Cerebrospinal fluid (CSF) and brain monoamine metabolites in the developing rat pup

Developmental Brain Research, 17 (1985) 225-232 Elsevier

225

BRD 50141

Cerebrospinal Fluid (CSF) and Brain Monoamine Metabolites in the Developing Rat Pup BENNETT A. SHAYWITZ 1, GEORGE M. ANDERSON 2 and DONALD J. COHEN 3

Departments of INeurology, 2Laboratory Medicine, 13Pediatrics, and 3Psychiatry, Yale University School of Medicine, P. O. Box 3333, New Haven, CT06510 (U.S.A.) (Accepted July 31st, 1984)

Key words: 5-hydroxyindoleaceticacid - - homovanillic acid - - serotonin - - norepinephrine - - dopamine - - tryptophan - tyrosine ----development - - rat - - cerebrospinal fluid - - brain

Concentrations of the neurotransmitters, serotonin (5-HT), dopamine (DA), and norepinephrine (NE) were measured in the developing rat brain at 12, 19, 26 and 42 days of age. The amino acid precursors, tryptophan (TRP) and tyrosine (TYR) were measured along with the 5-HT and DA metabolites, 5-hydroxyindoleaceticacid (5-HIAA) and homovanillic acid (HVA), in brain and cerebrospinal fluid (CSF) at the above ages. This first report of CSF HVA levels in the developing rat shows that it, like 5-HIAA, declines with age. In contrast, the ontogeny of the compounds in brain are dissimilar, with 5-HIAA remaining relatively constant with age while HVA declines markedly. Possible reasons for the differences and similarities in the ontogeny of 5-HIAA and HVA levels in brain and CSF are discussed. The persistence of the ontogenetic pattern for the neurotransmitters and acid metabolites after central DA depletion is also reported.

INTRODUCTION The importance of the central m o n o a m i n e neurotransmitters, d o p a m i n e ( D A ) , n o r e p i n e p h r i n e (NE), and serotonin (5-HT), has been well established. Changes in n e u r o t r a n s m i t t e r function can result in m a r k e d alterations in central nervous system (CNS) functioning and, while much of the evidence is derived from studies of animal behavior, investigations with human subject groups have implicated the m o n o a m i n e s in several serious neuropsychiatric disorders. Many of the studies in humans have d e p e n d ed upon the m e a s u r e m e n t of n e u r o t r a n s m i t t e r metabolites in cerebrospinal fluid (CSF) 9,15,21,23,24, 34,41,45.47. Alternatively, but less frequently, investigators have m e a s u r e d the neurotransmitters and their associated metabolites, enzymes, and receptors in p o s t m o r t e m h u m a n brain samples. It is apparent from both animal and clinical studies that, at least in the adult, C S F m e t a b o l i t e levels do reflect CNS levels to some extent. The nature and

meaning of changes seen in CSF m e t a b o l i t e levels in specific neuropsychiatric disorders are not entirely clear, and questions remain as to just how well brain and CSF levels are correlated. These uncertainties are c o m p o u n d e d when attempting to interpret CSF m e a s u r e s made in developing subjects. The develo p m e n t of n e u r o t r a n s m i t t e r synthesis, u p t a k e , storage, release, metabolism, and elimination have been extensively studied in animals; however, it is not clear how these various aspects of the maturing brain influence CSF metabolite levels. W e have m e a s u r e d levels of neurotransmitters, their amino acid precursors (tryptophan [TRP] and tyrosine [TYR]), and metabolites in brain and CSF of developing rat pups in o r d e r to further elucidate the normal ontogeny of the compounds. Of particular interest was .the ontogeny in CSF of homovanillic acid ( H V A ) and 5-hydroxyindoleacetic acid ( 5 - H I A A ) , metabolites of D A and 5-HT, respectively. These c o m p o u n d s have been widely m e a s u r e d in both adult and child 15,41 neuropsychiatric populations. Clarification of the on-

Correspondence: Bennett A. Shaywitz, Department of Neurology, Yale University School of Medicine, P.O. Box 3333, New Haven, CT 06510, U.S.A.

226 togenetic factors which affect the levels observed in CSF is required in order to meaningfully interpret measures obtained in children.

base) in 20 ~tl of solution. The 5,7-DHT was purchased from Regis Chemicals and dissolved in 0.9e;+ saline and kept on ice while in use.

MATERIALSAND METHODS

Technique of CSF collection in rat pups The procedure described here has been successfully employed in pups as young as 12 days of age. Rat pups were anesthetized with sodium pentobarbital (50 mg/kg), a midline scalp incision was made and the scalp reflected. Specially constructed ear bars were placed in the internal auditory meatus and the pups placed in a stereotaxic head-holder (Kopf). After 20-30 rain cervical spinal musculature was reflected and the atlanto-occipital membrane exposed in preparation for insertion of a micropipette into the cisterna magna. The micropipette was prepared from 1 x 150 mm glass tubing that had been drawn to a fine point (50 ~m tip o.d. with approximately 1 cm taper). Using a specially designed jig to hold the micropipette, the point was carefully advanced under direct vision with a dissecting microscope (Zeiss), the atlanto-occipital membrane punctured, and cisternal CSF collected in the capillary for 15-60 s (Fig. 1). CSF was blown into small plastic test tubes and frozen at -80 °C. Immediately after CSF collection, rats were sacrificed by decapitation, and their brains rapidly removed and frozen in dry ice.

Animals Sprague-Dawley rat pups with mother were obtained from Charles River, Inc., Wilmington, MA, at 24 h (_+12 h) of age and individually housed in clean plastic cages (30 x 32 x 10 cm) with sawdust bedding. Animals were housed under fluorescent lighting conditions (16 GE 40 W fluorescent bulbs) with 12 h of light (lights on 07.00 h) and 12 h of darkness at a temperature of 21 °C. Litters were culled to 8-9 pups at 5 days of age, and mothers and pups were housed together for the entire experimental period. Weights were recorded weekly beginning at 5 days of age. Food (Purina Chow) and tap water were available ad libitum to the dam and her pups. All experiments included approximately equal numbers of male and female rats. Preferential depletion of brain dopamine was accomplished in rat pups at 5 days of age using procedures described previously 12.42. Animals first were injected with desmethylimipramine (DMI, USV Pharmaceuticals, Tuckahoe, NY) at a dose of 20 mg/kg intraperitoneally (in 0.1 ml) and 1 h later received 100#g of 6-hydroxydopamine HBr (6O H D A , Regis Chemical Company, Chicago, IL, prepared as a free base) via an intracisternal (i.c.) injection. The 6-OHDA was prepared immediately prior to use in a 0.9% isotonic saline solution containing 0.4 mg/ml of ascorbic acid and kept on ice while in use. Intracisternal injections were administered by flexing the neck of the infant rat and injecting 20 ,ul of solution via a precalibrated microsyringe (Hamilton) with a 27-gauge needle inserted immediately beneath the occiput. Littermate controls received the saline intraperitoneally, followed 1 h later by the i.c. administration of 20 ~1 of saline containing 0.4 mg/ml of ascorbic acid. Preferential depletion of serotonin (5-HT) was accomplished using modifications of procedures described by Breese and CooperU and Chiondo j4 by pretreatment of 5-day-old rat pups with DMI intraperitoneally 1 h prior to intracisternal administration of 200 #g 5,7-dihydroxytryptamine (5,7-DHT as free

Fig. 1. Sampling of cisternal cerebrospinal fluid (CSF) from a 26-day-old rat. The capillary tip has punctured the exposed atlanto-occipital membrane, and CSF can be seen filling the tube. The animal is deeply anesthetized with pentobarbital.

227 in rat whole brain using a modified H P L C - a m p e r o metric m e t h o d 22. A f t e r the addition of dihydroxybenzylamine ( D H B A ) as an internal standard (100-1000 ng/g), the tissue was sonicated in 0.1 M perchloric acid (HC104). Protein precipitation was c o m p l e t e d by addition of 10% v/v 4 M HC104; after centrifugation, the supernatant was removed and split. One aliquot ( 5 0 - 7 5 % of the total volume) was adjusted to p H 8 . 4 - 8 . 6 with 3 M Tris buffer and the catechols a b s o r b e d on 10-25 mg of acid-washed alumina. The c o m p o u n d s were eluted in 100-500 ktl of 1 M acetic acid and 1 0 - 5 0 / A of this solution injected

Neurochemical analysis

All of the high-performance liquid chromatographic ( H P L C ) d e t e r m i n a t i o n s were p e r f o r m e d with systems comprised of A l t e x l l 0 A or W a t e r s M45 pumps, R h e o d y n 7125 or 7120 sample injectors, microparticulate H P L C columns and either a m p e r o metric (Bioanalytical Systems Controllers and thinlayer cells) or fluorometric (Perk±n-Elmer spectrop h o t o f l u o r o m e t e r or modified A m i n c o fluorometer) detectors. Standards were purchased from Sigma Chemical C o m p a n y . Stock solutions (usually 10 mg/100 ml) were m a d e up every month in distilled water (occasionally m e t h a n o l was a d d e d first to dissolve less hydrophilic c o m p o u n d s ) with 0.1% ascorbic acid added. D i l u t e d standards ( 0 . 1 - 1 0 ng//~l) were m a d e up daily in distilled water. M o b i l e phases were p r e p a r e d by mixing v/v, for at least 30 min, the appropriate quantity of m e t h a n o l or acetonitrile with the p r o p e r p H - a d j u s t e d buffer. CSF metabolites. A n H P L C m e t h o d was used to determine T Y R , T R P , and 5 - H I A A and H V A in 10-25/~1 of directly injected CSFS. The c o m p o u n d s were s e p a r a t e d on a 300 x 39 m m C18 B o n d a p a k (Waters Associates) column with a mobile phase of 85%, p H 4.25, 0.01 M sodium acetate/15% methanol, delivered at a flow rate of 2.0 ml/min. Fluorometric and a m p e r o m e t r i c detectors were used in series; absolute detection limits were in the 5 - 2 5 pg range. Brain D A and NE. The catechols were d e t e r m i n e d

into the H P L C system. A mobile phase of 50-100 mg/1 of octylsulfate, 50 mg/l of E D T A , 0.1 M p H 4.60 monobasic phosphate buffer with 5 - 1 5 % m e t h a n o l a d d e d was used with a reverse-phase C18 column. Optimization of the solvent system was easily accomplished to permit the most rapid analysis of both of the compounds. Brain indoles and H V A . The indoles, along with H V A , were d e t e r m i n e d by the direct injection of the supernatant o b t a i n e d from the H C 1 0 4 son±cation described in the previous section. The detection system and chromatographic conditions were similar to those described for the d e t e r m i n a t i o n of the m e t a b o lites in CSF 4. RESULTS Brain concentrations of the c o m p o u n d s studied are

TABLE I Mean neurochemical concentrations in brain control (ng/g ± S. E. M.)

Where means are significantly different from control animals, the value as the percent of control and the P value are indicated. Age (days)

NE

12 (n = 31) 19(n = 29,30) 26(n = 28,29) Adult (n = 7)

168 ± 250 ± 306 ± 346 ±

5.0 10 18 18

DA

5-HT

5-HIAA

HVA

344 ± 8.2 566 ± 21 648 ± 29 807±34

207 ± 303 ± 311 ± 527 ±

110 ± 15 (32%)** 110 ± 7.7 (19%)** 165 ± 46 (25%)**

TRP

10 22 12 20

292 ± 9.9 358 ± 13 290 ± 13 283±14

196 ± 179 ± 154 ± 76.9 ±

229 ± 22

281 ± 7.8

146 ± 37

232 ± 14 (77%)* 325 ± 10

331 ± 10

57.7 ± 13 (32%)** 38.4 ± 12 (25%)**

10 12 20 13

9410 ± 9540 ± 5190 ± 3790 ±

800 1160 380 140

Dopamine deplewd

12 (n = 17)

183 ± 10

19(n = 21)

212 ± 6.0 (85%)* 302 ± 16

26 (n = 6) * P < 0.01.

** P < 0.001.

330 ± 21

6780 ± 520 (72%)* 6130 ± 290 (64%)* 5170 ± 553

228 presented in Table 1. Levels of NE, DA, 5-HT, 5H I A A , HVA, and tryptophan (TRP) were measured in rat brain at 12, 19, 26, 42 and approximately 90 days of age. As has been observed previously )3,17,28,3(), levels of D A and NE increase rapidly in the developing rat pup and reach adult values at approximately 42 days (Fig. 2). A similar increase from 12 to 42 days was seen for 5-HT, as shown in Fig. 3. The increases in NE (98%), DA (139%), and 5-HT (155%) between days 12 and 42 are in marked contrast to changes observed for HVA, 5-HIAA, and TRP in brain. As shown in Fig. 2, HVA declines continually from 12 days to adulthood, with values at 42 days and, in adults, 55% and 39% of those observed at 12 days of age, respectively. While the 5-HT precursor, TRP, also exhibited a marked decline with age (see Table I), brain concentrations of 5-HIAA remained relatively stable. A slight, but significant, increase (P < 0.01) was seen at 19 days of age; otherwise 5-HIAA values were similar throughout development (Fig. 3). CSF concentrations of HVA, 5-HIAA, TRP, and tyrosine (TYR) in the same animals are given in Table If. The CSF levels of HVA and 5-H1AA have been plotted along with the brain concentrations in Figs. 2 and 3, respectively. The decline seen for CSF HVA, though slightly preceding that for brain HVA, was closely parallel. CSF HVA values observed at 42 days and in adults were 51% and 43% of those seen at 12 days of age. A marked age effect was also observed for CSF 5-HIAA, with adult levels declining to 52% of the levels present at

200

800

tO0

75 i

600

CSFHVA4 J

~

i

400

BrainDAi ~ t ~

~ z 25

200

0

I 12

I 19

2 6 AGE (days)

I ,~-- I 42 Adllll

~,

0

Fig. 2. Ontogeny of brain dopamine (DA) and homovanillic acid (HVA) and cisternal cerebrospinal fluid (CSF) HVA in Sprague-Dawley rats. Error bars indicate _+standard error of the mean (S.E.M.).

o

600

l Brain 5HT Brain 5HIAA

Eo)

:

o

~ i

i ~-'''~

,; 'i

5.,AA

i 4

i 5

400

/

200

~ ~ c~

0

I

q

12

19

I

26 AGE (days)

, I

4-2

//

I

_J

Adult

Fig. 3. Ontogenyof brain serotonin (5-HT) and 5-bydroxyindoleacetic acid (5-HIAA) and cisternal CSF 5-HIAA in rats. Error bars indicate _+standard error of the mean (S.E.M.).

12 days. Concentrations of the amino acid precursors, TRP and TYR, also declined with age. For TRP, no further decrease was observed after 42 days of age, when values were 24% of those observed at 12 days (Table II). The compounds also were measured in brain and CSF of 6-hydroxydopamine (6-OHDA)-treated rats at 12, 19, and 26 days of age. The developmental pattern of NE, 5-HT, and 5-HIAA was similar to that observed in the control animals. Concentrations of DA and HVA were significantly lowered in the treated animals at all ages, except for H V A at 12 days. In spite of these 6-OHDA-induced reductions in DA and HVA, both compounds exhibited ontogenies similar to those seen in the control animals. DA tended to increase and H V A to decrease over the 2week period studied. Although a significant decline in brain TRP was seen after 6 - O H D A treatment in 12- and 19-day-old rats, only in 19-day-old rats were 5-HT levels decreased (77% of controls). At neither age was brain 5-HIAA lowered, while a small decline ( - 8 0 % of controls) was seen in CSF 5-HIAA levels in the 19-day-old 6-OHDA-treated group. Correlations observed in control animals (12 days through adult) between selected pairs of compounds are presented in Table III. Because of the marked age effect on most of the measures, partial correlations were calculated, controlling for age. The 3 compounds measured in both brain and CSF (TRP, 5H I A A , and HVA) were all significantly correlated across the two tissues. Correlations calculated for brain and CSF H V A within age groups were signifi-

229

TABLE II

Mean neurochemical concentrations in CSF Data show ng/m _+ S.E.M. Control: sample number given in parentheses beneath mean for 12, 19, and 26-day-old groups. Dopaminedepleted group: where means are significantly different from control animals, the value as the percent of control and the P value are indicated.

Age (days)

5-HIAA

HVA

TRP

TYR

201 ± 12 (14) 225 ± 14 (19) 152 ± 7.8 (22) 111 ± 5.7

58.2 ± (13) 36.8 ± (14) 33.5 ± (22) 29.9 ±

1140 ± 152 (14) 853 ± 205 (19) 417 ± 41 (21) 275 ± 26

8160 ± 1820

104 ± 8.8

25.0 ± 2.9

276 ± 16

1020 ± 119

21.4 ± 2.6 (37%)* 17.6 ± 8.0 (48%)** 7.7 ± 3.0 (23%)***

971 ± 158

4790 + 1150 (59%)*

Control 12 19 26 42 (n = 13) Adult (n = 7)

6.8 5.5 2.5 4.3

(8) 4100 ± 1260

(7) 1790 ± 233

(3)

Dopamine depleted 12

232 ± 24 (n = 6,7)

19 (n = 17,18) 26

185 ± 11 (80%)* 159 ± 30

(n = 5)

620 ± 78 365 ± 36

* P < 0.05. ** P < 0.01. *** P < 0.001.

cant (19 days: r = 0.30, P = 0,02; 26 days: r = 32, P =

brain, levels of 5 - H T w e r e only slightly c o r r e l a t e d

0.05) only w h e n 6 - O H D A - t r e a t e d

animals w e r e in-

with t h o s e of its p r e c u r s o r , T R P . B r a i n T R P was

cluded. W i t h i n a g e - g r o u p c o r r e l a t i o n s for b r a i n and

m o r e highly c o r r e l a t e d with brain levels of the 5 - H T

C S F 5 - H I A A in the c o n t r o l g r o u p w e r e significant

m e t a b o l i t e , 5 - H I A A (r = 0.38, P = 0.001). P r e c u r -

only at 19 days (r = 0.61, P = 0.005, n = 19). In

sor a m i n o acids and their r e s p e c t i v e m e t a b o l i t e s w e r e also significantly c o r r e l a t e d in C S F . In a d d i t i o n , C S F c o n c e n t r a t i o n s of the a m i n o acids, T R P

TABLE III

and

T Y R , w e r e highly c o r r e l a t e d with o n e a n o t h e r . This

Inter-compound correlations in untreated animals

c o r r e l a t i o n within c o m p o u n d type also was s e e n for the two acid m e t a b o l i t e s , 5 - H I A A and H V A .

Age effect controlledfor r

P

N DISCUSSION

Brain-CSF TRP/TRP 5-HIAA/5-HIAA HVA/HVA Brain TRP/5-HT TRP/5-HIAA CSF TRP/5-HIAA TYPUHVA TRP/TYR 5-HIAA/HVA

0.72 0.34 0.31

0.0001 0.0001 0.001

124 122 129

0.17 0.38

0.02 0.001

191 191

0.35 0.49 0.74 0.30

0.001 0.001 0.001 0.001

132 64 70 129

S e v e r a l aspects of the d e v e l o p m e n t of central 5H T and D A systems can be m a d e c l e a r e r by c o m p a r ing the o n t o g e n i e s of t h e two systems in b r a i n and CSF. A s p r e v i o u s l y r e p o r t e d and d e m o n s t r a t e d h e r e , brain levels of 5 - H T 6,8,10,26,32,37,49 and D A (and N E ) 13,17,28,30 i n c r e a s e in a similar m a n n e r with age. H o w e v e r , the d e v e l o p m e n t a l p a t t e r n for their m e tabolites, 5 - H I A A and H V A , are strikingly differ-

230 ent. Levels of 5-HIAA at 12 days of age are very similar to those seen in the adult brain. This comparative constancy of 5-HIAA levels has been observed previously in rat 1°,26,37,49 and mouse 6 brain. A slight elevation of brain 5-HIAA was observed at 19 days; most previous studies 6,26,37,49 have also reported a peak in 5-HIAA levels at 15-20 days of age. This maximum in 5-HIAA levels and, presumably, in 5HT turnover occurs at a behaviorally significant period (transient increased motor activity) and might be a result of high levels of tryptophan hydroxylase activity and high TRP concentrations coinciding. It is fairly well established that early 5-HT synthesis is enzyme-limitedlS, 38, while adult turnover is controlled to a large degree by substrate availability 27.43. In contrast to the relatively constant 5-HIAA concentration, brain H V A fell continually with age from 12 days onwards, with adult values 39% of those at 12 days of age. This decline of H V A with age in whole rat brain has been reported by Keller and co-workers28; however, Nomura et al. 33 observed levels of HVA increasing with age in the striatum. While the rate-limiting enzyme for D A synthesis, tyrosine hydroxylase 25, is thought to reach adult levels sooner than tryptophan-hydroxylase 18,3s, levels of D A do not increase more rapidly than those of 5-HT. The development of neurotransmitter storage capacity is apparent early in both catecholamine 16 and 5-HT8,44 neurons, and similar declines in the respective amino acid precursors, TYR 29 and TRP (see Tables I - I I I and refs. 26, 37, 49) are observed. These similarities between the two systems suggest 5-HIAA and H V A may differ in their relationship to the parent amine. Homovanillic acid has been considered to originate to a large extent from extraneuronal (released) DA 35. On the other hand, approximately one-half or more of brain dihydroxyphenylacetic acid (DOPAC) is thought to arise from unreleased, intraneuronally metabolized D A 35,36. While this distinction between HVA as the extraneuronal metabolite and DOPAC, the intraneuronal metabolite, is not made with great certainty, differences in the ontogenies also support the idea that they arise from different D A pools. For, unlike the decline with age seen for HVA, brain DOPAC levels increase with age during development 28. Parenthetically, the NE metabolites, 3-methoxy-4hydroxyphenylglycol (MHPG) and dihydroxyphenylglycoi (DHPG) have also been suggested to be

predominantly of extra- and intraneuronal origin (respectively), and it would be of interest to determine if the ontogenies of these compounds paralleled those seen for HVA and DOPAC. It is important to note that the sum of brain HVA and DOPAC levels (estimated from ref. 28) shows little change over the age range examined here. The relatively constant levels of 5-HIAA are understandable in this light, as it is the one compound produced from both intra- and extraneuronal 5-HT. If intraneuronally produced 5-HIAA is assumed to parallel the rise in 5-HT (in the same way D O P A C increases in step with DA), it can be suggested that extraneuronal 5-HIAA declines with age. Unlike the situation with respect to H V A and DA, the age-related decline in released neurotransmitter is not reflected in lower brain metabolite levels (because of increasing intraneuronal production of 5-HIAA). Although substantial differences exist in the ontogenesis of brain 5-HIAA and HVA, both compounds undergo similar declines with age in CSF. The lowering of CSF 5-HIAA begins around day 19, when a maximum value of 225 ng/ml was observed. The maximum value for HVA was seen earlier, on day 12, and levels fell steadily from that time on. These first observations of CSF H V A levels in developing rat pups demonstrate, as with brain HVA, the presence of marked age effects. The H V A levels observed in cisternal CSF of untreated 42-day-old and adult animals (29.9 and 25.0 ng/ml, respectively) are substantially lower than levels seen in ventricular CSF of young adult rats (120-170 ng/ml) 19,20. The early increase and subsequent extended decline with age of CSF 5-HIAA has been previously reported26; however, the peak values observed here are higher and occur slightly later. The levels of 5-HIAA seen in cisternal CSF of adult (and 42-day-old) rats agree well with previous reports 4s. As with HVA, the cisternal levels are lower than those seen in ventricular CNF I9,20. The decline in CSF 5-HIAA occurs in spite of relatively constant brain concentrations. The lowering of CSF 5-HIAA with age might be due in part to increased bulk flow clearance of CSF and the development of active capillary clearance 7 of the acid metabolites from brain. Alternatively, CSF 5-HIAA could be considered to be a reflection of extraneuronal 5-HIAA (as discussed above). If this were the case, the fall with age of CSF 5-HIAA

231

H I A A . The parallel falls in CSF 5 - H I A A and H V A

model for the changes which occur in children's CSF levels3,39,41.

therefore both might be attributed to decreased ex-

At present, it can only be suggested that the age-

would be a result of a decrease in extraneuronal 5-

tracellular release of the parent neurotransmitter.

related declines in CSF metabolite levels in rats and

This is a crucial point in terms of the m e a n i n g accord-

children reflect a lowering of central production of

ed the fall of CSF 5 - H I A A and H V A seen in young

the compounds. Confirmation of this fact in animals would give increased weight to CSF measurements

animals and in children. It now appears that only a small percentage of rat brain 5-HIAA2, 31 and H V A 1 are eliminated via the CSF. It remains to be determined whether the age-related changes seen in CSF levels are a reflection of a shift in elimination routes

made in children and stimulate research into the functional significance of the marked changes seen in children from the neonatal period through approximately 10 years of age.

or a result of changes in brain production of the compounds. Careful studies of central 5-HT and D A

ACKNOWLEDGEMENTS

turnover and the rates and routes of D O P A C , H V A , and 5 - H I A A elimination over the crucial 12- to 42day-old age range should answer that basic question. It would appear that declines seen in rats3, 40 for CSF

This work

was

supported

by

NIMH

Grant

MH30929, N I H G r a n t MH24393, and the MacArthur Foundation.

5 - H I A A and H V A over that period serve as a good

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