The effect of two anesthetic agents on n

depletion ol central and peripheral catecholamme~ b~ administra- tion of reserpine or alpha-methyldopa potentlateb the action o!. guanethldme, has no effect on anesthetic requirements 15

,¢-, Elsewer SoentlfiC P u b h s h l p g C o m p a n y , A m s t e r d a m - Printed m The N e t h e r l a n d s

T H E E F F E C T OF T W O A N E S T H E T I C A G E N T S ON N O R E P I N E P H R I N E

A N D D O P A M I N E IN D I S C R E T E B R A I N N U C L E I , F I B E R T R A C T S ,

A N D T E R M I N A L R E G I O N S OF T H E R A T

MICHAEl_ F R O I Z E N , I R W I N J K O P I N , N G U Y E N B T H O A , J U S T I N Z I V I N , E R I C

A M U T H AND D A V I D M J A C O B O W 1 T Z

Laboratot ) of Chntcal St tence, Nattonal blstltute of Mental Health, Btnldmg lO/Room 2D-46, Bethe ~da,

Md 2 0 0 1 4 ( U S A )

(Accepted N o v e m b e r 24th, 1975)

SUMMARY

Catecholamlnerglc neurons have been implicated in the mechamsm of general anesthesia, but prewous attempts at measuring changes in adrenerg~c neuron function during anesthesia have been hmlted by techniques to whole brain Mlcrodlssectlon techniques and sensitive radlolsotoplc-enzymatlc assays were used to measure levels

of catecholamlnes in 20 different nuclei, fiber tracts or nerve terminal regions m brains of rats anesthetized for 90-105 mln with 1 ° o halothane or 18 o0 cyclopropane These two anesthetics were chosen because of their diverse effects on the electro- encephalogram and on the cardiovascular and respiratory systems Of the area~ examined, significant Increases m norepmephrme content with both anesthetic agents were found only in the nucleus accumbens, locus coeruleus and central gray cate- cholamlne areas Only m the nucleus accumbens was the dopamme level increased by both anesthetics, cyclopropane, but not halothane anesthesm, also increased the dopamme content of the caudate nucleus, while halothane, but not cyclopropane anesthesia, s~gmficantly decreased the dopamme level of the ventral nucleus of the thalamus Changes in levels of transmitters do not dlstlngmsh cause from effect of anesthesia, and further experiments are needed to dehneate what role, if any, the specific areas play m muscle relaxation, analgesla, sleep or anesthesia This study shows that a drug can affect one nucleus or regmn without slgmficantly affecting other regions that contain the same transmitter substance, and that changes m transmitter levels can occur selechvely m different regmns of brain even ff the nerve endings are demved from contiguous cell bodies

[ NTRODUCTION

Catecholammerg~c neuronal mechanisms are reputed to be revolved m the

imtlatmn and control ot paradoxical sleep 8 22 and m susceptibility to ,meqh~t~ action 7 l~ In dogs depletion ol central and peripheral catecholamme~ b~ administra- tion of reserpine or alpha-methyldopa potentlateb the action o! anesthetics, ,~lvle pretreatment with an agent that depletes only peripheral norepmephrmc s t o l ~ guanethldme, has no effect on anesthetic requirements 15 Conversely pretreatmcnt with a monoamlne oxldase mhibitm, which prevents the normal degradatwe met,ibo- hsm of neuronal monoamines and elevate, central and peripheral amine levels ~n- creases anesthetic drug requu ements, as does acute admimstrat~on of dextroamphet- amine, which increases release of monoamlnes 5,7 Evidence that anesthetic agents block amine release has recently accumulated Six different anesthetic agent~ ~eie found to decrease plasma level~ ofcatecholammes l~ For halothane and cyclopropane this effect wa~ due at least m part to a direct mhlbltlon of stimulation-reduced release of norepmephrme lrom peripheral bympathetic nerve~ "° Furthe~inole pentobarbltal, methoxyflurane and ether have been shown to block release ol cal~- cholamme8 fiom the adrenal medulla ~ ,.i~

In whole brain, changes na levels or turnover of catecholamlnes during anesth- esla have not been striking ~' The recent development of a mlcrodissectlon techni- quO ~', coupled w~th sensmve radiolsotop~c-enzymatic assays has enabled the measure- ment of the two major catecholammergic transmitters m individual brain nuclei, hbel tracts and nerve terminal regions Using these techniques, 20 different areab of brain have been examined m contl ol rats and in rats anesthetized with e~ther of two agent, which have different cardio, ascular and respiratory actions zl and which are at very different polnt~ on the electroencephalographlc spectrum o f inhalation anesthetic agents 1 2~

METHODS

At 7 15 a m , male Sprague-Dawley rats (250-300 g) (Hormone Assay) were either anesthetized with 3 oo halothane for 5 mln and then maintained w~th 1 15",, halothane m oxygen or kept in identical conditions without halothane administration

On either the same or alternate days, similar experiments were performed using 30 Oo cyclopropane in oxygen for induction of anesthesia and 18 o~ cyclopropane m oxygen for maintenance Between 95 and 105 mln after induction of anesthesia, anesthetized and control rats were alternately decapitated, and their brains removed and rapidly frozen on dry ice

Using a cryostat (at 10 °C), the brains were shced into 300-/~m coronal sec- tions beginning at the level of the nucleus accumbens and ending in mid-cerebellum Mlcropellets o f tissue from specific brain nuclei, fiber tract or terminal regions were removed with stainless steel cannulae of various Internal diameters using the method

of Palkovlts 16 The landmarks for ldentlficatmn in the unstained sections were the shape o f the brain and ventricles, and major nerve tracts A description of topograph~ and dissection parameters IS presented m Table 1 The approximate coordinates described pertain to the Konlg and Khppel 9 and Jacobowltz and Palkovlts ~' 17

TABLE I

punches (ram) coordinates ( /ml )

N mterstmahs strm termmahs

atlases In a d d i t i o n , a region in the central gray, rich in c a t e c h o l a m l n e fluorescent

v a r l c o s l t l e s , was Included in this s t u d y is

S a m p l e s f r o m b o t h right a n d left h o m o l o g o u s b r a i n regions o f each a n i m a l were l m m e d m t e l y b l o w n f r o m the d i s s e c t i n g needle Into 100/~1 ice-cold 0 1 N per-

c h l o r l c a c i d a n d h o m o g e n i z e d b y sonlficatlon ( K o n t e s u l t r a s o n i c cell d e s t r u c t o r , no 881440) A f t e r a h q u o t s for p r o t e i n d e t e r m i n a t i o n were r e m o v e d , the r e m a i n i n g s a m - ples were f r o z e n a n d k e p t at - - 1 0 ' C for u p to two weeks b e f o r e being a s s a y e d f o r

n o r e p l n e p h r l n e a n d d o p a m l n e P r o t e i n c o n t e n t in 10-/~1 a h q u o t s o f the h o m o g e n a t e was m e a s u r e d b y the m l c r o m e t h o d o f L o w r y e t a l l z

A f t e r the f r o z e n s a m p l e s were t h a w e d , v o r t e x e d a n d c e n t r i f u g e d a t 8000 y g for

30 sec, 25-#l a h q u o t s were a s s a y e d for c a t e c h o l a m l n e s as d e s c r i b e d by C o y l e a n d

H e n r y 2 This m e t h o d is b a s e d on the e n z y m a t i c c o n v e r s i o n o f c a t e c h o l a m l n e s to their

O - m e t h y l a t e d d e r i v a t i v e s in the presence o f the t r l t l a t e d m e t h y l d o n o r ([~H-methyl]S-

a d e n o s y l m e t h l o n l n e , N e w E n g l a n d N u c l e a r C o r p , Boston, M a s s ) T h e O - m e t h y l a t e d

~ - O H d e n v a t e s [ 3 H - m e t h y l ] m e t a n e p h r m e a n d [ a H - m e t h y l ] n o r m e t a n e p h r l n e are

o x i d i z e d by p e r l o d a t e to v a m l h n w h i c h is then e x t r a c t e d s e p a r a t e l y into t o l u e n e - l s o a m y l

a l c o h o l (3 2) a n d the t r i t l a t e d 3 - m e t h o x y d o p a m l n e IS a s s a y e d b y h q m d s c l n t d l a t l o n

s p e c t r o m e t r y E p i n e p h r i n e a n d n o r e p i n e p h r l n e are n o t d i s t i n g u i s h e d b y this a s s a y

p r o c e d u i e , a n d levels o f these c a t e c h o l a m l n e s m b r a i n are r e p o r t e d as n a n o g r a m s o f

n o r e p l n e p h r l n e p e r m i l l i g r a m o f p r o t e i n , d o p a m l n e is r e p o r t e d as n a n o g r a m s o f

d o p a m l n e p e r m l l h g r a m o f p r o t e i n W h e n d o p a m m e c o n t e n t was f o u n d to be less t h a n

T A B L E 11

I ~ F I ~ I ' ( T O F k N I S J H L T I ( k ( ~ f ' N [ S ( l ~ N O R I q ~ I N I P H I ¢ I N I " ( ( ) N T F N T o l ~ SPI~( I P I ( B R A I N N b t I [ I A N D R I ~ H I , ' ~ , ~

T H E R ~ , T

n - 6 8

\ ot epmel)ht the c oHtt HI t ng/tttg p t o t e m ~ 5 ~ M ( ontt )l / ',, H a l o t h a n e (_ ontt o/ 0 ,, C~ch~-

pl opalle ( A ) A l e a s m t , h w h lel,el~ o / n o t e p m e p h t m e were a l t e l e d b~ b o t h anesthetic agent~

C e n t r a l g r a y

c a t e c h o l a r n m e a r e a ~ 5 7 2 7 8 9 6 4 j 1 0 2 " * 5 7 2 A 7 8 1 0 0 4 £ 8 1 " *

L o c u s c o e r u l e u s ~ 41 ~; 7 0 6 3 7 ~ 4 1 " 4 1 3 ~ 7 0 7 3 9 9 4 ~

( B ) A t e a ~ m t~ht~h lel,el~ o / n o 1 e p t n e p l w m e ~el e n o t a l t e r e d b~ ettller a g e n t

N u c l e u s m t e r s t l t m h s

P a r a v e n t r l c u l a r

V e n t r a l n u c l e u s oi' t h e

N ucleu~

S u b s t a n t t a m g r a

N u c l e u s r a p h e d o r s a h s ~ 2 0 9 ~ ~ 4 21 1 t 2 3 2 0 9 + 3 4 1 8 5 ~ ~t)

* S ~ g m f i c a n t l y d i f f e r e n t f r o m c o n t r o l , P " 0 0 5 , b y g r o u p ' F - t e s t a n d t w o - w a y analyb~s ol v a r i a n c e

** S~gnxficantly d i f f e r e n t f r o m c o n t r o l , P 0 0 1 , b y g r o u p ' F - t e s t a n d t w o - w a y analys~s o f v a r i a n c e

*** A m m a [ s w e r e a n e s t h e t i z e d a n d d e c a p i t a t e d as d e s c r i b e d m M e t h o d s E x p e r i m e n t s w i t h h a l o t h a n e

a n d c y c l o p ~ o p a n e f o r t h e s e r e g i o n s w e r e p e r f o r m e d o n s e p a r a t e d a y s , a n d t h e r e f o r e l w o sets ot

c o n t r o l s ~ e r e u s e d

§ A n i m a l s w e r e a n e s t h e t i z e d a n d d e c a p i t a t e d as d e s c r i b e d in M e t h o d s E x p e r i m e n t s w i t h h a l o t h a n e

a n d c y c l o p r o p a n e f o r t h e s e r e g i o n s w e r e p e r f o r m e d o n t h e s a m e d a y , a n d t h e r e f o r e , o n e set o f

c o n t r o l s w a s u s e d

20 ~ o f the noreplnephrme content, the results were considered to be eqmvocal and

are n o t reported

RESULTS

Halothane

Halothane anesthesm produced a slgmficant elevatmn o f norepmephrme con- tent in the nucleus accumbens, central gray catecholamme area and locus coeruleus

T A B L E 11I

E I F E C T O F A N E S T H E T I C A G E N T S O N D O P A M I N E C O N T E N T O F S P E C I F I C B R ~ I N N U C L E I A N D R E G I O N S O F T H E

R &T

n = 6-8

D o p a m m e ~ontent (ngrmg J~ S E ~1 )

Control 1% Halothane Control 20 ° o C~clo-

propane

CA) Areas m whu h level~ o / d o p a m m e were altel ed b) both anesthetic agents

N u c l e u s a c c u m b e n s * * 6 8 6 ± 1 0 3 1 4 6 7 - L 2 5 9 * 7 3 7 , 6 8 9 4 7 ± 5 8 "

( B) Areas tit which levels o / d o p a m t n e were altered b) one but not both anesthetic agent~

C a u d a t e n u c l e u s * * * 1 0 7 4 , 7 4 1 3 7 0 ~ 120 1 0 7 4 , 7 4 1 4 8 4 , 8 4 " Ventral nucleus o f the

( C ) Atea~ m whwh levels o f noJepmephtme were not

0 1 " 1 1 :L 0 2

atteted b~ either agent

147 6 9 6 ± 132 Nucleus lnterstltlahs

S t l m t e r m l n a h s * * 9 5 " 13 136 ¢ 4 7 9 1 , 3 4 1 1 0 , 3 3

C l n g u l a t e c o r t e x * ~ 17 ~ 0 2 2 0 ± 0 3 1 2 , 0 1 I 1 , 0 6

P a r a v e n m c u l a r

M e d l a n e m m e n c e * * 3 7 5 A 3 7 401 -L 2 2 5 8 5 5 ~ 7 0 7 3 0 _ ~ 4 0

A t c u a t e n u c l e u s * * * 1 6 4 2 - 2 2 1 9 4 - i 2 6 1 6 4 , 2 2 1 9 6 , 4 0

H a b e n u l a * * 1 7 - 0 1 1 6 ~ _ 0 1 2 7 , 0 5 1 9 - ~ 0 5

S u p e r l o r c o l h c u l u s * * 1 7 ¢ 0 3 1 5 , 0 I 18 t 0 6 2 2 ~ 1 0

M e d l a l g e n l c u l a t e * * 1 7 : 4 0 6 I 6 ~ 0 3 I 7 , 0 9 2 0 ~ 1 0 Substantla nlgra

c o m p a c t a * * * 3 3 5 ~ 2 2 3 4 3 ± 4 3 3 3 5 , 2 2 3 7 5 , 4 1 Nucleus raphe

d o r s a h s * * * 4 2 ± l 0 4 1 + 0 8 4 2 , 1 0 5 8 , 1 3

Cerebellum **~ 2 0 , 0 4 I 5 2- 0 3 2 0 5 0 4 1 1 _[ 0 6

Significantly different from control P 0 0 5 by group ' f - t e s t a n d by two-way analysl~ o f variance

~* Animals ~ e r e anesthetized and decapitated as described in Method5 Experiments with halothane and cyclopropane for these regions were performed on separate days, and therefore, two sets o f controls ~ e r e used

** Animals were anesthetized a n d decapitated as described in M e t h o d s Experiments with halothane and cyclopropane for these regions ~ e r e performed on the same day, and, therefore, one set o f controls was used

In all other areas examined levels o f norepmephrme during anesthesia were not different from levels found in control ammals (Table 11) D o p a m m e content o f the nucleus accumbens was also increased during halothane anesthesia, while that m the ventral nucleus o f the thalamus decreased In all other areas examined, levels o f dop- amine during anesthesia were unchanged from control values (.Table 111)

Cyclopropane

Cyclopropane anesthesm produced significant elevatmns m n o r e p m e p h r m e content in the nucleus accumbens, central gray catecholamme area and locus coeru-

leub, while no change was detected m all other areas examined (Table II) The dop- amine contents of the nucleus accumbens and caudate nucleus were also increased during cyclopropane anesthebla in all other areas examined during c~clopropdne anesthesia, levels ol dopamme remained unchanged from control values (Table Ill)

DISCUSSION

Previous investigations of the role of adrenerg~c neurons in anesthesm have either revolved pharmacological tools to increase or decrease levels of catecholammes

or other putatwe neurotransmltters throughout the brain 4,7,13,1~ or have momtored changes m total brain neurohormone levels 15 Such studies could not detect changes induced by anesthettc agents that might occur only m a few d~screte regions The purpose of this study was to determine ]f an anesthetic agent could d~fferentlally affect catecholamme content m d~screte areas of the brain The results show that two dxverse anesthetic agents are assocmted w~th selectwe increases m catecholamme levels m the nucleus accumbens, locus coeruleus, and central gray catecholamme area and not m

17 other catecholamme-contamlng regaons examined

Concentrations o f a transmitter may be elevated by increased synthes~s, m- creased reuptake or decreased release A decrease m release, if ~t occurs, may reflect a direct action of a drug m preventing release or an redirect effect medmted by other neurons which result m mact~wty of the ammerg~c neurons and decreased release ot catecholamme The fact that anesthesm w~th e~ther halothane or cyclopropane, two agents at very d~fferent points of the chmcal and electroencephalographlc spectrum of inhalation anesthetics 1,2t,-'~, is assocmte0 w~th locahzed increases m levels of n m - epinephrine m the locus coeruleus and central gray catecholamme area does not necessarily mean that an5' of these regions are revolved m ruination or maintenance ot the state of anesthesm, analgesm or sleep Slmdarly, the anesthesm-mduced increase

of dopamme content m the nucleus accumbens does not warrant the conclusion that mh~bmon of dopamme release m th~s area promotes anesthesia

Anesthesm w~th ezther halothane or cyclopropane was associated with an m- crease m norepmephrme content m the central gray catecholamme area which Js located at the lateral aspect of the serotonm-contammg nucleus raphe dorsahs cell bodies ~s and contains a large number of fluorescent catecholamlne terminals Cate- cholammerg~c neurons m th~s area emanate from the locus coeruleus and the ventral noradrenerglc pathway Is

In the nucleus accumbens, terminating dopammerg~c axons are believed to arnve wa the mesohmblc dopamlne system from cell bodies (A10) originating dorsal and lateral to the nucleus mterpedunculans za Lesions made in this nucleus produce slgmficantly Iowerjump threshold to electric footshock m rats 11, suggesting a possible

~ole of the nucleus accumbens m analgesm The mesohmblc dopamme system terminates not only m the nucleus accumbens, but also m the olfactory tubercle where the dopamme content of the anesthetized and cot~trol ammals was slmdar (Table Ill) Thus, terminal areas derived from contiguous cell bodies do not necessar- fly react s~mflarly to the same drug

T h e i n c r e a s e o f c a t e c h o l a m l n e s m t h e t w o a r e a s c o n t a i n i n g n e r v e t e r m i n a l s ,

c e n t r a l g r a y c a t e c h o l a m l n e a r e a a n d n u c l e u s a c c u m b e n s , a n d t h e cell b o d y r e g i o n o f

t h e l o c u s c o e r u l e u s m a y r e f l e c t a d e c r e a s e m r e l e a s e o f t h e c a t e c h o l a m l n e S u c h a n

i n c r e a s e m a m i n e c o n c e n t r a u o n w o u l d b e e x p e c t e d t o b e g r e a t e s t in a r e a s in w h i c h

t h e r e ~s a r a p ~ d t u r n o v e r

T h e e x p e r i m e n t s r e p o r t e d h e r e s h o w t h a t a n e s t h e s i a w~th e ~ t h e r o f t w o d i v e r s e

i n h a l a t i o n a n e s t h e t i c a g e n t s ~s a s s o c m t e d w~th i n c r e a s e s in c a t e c h o l a m m e l e v e l s m t h e

n u c l e u s a c c u m b e n s , l o c u s c o e r u l e u s , a n d c e n t r a l g r a y c a t e c h o l a m m e a ~ e a a n d n o t in

17 o t h e r c a t e c h o l a m l n e - c o n t a m l n g r e g i o n s e x a m i n e d F u r t h e r s t u d y o f t h e s e a r e a s ~s

n e c e s s a r y t o d e h n e a t e t h e i r r o l e a n d r e l a t i v e ~ m p o r t a n c e , ff a n y , m p r o m o t i n g a n a l -

g e s m , m u s c l e r e l a x a t i o n , s l e e p o r a n e s t h e s i a

R E F E R E N C E S

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38 (1973) 564 582

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Brlt J Pharmacol, 49 (1973) 205 213

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59 (19T~) 449 450

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J cO;~lp Neu;ol, 157 (1974) 29 42

area bordering on the nucleus raphe dorsahs, Brain Research, 101 (1975) 561-568

19 ROIZEN M I~,Mos,',,J HI'qR~ D P '~NI3KoPIN, I J , L f f e c t s o t h a l o t h a n c o n p l a s m a t a t t ~ h o f amme~ 4ne~thesu~h~¢~ 41 (1974)432 439

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n o r e p m e p h r m e but not oi dopamme-beta-hydroxylase, from gumea-p~g vas deferens Lmo ~ /

Pha;ma¢ol, ~1 (1975) 311 ~f

2 l SH1MOSATO % , A N D [~'ISTFIN [] E E~'eCl O[ anesthetzc drugs on the heart a c~Jticat i e ~ e ~ oi myocardial contractdtt> and ~ts lelatlonshlp to hemodynam~cs, Chn 4nesth 3 (1969) 1- 74

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23 UNGVRSTEDT U , Stc~eota\~c m a p p i n g of m o n o a m m e path,says m the rat brain, ,,l~ta ph~ sud stand,Suppl 367 (1971) [ 48

24 WINTERS, W D , ElYRRAP.-Au '~DO, T , GUZMAN-FLORES, C , AND ALCARAZ, M , The catalcpu~ state reduced by ketamme a tewew of the n e u r o p h a r m a c o l o g y of anesthesia, lVeutopharma¢olog~

11 (1972) q03 qlS