Btam Research, 110 (1976) 515-522 515 ,¢-, Elsewer SoentlfiC Pubhshlpg Company, A m s t e r d a m - Printed m The Netherlands T H E E F F E C T OF TWO 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 TRACTS, A N D T E R M I N A L R E G I O N S OF THE 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 A M U T H AND D A V I D M J A C O B O W T Z B T H O A , J U S T I N ZIVIN, ERIC Laboratot ) of Chntcal St tence, Nattonal blstltute of Mental Health, Btnldmg lO/Room 2D-46, Bethe ~da, Md 0 ( 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 ° o halothane or 18 o0 cyclopropane These two anesthetics were chosen because of their diverse effects on the electroencephalogram 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 catecholamlne 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 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 516 imtlatmn and control ot paradoxical sleep 22 and m susceptibility to ,meqh~t~ action l~ In dogs depletion ol central and peripheral catecholamme~ b~ administration of reserpine or alpha-methyldopa potentlateb the action o! anesthetics, ,~lvle pretreatment with an agent that depletes only peripheral norepmephrmc stol~ guanethldme, has no effect on anesthetic requirements 15 Conversely pretreatmcnt with a monoamlne oxldase mhibitm, which prevents the normal degradatwe met,ibohsm of neuronal monoamines and elevate, central and peripheral amine levels ~ncreases anesthetic drug requu ements, as does acute admimstrat~on of dextroamphetamine, 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 anesthesla have not been striking ~' The recent development of a mlcrodissectlon techniquO ~', coupled w~th sensmve radiolsotop~c-enzymatic assays has enabled the measurement 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 of inhalation anesthetic agents 2~ METHODS At 15 a m , male Sprague-Dawley rats (250-300 g) (Hormone Assay) were either anesthetized with oo halothane for mln and then maintained w~th 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 sections beginning at the level of the nucleus accumbens and ending in mid-cerebellum Mlcropellets of 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 of the brain and ventricles, and major nerve tracts A description of topograph~ and dissection parameters IS presented m Table The approximate coordinates described pertain to the Konlg and Khppel and Jacobowltz and Palkovlts ~' 17 517 TABLE I AJea Number oJ punches Stze cannuht (ram) Approxtmate coordinates ( /ml ) Nucleus accumbens Caudate nucleus Olfactory tubercle N mterstmahs strm termmahs (ventrahs) Cmgulate cortex Paravenmcular nucleus Ventral nucleus of the thalamus Medmn eminence Arcuate nucleus Nucleus dorsomedmhs Habenula nucleus Htppocampus Dorsal bundle Superior colhculus Medml gemculate body Substantm mgra, zona compacta Nucleus raphe dorsahs Central gray catecholamme area Locus coeruleus Cerebellum 4 05 05 05 A9410 A9410, A8920 A9410, A8920 4 4 4 4 4 4 4 05 10 05 10 05 03 05 10 I0 10 10 10 05 05 05 05 10 A6860, A6670 A6860-A5780 A5660, A5340 A5150, A4620 A4890 A3750 A4890-A3750 A4620, A4380 A3990, A3750 A3990, A3750 A3180, A2790 A1760, A1270 A2180, AI760 A2180 A620- P290 Pl0~2620 P2300-P2800 P2000 P2800 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 varlcosltles, was Included in this study 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 animal were l m m e d m t e l y b l o w n f r o m the dissecting needle Into 100/~1 ice-cold N perchlorlc acid a n d h o m o g e n i z e d by sonlficatlon ( K o n t e s ultrasonic cell destructor, 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 removed, the r e m a i n i n g samples were frozen a n d k e p t at - - ' C for up to two weeks before being assayed for 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 by the m l c r o m e t h o d o f L o w r y et al lz A f t e r the frozen samples were t h a w e d , v o r t e x e d a n d centrifuged at 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 described by Coyle a n d H e n r y 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 derivatives in the presence o f the t r l t l a t e d methyl d o n o r ([~H-methyl]Sa 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 ) The O - m e t h y l a t e d ~ - O H d e n v a t e s [ 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 oxidized by p e r l o d a t e to v a m l h n which 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 - m e t h o x y d o p a m l n e IS assayed by h q m d sclntdlatlon 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 distinguished by this assay 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 per 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 per 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 518 TABLE I~FI~I'(T OF THE n 11 kNISJHLTI( k(~f'N[S(l~ NORIq~INI PHI¢INI" (()NTFNT o l ~ SPI~( I P I ( BRAIN Nbt I [I AND RI~HI,' ~,~ R~,T -68 \ ot epmel)ht the c oHtt HI t ng/tttg p t o t e m ~ ~ M ( ontt )l / ',, H a l o t h a n e (_ ontt o/ ,, 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 Nucleusaccumbens*** 40 02 Central gray catecholarnmearea~ 572 78 Locuscoeruleus~ 41 ~; 70 ( B ) Atea~ m t~ht~h lel,el~ Caudatenucleus*** Olfactorytubercle~ Nucleus mterstltmhs strmtermlnahs~ C l n g u l a t e c o r t e x **~ Paraventrlcular nucleus*** Medlanemmenee*** Arcuate nucleu~ V e n t r a l n u c l e u s oi' the thalamus *~° N ucleu~ dorsomedmhs *~ Habenula*** H J p p o c a m p u s **~ Dorsal bundle**~ S u p e n o r c o l h c u l u s ~** Medlalgen~culate*'* Substantta mgra compacta§ Nucleusraphedorsahs~ Cerebellum§ were a l t e l e d b~ both anesthetic agent~ 73 ~* 39J 07 964 637 j 102"* ~ 41" 572 A 413 ~ 78 70 o / n o eptneplwme ~el e not a l t e r e d b~ ettller agent 2~ , 03 28 I 05 23 t 03 ]7 05 ~8 ~ 03 ~4 [ 0~ ~57 ~9 52 ~ 08 389 4"; 04 357 ~ ~7 _ 461 432 288 - 114 ~ 44 28 462 ~37 269 26 75 ~7 448 ~ 63 432 44 288 ~ 28 ~ , 02 ~;35 36 "70 ~ 53 ] 64 F ~;9 03 04 08 07 06 12() ~ ( ) 209 ~ ~4 I ' 07 ~ ~4 t _81 ~9 66 80 00 68 02 t 60 } 03 ~ 0"~ 04 ~ 04 145 ~ 16 21 t 24 I 05 ~0 52 03 ~ 05 65 07 ~ 1004 £ 739 81"* 94 ~ 24 30 03 05 ~77 46 28 06 62 :t 524 :~98 69 6"~ 64 I 346 138 { 08 106% 09 116 t 30 86 08 11 :~Ss z 11 s 34 ¢ 05 98 02 11 _ _ I ~, 1(12 ' 98 06 120~ 209+ 31 t 103 185 55 06 34 07 ~ ~ I ~t) 19 * S~gmficantly different f r o m c o n t r o l , P " 0 , by 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 different f r o m c o n t r o l , P 0 , 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 for 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 days, 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 used § 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 for 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 the s a m e day, 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 used 20 ~ of the noreplnephrme content, the results were considered to be eqmvocal and are n o t reported RESULTS Halothane Halothane anesthesm produced a slgmficant elevatmn of norepmephrme content in the nucleus accumbens, central gray catecholamme area and locus coeruleus 519 T A B L E 11I EIFECT OF ANESTHETIC AGENTS ON DOPAMINE CONTENT OF SPECIFIC BR~IN NUCLEI AND REGIONS OF THE 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~clopropane CA) Areas m whu h level~ o / d o p a m m e were altel ed b) both anesthetic agents Nucleusaccumbens** 686±103 1467-L * 737, 68 947±58" ( B) Areas tit which levels o / d o p a m t n e were altered b) one but not both anesthetic agent~ Caudatenucleus*** 1074, 74 ~ 120 1074, 74 1484,84" Ventral nucleus of the thalamus** 09i 02 05, 06 L02 01" 1 :L ( C ) Atea~ m whwh levels Olfactory tubercle** Nucleus lnterstltlahs Stlmtermlnahs** Clngulatecortex* ~ Paravenmcular nucleus** Medlanemmence** Atcuatenucleus*** Habenula** Superlorcolhculus** Medlalgenlculate** Substantla nlgra compacta*** Nucleus raphe dorsahs*** Cerebellum **~ o f noJepmephtme were not atteted b~ either agent 6 :~ 124 678 147 ± 132 95" 17 ~ 13 02 136 ¢ 20± 03 149± 375 A 16421717 ¢ 7:4 44 37 22 01 03 06 125_t: 401 -L 194-i 16~_ 15, I6 ~ 335 ~ 22 343 ± 42± 20, l0 04 707,8 91, 2, 34 01 110,33 I 1,06 10 22 26 01 I 03 84, 5855~ 164, 27, 18 t I 7, 26 70 22 05 06 09 91,26 730_~40 196,40 19-~05 2 ~ 10 20~ 43 335, 22 375 , 41+ 08 I 2- 42, 205 10 04 58,13 1 _[ Significantly different from control P 0 by group ' f - t e s t and by two-way analysl~ of variance ~* Animals ~ere anesthetized and decapitated as described in Method5 Experiments with halothane and cyclopropane for these regions were performed on separate days, and therefore, two sets of controls ~ere used ** Animals were anesthetized and decapitated as described in Methods Experiments with halothane and cyclopropane for these regions ~ere performed on the same day, and, therefore, one set of controls was used In all other areas examined levels of norepmephrme during anesthesia were not different from levels found in control ammals (Table 11) D o p a m m e content of the nucleus accumbens was also increased during halothane anesthesia, while that m the ventral nucleus of the thalamus decreased In all other areas examined, levels of dopamine during anesthesia were unchanged from control values (.Table 111) Cyclopropane Cyclopropane anesthesm produced significant elevatmns m norepmephrme content in the nucleus accumbens, central gray catecholamme area and locus coeru- 520 leub, while no change was detected m all other areas examined (Table II) The dopamine 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 of a transmitter may be elevated by increased synthes~s, mcreased 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 nmepinephrine 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 mcrease 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 Catecholammerg~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 not necessarfly react s~mflarly to the same drug 521 The increase of catecholamlnes m the two areas containing nerve terminals, 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 reflect 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 increase m amine concentrauon Such an 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 rap~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~ther 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 levels m t h e nucleus accumbens, locus coeruleus, and central gray catecholamme 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 regions examined a~ea a n d n o t in 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 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