Early induction of rat brain tryptophan hydroxylase (TPH) mRNA following parachlorophenylalanine (PCPA) treatment

Molecular Brain Research, 22 (1994) 20-28 © 1994 Elsevier Science B.V. All rights reserved 0169-328X/94/$07.00

20

BRESM 70706

Early induction of rat brain tryptophan hydroxylase (TPH) mRNA following parachlorophenylalanine (PCPA) treatment D.H. Park *, D.M. Stone, H. Baker, K.S. Kim, T.H. Joh Laboratory of Molecular Neurobiology, Cornell Uniuersity Medical College, The W.M. Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, N Y 10605, USA (Accepted 10 August 1993)

Key words: In situ hybridization; mRNA; Immunohistochemistry; Serotonin; p-Chlorophenylalanine; Tryptophan hydroxylase; Gene regulation

Tryptophan hydroxylase (TPH) is the first and presumably rate-limiting enzyme in serotonin (5-HT) biosynthesis. End-product inhibition of rate-limiting enzymes is common and 5-HT is known to inhibit TPH activity in vivo. However, it is not known whether levels of 5-HT could also be involved in the regulation of the TPH gene. In order to determine whether TPH gene regulation is dependent on the 5-HT concentration, 5-HT levels were reduced by the administration of parachlorophenylalanine (PCPA). PCPA is a potent, specific and irreversible inhibitor of TPH activity which drastically reduces 5-HT concentration in the 5-HT neurons and terminals. When PCPA was administered, TPH activity in both cell bodies and nerve terminal areas, was reduced to 10% of control values and recovered to the control levels by day 7 in raphe nucleus, and within 14 days in the hypothalamus. In serotonergic terminal areas, 5-HT could not be detected immunohistochemically at day 1, but slowly recovered within 2 weeks. At all time points examined, aromatic e-amino acid decarboxylase (AADC) levels were not changed either in the cell body or terminal areas. The steady state levels of TPH mRNA estimated by in situ hybridization increased at day 1 and returned to control levels by day 4. AADC message levels were not altered throughout the periods. These data suggest that a decrease in 5-HT concentration may lead to an up-regulation of TPH gene transcription, by an, as yet, unknown mechanism. Taken together, our results suggest that feedback control of the rate-limiting enzyme, TPH, may occur not only through changes in enzyme activity, but also through changes in gene transcription (Preliminary results were published by Park et al. [Soc. Neurosci. Abstr., 17 (1991) 1176] at the Neuroscience Meeting in 1991).

INTRODUCTION Tryptophan hydroxylase (TPH, EC 1.14.16.4) is the first and rate-limiting enzyme in serotonin (5-HT) biosynthesiss'1°'17. A hallmark of the functional control of enzymatic activity of the rate-limiting enzyme in a particular biosynthetic pathway is that activity is inhibited by the end-product(s). For instance, the enzyme activity of tyrosine hydroxylase (TH), considered the rate-limiting enzyme in the biosynthetic pathway of catecholamines 16'2°, is inhibited by its end-products, dopamine and norepinephrine 21'29. Chronic administration of L-dihydroxyphenylalanine (L-DOPA), a direct product of TH enzyme and a precursor of catecholamines, reduces TH activity both in brain 28 and adrenal 34. A similar negative feedback by the endproduct at the rate-limiting step has been observed for the 5-HT biosynthetic pathway. When a monoamine oxidase inhibitor was administered, 5-HT levels in-

* Corresponding author. Fax: (1) (914) 948-9541.

SSD1 0 1 6 9 - 3 2 8 X ( 9 3 ) E 0 1 4 5 - P

creased at the 5-HT nerve terminals and the conversion of [3H]tryptophan to [3H]5-HT was markedly decreased 5'9'19. Although there are controversial reports indicating that high concentrations of 5-HT were unable to inhibit TPH activity in vitro 12'37, it is generally believed that 5-HT inhibits TPH activity in vivo9. An intriguing question is whether the end-product is ever involved in the regulation of gene for the rate-limiting enzyme, possibly at the transcriptional level. Accumulation of 5-HT by the inhibition of monoamine oxidase 5'9'19 was observed in the 5-HT nerve endings; however this treatment may not be an effective means for increasing 5-HT in the cell bodies. Therefore, we elected instead to deplete 5-HT in vivo, and look for possible consequent changes in TPH gene expression. Parachlorophenylalanine (PCPA) is a potent and specific inhibitor of TPH activity14. When this drug is administered, TPH activity is drastically inhibited and the brain 5-HT levels are severely reduced 14'27'36. Thus, we administered PCPA and investigated the levels of TPH in 5-HT cell bodies and nerve terminals, the levels of 5-HT in nerve terminals and the steady-state

21

c

~,~(11)[

4-

"~ 16.

"" c

12'

o.

[Z

E

E

8

3J

2.

MAM)C

E =~

*(7)

*(7)

2

4 ~ 0 "1-

o 0

.

0

o

k

B

I~

;

,s

(days) Fig. 1. TPH and AADC activities in the dorsal raphe nucleus (DRN) following PCPA treatment. The numbers in the parentheses indicate the number of animals used for the experiment. The bars indicate m e a n + S.E.M. * P < 0.001 when compared to saline-injected control. Note that PCPA treatment led to a 90% decrease in TPH activity in the DRN between 1 and 2 days post-treatment followed by gradual recovery to control level by 7 days, while AADC activity was not noticeably changed during the period. time

levels of TPH mRNA in cell body regions by in situ hybridization using a TPH cDNA t3. We report here that depletion of 5-HT by PCPA up-regulates TPH gene expression as judged by the increase in the steady state levels of TPH mRNA (Preliminary results were presented at the Neuroscience Meeting, 1991) 24. These data suggest that the level of 5-HT may influence gene transcription of TPH in vivo. MATERIALS AND METHODS Materials D,L-Parachlorophenylalanine methyl ester (PCPA) was obtained from Sigma Chemical Co. (St. Louis, MO), T7 RNA polymerase from New England Biolabs (Beverly, MA), Bluescript II plasmid vector from Stratagene Cloning Systems (La Jolla, CA), and RNase A from Boehringer Mannheim Biochemicals (Indianapolis, IN). D,L[1-14C]DOPA, and [3SS]uridine 5'-(a-thio)tripbosphate were purchased from Dupont-NEN (Boston, MA) and L-[1-z4C]tryptophan from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Animals Sprague-Dawley male rats weighing 125-150 g, obtained from Charles River (Boston, MA), were subcutaneously injected with PCPA (300 m g / k g / m l of saline) or vehicle (saline). For biochemical assays, animals were anesthetized by inhalation of CO 2 (a few min on dry ice) and killed by decapitation at 1, 2, 4, 7, and 14 days post-treatment and dorsal raphe nucleus (DRN), caudal brainstem and hypothalamus were immediately dissected for biochemical measurements. The landmarks utilized in the dissection of these structures 25 were as follows: for the dorsal raphe nucleus, the cerebellum was removed and the brain coronally transected at the level of the inferior colliculus. The anterior border was defined by the mammillary bodies. Two mm of tissue ventral to the aqueduct and dorsal to the peduncles was then dissected. The block was further trimmed by removal of 1 mm from each lateral side. The caudal brainstem was comprised of the remaining caudal tissue anterior to the spinal cord. The hypothalamus was dissected using the optic chiasm as the anterior border, the hypothalamic sulci as the lateral borders and the mammillary bodies as the caudal border.

3

lb

is

0

tlme (days)

Fig. 2. TPH and AADC activities in the caudal brainstem of rats treated with PCPA. The numbers in the parentheses, the bars and * P are the same as described in Fig. 1 legend. PCPA administration reduced TPH activity by 90% between 1 and 2 day post-treatment followed by full recovery to control level by 1 week, while AADC activity was not altered.

Enzyme assays For determination of enzyme activity, tissues were homogenized in 50 mM Tris-acetate buffer, pH 7.5 containing 2 mM dithiothreitol (DTT), as follows: dorsal raphe nucleus in 500/zl; caudal brainstem in 1200 tzl; and hypothalamus in 400/zl. Following centrifugation at 10,000 × g for 10 min, the supernatant was used for enzyme assays and determination of protein concentration. TPH activity was determined as described elsewhere in detail 23, using 37.5 nmol of tryptophan as a substrate in a total volume of 150/~1. Aromatic L-amino acid decarboxylase (AADC) activity was assayed by a minor modification 22 of the method of Lamprecht and Coyle is, using 30 mM potassium phosphate buffer, pH 7.0. Protein concentration was measured according to Lowry et al. TM, using bovine serum albumin as standard. lmmunocytochemistry and in situ hybridization At 0, 12 h, 1, 2, 4, 7, and 14 days after PCPA administration, 2-4 rats from each group were processed for both immunocytochemistry and in situ hybridization histochemistry, using adjacent tissue sections. Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and rapidly perfused transcardially with 50-100 ml heparinized saline containing 0.5% sodium nitrite, following by 400 ml of 0.1 M sodium phosphate buffer, pH 7.2, containing 4% formaldehyde generated from paraformaldehyde. The brain was removed and a coronal tissue block containing the midbrain raphe nuclei was post-fixed in 4%

2.=i

I0.0 8o ;~-

el

5" 3 o

-~ 1.0(3. 5.0

AAAI~

} o -I0.00 0

time (dG~e~)

15

0.0 ~

Fig. 3. TPH and AADC activities in hypothalamus after PCPA treatment. See the legend of Fig. 1 for the numbers in the parentheses, the bars and * P. Note that TPH activity was decreased markedly 1 day post-PCPA but its full restoration to saline-injected control level was slower than that in cell body areas. AADC activity was unchanged.

22

Fig. 4. In situ hybridization to TPH mRNA in the dorsal raphe nucleus (DRN). Dark-field photomicrographs illustrate hybridization signals for TPH message of saline-injected control animals (A) and those at 12 h (B), 1 day (C) and 4 days post-PCPA (D). Note that the hybridization signal for TPH mRNA increased almost 2-fold by 1 day post-PCPA and returned to control level by 4 days. Bar = 250/xm; V, ventricle.

23 buffered formaldehyde for 1 h, then rinsed in buffer and placed overnight in 30% sucrose (4°C). Coronal tissue sections of 36 p.m thickness were cut on a sliding microtome and collected in either standard wells filled with 0.1 M sodium phosphate buffer (for immunocytochemistry) or stored in 20-ml glass vials filled with 2 × SSC (1 X SSC; 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) and 20 mM DTT at 4°C (for in situ hybridization). Serial sections were taken in groups of four: one each for 5-HT immunostaining, AADC immunostaining, and for both AADC and TPH in situ hybridization. Immunocytochemical staining procedures were as previously described 2s'32. The tissue sections were incubated with specific antiserum to 5-HT (diluted 1/30,000) and AADC (diluted 1/15,000). The specificity of the 5-HT antiserum and AADC antiserum was determined as described by Towle et al. 33 and Baker 2, respectively. For in situ hybridization, 2:< SSC and 20 mM D'Iq" was removed from vials and replaced with prehybridization buffer (75% formamide, 10% dextran sulfate, 2 x SSC, 1 × Denhardt's, 50 mM DTI', 0.5 m g / m l yeast tRNA and 0.5 m g / m l sonicated denatured salmon sperm DNA). As previously described 25'32, tissue sections were prehybridized for 2-3 h (42°C), appropriate 35S-labeled denatured probe

was added to each vial (1 × 106 cpm per section), and sections were hybridized overnight (48"C for TPH and AADC probe) and washed. Tissue sections from different treatment groups were processed in the same vial to ensure identity of probe hybridization and washing conditions (sections from the different groups were identified by differentially placed needle holes). Sections were mounted onto acid-washed, gelatin-subbed slides, which were subsequently air-dried and dehydrated through graded ethanols (70, 95, 100%). Slides were apposed to Kodak XAR-5 film for 2-4 days, then dipped in Kodak NTB-2 emulsion and exposed for 1-4 weeks at 4°C. After developing slides in Kodak D-19, sections were counterstained with Cresyl violet and slides were dehydrated and coverslipped with permount (Fisher Scientific). The probe for TPH was a mixture of two 35S-labeled TPH RNA transcripts, antisense to bases 1-493 and 493-1335 of rat TPH cDNA 13'25, The two fragments were subcloned separately into the EcoRI site of Bluescript plasmid; after confirming insert orientation, the plasmids were linearized with EcoRV, and RNA probes were synthesized using T7 RNA polyrnerase and [35S]-UTP, to a specific activity of 1 x 108 cpm//~g. TPH probe specificity was established based on the correspondence between regions of dense probe labeling (a strong hybridization signal for TPH was detected in pineal

Fig. 5. Bright-field photomicrographs of in situ hybridization signals for TPH mRNA in the dorsal raphe nucleus (DRN). Note that grain density on cell in the DRN was markedly increased on 1 day post-PCPA (B), compared to those of saline-injected control (A) as shown in high power bright-field photomicrographs. Bar = 50/~m. mlf, medial longitudinal fasciculus.

24 gland with much weaker signals in the raphe nuclei) and 5-HT immunoreactivity~3'z5.The probe for AADC was a 600 bp fragment of an AADC clone subcloned in Bluescript, linearized with Hindltl and transcribed with SP6 25'32.All other procedures were identical to those for TPH. For quantitative analysis of in situ autoradiograms, an optical density measurement of 4 to 5 sections of the most heavily-labelled dorsal raphe region from each rat was determined using a Microscan Image Analysis System (Technology Resources, Inc.). RESULTS

TPH and AADC activities T P H activity in both cell body regions and the nerve endings of 5-HT neurons was inhibited by the administration of PCPA. Enzyme activity was reduced to 10% of the corresponding saline-injected control within 1 day post-PCPA and recovered within 7 days in dorsal raphe nucleus and caudal brainstem (Figs. 1 and 2). The recovery of T P H activity in hypothalamus was slower than in the other two areas and returned to the saline-injected control level within 14 days (Fig. 3). In contrast, A A D C activity in these areas was unchanged throughout the experimental period (Figs. 1, 2 and 3). In situ hybridization and immunocytochemistry T P H m R N A level in dorsal raphe nucleus following PCPA treatment was assessed by in situ hybridization

histochemistry. Fig. 4 illustrates hybridization signals for T P H m R N A in the D R N of control animals (4A) and those at 12 h (4B), 1 day (4C) and 4 days (4D) post-PCPA. The level of T P H message in the D R N increased markedly by 1 day post-PCPA and returned to control levels by 4 days. In addition, grain density on cells in the D R N was markedly increased on 1 day post-PCPA (Fig. 5B), compared to those of control (Fig. 5A). However, no increase was observed in A A D C m R N A at 1 day post-PCPA [Fig. 6 control (A), 1 day (B)]. Densitometric estimation of in situ autoradiograms for T P H m R N A of D R N shows that hybridization signal at 1 day post-PCPA was increased about two-fold over control. At all other time points examined, there was no noticeable alteration observed in the hybridization signal for T P H m R N A . A similar comparison for A A D C m R N A revealed no significant difference between control (Fig. 6A) and 1 day postPCPA (Fig. 6B) groups. One day following PCPA administration, fibers containing 5-HT immunoreactivity were almost undetectable in the superior colliculus (Fig. 7B) and other brain regions (not illustrated) whereas large number of serotonergic terminals were observed in saline-injected control animals (Fig. 7A). Immunostaining in the superior colliculus was still dramatically reduced at 4 days

Fig. 6. In situ hybridization to AADC mRNA in the dorsal raphe nucleus. Dark-field photomicrographs illustrate hybridization signals for AADC message of saline-injected control animals (A) and those at 1 day (B) post-PCPA. Note that there is no increase in the hybridization signal for AADC mRNA between control and 1 day post-PCPA groups. Bar = 250/xm. V, ventricle.

25

Fig. 7. Dark-field photomicrographsof serotonin immunostaining in superior coUiculus(SC). Note complete absence of 5-HT-labelled terminals 1 day (B) and 4 day (C) post-PCPAand complete labelling recovery2 weeks (D) to control level (A). Bar = 100/~m. (Fig. 7C) but within 2 weeks fully returned to control levels in both number as well as staining intensity (Fig. 7D). A A D C immunostaining in the dorsal raphe was unchanged at all time points (data not shown).

DISCUSSION The steady-state levels of neurotransmitters in neurons are controlled by many biochemical and physio-

26

logical factors. One of these factors is the activity of the rate-limiting enzyme in the neurotransmitter biosynthetic pathway. The regulation of the rate-limiting enzyme directly affects the levels of neurotransmitters. In vivo, TPH is presumably the rate-limiting enzyme8'17 in 5-HT biosynthesis, and thus, TPH activity primarily dictates 5-HT levels. Because of the lack of appropriate probes, previous studies of the regulation of TPH have been at the protein level. The isolation of a specific cDNA for TPH and the availability of a sensitive in situ hybridization technique permitted us to characterize changes in the levels of mRNA following the administration of an irreversible inhibitor of TPH enzyme, PCPA. As previously observed, PCPA reduced TPH activity in both cell body and nerve terminal regions. Within 1 day of treatment, TPH activity was reduced to 10% o f control levels in both areas and did not return to control levels until day 7 in the dorsal raphe nucleus where 5-HT cell bodies are located. In the hypothalamus, where the nerve endings reside, the return of activity was slower requiring between 7 and 14 days. Since PCPA is an irreversible inhibitor of TPH, the recovery of TPH activity most likely requires the synthesis of new TPH enzyme. The slower recovery period in the nerve ending regions is probably a result of the need to transport newly synthesized TPH from the cell bodies to the terminals. The time-course of the recovery of TPH activity in caudal brainstem resembles that in DRN and may be attributed to the proximity of this area to the cell bodies in the caudal raphe nuclei. Staining with antisera to 5-HT supported the changes in TPH activity in the terminal regions. In contrast to the decrease in TPH activity, the steady state levels of TPH mRNA were increased at 1 day post-treatment. Recently, Cortes et al. 6 also reported that TPH message was increased one day postPCPA. Although these results are in agreement with ours the precise distribution and identity of the neurons with altered mRNA levels was difficult to discern in their photomicrographs. Perhaps, the oligonucleotides probes ( - 5 0 bp) they used may not be long enough to demonstrate all the TPH message present in this region. In contrast, our current study as well as previous in situ hybridization studies demonstrated an identical distribution of TPH message and either TPH protein or 5-HT in cells of the dorsal raphe nuclei3'7'l 1,13,25,31,35. Presently, the mechanisms underlying the regulation of TPH mRNA levels is not known. At least two possible mechanisms could be postulated for the observed elevation in TPH mRNA. First, TPH gene transcription may increase, that is a decrease in the

intracellular 5-HT concentration may lead to an upregulation of TPH mRNA levels through synthesis of new message. Alternatively, stabilization of TPH mRNA already present in 5-HT neuronal cell bodies may occur. The increase in mRNA is relatively short lived (less than 4 days). It is possible that the increase in TPH mRNA immediately results in new protein synthesis, and as mentioned above an increase in the levels of the product, 5-HT. Regardless of the mechanism, higher levels of product would result in a return to normal levels of message. To distinguish between these mechanisms requires further experimentation. However, gene regulation for other rate-limiting monoaminergic enzymes has been investigated. For tyrosine hydroxylase, a monooxygenase with sequence homology to TPH, treatments such as reserpine and stress are postulated to increase activity through a gene transcription mechanism 4'3°. The regulation of TPH may be analogous with that observed for TH, supporting the hypothesis that the increase in steady-state levels of TPH occurs through alterations in gene transcription. Relative estimation of the message in these studies was performed by in situ hybridization analysis. The more precise quantitative procedure of Northern blot analysis could not be applied because of the relatively low levels of TPH mRNA in the central nervous system 7'13. The relatively low levels of TPH mRNA is in contrast with the high enzyme activity observed in the CNS especially as compared to the pineal gland which contains high mRNA levels and relatively low enzyme activity. Dumas et al. 7 and Kim et al. t3 have estimated that 100 to 150 times more mRNA exists in the pineal gland than in the brainstem. Since the same gene appears to code for TPH in both brainstem and pineal, the data suggest that regulation probably occur posttranscriptionally. While differential translational efficiency has been suggested as a mechanism7'~3, alternative strategies such as phosphorylation efficiency or end-product inhibition of enzyme activity could be postulated. In these studies, PCPA administration did not alter the enzyme activity and the mRNA levels of the second enzyme in 5-HT biosynthesis, AADC. The data suggest that not all enzymes in the pathway are similarly regulated. Previous studies also indicate that under some experimental conditions when other enzymes are altered either protein or mRNA levels for AADC are not changed 1'2'26"32. From the results of the present study we are tempted to speculate that the end-product, 5-HT, may play a role in gene transcription of TPH, the rate-limiting enzyme for 5-HT synthesis. Previously, various bio-

27

chemical studies suggest that the end-product usually inhibits either the first a n d / o r the rate-limiting enzyme in a biosynthetic pathway, a process known as feedback inhibition. However, the generality of endproduct participation in gene regulation of rate limiting enzymes remains to be established. In addition, further investigation is required to understand the precise molecular mechanisms underlying feed-back control of neurotransmitter genes. Acknowledgements. We would like to extend our appreciation to Mr. Charles Carver and Kimberly Morel for their outstanding work in preparing the figures and the photographs. This work was supported by National Institute of Health Grant MH 44043. REFERENCES 1 Baker, H., Kawano, T., Albert, V., Joh, T.H., Reis, D.J. and Margolis, F.L., Olfactory bulb dopamine neurons survive deafferentation-induced loss of tyrosine hydroxylase, J. Neurosci., 11 (1984) 605-615. 2 Baker, H., Unilateral, neonatal olfactory deprivation alters tyrosine hydroxylase expression but not aromatic amino acid decarboxylase or GABA immunoreactivity, Neuroscience, 36 (1990) 761-771. 3 Bendotti, C., Servadio, A., Forloni, G., Angeretti, N. and Samanin, R., Increased tryptophan hydroxylase mRNA in raphe serotonergic neurons spared by 5,7-dihydroxytryptamine, Mol. Brain Res., 8 (1990) 343-348. 4 Biguet, N.F., Buda, M., Lamouroux, A., Samolyk, D. and Mallet, J., Time course of the changes of TH mRNA in rat brain and adrenal medulla after a single injection of reserpine, EMBO J., 5 (1986) 287-291. 5 Carlsson, A. and Lindqvist, M., The effect of L-tryptophan and some psychotropic drugs on the formation of 5-hydroxytryptophan in the mouse in vivo, J. Neural Transm., 33 (1972) 23-43. 6 Cortes, R., Mengod, G., Celada, P. and Artigas, F,, P-chlorophenylalanine increases tryptophan 5-hydroxylase mRNA levels in the rat dorsal raphe: a time course study using in situ hybridization, J. Neurochem., 60 (1993) 761-764. 7 Dumas, S., Darmon, M.C., Delort, J. and Mallet, J., Differential control of tryptophan hydroxylase expression in raphe and in pineal gland: evidence for a role of translation efficiency, J. Neurosci. Res., 24 (1989) 537-547. 8 Grahame-Smith, D.G., Tryptophan hydroxylation in brain, Biochem. Biophys. Res. Commun. 16 (1964) 586-592. 9 Hamon, M., Bourgoin, S., Morot-Yaudry, Y. and Glowinski, J., End product inhibition of serotonin synthesis in the rat striatum, Nature (New Biol.), 237 (1972) 184-187. 10 Ichiyama, A., Nakamura, S., Nishizuka, Y. and Hayaishi, O., Enzymatic studies on the biosynthesis of serotonin in mammalian brain, J. Biol. Chem., 245 (1970) 1699-1709. 11 Ishimura, K., Takeuchi, Y., Fujiwara, K., Tominaga, M., Yoshioka, H. and Sawada, T., Quantitative analysis of the distribution of serotonin-immunoreactive cell bodies in the mouse brain, Neurosci. Lett., 91 (1988) 265-270. 12 Jequier, E., Robinson, D.S., Lovenberg, W. and Sjoerdsma, A., Further studies on tryptophan hydroxylase in rat brainstem and beef pineal, Biochem. Pharmacol., 18 (1969) 1071-1081. 13 Kim, K.S., Wessel, T.C., Stone, D.M., Carver, C.H., Joh, T.H. and Park, D.H., Molecular cloning and characterization of cDNA encoding tryptophan hydroxylase from rat central serotonergic neurons, Mol. Brain Res., 9 (1991) 277-283. 14 Koe, B.K. and Weissman, A., p-Chlorophenylalanine: a specific depletor of brain serotonin, J. Pharmacol. Exp. Ther., 154 (1966) 499-516.

15 Lamprecht, F. and Coyle, J.T., DOPA decarboxylase in the developing rat brain, Brain Res., 41 (1972) 503-506. 16 Levitt, M., Spector, S., Sjoerdsma, A. and Udenfriend, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmacol. Exp. Ther., 148 (1965) 1-8. 17 Lovenberg, W., Jequier, E. and Sjoerdsma, A., Tryptophan hydroxylases: measurement in pineal gland, brain stem, and carcinoid tumor, Science, 155 (1967) 217-219. 18 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 19 Macon, J.B., Sokoloff, L. and Glowinski, J., Feedback control of rat brain 5-hydroxytryptamine synthesis, J. Neurochem., 18 (1971) 323-331. 20 Nagatsu, T., Levitt, M. and Udenfriend, S., Tyrosine hydroxylase, the initial step in norepinephrine biosynthesis, J. Biol. Chem., 239 (1964) 2910-2917. 21 Neff, N.H. and Costa, E., The influence of monoamine oxidase inhibition on catecholamine synthesis, Life Sci., 5 (1966) 951-959. 22 Park, D.H., Woo, J.I., Hwang, O., Ehrlich, M,, Abate, C. and Job, T.H., Different charge forms of aromatic L-amino acid decarboxylase, Brain Res., 370 (1986) 375-377. 23 Park, D.H., Park, H.S., Joh, T.H., Anwar, M. and Ruggiero, D.A., Strain differences between albino and pigmented rats in monoamine-synthesizing enzyme activities of brain, retina and adrenal gland, Brain Res., 508 (1990) 301-304. 24 Park, D.H., Stone, D.M., Baker, H., Kim, K.S. and Joh, T.H., Early induction of rat brain tryptophan hydroxylase (TPH) mRNA following parachlorophenylalanine (PCPA) treatment, Soc. Neurosci. Abstr., 17 (1991) 1176. 25 Park, D.H., Stone, D.M., Baker, H., Wessel, T.C., Kim, K.S., Towle, A.C. and Joh, T.H., Changes in activity and mRNA for rat tryptophan hydroxylase and aromatic L-amino acid decarboxylase of brain serotonergic cell bodies and terminals following neonatal 5,7-dihydroxytryptamine, Brain Res., 609 (1993) 59-66. 26 Reis, D.J., Joh, T.H. and Ross, R.A., Effects of reserpine on activities and amounts of tyrosine hydroxylase and dopamine fl-hydroxylase in catecholamine neuronal systems in rat brain, J. Pharmacol. Exp. Ther., 193 (1975) 775-784. 27 Richard, F., Sanne, J.L., Bourde, O., Weissmann, D., Ehret, M., Cash, C., Maitre, M. and Pujol, J.F., Variation of tryptophan 5-hydroxylase concentration in the rat raphe dorsalis nucleus after p-chlorophenylalanine administration, I. A model to study the turnover of the enzymatic protein, Brain Res., 536 (1990) 41-45. 28 Roberge, A.G. and Poirier, L.J., Effect of chronically administered L-DOPA on DOPA/5-HTP decarboxylase and tyrosine and tryptophan hydroxylase in cat brain, J. Neural Transm., 34 (1973) 171-185. 29 Spector, S., Gordon, R., Sjoerdsma, A. and Udenfriend, S., End-product inhibition of tyrosine hydroxylase as a possible mechanism for regulation of norepinephrine synthesis, Mol. Pharmacol., 3 (1967) 549-555. 30 Stachowiak, M.IC, Fluharty, S.J., Stricker, E.M., Zigmond, M.J. and Kaplan, B.B., Molecular adaptations in catecholamine biosynthesis induced by cold stress and sympathectomy, Z Neurosci. Res., 16 (1986) 13-24. 31 Steinbusch, H.W.M., Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals, Neuroscience, 6 (1981) 557-618. 32 Stone, D.M., Wessel, T., Joh, T.H. and Baker, H., Decrease in tyrosine hydroxylase, but not aromatic L-amino acid decarboxylase, messenger RNA in rat olfactory bulb following neonatal, unilateral odor deprivation, Mol. Brain Res., 8 (1990) 291-300. 33 Towle, A.C., Breese, G.R., Mueller, R.A., Coyle, S. and Lauder, J.M., Early postnatal administration of 5,7-DHT: effects on serotonergic neurons and terminals, Brain Res., 310 (1984) 67-75. 34 Udenfriend, S. and Dairman, W., The regulation of norepinephrine synthesis. In G. Weber (Ed.), Advances in Enzyme Regulation, Pergamon, New York, 1971,pp. 145-165. 35 Weissmann, D., Belin, M.F., Aguera, M., Meunier, C., Maitre,

28 M., Cash, C.D., Ehret, M., Mandel, P. and Pujol, J.F., Immunohistochemistry of tryptophan hydroxylase in the rat brain, Neuroscience, 23 (1987) 291-304. 36 Weissmann, D., Chamba, G., Debure, L., Rousset, C., Richard, F., Maitre, M and Pujol, J.F., Variation of tryptophan 5-hydroxylase concentration in the rat raphe dorsalis nucleus after p-chloro-

phenylalanine administration. 11. Anatomical distribution of the tryptophan 5-hydroxylase protein and regional variation of its turnover rate, Brain Res., 536 (1990) 46-55. 37 Youdim, M.B.H., Hamon, M. and Bourgoin, S., Properties of partially purified pig brain stem tryptophan hydroxylase, J. Neurochem., 25 (1975) 407-417.