Age-related changes in the metabolism of neurotransmitters and the effect of scavengers: an in vivo microdialysis study

Arch. Gerontol. Geriatr. suppl. 4 (1994) 171-176 9 1994 Elsevier Science Ireland Ltd. All rights reserved. 0167-4943/94/$07.00

171

AGE-RELATED CHANGES IN THE METABOLISM OF NEUROTRANSMITTERS AND THE EFFECT OF SCAVENGERS: AN IN VIVO M I C R O D I A L Y S I S STUDY

M. N A K A N O , E. HITOMI and T .

MIZUNO

I n s t i t u t e for Medical Science of A g i n g , A i c h i - k e n , 480-11, Japan

Aichi Medical U n i v e r s i t y ,

Nagakute-cho,

SUMMARY Brain microdialysis is a method to s t u d y the in vivo release and metabolism of n e u r o t r a n s m i t t e r s using f r e e l y - m o v i n g animals. We studied the changes in n e u r o t r a n s m i t t e r metabolism from v e r y y o u n g to tile old rats ( S p r a g u e - D a w l e y ) . For brain microdialysis, a guide cannula was implanted in the striatum (A + I . 0 , L +2.5, V - 4 . 0 ) . E x t r a c e l l u l a r c o n t e n t of n e u r o t r a n s m i t t e r s and t h e i r metabolites were detected with an electrochemical detector (ECD-100, EICOM C o . ) . The cont e n t was affected by KCI (I00 mM), calcium-free Ringer and t e t r o d o t o x i n in a p e r f u s i o n medium. Dopamine and its metabolites such as d i h y d r o x y p h e n y l a c e t i c acid and homovanillic acid were s i g n i f i c a n t l y changed with age. The c o n t e n t of these metabolites were maximal around 2-3 months of age. In the case of serotonin metabolism, statisticaly no s i g n i f i c a n t change was o b s e r v e d in the e x t r a cellular content of 5 - h y d r o x y i n d o l e a c e t i c acid a f t e r sexual maturation (I .5 months of age). Potassium-induced dopamine release which is newly s y n t h e s i z e d one, was maximum around 1.5 years of age and the content was about 50 % lower in the old. The basal level of dopamine s i g n i f i c a n t l y decreased with age. M o r e o v e r , the i n h i b i t o r y e f f e c t of p a r g y l i n e , a monoamine oxidase i n h i b i t o r , was also decreased with age. On the basis of these r e s u l t s , it is suggested that the s y n a p t i c function shows postnatal development and reaches maximum at sexual maturation. Microdialysis could be a useful p r o c e d u r e for s t u d y i n g the in v i v o metabolism of n e u r o t r a n s m i t t e r s . Keywords: pargyline

brain microdialysis,

neurotransmitters,

monoamine oxidase, dopamine,

INTRODUCTION T h e r e are s i g n i f i c a n t during

aging.

changes

These a g e - r e l a t e d

in the system of striatal alterations,

species of animals and the conditions

however,

neurotransmitters

are d e p e n d e n t

of the e x p e r i m e n t s .

Brain

on the

catecholamine

levels and synthesis have been found to be decreased in s t r i a t u m ,

brain stem,

and hypothalamus of old animals (Rouben et a l . ,

1986; Morgan and May, 1990).

So f a r , age-related changes in n e u r o t r a n s m i t t e r s

were usually examined with the

slices of b r a i n .

However, this method could not d e t e c t the dynamic changes of

neurotransmitters

in the b r a i n .

Thus,

we used an in v i v o

for measuring the metabolism of n e u r o t r a n s m i t t e r s Brain

microdialysis

has developed

extracellular

fluid of the brain

Benveniste,

1989).

the

perfusion

for

through

as a method

in e x t r a c e l l u l a r

dialysis

probes

microdialysis

in striatum with age.

neurotransmitters

Neurotransmitters

medium

rapidly

brain for

sampling

(Westerink, fluid

implanted

et a l . ,

the 1987;

were collected into

the

striatum

into or

o t h e r b r a i n regions. It seems celar that dopamine (DA) in n e r v e terminals

(Groppetti

et a l . ,

is stored

in at least two compartments

1977; Raiteri et a l . ,

1979).

Nurotransmit-

172 ter

release from central

agents, synaptic

receptors.

veratrine,

by various

t h e r et al.

pharmacological

on the mechanism of release or i n d i r e c t l y

Several agents such as potassium,

tyramine,

and ouabain cause dopamine release ( H e r d o n ,

1988; A r b u t h n o t t , stores,

neurons can be influenced

which act d i r e c t l y

1990; F a i r b r o t h e r et a l . ,

via p r e -

amphetamine,

1985, B u t c h e r

1990; Dluzen et a l . ,

et a l . ,

1991). F a i r b r o -

(1990) r e p o r t e d that tyramine induced a release of DA from vascular

whereas the e f f e c t of potassium

t h e y suggested that e x t r a c e l l u l a r

involved newly s y n t h e s i z e d

3,4-dihydrosyphenylacetic

acid

DA.

Thus,

(DOPAC)

was

d e r i v e d from a newly s y n t h e s i z e d pool(s) of DA. It is important

to s t u d y

the a g e - r e l a t e d changes in the magnitude of DA

release using release promoting a g e n t , and a g e - r e l a t e d changes in the alteration of DA metabolism. rotransmitters

Little is known about the c o n t e n t and the metabolism of n e u -

from v e r y young to old animals.

b r a i n microdialysis

Thus,

we employed the in v i v o

in f r e e l y moving animals to measure the a g e - r e l a t e d changes

in the metabolism of DA and the magnitude of the potassium-evoked DA release. Rooibos tea

{Aspalathus

linearis)

is o r i g i n a t e d

in

South

Africa.

The tea

e x t r a c t contains some flavonoids,

and volatile components such as guaiacol and

geranylacetone,

Rooibos tea shows high

but

no caffeine.

antioxidant

activity.

Neuronal f u n c t i o n was affected by a n t i o x i d a n t s / s c a v e n g e r s . We studied a g e - r e l a t e d changes and the effect of Rooibos tea e x t r a c t s the metabolism of n e u r o t r a n s m i t t e r s microdialysis

with f r e e l y - m o v i n g

on

from v e r y young to the old rats using b r a i n

animals.

Preliminary

results have been r e p o r t e d

eleswhere (Nakano and Mizuno, 1992}. ~,~ATERIALS AND METHODS Animals. Male S p r a g u e - D a w l e y rats of various ages from weanling were obtained from Japan SLC, Inc. (Hamamatsu, J a p a n ) . The animals were k e p t with standard commercial food and water ad libitum u n d e r a l i g h t - d a r k cycle of 12 h o u r s ( l i g h t at 7:00 a . m . } . E x p e r i m e n t s were started between 9:00-10:00 a.m. The animal experiments were c o n d u c t e d with the line of the g u i d e l i n e for animal e x p e r i m e n t s , Aichi Medical U n i v e r s i t y . Microdialysis. Rats were anesthesized with sodium p e n t o b a r b i t a l (Nembutal; 50 m g / k g body weight) and placed in a stereotaxic a p p a r a t u s . The skull was e x p o s e d , and a small hole was d r i l l e d to allow implantation of a g u i d e cannula in the s t r i a t u m . Stereotaxic coordinates from bregma were A +1.0, L +2.5, and V - 4 . 0 ( d u r a ) according to the atlas of Paxinos and Watson (1986). The g u i d e cannula was secured to the skull using skull screw and dental cement. Microdialysis probes ( B D P - I - 8 - 0 3 ; molecular c u t o f f 50,000; EICOM C o . , Kyoto, Japan) were designated to e x t e n d 3 rnm beyond the tip of the guide cannula. HPLC analysis. Brain microdialysis and HPLC analysis were performed with a f u l l y automated system. E x t r a c e l l u l a r contents of n e u r o t r a n s m i t t e r s and its metabolites were detected with electrochemical detector (ECD-100, EICOM C o . , K y o t o ) . The dialysis probe was p e r f u s e d with a Ringer solution {NaCI 148 raM, KCI 4.0 mM, CaCI 2 2.3 mM) at a constant flow rate of 2 l~l/min by means of a high precision s y r i n g e pump. When calcium-free Ringer solution or high potassium Ringer solution was used, CaCI 2 or KCI was replaced by NaCI. Perfusions

173 were s t a r t e d 20-24 hrs a f t e r operation. The column used was MA5ODS (4.6 x 150 mm), and the mobile phase was 0.1 M sodium c i t r a t e - a c e t a t e b u f f e r (pH 3.5) containing 0.1 mM EDTA and 17 % methanol. The dialysates were collected for 30 min i n t e r v a l s , and was d i e r e c t l y appl~ed on the HPLC-ECD a p p a r a t u s . Preparation of Rooibos tea e x t r a c t . Two g of dried tea leaf was boiled with 250 ml of water for 15 min and designated as a tea e x t r a c t . The tea e x t r a c t (0.1 mll100 gr body weight) was administered i n t r a p e r i t o n e a l l y . HPLC f r a c t i o n a t i o n of the tea e x t r a c t . Waters HPLC system was used for the analysis of tea e x t r a c t . Column used was micro Bondasphere C18 (3.9 x 150 ram). The mobile phase was 8 % to 40 % a c e t n i t r i l e in 2 % acetic acid in a lenear g r a d i e n t . Flow rate was 2.5 m l l m i n , and column eluate detected at 280 nm using UV detector. Autooxidation of linoleic acid in water-alchohol system. A u t o o x i d a t i o n of linoleic acid in water-alchohol solution has been measured by the thiocyanate and TBA methods (Osawa and Namiki, 1985). Each sample was added into a m i x t u r e of linoleic a c i d - e t h a n o l - 0 . 2 M phosphate b u f f e r (pH 7 . 0 ) . The m i x u t r e was incubated at 40 C and the p e r o x i d e value was determined at 500 nm a f t e r color reaction with FeCI 3 and ammonium thiocyanate. The formation of TBA reacting substances was measured at 532 nm a f t e r the reaction with t h i o b a r b i t u r i c acid. RESULTS AND DISCUSSION Age changes in dopamine metabolism. Metabolites of dopamine such as DOPAC and

homovanillic acid

(HVA)

in the

perfusates

showed maximum at 2-3-

month-old r a t s , t h e r e a f t e r the content decreased with age. Serotonin metabolite, 5-hydroxyindoleacetic

acid

(5-HIAA)

increased

to

a

statistically

significant

e x t e n t when compared to I month of age (immature a n i m a l s ) , b u t a f t e r the mat u r a t i o n no s t a t i s t i c a l l y s i g n i f i c a n t change was o b s e r v e d . As can be seen in F i g u r e high

concentration

of

KCI.

I,

dopamine content

increased

Potassium-evoked dopamine

(DA)

e x t r e m e l y with release and

basal level of DA were maximum around

1.5 months of age,

lower levels were seen in the old ages.

The ratio of DA content in high

solution

to the content

Furthermore,

in

Ringer

solution

(KCl/Ringer)

i n h i b i t o r y e f f e c t of monoamine oxidase

and s i g n i f i c a n t l y

decreased w i t h

(MAO)

the

KCI age.

a c t i v i t y showed a

maximum around 1.5 months of age. Present s t u d y demonstrates a g e - r e l a t e d changes in s t r i a t a l DA metabolism, which is determined by measuring DA, DOPAC and HVA using m i c r o d i a l y s i s with f r e e l y moving r a t s .

Postnatal ontogenic development of dopaminergic neuron has

been reported to be adult level around one month a f t e r b i r t h . of

neurotransmitter

shown in Figure I , around

metabolites

reflects

a

functional

status

The concentration of

neurons.

As

the magnitude of potassium-induced DA release was maximum

1.5 month of age, and then decreased with age.

Dopamine can be re-

leased from dopaminergic terminals via two mechanisms: c a r r i e r - d e p e n d e n t p r o cess which

is i n s e n s i t i v e to e x t r a c e l l u l a r calcium and c a r r i e r - i n d e p e n d e n t p r o -

cess which is dependent on e x t r a c e l l u l a r calcium. ported

that

potassium-induced

F a i r b r o t h e r et al.

(1990) r e -

DA release involves a c a r r i e r - i n d e p e n d e n t ,

and

174 2000

O W

~'

1000

l0 0

1

1.5

2.5

6

.

18

22

Age (Months)

F i g u r e I . A g e - c h a n g e s in the magnitude of potassium-evoked release of dopamine. DA c o n t e n t in the case of high KCI (100 mM) ( - o - ) and in R i n g e r solution (~). Bars show + S . D . One or two asterisks indicate s i g n i f i c a n t d i f f e r e n c e s at p < 0.05 or 0 . 0 1 , - r e s p e c t i v e l y . this process involves newly s y n t h e s i z e d DA. Striatal DA synthesis from t y r o s i n e is decreased

in aged rats

(Ponzio et a l . ,

duced t y r o s i n e h y d r o x y l a s e a c t i v i t y Therefore,

it is i n f e r r e d

1978).

T h i s could connect with

(McGeer et a l . ,

1971; A l g e r i et a l . ,

level of newly s y n t h e s i z e d

Sexual maturation of rats is a r o u n d 1.5 month of age (Nakano, 1990). T h u s ,

ration.

1977).

that decrease in the magnitude of potassium-induced

DA release is due to the decreased al.,

re-

DA with

age.

1991; Nakano et

the s y n t h e t i c a c t i v i t y of DA is maximum a r o u n d sexual matu-

From these r e s u l t s ,

it is suggested that neuronal f u n c t i o n

of dopamin-

e r g i c neurons is maximum around 1.5 to 2 months of age, and that s e r o t o n e r g i c system appears to be stable a f t e r sexual maturation. Effects of a n t i o x i d a n t s / s c a v e n g e r s fects on the n e u r o t r a n s m i t t e r activity

and

scavanger

of plant o r i g i n .

metabolism.

activity

of

At first,

Rooibos

tea.

We examined

these e f -

we checked the a n t i o x i d a n t

Antioxidant

activity

was mea-

s u r e d by the isothiocyanate and T B A methods using linoleic acid as the lipid for autooxidation.

The e x t r a c t shows high a n t i o x i d a n t a c t i v i t y

We examined the e f f e c t of c r u d e transmitters. ficantly weight).

tea e x t r a c t on the metabolism of n e u r o -

The e x t r a c e l l u l a r c o n t e n t of DOPAC, HVA and 5 - H I A A were s i g n i -

changed

with

the

i.p.

injection

of

As shown in Figure 2A and 2B,

metabolism

(data not s h o w n ) .

was s i g n i f i c a n t l y

jection of the e x t r a c t ,

suppressed

a significant

tea e x t r a c t s

respectively,

(0.1

Shortly

increase was observed

e x t r a c e l l u l a r c o n t e n t of n e u r o t r a n s m i t t e r

g

body

dopamine and serotonin

by tea e x t r a c t s .

c o n t e n t of DOPAC and 5 - H I A A in old rats.

ml/100 after

the in-

in an e x t r a c e l l u l a r

However, a g e - r e l a t e d changes in the

metabolites were not o b s e r v e d at more

than 10 hours a f t e r the injection of tea e x t r a c t s . As described tea e x t r a c t .

Thus,

above,

metabolism of n e u r o t r a n s m i t t e r s

it is important

was affected

by

the

to determine what active substances change

175 4

=

s

.B

3

14 2

2 2

12

22

2

12

Age (months)

Age

22

(months)

F i g u r e 2. Effect of Rooibos tea e x t r a c t on n e u r o t r a n s m i t t e r metabolism. A" DOPAC c o n t e n t , B: 5 - H I A A c o n t e n t . Control ( w i t h o u t tea e x t r a c t : 1 ) , three hours ( ~ ) and 12 hours (x~,~) a f t e r the i . p . injection of the tea e x t r a c t . Bars show + S . D . One or two a s t e r i s k s indicate s i g n i f i c a n t d i f f e r e n c e s at p < 0.05 or 0.01, "~espectively.

8 o

3

F i g u r e 3. HPLC p a t t e r n of the f r a c t i o n associated with ConA-Sepharose column. the metabolism of n e u r o t r a n s m i t t e r s .

The c r u d e tea e x t r a c t contains sugars and

peptides ( L o w r y - p o s i t i v e s u b s t a n c e s ) .

In the e x t r a c t t h e r e are s u g a r - c o n t a i n i n g

substances which pass t h r o u g h a v i s k i n g t u b e . The s u g a r - c o n t a i n i n g

substances

(low molecular w e i g h t ) were f r a c t i o n a t e d with Con A - s e p h a r o s e column chromatography

(Nakano et a l . ,

1992). Con A - s e p h a r o s e associated substances were f u r -

t h e r fractionated w i t h HPLC. The IIPLC p a t t e r n of Con A-associated substances showed t h r e e major peaks and t h r e e minor peaks. These peaks contain o r i e n t i n , homo-orientin and u n k n o w n substances ( F i g u r e 3). activity

of various

fractions

of the e x t r a c t s .

a n t i o x i d a n t and scavenge a c t i v i t y .

We examined the a n t i o x i d a n t

Two u n k n o w n

From these r e s u l t s ,

peaks show

Rooibos tea e x t r a c t could

contain a n t i o x i d a n t substances which are small molecules, substances.

major

and s u g a r - c o n t a i n i n g

It is now in p r o g r e s s for d e t e r m i n i n g a chemical s t r u c t u r e of active

substances and also physiological f u n c t i o n of the substances.

176 REFERENCES A r b u t h n o t t , G.W., F a i r b r o t h e r , I.S. and B u t c h e r , S.P. (1990): Brain microdialysis studies on the control of dopamine release and metabolism in vivo. J. Neurosci. Methods, 34, 73-81. A l g e r i , S., Bonati, M., Brunello, N. and Ponzio, F. (1977): D i h y d r o p t e r i n e reductase and t y r o s i n e h y d r o x y l a s e activities in rat brain d u r i n g development and senescence: a comparative s t u d y . Brain Res., 132, 569-574. Benveniste, 11. (1989): Brain microdialysis. J. Neurochem., 52, 1667-1679. B u t c h e r , S . P . , F a i r a b r o t h e r , I . S . , Kelly, J . S . , A r b u t h n o t t , G.W. (1988): Amphetamine-induced dopamine release in the rat striatum: An in vivo microdia!ysis s t u d y . J. Neurochem., 50, 346-355. Dluzen, D . E . , McDoermott, J . L . and Ramirez, V . D . (1991): Changes in dopamine release in v i t r o from the corpus striatum of young versus aged rats as a function of infusion modes of L-DOPA, potassium, and amphetamine. Expt. Neurol., 112, 153-169. F a i r b r o t h e r , I . S . , A r b u t h n o t t , G.W., Kelly, J.S. and Butcher, S.P. (1990): In vivo mechanisms u n d e r l y i n g dopamine release from rat n i g r o s t r i a t a l t e r minals: Studies using potassium and tyramine. J. Neurochem., 54, 18441851. Gropetti, A . , Algeri, S., Cattabeni, F., Di Giulio, A . , Gall, C . L . , Ponzio, F. and Spano, P.F. (1977): Changes in specific a c t i v i t y of dopamine metabolites as evidence of multiple compartmentation of dopamine in striatal neurons. J. Neurochem., 28, 193-197. Herdon, H . , S t r u p i s h , J. and Nahorski, S.R. (1985): Differences between the release of radiolabelled and endogeneous dopamine from superfused rat brain slices: Effects of depolarizing stimuli, amphetamine and synthesis i n h i b i t i o n . Brain Res., 348, 309-320: McGeer, E . G . , Fibiger, H . C . , McGeer, P.L. and Wickson, V. (1971): Aging and brain enzymes. Exp. G e r o n t . , 6, 391-396. Morgan, D.G. and r•ay, P.C. (1990): Age-related changes in synaptic neurochemistry. In: Handbook of the Biology of A g i n g , 3rd ed. pp. 219-254. Editors: E.L. Schneider and J.W. Rowe, Academic Press, New Y o r k . Nakano, M. (1991): Beginning and progress of aging: is there an aging gene? Seikagaku, 63, 359-362. (in Japanese). Nakano, M. and Mizuno, T. (1992): Age-related changes in n e u r o t r a n s m i t t e r s : Analysis of in vivo brain microdialysis. Biomed. Gerontol., 16, 152-153. Nakano, M., Mizuno, T. and Gotoh, S. (1990): Accumulation of cardiac lipofuscin in mammals: Correlation between sexual maturation and the f i r s t appearance of lipofuscin. Mech. Ageing D e v . , 52, 93-106. Nakano, M., Mizuno, T . , Kator, K. and Shibata, Y. (1992): Alteration of brain metabolism with the e x t r a c t of Rooibos tea (Asparathus l i n e a r i s ) . B i r y o Eiyoso-Kenkyu, 9, 53-56 (in Japanese). Osawa, T. and Namiki, M. (1985): Natural antioxidants isolated from Eucalyptus leaf waxes. J. A g r i c . Food Chem., 33, 777-780. Paxinos, G. and Watson, C. (1986): The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, New Y o r k . Ponzio, F., Brunello, N. and A l g e r i , S. (1978): Catecholamine synthesis in brain of aging rat. J. Neurochem., 30, 1617-1620. Raiteri, M., Cerrito, F., Cervoni, A.M. and Levi, G. (1979): Dopamine can be released by two mechanisms d i f f e r e n t i a l l y affected by the dopamine t r a n s port i n h i b i t o r nomifensine. J. Pharmacol. Exp. T h e r . , 208, 195-202. Roubein, I . F . , Embree, L.J. and Jackson, D.W. (1986): Changes in catecholamine levels in discrete regions of rat brain d u r i n g aging. Exp. Aging Res., 12, 193-196. Westrink, B . H . C . , Damsma, G . , Rollema, H . , DeVries, J . B . and Horn, A . S . (1987): Scope and limitations of in vivo brain dialysis: A comparison of its application to various neurotransmitter systems. Life Sci., 41, 1763-1776.