Lifetime brain serotonin: Regional effects of age and precursor availability

Neurobiology ¢~f'A~.,in~,,,Vol. 5, pp. 23,5--242,1984. ~ Ankho InternationalInc. Printed in the U.S.A.

0197-4580/84$3.00 + .00

Lifetime Brain Serotonin: Regional Effects of Age and Precursor Availability P. S. T I M I R A S , D. B. H U D S O N A N D P. E. S E G A L L D e p a r t m e n t Physiology-Anatomy, University o f Calif'ornia, Berkeley, CA 94720 R e c e i v e d 20 M a y 1983 TIM1RAS, P. S., D. B. HUDSON AND P. E. SEGALL. Lifetime brain serotonin: Regional eff'eets ~l'age and precursor availability. NEUROBIOL AGING 5(3) 235-242, 1984.--In the rat, regional brain serotonin levels which do not change from 2-30 months of age are increased at 36 months. Corresponding catecholamine levels progressively decrease. Feeding a diet restricted in the amino acid tryptophan (the precursor of serotonin) from weaning to two years of age markedly reduces serotonin levels in all brain regions and lowers norepinephrine levels in the cerebral hemispheres. Regional activity of synthesizing (tyrosine and tryptophan hydroxylases) and catabolizing enzymes (MAO-A) does not change markedly with age or dietary manipulation except for sporadic increases in tryosine hydroxylase activity in pair-fed animals. Returning the tryptophan-deficient animals to a normal diet produces a certain degree of rehabilitation the effectiveness of which varies with the function considered: Impaired brain serotonin levels recover moderately but remain lower than controls as late as 36 months, growth is never completely compensated, and norepinephrine levels show a rebound increase. Brain serotonin changes

Tryptophan-restriction

Aging

Brain monoamine imbalance with aging

precursors in the diet [3, 5, 11, 12, 39], we wanted to investigate whether lowering the dietary intake of the serotonin precursor, tryptophan, early during development and throughout adulthood might later influence the timetable of aging of serotonergic and, indirectly, catecholaminergic systems. Low levels of tryptophan in the diet caused voluntary restriction of food intake and reduced body growth, therefore we included an additional group of animals (pair-fed) fed a qualitatively complete diet but in reduced amounts to match those consumed under tryptophan restriction.

WITH aging the levels and metabolism of cholinergic and catecholaminergic systems in discrete brain regions decline progressively while the incidence and severity of certain neurologic and mental disorders increase (e.g., loss of striatal dopamine in Parkinson's disease and loss of cortical acetylcholine in Alzheimer's disease and some types of senile dementia) ]3, 11, 23]. In contrast, chemical and morphologic studies of the rat brain suggest that serotonin levels remain unchanged with normal aging [1, 8, 32-36]. Likewise, in humans, aging alone does not result in reduced brain serotonin levels and, in abnormal aging (e.g., Alzheimer's disease), serotonin receptors are decreased in younger but not in older patients in whom the prevalent loss is cholinergic [3,6]. While preweaning development of brain serotonin has been well described, little is known of its regional distribution and metabolism in maturity or with aging. The primary purpose of this study was to obtain a normal profile of brain serotonin throughout the lifespan. In our experiments, brain serotonin as well as norepinephrine and dopamine were measured in rats at progressive ages: 2 months, (upon achievement of sexual maturity), 4, 6, and 12 months (during adulthood) and 24, 30 and 36 months (progressively older ages). On the assumption that regional differences in the timetable of development may persist as well in aging, we compared regions with high densities of serotonergic cell bodies (e.g., pons-medulla) with those rich in serotonergic terminals (e.g., cerebral cortex), or areas involved with endocrine activity (e.g., hypothalamus). A secondary purpose was to study the effects of longterm dietary tryptophan restriction in developing, adult and senescent rats. On the premise that some neurotransmitter levels in the brain may be modified by manipulation of their

METHOD

Animals At 21 days of age 240 female Long-Evans rats were assigned to five groups: (1) control group fed Purina rat chow ad lib; (2) a group fed ad lib a specially formulated diet supplemented with an amount of tryptophan equivalent to the amount in Purina rat chow; (3 and 4) two groups (T30% and T40%) receiving ad lib the specially formulated diet containing progressively lower levels of tryptophan; and (5) a (pairfed) group fed Purina rat chow equal in weight to the food consumed by groups 3 and 4 (Table 1). Comparison of the effects of diet in control (1) (Purina) and control (2) (Teklad diet with normal tryptophan levels) shows no differences within any of the parameters considered. Therefore, all values from both groups were added in calculating the mean and standard error for control values. Water was available ad lib to all groups. Temperature was maintained at 22°C-+2 ° and lighting was on a 12/12 schedule. At 2, 4, 6, 12 and 24 months of age five animals from each group were sacrificed. At 24 months of age most

235

236

TIMIRAS, HUDSON ANI) SEGA[~[, TABLE 1 TRYPTOPHAN CONTENTOF DIETS AND AMOUNTSINGESTEDDAILY BY ADULT FEMALE RATS* Diet Group Designation

Tryptophan$ Content Daily food intake Daily tryptophan intake

T30%

T4W/?,

Control? (T 10~)

Pair-fed (T 1~ )

0.62 mg/g 5g 3. Img

0.81 mg/g 5g 4.05 mg

2.07 mg/g 15 g 31.05 mg

2.07 mg/g 5g 10.35 mg

*All experimental diets were prepared according to our instructions by Teklad Test Diets, Madison, WL They consisted: caseine hydrolysate acid (salt free) 150 g/kg; sucrose, 4 g/kg; corn oil, 50 g/kg; mineral mix, Jones Foster 50 g/kg; vitamin mix, Teklad, l0 g/kg. Tryptophan, an essential amino acid is particularly low in corn which was selected as a primary protein source. The progressive restoration of tryptophan was accomplished by an equivalent reduction in the amount of ground corn which varies from 722 g/kg in T30%, 721.85 g/kg in T40% and 720.5 g/kg in TI00% and the addition of tryptophan: 0.0 g/kg in T30%; 0.15 g/kg in T40% and 1.5 g/kg in T100%. The tryptophan content of the T100% diet was the same as that of Purina Rat Chow (Ralston Purina Co., St. Louis, MO.) ?Controls were fed ad lib either Purina Rat Chow or TI00% diet, the tryptophan content being equivalent. Pair-fed animals were fed Purina Chow in amounts similar to those consumed by the rats fed the tryptophan restricted diets. :~Tryptophan content of all diets was assayed microbiologically according to the method of Wooley and Sebral, J. Biol. Chem. 157:141, 1945 by Raltech Scientific Services, Division Ralston Purina Co.

tryptophan-restricted rats were returned to the control diet; surviving control and refed rats were also sacrificed at 30 and 36 months. Pair-fed rats were not studied beyond 24 months. Females were used because of our interest in reproduction, results of this part of the work have been reported elsewhere [30]. Data on a restricted number of 30--36 month animals are included to indicate the trend at these later ages, not for statistical purposes. Animals in all experiments were sacrif'med by decapitation, the brain was immediately placed on ice, and rapidly divided into five areas: cerebral hemispheres (minus olfactory lobes and caudate nucleus), caudate nucleus, hypothalamus, mesodiencephalon and pons-medulla.

Neurochemical Analyses The monoamines, serotonin, norepinephrine, and dopamine, were extracted and assayed by our modification of the method of Chang [9] utilizing half of each brain region, except the hypothalamus, where the entire tissue was used. Extraction. Tissue (280 mg or less) was sonicated in 2.8 ml acidified n-butanol (0.85 ml concentrated HCI/L) and centrifuged at 900 g for 15 minutes. The supernatant (2.5 ml) was extracted with 5 ml heptane and 1.2 ml glass-distilled water by shaking for 20 minutes. After centrifugation at 300 g for 10 minutes, the upper organic phase and tissue interface were aspirated and the water phase aliquotted for the assays. Blanks and standards of 25-100 ng of each monoamine were extracted as above in similiar volumes. Sample concentrations, in nanograms, were determined by comparison to these standards and calculated per milligram wet tissue weight. Serotonin. An aliquot (0.4 ml) of the water phase was reacted with 1.0 ml O-phthaldehyde (OPT, Regis Chemical Corp., 10 mg/100 ml 10 N HCI) and heated in water at 100°C

for 10 minutes. After cooling, the resulting fluorescence was read at 355/470 nm on a Perkin-Elmer spectrofluorometer Model MPF-44A. Norepinephrine-Dopamine. An aliquot (0.6 ml) of the water phase was added to 3 ml 2 N sodium acetate and 200 mg activated acid aluminum oxide IAcid Alumina, Bio-Rad), shaken for 20 minutes, centrifuged at 300 g for 10 minutes and the water phase aspirated. The alumina was washed two times with 5 ml water. The monoamines were eluted with 1.2 ml 0.1 M acetic acid. The eluate (1.0 rot) was adjusted to pH 6.5 with a sodium acetate-EDTA buffer and oxidized for exactly 2 minutes with an iodine-potassium iodide solution. The reaction was stopped with alkaline sulfite and the pH adjusted to 5.4 with glacial acetic acid. Samples were heated in water for 2 minutes at 100°C. cooled, and the norepinephrien fluorescence read at 385/485 nm. The samples were heated for an additional 10 minutes to develop the dopamine fluorescence which was read at 325/380 nm.

Enzyme Assays Enzyme assays were performed in duplicate immediately after the supernatant preparation: optimum conditions for enzyme reactions had been previously assessed. Tryptophan hydroxylase. Tryptophan hydroxylase was assayed by our modification of the method of Ichiyamaet al. [16]. Tissues were assayed fresh, immediately after sacrifice. A 10% homogenate was prepared in 10 mM tris acetate pH 7,4 buffer containing 1 mM DTT and centrifuged at 50,000 g for 30 minutes. Duplicate 500/zl aliquots of the supernatant were incubated at 37°C for 30 minutes with a substrate solution at pH 7.4 containing 1.4 mM L-(i-14C) tryptophan (58 mCi/nmole SPA., NEN) and 100 /xM L-tryptophan (final concentrationsL The reaction was stopped by the addition of 6 N PCA and the incubation, at 37°C. continued for 2 hours.

BRAIN SEROTONIN,

LOW TRYPTOPHAN

AND AGING

237

TABLE 2 REGIONAL MONOAMINE LEVELS IN ADULT AND AGED FEMALE RATS ON CONTROL DIETS*

Age

2 months

4 months

6 months

1 year

2 years

2.5 years

3 years

Serotonin (ng/g) Cerebral Hemispheres Corpus Striatum Hypothalamus Mesodiencephelon Pons Medulla

425 ± 922 1994 1193 1081

_+ _+ +_ _+

16+ 47 68 79 91

465 _+ 33 1179 1898 1202 1051

± ± ± ±

60 92 45 91

528 + 1003 1861 1194 1103

28

± 39 ± 117 + 53 ± 39

586 +

37

1378 ± 118 --~ 1245 + 31 1303 ± 56

522 ± 1083 1830 1402 1170

_+ _+ ± +

23 60 85 77 75

501 +

15

742

± + + ±

99 139 92 18

1866 2669 1779 1618

220 _+

24

181

1394 + 508 + 563 ±

113 15 109

1280 392 361

599 ±

I1

575

1039 1934 1163 1(/25

Norepinephrine (ng/g) Cerebral Hemispheres Hypothalamus Mesodiencephelon Pons Medulla

229 +

18

2395 +_ 107 539 + 37 447 + 38

238 _+ 16 1771 ± 440 ± 466 +

96 38 64

273 +

20

237 ±

20

242 _+ 11

1849 _+ 130 403 ± 45 373 ± 16

-533 ± 521 +

18 32

1429 ± 111 456 ± 18 487 ± 18

Dopamine (ng/g) Cerebral Hemispheres Corpus Striatum

674 +

41

837 ± 134

10520 + 414

13666 ± 871

806 +

98

12274 ± 954

421 ±

41

9235 + 633

468 ±

23

8892 ± 359

10928 _+ 1158

7984

*Diets (Purina and Teklad described in Table 1) contain normal amounts of tryptophan. +Average ±SE for 10 animals per group, sacrificed 1000-1200 hr. Two animals for 3-year-old group. :~Serotonin not measured in hypothalamus at one year of age.

T h e ~4CO~ e v o l v e d in the r e a c t i o n w a s t r a p p e d by a folded filter p a p e r e m b e d d e d with 200 ~1 H y a m i n e ( N E N ) in a plastic c e n t e r well. C e n t e r well w a s t r a n s f e r r e d to a scintillation vial c o n t a i n i n g 10 ml D i m i s c i n t ( N a t i o n a l Diagnostics). T h e s a m p l e s w e r e c o u n t e d for 10 m i n u t e s after d i s s i p a t i o n of the initial c h e m i l u m i n e s c e n c e effect. Tryosine hydroxylase. T y r o s i n e h y d r o x y l a s e w a s a s s a y e d a c c o r d i n g to the m e t h o d o f W a y m i r e et al. [37]. D u p l i c a t e 50 /zl a l i q u o t s o f a 10% h o m o g e n a t e in 0.32 M s u c r o s e w e r e a d d e d to a s u b s t r a t e at pH 7.4 c o n t a i n i n g a final c o n c e n t r a tion o f 20 n M L-(I-14C)-tyrosine (50 m C i / n m o l e S P A . , N E N ) a n d 80 n M L - t y r o s i n e . S a m p l e s w e r e i n c u b a t e d at 37°C for 20 m i n u t e s in a s h a k e r b a t h . T h e r e a c t i o n w a s s t o p p e d by the a d d i t i o n o f 10% T C A and the i n c u b a t i o n at 37°C w a s c o n t i n ued for two hours. T h e ~4CO~ d e v e l o p e d in the r e a c t i o n was t r a p p e d by a folded filter p a p e r c o n t a i n i n g 200/xl H y a m i n e I N E N ) in a plastic c e n t e r well. T h e well w a s t r a n s f e r r e d to a scintillation vial c o n t a i n i n g 10 ml D i m i s c i n t ( N a t i o n a l Diagnostics). T h e s a m p l e s w e r e c o u n t e d for 10 m i n u t e s after dissipation o f the initial c h e m i l u m i n e s c e n c e . Monoamine oxidase-A. M o n o a m i n e o x i d a s e - A was ass a y e d with o u r m o d i f i c a t i o n o f the m e t h o d of G a b a y et al. [13]. D u p l i c a t e 25/zl a l i q u o t s o f a 10% h o m o g e n a t e in 0.32 M s u c r o s e w e r e i n c u b a t e d at pH 7.4 at 37°C for 30 m i n u t e s with 4 n m o l e s 5 - h y d r o x y (side chain-2-14C) t r y p t a m i n e (50 mCi/nmole SpA, Amersham) and 36 nmoles 5 - h y d r o x y t r y p t a m i n e (final c o n c e n t r a t i o n s ) . T h e r e a c t i o n was s t o p p e d on ice w i t h cold 2 N HCI a n d the r a d i o a c t i v e p r o d u c t w a s e x t r a c t e d with 5 ml cold ethyl a c e t a t e . A 2 ml aliquot of the s u p e r n a t a n t was a d d e d to 10 ml A q u a s o l I N E N ) a n d c o u n t e d for 10 m i n u t e s in a scintillation c o u n t e r .

Protein Assay P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by the m e t h o d of L o w r y et al. [19]. A l i q u o t s c o n t a i n i n g 1-2 m g o f tissue w e r e assayed.

Statistics All results were a n a l y z e d b y o n e - w a y a n a l y s i s o f v a r i a n c e ( A N O V A ) followed by S t u d e n t ' s t test for b e t w e e n - g r o u p c o m p a r i s o n s . A N O V A statistics include c o n t r o l , T4W~, T30%, a n d pair-fed g r o u p s . RESULTS

Growth and Lff'espan A n i m a l s w e r e f o l l o w e d in t e r m s o f w h o l e b o d y a n d o r g a n w e i g h t s , r e p r o d u c t i v e ability, m o r b i d i t y a n d m o r t a l i t y t h r o u g h o u t the lifespan a n d m o s t of the d a t a c o l l e c t e d are available e l s e w h e r e [22, 27-30]. W e r e p o r t here briefly only o n b o d y weight, as a gross i n d i c a t i o n o f h e a l t h , a n d o n mortality, as a guideline for lifespan d u r a t i o n . B o d y w e i g h t s o f c o n t r o l s (either fed P u r i n a rat c h o w o r the 100%. t r y p t o p h a n s u p p l e m e n t e d T e k l a d diet, a d lib) follow the well e s t a b l i s h e d g r o w t h c u r v e s for L o n g - E v a n s rats. B o d y w e i g h t s o f the e x p e r i m e n t a l a n i m a l s lagged b e h i n d [40-60%] t h o s e of controis t h r o u g h o u t life, e v e n a f t e r the t r y p t o p h a n - d e f i c i e n t a n i m a l s were t r a n s f e r r e d to the c o m p l e t e diet at t w o years of age. At a b o u t 30 m o n t h s o f age, the b o d y w e i g h t s o f c o n t r o l s d r o p p e d rapidly (3(1%, in six m o n t h s ) while the w e i g h t s o f the e x p e r i m e n t a l a n i m a l s were only slightly r e d u c e d . T h e s h a r p decline in weight a m o n g c o n t r o l s a c c o m p a n i e d a m a r k e d in-

238

TIMIRAS, H U D S O N AND S E G A L I ,

I

.p_Loo5 "~

Serofonin

Control

[]

!

.p &oo5

T40%

o--, Conlrol

[] T30%

+50.

,

CEREBRAL HEMISPHERES

~

Pg~r fed

Norepinephrine CEREBRALHEMISPHERES

[ ] T4oojo [ ] T3O%

754 " 646

,oo " N ,.

n

L

#

°

,

.

HYPOTHAL A M US +100 F

~[

i

CORPUS STRIATUM

/"

120~

80

o4

]2048

+3o;

i

+50

1,792 1530

-

g ,ooi

~-

-~

I \_L_.JI

liit

liiF i "

o i

0

I ..........

L_

HYPOTHALAMUS

(.3 +30F 100:

- ,:.~

,a

"~

_

i

L___~

.

-

.~_

] 2843 E

[

0E::

MESODIENCEPHELON

.13

80~

I

2031

0

MESODIENCEPHELON

-/

0

yr

1825 (/)

c

~

(~ +100, o

PONS MEDULLA

~

f20

c

CL , I:.:

100

|:.IVA

131 I

+5C

&_

567

I00

-50~

L

1

I

I

I __

I

.

L____ ~---J

-512

PONS M E D U L L A

2mos

+50

1oo

i

-50

2mos. 4mos 6mos

Lyr

473 379

-

2yrs. 2.5yrs 3yrs.

Age FIG. 1. Diet- and age-associated changes in brain serotonin levels. Tryptophan-restricted diets (containing 40% or 30% optimal tryptophan) were fed from weaning to 2 years of age, thereafter normal (optimal tryptophan) diet was fed. Pair-fed animals were given norreal diet equal in weight to the tryptophan-restricted diet consumed by their counterparts. The connected lines indicates the mean control levels (ng/g brain tissue) of serotonin at different ages. Bars indicate the per cent difference from control level (100%) of serotonin in cerebral hemispheres, corpus striatum, hypothalamus, mesodiencephalon, and pons-medulla at ages from 2 months to 3 years. Each bar represents 5 animals except beyond 2 years when only 1-2 animals are represented. Standard errors have been omitted for clarity. Significant differences (p<0.05) are indicated with *

crease in mortality: from no mortality at one year to 18% at 18 months and 91% at 36 months. The median length o f life of control rats in this study was about 23.5 months, the same as reported for male SPF Fisher 344 rats [40]. In the Tryptophan-restricted rats, the mortality was high at early ages, 25% for T40% and 36% for T30% at one year, however, the mortality rate decreased thereafter. Maximal lifespan in

4mos

6mos

lyr

2yrs

25yrs

3yrs

Age FIG. 2. Diet- and age-associated changes in brain norepinephrine levels. Tryptophan-restricted diets (containing 41:)% or 30% Optimal tryptophan) were fed from weaning to 2 years of age, thereafter normal (optimal tyrptophan) diet was fed. Pair-fed were fed n o e l diet equal in weight to the tryptophan-restricted diet consumed by their counterparts. The connected line indicates the mean control levels (ng/g brain tissue) of norepinephrine at different ages. Bars indicate the percent difference from control level (100%) of norepinephrine in cerebral hemisphere s, hypothalamus, mesodiencephalon and pons-medulla at ages from 2 months to 3 years. Each bar represents 5 animals except beyond 2 years when only l-2animals are represented. Standard errors have been omitted for clarity. Significant differences (0<0.05) are indicated with *

these restricted animals were longer (1,347 days in T40% and 1,527 days in T30%) as compared to controls (1,246 days). In pair-fed groups, mortality was less than for controls at two years of age when this portion of the study was concluded.

Brain Monoamine Levels In controls (Purina chow or 100% tryptophan Supplemented Teklad diet) monoamine levels were the same and, therefore, values shown in Table 2 are the mean of both groups. Serotonin levels, generally, remained unchanged until advanced age when they increased in the hyp0thalamus, mesodiencephalon and pons-medulla (Fig: 1). In the cerebral hemispheres, levels increased up to one year of age, declined between one and two-and-a-half years and then in-

BRAIN SEROTONIN, LOW TRYPTOPHAN AND AGING

p ~ 0 l05

~.

Control

DopQrnine

[]

CEREBRAL HEMISPHERES ~ ~

o~ +5°F

T40%

[ ] T30% [ ] Pairfed -3878

JEI

o +5o

-0

CORPUS STRIATUM

o'~

l

,

8400

~-~o ®

I

2mos

4 m o s 6mos

lyr

2yr8

25yrs 3yr&

Age FIG. 3. Diet- and age-associated changes in brain dopamine levels.

Tryptophan-restricted diets (containing 40% or 30% optimal tryptophan) were fed from weaning to 2 years of age, thereafter normal (optimal tryptophan) diet was fed. Pair fed rats were given normal diet equal in weight to the tryptophan-restricted diet consumed by their counterparts. The connected line indicates mean control levels (ng/g brain tissue) of dopamine at different ages. Bars indicate the percent difference from control level (100%) of dopamine in cerebral hemispheres and corpus striatum at ages from 2 months to 3 years. Each bar represents 5 animals except beyond 2 years when only 1-2 animals are represented. Standard errors have been omitted for clarity, significant differences (p<0.05) are indicated with *

creased to three years. In the striatum, serotonin levels increased progressively from two to 12 months, and then again at three years. Norepinephrine and dopamine levels decreased with age in all areas where measured (Figs. 2, 3). This decrease was not uniform: norepinephrine in the hypothalamus decreased by 50% between two months and two years but remained practically unchanged in the cerebral hemispheres over the same age period. Dopamine levels increased between two and six months, a period when the axonal endings are still proliferating in this region, and then decreased in old rats. In the tryptophan-deficient animals, serotonin levels were lower (except in the 3-year old pons-medulla) than controls at all ages and in all brain areas (Fig. 1). ANOVA revealed marked differences in serotonin for all brain areas at early ages. These differences were less, but still significant, at later ages when fewer areas were affected. For example in the cerebral hemispheres at two months, F(3,21)=55.75; at four months, F(3,17)=19.42; at six months, F(3,21)=15.33; at one year, F(3,17)=2.28; and at two years, F(3,27)=8.41. In the hypothalamus the values of two months were, F(3,21)=18.07; at four months, F(3,17)=11.51) at six months, F(3,19)=5.92; at one year not assayed; and at two years, F(3,27)=2.02. The degree of reduction, greater in some areas than in others (e.g., at two months, 80% in ponsmedulla, 2(Y~, in the hypothalamus), is related in most cases to the severity of restriction, the most restricted having the lowest serotonin content. Dietary rehabilitation at approximately 24 months of age did not significantly alter the reduced serotonin levels. At three years, some regions showed a small degree of recovery and the pons-medulla definitely increased in serotonin in the T40%, animals.

239 The effects of tryptophan dificiency were more variable on catecholamine than on serotonin content (Figs. 2, 3). Dopamine levels were lower or unchanged at younger ages in tryptophan-deficient animals, then significantly higher at two years of age (for example, in cerebral hemispheres, F(3,27)=5.97) and dropped below control levels again at later ages (Fig. 3). The most striking changes were a reduction of norepinephrine in the cerebral hemispheres especially at early ages (two months, F(3,21)=8.01, four months, (3,17)=3.32, six months, F(3,21)=4.50, one year, F(3,17)=4.03, two years, F(3,27)=4.55). Sporadic changes were observed in other areas, such as the mesodiencephalon rich in adrenergic neurons (at one year, F(3,20)=23.71) and in the hypothalamus, norepinephrine responsive tissue for release of neurohormones (at six months, F(3,19)=5.56). Norepinephrine markedly increased in the hypothalamus, mesodiencephalon and pons-medulla at 3 years of age after food rehabilitation (Fig. 2). A striking difference between control and pair-fed animals was reflected in serotonin levels which were all significantly higher in pair-fed than controls (Fig. 1). Similarly, norepinephrine levels in these animals were increased overall, although the increase was not as uniform or marked as that of serotonin (Fig. 2), Dopamine levels were essentially unchanged or increased. (Fig. 3).

Monoaminergic Enzyme Activity Studies of the various metabolic enzymes were carried out as permitted by the available amounts of tissue and activity of the individual enzymes. Due to these limitations all enzymes could not be studied in all areas at all ages. In controls, tyrosine hydroxylase activity reached adult values by two months of age and remained unchanged up to three years of age except for large increases at this late age in the corpus striatum and ports medulla in the few remaining animals (Table 3). Tryptophan restriction did not significantly alter the enzyme activity at any age. A general overview of the effects of pair-feeding showed an increase in tryosine hydroxylase, significant in the cerebral hemispheres and mesodiencephalon at four months of age, in the corpus striatum at one and two years and in the hypothalamus at one year, the only age studied. Tryptophan hydroxylase activity was assayed in fresh tissue in those brain regions most involved in serotonergic transmission [26]. The activity was highest at two months in the mesodiencephalon and pons-medulla, at one year in the cerebral hemispheres and declined thereafter (Table 4). Despite the marked decrease in serotonin levels in the tryptophan-deficient animals the enzyme activity was unaltered. When measured at two years of age (Table 5), the activity of monoamine oxidase-A, involved in serotonin catabolism, was unchanged by the tryptophan deficiency and associated low levels of brain serotonin. Pair feeding did not change the monoamine-oxidase A activity. DISCUSSION The picture of the aging brain emerging from current and earlier data as well as those of other investigators [21, 24, 31-36] is one of age, region, and neurotransmitter specificity. While catecholamine levels decline progressively with aging in most brain areas, serotonin levels, initially decreasing in the cerebral hemispheres, remain unchanged or increase in

240

TIMIRAS. HUDSON AND SEGAI,I "FABLE 3 TYROSINE HYDROXYLASE ACTIVITY IN BRAINS OF FEMALE LONG-EVANS RATS Tyrosine Hydroxylase (nmoles/hr/mg protein) Age

2 months

Cerebral Hemispheres Control (10)* 0.46 ± 0.07t T40% (5) 0.65 ± 0.26 T30% (5) 0.60 ± 0.14 Pair-fed (5) 0.36 ± 0.02 Mesodiencephelon Control 0.61 _+ 0.06 T40% 0.58 ± 0.09 T30% 0.46 -+ 0.12 Pair-fed 0.81 ± 0.09

1 year Control T40% T30% Pair-fed

3.85 5.52 3.65 5.90

± ± ± ±

0.25 0.99 0.29 0.46:~

4 months

6 months

l year

2 years

3 years

0.39 0.51 0.37 0.77

--- 0.08 ± 0.09 ± 0.11 ± 0.08~

0.58 0.70 0.79 0.73

± 0.08 m 0.21 ± 0.17 ± 0.03

0.51 0.71 0.94 0.84

__+0.11 ± 0.15 +_ 0.22 ± 0.06~

0.44 0.65 0.61 0.48

± 0.04 ± 0.07 ± 0.04 _+ 0.09

0.46 0.73 0.64

0.53 0.66 0.48 1.12

± 0.07 _+ 0.10 ± 0.04 ± 0.08,

0.68 0.64 0.65 1.17

± ± ± ~

0.84 0.83 0.61 1.21

± ± ± ±

0.60 0.73 0.66 0.59

± ± ± ±

0.54 0.31 1.10

Corpus Striatum 2 years 4.02 5.05 4.32 6.69

± 0.34 ~ 0.38~: ± 0.52 -+ 0.655

0.07 0.08 0.05 0.12;t

3 years 6.72 10.76 8.93 --

1 year 0.23 0,33 0.18 0.26

0.05 0.04 0.14 0.24

Pons Medulla 2 years

_+ 0.03 _+ 0.04 ± 0.02 ± 0.02

0.18 0.18 0.19 0.18

_+ 0.01 + 0.01 ± 0.02 -+ 0.01

0.05 0.09 0.09 0.03

3 years 0.40 0.30 0.34

H ypothalamus 1 year 0.67 0.59 0.58 0.98

± ± ± ±

0.05 0:04 0.05 0.07$

*Numbers in ( ) indicate number of animals. Two animals for 3-year-old group. tAverage -+SE. ~:p<0.05 between control and experimental animals.

m o s t brain areas at a d v a n c e d age [32]. The relative susceptibility to aging o f brain c a t e c h o l a m i n e s as o p p o s e d to the relative stability o f brain serotonin is supported by morphologic studies which show a loss o f catecholaminergic neurons and/or alterations in their structure with aging while serotonergic neurons s e e m less affected [8]. If, as postulated recently [5], c a t e c h o l a m i n e s stimulate the synthesis o f choline, the acetylcholine precursor, then, with aging, reduction in brain c a t e c h o l a m i n e s m a y be responsible in part, t o g e t h e r with the regional loss of cholinergic neurons [3,11], for the progressive cholinergic deficits in specific brain areas. The resulting imbalance, perhaps, m o r e than a global n e u r o t r a n s m i t t e r reduction m a y be instrumental in inducing functional neurologic and behavioral deficits o f the elderly. Certain characteristics o f the serotonergic p a t h w a y s [2] may help explain their apparent stability into old age: (a) specialized c o n t a c t s with persistently dividing glial cells [7,17] provide metabolic [15,18] support for the aging n e u r o n s ; (b) multiple m o d e s o f transmission (synaptic contact, extrasynaptic diffusion, small attachment plaques) minimize the c o n s e q u e n c e s on neurotransmission of the age-related reduction in dendritic domain [10,25]; and (c) ability to sprout s u b s e q u e n t to n e u r o t o x i c (and perhaps age-related) lesions is greater than that of the other m o n o a m i n e r g i c neurons [4]. F u r t h e r support for the p r e s e r v a t i o n o f brain serotonin levels until late age is provided by the sustained efficiency o f metabolic e n z y m e activity reported here and the ability to a c c u m u l a t e serotonin after inhibition of catabolism by M A O blockade [32]. L o n g - t e r m , dietary t r y p t o p h a n restriction from weaning to old age r e d u c e s serotonin levels in most brain areas proportionally to the severity o f the restriction. Without entering in the c o n t r o v e r s y o f the source of brain tryptophan and c o n s e q u e n t l y brain serotonin levels [14, 24, 39], the present

TABLE 4 TRYPTOPHAN HYDROXYLASE ACTIVITY IN BRAINS OF FEMALE LONG-EVANS RATS Tryptophan Hydroxylase Inmoles/hr/mgprotein) Age Cerebral Hemispheres Control (10)* T40% (5) T30% (5) Mesodiencephelon Control T40% T30% Pons Medulla Control T40% T30%

2 months

I year

16-18 months

0.48 -- 0.06t

0.70 -~ 0.07 0.44 z 0.11 0.56 -- 0.12

0.60 _ 0,01 0 55 ± 0.04

1.09 = 0.15 0.91 ~ 0.14 1.08 ~ 0.43

1.38 ~ 0.05 128 +- 0.11

1.68 m 0.22 1.47 ± 0.33 2.20 --- 0.62

1.55 _+_0.05 1.46 ± 0.16

0.43 ~ 0.05 3.41 _~ 0.37 2.45 _ 0.38 2.16 _* 0.31 2.10 _-z 0.42

*Numbers in ( ) indicate number of animals. tAverage ±SE.

data show that dietary restriction of this amino acid induces generalized, s e v e r e and persistent reduction in brain serotonin levels; this is specific for serotonin and for tryptophan restriction. Indeed, the pair-fed animals, although retarded in growth, show brain serotonin l e v e l s - - a s well as c a t e c h o l a m i n e s - - n o r m a l or elevated, an observation in a g r e e m e n t with reports that acute food deprivation increases brain tryptophan [12]. The manipulation o f naturally occur-

BRAIN SEROTONIN,

LOW TRYPTOPHAN

AND AGING

241

TABLE 5 MONOAMINE OXIDASE-A ACTIVITY IN BRAINS OF TWO-YEAR-OLD FEMALE LONG-EVANS RATS Cerebral Hemispheres Control T40C'~ T30C~ Pair-fed

(10)* (5) (5) (5)

78.4 79.6 81.3 75.7

_+ 1.1+ _+ 0.7 _+ 1.5 ± 3.9

Corpus Striatum 70.9 72.8 65.4 71.3

___ 2.4 ± 2.9 ± 0.7 ± 3.7

Mesodiencephelon 89.4 90.9 96.2 89.4

_+ 3.7 _+ 2.1 _+ 1.7 ± 1.9

Pons Medulla 67.1 65.0 72.8 70.4

_+ 1.8 +_ 1.6 _+ 1.6 _+ 2.5

*Numbers in ( ) indicate number of animals. tAverage ±SE, nmoles/hr/mg protein.

ring d i e t a r y c o n s t i t u e n t s t h a t are p r e c u r s o r s o f n e u r o t r a n s m i t t e r s has b e e n a d v o c a t e d for the t r e a t m e n t o f a n u m b e r o f b r a i n d i s o r d e r s i n c l u d i n g senility. A m u c h used p r e c u r s o r , a l t h o u g h its efficacy is c o n t r o v e r s i a l , is c h o l i n e , or lecithin. S u c h a d m i n i s t r a t i o n e n h a n c e s regional (e.g., h i p p o c a m p u s ) a c e t y l c h o l i n e s y n t h e s i s a n d is s t a t e d to r e d u c e the c o g n i t i v e deficits (e.g., m e m o r y loss) c h a r a c t e r i s t i c of t h e elderly, p a r t i c u l a r l y t h o s e a f f e c t e d by senile d e m e n t i a o f the AIz h e i m e r t y p e [3,11 ]. B e c a u s e of s e r o t o n i n m a n y physiological a c t i o n s (e.g., e n d o c r i n e , b e h a v i o r a l ) , it m a y be a n t i c i p a t e d t h a t m a n i p u l a t i o n of b r a i n s e r o t o n i n m a y influe n c e a n u m b e r o f physiological p a r a m e t e r s . I n d e e d , topical (in t h e h y p o t h a l a m u s ) o r s y s t e m i c a d m i n i s t r a t i o n o f serotonin a g o n i s t s or a n t a g o n i s t s as well as d i e t a r y t r y p t o p h a n restriction m a y m o d i f y t h e t i m e t a b l e of r e p r o d u c t i v e d e v e l o p m e n t a n d aging [30], m a y m e d i a t e m e m o r y a n d l e a r n i n g [38],

m a y p r o l o n g t h e r m o r e g u l a t o r y c o m p e t e n c e [28], a n d inc r e a s e the lifespan [29]. W h e t h e r t h e s e a c t i o n s r e s u l t i n g from d i e t a r y o r p h a r m a c o l o g i c s e r o t o n i n m a n i p u l a t i o n s are due directly to the c h a n g e s in b r a i n s e r o t o n i n levels, o r t h e acc o m p a n y i n g r e d u c t i o n in food intake, o r indirectly result f r o m c h a n g e s in c a t e c h o l a m i n e levels r e m a i n s to be elucid a t e d . F o r e x a m p l e , caloric r e s t r i c t i o n p r o l o n g s the lifespan [20,40]. T h e i n c r e a s e d c a t e c h o a l m i n e r g i c levels in the reh a b i l i t a t e d t r y p t o p h a n r e s t r i c t e d rats and the p o t e n t i a l seco n d a r y i n c r e a s e in c h o l i n e r g i c levels 15] m a y explain s o m e o f the o b s e r v e d physiological i m p r o v e m e n t d u r i n g aging.

ACKNOWLEDGEMENT The authors gratefully acknowledge the technical assistance of Carole Miller and the support of NIH AG00043.

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