u a l l y (2 weeks o r more a f t e r t h i s type o f a c t i v i t y  i s g r a d u a l l y r e p l a c e d by low v o l t a g e  operation) fast  a c t i v i t y o n l y 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 p e r i o d s o f d e a c t i v a t i o n . B a t s e l s u g g e s t s t h a t these f a c t s o f f e r p r e s u m p t i v e e v i d e n c e t h a t 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 assume r a t e s o f 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. o b s e r v e d the s p o r a d i c development o f r e l a t i v e l y spontaneous EEG  " a c t i v a t i o n " patterns  We,  can  t o o , have  l o n g - l a s t i n g (£ to I hour)  i n our p r e p a r a t i o n s as l o n g 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 d u r i n g i n s e r t i o n o f stomach tube p r i o r to f e e d i n g and accompanying d e f e c a t i o n and p r e s e n c e o f sudden a l t e r a t i o n s i m e x t e r n a l environmental  the  i n the  factors  (changes i n the n o i s e l e v e l of the room, v a r y i n g degrees o f  lighting,  personnel  patterns  a l i e n to the p r e p a r a t i o n , e t c . ) .  However, these  always e v e n t u a l l y s u b s i d e d 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  always been o b t a i n e d  f o r prolonged  p r e p a r a t i o n s were f o l l o w e d f o r as  r e l i a b l e d e a c t i v a t i o n has  p e r i o d s o f time ( o c c a s i o n a l l y these long as 1-3 months).  Hence, o u r  - ]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. experience (with extra-cranial partial trigeminal  Our  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 ( 3 1 ) .  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 peripheral 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 , 6 3 ) 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 ( 1 3 9 ) , 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 significant 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 unilateral 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  s t i l l 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 hypothalamicmid-brain activity may be functionally inter-related, and that the hypothalamus 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 administration 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 intracarotid 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 bearing 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 observation 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 ( 1 1 7 ) .  It has been reported that a substance clearly  distinguishable from epinephrine and norepinephrine both pharmacologically 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 ( 1 1 0 ) .  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 ( 1 5 4 ) , 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 cardiovascular 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 phenoxybenzamine 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 isoproterenol 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 atropineinduced 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 conforms 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  Phenoxybenzamine  Chlorpromazine  Dichloroisoproterenol  Atropine  Epinephrine  +  4-  -  Norepinephrine  4-  -t-  -  +  Amphetamine  +  +®  -  +  IsopropylNorepinephrine  +  +  r®  +  Histamine  •+  +  -  4-  Serotonin  -  +  -  +  Acetylcholine  -  -  -  4-  Eserine  —  -  -  Key:  + — ®  = = =  blockade no blockade l a r g e doses may overcome blockade PLATE  E  +  ®  - 160 -  and blocked only by chlorpromazine and atropine, and a t h i r d which is responsive to histamine and the short-acting adrenergic amines and which is blocked by phenoxybenzamine as well as chlorpromazine and atropine. The p o s s i b i l i t y of a further d i f f e r e n t i a t i o n within this adrenoceptive category may be indicated by the very s e l e c t i v e blocking action of DCI for isoproterenol.  Similar observations of the e f f e c t s of these agents  on relaxation of the intestine has prompted Ahlquist (4) inference for this  to make this  tissue.  These observations furnish a basis for some conclusions with regard to the sequence of these links form the pathway for EEG " a c t i v a t i o n " .  in the chain of neurones which The fact that adrenergic,  " s e r o t i n e r g i c " and "histaminergic" EEG " a c t i v a t i o n " a l l are blocked by atropine would seem to suggest that neurones responsive to these agents may feed Into c h o l i n e r g i c - s e n s i t i v e neurones and that the l a t t e r may be the f i n a l  links  "activation".  in the neuronal chain leading to drug-Induced EEG The observation that " s e r o t i n e r g i c " a c t i v a t i o n is not  blocked by phenoxybenzamine suggests the p o s s i b i l i t y  that serotonin  s e n s i t i v e neurones may occupy a position intermediate between adrenoceptive and cholinoceptive components. The e f f e c t s of blocking compounds on EEG "activation"produced by sensory inflow are of i n t e r e s t .  Changes in the e l e c t r i c a l a c t i v i t y 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 a c t i v a t i o n ) . Such observations have been made by other investigators of atropine and chlorpromazine (22,1(^0,178).  in the case  - 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 following 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 s t i l l 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 "Activation", 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,  - 165-  but d i f f e r s 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 p o s s i b i l i t y that dibenamine may have a t a r a c t i c actions and feel  that "the demonstration of prolonged central neuronal alterations  does raise the p o s s i b i l i t y that less toxic agents with greater tranquil izing potency may be found among some of the interesting chemical analogues of dibenamine."  - 166  V.  1.  SUMMARY AND CONCLUSIONS  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 -  a m i n e and an  norepinephrine  the  intermediate position.  l a s t i n g EEG  least potent, with epinephrine  A m p h e t a m i n e and  e s e r i n e both  " a c t i v a t i o n " , w i t h amphetamine h a v i n g  occupying  produce  long-  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  drug-induced  t h i s p r e p a r a t i o n i s the the c e n t r a l  n e r v o u s s y s t e m and  (a)  drug-induced fluctuations,  (b) d r u g - i n d u c e d  h i s t a m i n e ) , and  (c)  cephalic  Unilateral  c o r t e x b u t do  suggested  one  t e r m i n a t i o n of  i n blood  pressure not  ( v a s o p r e s s i n , low d o s e s o f typical  EEG  "activation"  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 which  not a f f e c t  cholinergic  activation  t h e mesen-  i n the  ipsi-  activation.  the 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 of of  responsive  converging  on  the f i n a l  three types  p a t h w a y f o r EEG  t o c h o l i n e r g i c compounds and  which f$ responsive  and  a t r o p i n e ; and  t o s e r o t o n i n and  a t h i r d which  and  which  atropine.  blocked  blocked only  is responsive  s h o r t - a c t i n g a d r e n e r g i c a m i n e s and chlorpromazine  both  denervated.  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  one  as  and  of  Results obtained w i t h various s y n a p t i c b l o c k i n g agents  receptors capable  as w e l l  of  and  on  the  i n blood pressure are  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  5.  tion";  "activation"  sinuses are completely k.  lateral  EEG  activity  alterations  the d e m o n s t r a t i o n  p a t t e r n s f o l l o w i n g drug carotid  i s based upon s e v e r a l l i n e s  in electro-cortical  in  t h e s e compounds  independence between the o n s e t  u n i f o r m l y a c c o m p a n i e d by  have o b s e r v e d  i s not mediated v i a r e f l e x e s from  This view  temporal changes  " a c t i v a t i o n " w h i c h we  r e s u l t of a d i r e c t a c t i o n of  c a r d i o v a s c u l a r system. evidence;  EEG  to h i s t a m i n e  is blocked The  by  by  responses  have  of "activa-  by a t r o p i n e ; chlorpromazine and  the  phenoxybenzamine of  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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