Pharma¢,~lo~.,.~ Bio(hemAtr.~ & Behuvior. Vol. 9. pp. 559--561. Printed in the U.S.A.

BRIEF COMMUNICATION Effects of Anisomycin and CNS Stimulants on Brain Catecholamine Synthesis'

PAUl. LUNDGREN

AND LAURENCE

A. C A R R

l ) e p a r t m e n t o f Pharmacoh~,t,y a n d Toxico/o.t,,y, U n i v e r s i t y o.l" Louisville, Louisvilh', K Y 40202 R e c e i v e d 14 J u n e 1978 LUNDGREN, P. AND L. A. CARR. k~[]'ect.~~[" anAomycin aml C..V3' ~timuhmt.~ on hram catecholamim" ~ynthe~i.~. PHARMAC. BIOCHEM. BEHAV. 9~4~ 55%561, 1978.--Mice were injected with anisomycin, an inhibitor of cerebnd protein synthesis; d-amphetamine, strychnine, or caffeine was administered 30 rain later. Fifteen rain before sacrifice at I or 2 hr after injection of anisomycin, :~H-tyrosine was injected intravenously, and catecholamine synthesis rates were estimated by measurement of the specific activity of ::H-tyrosine and the accumulation of 'H-norepinephrine and ~Hdopamine. Anisomycin decreased synthesis rates of catecholamines, but this effect was not significantly affected by any of the CNS stimulants. "[hese results suggest that the recently reported reversal of anisomycin-induced amnesia by these stimulants is not due to an attenuation of brain catecholamine synthesis inhibition. Anisomycin

Memory

Catecholamines

CNS stimulants . . .

IN S T U D I E S attempting to identify the neurochemical systems involved in storage and retrieval of memory, much attention has been focused on the effects of inhibitors of protein synthesis, particularly cycloheximide, acetoxycycIoheximide, and anisomycin (ANt). It has been shown that these drugs produce amnesia for a specific learning task when given shortly before or after training, and it is generally believed that this action is due to inhibition of cerebral protein synthesis. However, it has recently become apparent that these drugs have effects on central catecholaminecontaining neurons which may account for the disruption of long-term memory. For example, inhibitors of protein synthesis have been found to inhibit tyrosine hydroxylase 131 and to inhibit catecholamine synthesis 12,61. Furthermore, the amnesic effect of these agents may be prevented by drugs which would be expected to enhance catecholamine neuron function 18, 9. 10, 13]. Although these studies clearly show that treatments affecting catecholamine neuron function may prevent the amnesic effect of protein synthesis inhibitors, this may not necessarily be a conclusive argument that impairment of adrenergic function is the primary effect responsible for the amnesia produced by these drugs, since similar effects have been reported alter administration of CNS stimulants which may not act solely on catecholamine neurons. Flood and co-workers 151 have shown that strychnine, caffeine, nicotine, picrotoxin, and d-amphetamine protect against amnesia induced by AN1,

whereas chloral hydrate and sodium phenobarbital enhanced the amnesia. Since these treatments would not be expected to affect any single neurotransmitter system in particular, it was concluded that modulation of CNS arousal by these drugs is the mechanism by which they affect memory. The purpose of the experiments reported here was to study the effects of anisomycin on catecholamine synthesis and to determine whether these effects are modified by a number of CNS stimulants. METHODS Animals used were male C57/BI,6 mice (Jackson [,abs) between 9 and 14 weeks old and weighing 18-29 g. '|'hey were housed in small groups of 5-6 animals and provided with water and Purina Rat Chow ad lib. All drugs were dissolved in 0.14 M NaCI buffered to pH 7 with 0.01 M sodium phosphate. Anisomycin (2-p-methoxyphenyl-3-acetoxy-4hydroxypyrrolidine) was a gift from Dr. Nathan Belcl-er of Pfizer Pharmaceuticals and was injected subeutaneousl.\. I_'~ mg/kg) on the back of the neck. Thirty min after ANI was administered, saline, strychnine sulfate (0.3 mg/kg, Merck), caffeine (anhydrous, 100 m,g/kg, Sigma Chemical Co.I, or d-amphetamine sulfate (5 mg/kg, Smith, Kline, and French, Inc.) was injected intraperitoneally. The injection volume for all drugs was 0.01 ml/g body weight. :~H-tyrosine (3,5-:'Htyrosine, specific activity 40-60 Ci/mmole, New England Nuclear) was evaporated to dryness t.,nder nitrogen and re-

'Supported by a grant (NS-12129) from the National Institute of Neurological and Communicative Disorders and Stroke.

C o p y r i g h t ' 1978 A N K H O

I n t e r n a t i o n a l lnc.--(X~91-3057/78/100559-0350(Lg0/0

LUNDGREN ANDCARR

56O 1 ABLE I

TABI.E 3

t'~It"FEC'I'S OF ANISOMYCIN AND STIMUI.ANTS ON ENIX)GENOUS TYROSINE CONCENTRA'IION

E F F E C T S Or: ANISOMYCIN AND S'I'IMUI,ANTS C'N DOPAMINE SYNTHESIS

I Saline ANI.Saline ANt{D-AMP ANI4("AFF ANI~-,~'I'R

33.1 54.7 54.4 56.2 48.5

hour

2

28.4 50.6 50.8 47.6 60.6

' 0.8(4)*

, 1.9(4)4. , 4.1(4)+ * 1.1(4) + ~- 4.5(4) +

hour', : 1.7(4) • 2.1(4)+ * 3.5(5)t .+_ 1.4(4)+ * 8.2(4) +

*p,g t y r o s i n e / g brain t i s s u e -" S E M . N u m b e r o f a n i m a l s it s h o w n in p a r e n t h e s e ' < S t i m u l a n t s w e r e i n j e c t e d 30 m i n a r i e l A N I . and animails w e r e s a c r i f i c e d at 1 or 2 hr after A N I . Drug d o s e s w e r e as t b l l o w s : A N I - 2 5 mg,'kg, D-AMP- 5 mg.'kg. S T R - 0 . 3 m g / k g . C A F F I(R) mg.kg. ~ S i g n i ~ c a n l l y g r e a t e r t h a n c o n t r o l p. 0.01 TABLE

2

I:,FI:I{("IS ()t: ANISOMYCIN AND STIMUI,ANTS ON NOREPINEPHRINE SY Nrl.t ESIS

1 hour

2 hours

Saline

0 . 0 3 0 -'- 0.(IO5(4)*

0.034

-

AN I • Salirm

0 . 0 1 7 - O.(R)3(4F{

0.012

• ().(R)214):i:

ANI 4 I)-AMP

t).OlO _-t 0.001(4)!:

O.(X)6 " ().(R)l(5):i:

ANI

- CAFF

(I.014

':- 0.002(4F!.

0.011

ANI. S'FR

0.018

,

0.010--0.(X)2(41:!:

Refer to legend of lahlc

I

0.004(4)~

0.007141

• 0.(RH(4):!:

I hour

2 hours

Saline ANI-Saline

0.026

* 0.0,tl4(4)*

0.040

~ 0.007(4)

0.012

• 0.002(4)+

0.(113

. 0.00214P

ANI.D-AMP

0.010

, 0.(IO2(4)+

0.OII

, 0.001(4)+

ANI-CAFF

0.013

• 0.003(4)t

0.013

•

ANI-STR

0.012

* 0.(X)2(4)+

0.013

"_ 0.(X)2(4l +

0.(X)2(4) +

Refer to legend of Table I for detaib,. * /zg/g brain tissue:15 rain .-_ SEM. ~ Significantly decrea,,ed from cor~trol p. 0.t)l

addition of stimulanls did not aller the effect of AN! on tyrosine. The specific aclivily of :~H-tyrosine was increased by all drug trealments at 2 hr due to a greater relative increase in :~H-tyrosine concentration than in e n d o g e n o u s levels of tyrosine. ANI decreased the synthesis rates of norepinephrine and dopamine at both time intervals studied (Tables 2 and 3). ANI and d-amphetamine seemed to have a greater effect on norepinephrine synthesis than AN! alone, hut statistical analysis of these 2 treatments did not reveal a significant difference. N o n e of the stimulants used in this study significantly modified lhe inhibition of c a l e c h o l a m i n e synthesis by ANI.

for details. ~L~Cus~io~

:~: ,u,g,'g brain tissue,15 rain • S E M .

: Significantly decreased from control :i: Significan0y decreased from control

p. p.

0.05 0.01

dissolved in phosphate buffered saline to a concentration of 5(X) /zCi/ml. Each mouse received {).2 ml (100 /.tCi) of this solution intravenously via the latend tail vein either 45 or 105 rain after the injection of ANI and sacrificed 15 rain later by cervical dislocation. Immediately after sacrifice, each mouse was decapitated, the bndn was r e m o v e d , and the cerebellum, olfactory bulbs, and lower brain stem were dissected off. The remaining tissue was weighed and homogenized in 3 ml of ice-cold 0.4 N HC10,, and the homogenate was centrifuged for 5 rain at IO,0{R) X g. The supernalant was then decanted and the pellet rehomogenized and centrifuged as before, and the 2 supernatants were combined and frozen until assayed. E n d o g e n o u s tyrosine was measured fluorometrically by the method of Waalkes and Udenfriend [I 7]. The concentralions of :H-tyrosine, ::H-norepinephrine, and :~H-dopamine were measured in the brain h o m o g e n a t e s by methods previously reported [ 16]. The rates of catecholamine synthesis were calculated by dividing the brain concentration of :{Hcatecholamine by the specific activity of tyrosine [20]. Data were analyzed by' a one-way A N O V A across each time level, and where c o m p a r i s o n of individual means to one another was desired, this was done by the l)uncan Studentized range test [19]. RES;UI.TS

Administration of ANI alone increased the bndn concentration of e n d o g e n o u s tyrosine at I and 2 hr lTable I). The

The effects of A N I on endogenous tyrosinc levels in the brain :ire in agreemenl with olher studies [21 and are probably due Io a reduction of tyrosine incorporation inlo protein. In preliminary studies, i~ was tbund that lhe dose of AN I u~ed in the pre~ent report inhibited protein synthesN by ~ ; ~ al ~/z hr, 8~:~ at 1 hr, and 85(~ at 2 hr. Spanis and Squire 114] have shown thal lhe increase in e n d o g e n o u s tyrosine levels alone cannot accounl for the amnesia resulting from administration of protein synthesis inhibitors. It is also apparent from the presenl results that Ire reported protective effects of C N S stimulanls on amnesia induced by ANI [4,5] are not due to an alteralion of endogenous tyrosine levels. AN1 inhibiled catecholamine synthesis for at least 2 hr, a~ was also reporled by Flcxner and G o o d m a n [2 ~. Considering the reports implicating brain catecholamine neuron systems in m e m o r y formation [ I, 8, I I l, it is likely that inhibition of catecholamine synthesis may be at leasl pa~ly responsible for the amnesia caused by ANI. A conlrasling view has been proposed by Squire el a/.. ] 15], who reporled lhal a dose of a-methyl-p-tyrosine I A M F F ) thal inhibited tyrosine hydroxylase to the same degree as AN! had no amnesic eft~ct. Since ~hese authors ~11 thal it was unlikely that inhibition of catecholamine synthesi~ by ANI was responsible for its amnesic effect, this action was a t m b u t e d to inhibition of protein synthesis. H o w e v e r , other sludies argue against this mechanism since drugs which p r e v e m or attenuate the amnesia do nol aller lhc degree of protein ~ynlhesis inhibition [51. It is also possible that the inhibition of c a t e c h o l a m i n e synthesis by ANI is an indirect result of an action on some olher dynamic aspecl of calecholamine neuron function. For example, if ANI interferes with neuronal release of

ANISOMYCIN,

CNS STIMULANTS

AND BRAIN AMINES

c a t e c h o l a m i n e s , as s u g g e s t e d by F l e x n e r a n d G o o d m a n 12], t h e n r e d u c t i o n o f c a t e c h o l a m i n e s y n t h e s i s r a t e s by A N I m a y be due to f e e d b a c k inhibition o f t y r o s i n e h y d r o x y l a s e by i n c r e a s e d i n t r a n e u r o n a l levels o f c a t e c h o l a m i n e s 1181. If this is the case, t h e n the effects o f A M P T on c a t e c h o l a m i n e n e u r o n s y s t e m s would not be a n a l o g o u s to t h o s e of A N I , so the c o n c l u s i o n r e a c h e d by S q u i r e et a l . . m a y be in d o u b t . T h i s p r o p o s e d f e e d b a c k m e c h a n i s m is also s u p p o r t e d by the o b s e r v a t i o n in the p r e s e n t s t u d y that the inhibition of c a t e c h o l a m i n e s y n t h e s i s by A N I s e e m s to be p o t e n t i a t e d by d - a m p h e t a m i n e . T h i s d o s e o f d - a m p h e t a m i n e h a s b e e n rep o r t e d to inhibit c a t e c h o l a m i n e s y n t h e s i s [12h p r e s u m a b l y due to f e e d b a c k inhibition o f t y r o s i n e h y d r o x y l a s e as a result o f d e c r e a s e d i n t r a n e u r o n a l binding o f c a t e c h o l a m i n e s . If A N I c a u s e s a d e c r e a s e d release o f c a t e c h o l a m i n e s , t h e n the inhibition o f c a t e c h o l a m i n e s y n t h e s i s ~ffter A N I m a y be p o t e n t i a t e d a f t e r d - a m p h e t a m i n e by a f u r t h e r i n c r e a s e in i n t r a n e u r o n a l c a t e c h o l a m i n e levels.

561

T h e s t i m u l a n t s used in this s t u d y did not p r e v e n t the inhibition of c a t e c h o l a m i n e s y n t h e s i s by ANI. T h e r e f o r e , reversal o f A N l - i n d u c e d a m n e s i a by t h e s e d r u g s is a p p a r e n t l y not due to a direct effect on c a t e c h o l a m i n e s y n t h e s i s . Howe v e r , this finding d o e s not n e c e s s a r i l y w e a k e n the h y p o t h e s i s that p r o d u c t i o n o f a m n e s i a by A N I is due, in part, to effects on c a t e c h o l a m i n e n e u r o n s y s t e m s . It is possible that the s t i m u l a n t s used in this s t u d y c o m p e n s a t e for i m p a i r m e n t o f a d r e n e r g i c f u n c t i o n by a c t i o n s on o t h e r n e u r o n a l s y s t e m s . Flood et , I . . 141 h a v e p r e s e n t e d e v i d e n c e thai r e s t o r a t i o n o f n o r m a l m e m o r y f u n c t i o n by a d m i n i s t r a t i o n o f s t i m u l a n t s a f t e r A N I is due to i n c r e a s e d C N S arousal. In normal animals, c a t e c h o l a m i n e n e u r o n s y s t e m s may m o d u l a t e levels o f arousal as part of the m e m o r y storage p r o c e s s 171, so p e r h a p s the general C N S s t i m u l a n t s f o u n d to p r e v e n t a m n e s i a comp e n s a t e for lowered levels of arousal c a u s e d by i m p a i r m e n t of a d r e n e r g i c s y s t e m s by inhibilors o f protein s y n t h e s i s .

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I0. Quinton, E. E. and A. S. Bloom. Effects old-amphetamine lind strychnine on cycloheximide- and diethyldithiocarbamateinduced amnesia in mice. J. ((,rap. physiol. Psych,/. 91: 139(~ 1397. 1977. I I. Randt, C. T., D. Qua~ermain, M. Goldstein and B. Anagnoste. Norepinephfine biosynthesis inhibition: Effecls on memory in mice. Science 172: 4 9 ~ , 1971. 12. Riffee, W. H. and M. C. Gerald. Determinution of endogcnoux concentrations of norepinephfine and dopamine and their rates of synthesis in a single mouse brain: Biphusic effects of ~ • ~amphetamine. Arch,. int. I'harmacodvn. ltu'r. 219: 7~78, 1976. 13. Scrota, R. G., R. B. Roberts and L. B. Flexner. Acetoxycycloheximide-induced trunsient amnesiu: Protective effects of udrenergic stimulants. I%~c. IHIIlI. A('tld. ,~'ci. I.l..~.a. 69: 3 ~ 3 4 2 , 1972. 14. Spanis, C. W. and I,. R. Squire. Elevation of brain tyrosinc b~ inhibitors of protein synthesis is not responsible for their amnesic effect. Brain Re.~. 139: 38~388. 1977. 15. Squire. L. R., R. Kuczenski and S. H. Barondes. Tyrosine hydroxylasc inhibition by cycloheximide and anisomycin is not responsible for their anmesic effect. Ilrai, R~'~. 82: 241-248. 1974. 16. Voogt, J. L. and L. A. Carr. Plusma prolactin levels and hypothalamic catecholamine synthesis during suckling. ,Veuroendocrin,log.v 16:10~118, 1974. 17. Waalkes, T. P. und S. Udenfriend. Fluorometric method for estimation of lyrosine in plasmu und tissues. ,I. I , h c/in. ~h'd. 50: 733-736, 1957. 18. Weiner. N., F. C. Bove, R. Bjur. G. Cloutier and S. Z. l,angcr. Norepincph6ne biosynthesis in relationship to neural activalion. In: N e w ('ollc~'pl,~ itt :%'~'ttrolrt~tt~nlitt¢'r R~',k,ulalion, edited by A. Mandell. New York: Plenum Press, 1973. pp. 8~113. 19. Winer, B. J. A'talAtical Principlc~ in E~pcrimcnlal I)~'~i.gn. New York: McGraw Hill Inc.. 1971• 20. Zschaek, 1,. 1,. and R. J. Wu~man. Bruin :H-calechol synthesis and the vaginal estrous cycle. ,Vctmwml~crinoh~.e~ I I: 14~ 149. 1973.