- Email: [email protected]
Antisense oligonucleotide inhibition of tryptophan hydroxylase activity in mouse brain
ELSEVIER
Regulatory Peptides 59 (1995) 163-170
Antisense oligonucleotide inhibition of tryptophan hydroxylase activity in mouse brain M.M. McCarthy a,*, D.A. Nielsen b, D. Goldman b a Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD, USA b Laboratory ofNeurogenetics, NIAAA, NIH, Rockville, MD, USA
Received 10 March 1995; accepted 27 June 1995
Abstract To examine in vivo effectiveness of antisense oligonucleotides against tryptophan hydroxylase (TPH) mRNA, adult male swiss-NIH mice were implanted with in-dwelling cannula into the 4th ventricle and after recovery infused with either antisense oligonucleotide to TPH, scrambled control oligo or saline vehicle for four consecutive days. An additional group of animals bearing cannu'~la were injected a single time i.p. with the TPH inhibitor para-chlorophenylalanine (PCPA; 300 mg/kg). All animals were sacrificed on the afternoon of the 4th day of treatment. TPH activity was measured by enzymatic assay and HPLC quantification of end-product synthesis. There was a significant decrease ( > 50%) in TPH activity in both the PCPA-treated and antisense-oligo infused animals compared to either scrambled-oligo or saline-infused subjects (ANOVA; P < 0.05). There was no difference between saline and scrambled oligo-infusion. In a separate group of animals treated in the same way, behavioral tests were conducted on the afternoon of the 4th day. Two tests of anxiety, the hole-board apparatus and the elevated plus-maze, indicated some significant effects of PCPA treatment and/or antisense oligo-infusion but confounding effects due to alterations in locomotion could not be ruled out. However, tests on a rotorod apparatus indicated that antisense oligo-infused animals retained good balance and coordination in that their performance significantly improved ,an the second test, as did that of scrambled-oligo infused animals. In contrast, PCPA-treated animals did not improve, suggesting that locomotor performance had been impaired. These data support the notion that antisense oligo blockade may offer advantages over pharmacological manipulations of enzyme activity. Keywords: Tryptophan hydroxylase;Antisense oligonucleotide;Mouse brain; Inhibition
1. Introduction Tryptophan hydroxylase (TPH; EC 1.14.16.4) is the rate-limiting enzyme in the biosynthesis of serotonin [2]. It catalyzes the biopterin-dependent, monooxygenation of tryptophan to 5-hydroxytryptophan (5-HTP), which is subsequently decarboxyl-
* Corresponding author. Fax: + 1 (410) 7068341.
ated to form the neurotransmitter serotonin, also called 5-HT. The substrate for TPH, tryptophan, is present in brain at close to saturating concentrations so it is the amount of TPH and its level of activity that regulates 5-HT levels. Serotonergic neurons are predominantly found in the seven raphe nuclei of the brain stem and coalesce into three major ascending projections to the forebrain. A clear distinction is made between two pathways projecting to the nigro-striatal system, and
0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0167-0115(95)00102-6
164
M.M. McCarthy et al./ RegulatoryPeptides59 (1995) 163-170
arising exclusively from the dorsal raphe nucleus, and a ventral pathway arising from the ventral raphe and projecting to limbic and cortical systems (see [6]). A full length, murine TPH cDNA has been cloned [13,14] and the sequence encodes a 447 amino acid protein. Sequence analysis of the 5' upstream region indicates the presence of many putative regulatory elements. In addition to the conserved TATA and CCAAT boxes, there are sequences related to the consensus binding sequences of AP-1, AP-2 and AP-3. Regulatory sites are present for cyclic AMP regulation, Spl, NF-1, metals, glucocorticoid and octomer binding sites (Nielsen, unpublished observations). Thus there is considerable potential for regulation of TPH gene expression. The role of 5-HT in various behaviors has been investigated pharmacologically as well as by inhibition of 5-HT by para-chloroamphetamine (PCPA) and lesioning of serotonergic neurons by the toxin, 5,7-dihydroxytryptamine (5,7-DHT). In the rat, TPH concentration has been found to decrease between 44 and 63% two days after administration of PCPA [18]. We report here the use of antisense oligonucleotides against TPH mRNA administered directly into the mouse brain to alter TPH activity and serotonin synthesis, resulting in modulation of anxiety-related behaviors. The antisense oligo treatment was equally effective at reducing TPH activity to that seen with PCPA but possibly with fewer side effects.
2. M e t h o d s
Animals Adult male NIH-Swiss mice were cannulated into tlae 4th ventricle using PE-10 tubing pre-cut to the appropriate length and secured in place by an additional piece of plastic tubing around the cannula that was super-glued to the skull. Cannula were kept sealed until the time of infusion with a small piece of stainless steel wire. Surgery was performed under pentobarbital anesthesia (50 mg/kg/i.p.), after which animals were individually housed on a 12/12 h light/dark cycle with food and water available ad lib. The mice weighed between 22 and 28 g at the time of testing and were naive to the testing apparatuses.
Drug treatments Oligonucleotides. The oligonucleotides were obtained from a commercial source (Oligos Etc., Wilsonville, OR) and were HPLC purified. The sequence of the antisense oligo was: 5'-ATCATGGTGAATCTG-3' and was in the region of the translation start codon. The control oligo was a scrambled sequence, 5 ' - T C G A A T C G T T A A T C G - ' 3 with roughly the same nucleotide content as the antisense oligo and with no obvious homology to cDNA sequences available in the Genbank database. Oligos were dissolved in saline at a concentration of 1 /xg//.*l and infusion volume was 4 /zl, for a total dose of 4/xg. Control infusions consisted of an equal volume of saline. Para-chlorophenylalanine (PCPA). As an additional comparison, animals with in-dwelling cannula received a single i.p. injection of PCPA (Sigma) at a dose of 300 m g / k g BW coincident with the first infusion of oligonucleotides. Intraventicular infusions All infusions were performed in the morning during the light portion of the light/dark cycle. Animals were lightly anesthetized with the inhalant anesthetic Flurothane, the stainless steel wire removed, and infused with 4 / z l of saline using a 10-/~1 Hamilton syringe connected to PE-10 tubing and a 33-g syringe needle. Infusions lasted approximately 2 min with the needle left in place an additional 30 s before removal and replacement of the stainless steel wire. Animals were infused on four consecutive days and either sacrificed or behaviorally tested on the afternoon of the 4th day. Behavioral tests Holeboard testing. The holeboard consisted of a Plexiglas box (40 × 40 × 30 cm) the floor of which had four equally spaced holes, 3 cm in diameter. In two opposite walls, 2 cm above the floor, were four equally spaced infra-red photobeams to measure movement in the box. There were also photobeams beneath each hole to measure the number and duration of head-dips. The apparatus was interfaced with a PDP- 11 computer running SKED-11 software (State Systems Inc., Kalamazoo, MI). The test was conducted in a dimly lit quiet room and involved placing a mouse in the center of the floor and allowing it to
M.M. McCarthyet al./Regulatory Peptides59 (1995) 163-170
explore for 5 min. This test was always conducted prior to the elevated plus-maze test. Elevated plus-maze. The plus-maze was made of black Plexiglas and consisted of two opposite facing open arms (30 x 5 cra) and two closed arms (30 × 5 cm) with a 5 × 5 cm central area. The closed arms had clear 15 cm high Plexiglas walls which were open at the ends. The whole plus-maze apparatus was mounted on a base which raised it 38.5 cm above the floor. The testing involved placing the mouse in the center of the maze and allowing it to move around the apparatus for 5 min. The number and duration of entries into either the open or closed arms were recorded using an event recorder interfaced with a PDP-11 computer running SKED-11 software. The principal measures taken were: (1) the percentage of the total number of entries which were into open arms; (2) the percentage of the total time spent on the arms that was spent on the open arms; and (3) the total number of arm-entries. Testing was conducted in a dimly lit quiet room immediately after the holeboard procedure and was based on previous procedures used in this laboratory [4]. Rotorod. Animals were tested on an automated rotorod apparatus (Harvard Apparatus) for two trials separated by 1-3 h. These were conducted on the same day and after the hole-board and elevated plus-maze behavioral tests. The animal was placed on the immobile rod and the apparatus turned on. The speed of rotation accelerated at a constant rate during the 5 min te:st. The number of revolutions successfully completed by the animal was counted automatically with a cutoff of 300 revolutions. Tissue On the afternoon of the 4th consecutive day of oligo infusions, some animals were sacrificed by cervical dislocation, tire brains removed and the midbrain region containing the raphe nuclei rapidly dissected and frozen on dry ice. Tissue was weighed and then homogenized in 1:2 weight/volume of buffer. Tryptophan hydroxylr, tse activity assay Supematant extracts of frozen tissue were prepared and the tryptophan hydroxylase activity was
165
measured the same day. The enzyme assay is based on that of [1] and involved conversion of tryptophan to 5-HTP measured in the presence of a decarboxylase inhibitor to prevent further metabolism to 5-HT. Tissue samples were homogenized in 2 volumes Tris-HC1 buffer (50 /~M, pH 7.4) with a Tissue Tearer (45 s at setting 5; Biospec Product Inc., Racine, WI). The homogenate was centrifuged at 33,000 g for 30 rain at 4°C. Dithiotriotol, in a final concentration of 2 mM was added to the resulting supematant fraction which was then run through a Sephadex G-25M column (Pharmacia) equilibrated with 0.05 M Tris-HC1 (pH 7.4). This filtration step removed small molecules such as endogenous tryptophan. The pink-colored protein fraction which appeared in the void volume of the column was collected and 50/.d aliquots were assayed. The reaction was carried out in a total volume of 300/~1 in buffer containing Tris-HC1 (20 ~M), NADPH, 6MPH4 (50 /zM), D T r (2raM) brocresine (40 nmoles) and catalase (1000 U). After a 10 min preincubation at 37°C, the reaction was initiated by addition of 0.06/~moles L-tryptophan (D-tryptophan was added to the blanks and standards) and incubated at 37°C for 20 min. The final concentration of L-tryptophan was 200 /.~M. The enzyme reaction was terminated with 30 /.d of 70% perchloric acid and the precipitated protein removed by centrifugation for 30 s at 12,000 g in an Eppendorf microfuge. The 5-HTP formed was separated from the other assay constituents by high-pressure liquid chromatography (HPLC) with a mobile phase of 0.075 M sodium phosphate buffer, 0.038 mM EDTA, 0.646 mM octyl sodium sulfate and 0.01% triethylamine. The pH was adjusted to 3.0 with phosphoric acid and acetonitrile added to a final concentration of 13%. The 5-HTP was then quantified in a 20 /zl injection volume by electrochemical detection using a Coulochem detector (Model 5100A) set at a 0.5 ml/min flow rate. A set of standards in the range of 220 to 1400 picograms of 5-HTP were run with each assay. Protein was quantified using the DC Protein Assay kit (Bio-Rad). Data are expressed as pmol 5 - H T P / m g protein/min. Statistics Data were analyzed by one-way ANOVA with a post-hoc Neuman-Keuls to establish a value of P <
M.M. McCarthyet al. /Regulatory Peptides59 (1995) 163-170
166
Experiment two: effect of antisense oligos, scrambled oligo and P C P A on anxiety and locomotor behavior.
0.05. In the case of the rotorod apparatus, the same animals were tested twice and so data were analyzed by paired t-test to compare performance on test # 1 and test # 2 .
Behavioral tests of anxiety There were no effects of antisense or scrambled oligos on behavior in the hole-board apparatus. However, animals treated with P C P A exhibited a significantly decreased amount of time spent head-dipping compared to the other two groups ( A N O V A ; F[2,9] = 6.27; P < 0.05; see Fig. 2). There was no effect of P C P A treatment on the number o f beams crossed or the number of head dips. On the elevated plus-maze there were significantly fewer entrances onto the open arms by antisense oligo-infused and PCPA-treated animals compared to scrambled oligo-infused mice ( A N O V A ; F[2,9] = 5.7; P < 0.05). There were no other significant differences but a notably high level of variability (S.E.M. was greater than the mean) in the time spent on the open arm in animals treated with" P C P A (see Fig. 3).
3. Results
3.1. Administration of antisense and scrambled oligos in vivo Experiment one: effect of antisense oligos, scrambled oligo and P C P A on tryptophan hydroxylase activity in brain tissue. There was a significant decrease in tryptophan hydroxylase activity as indicated by a decreased rate of synthesis of 5-HTP in brain tissue microdissected from the region of the dorsal raphe in animals infused daily with antisense oligonucleotide or given an i.p. injection of P C P A compared to either animals infused with saline or scrambled oligo ( A N O V A ; F[3,14] = 3.90; P < 0.05; see Fig. 1). There was no difference in the mean mg P / s a m p l e across groups.
Locomotor behavior A l l animals performed equally well on the first rotorod test. However, on the second test both the
30-
E 20
¸
10
¸
T
T
[] [] P'] []
SALINE(n=6) PCPA(n--4) SCRAMBLEDOLIGO(n=5) ANTISENSEOLIGO(n=5)
r~ [...
Fig. 1. Tryptophan hydroxylase activity in dorsal medulla. Animals were either injected i.p. with PCPA (300 mg/kg) once or were infused daily with antisense oligo, scrambled oligo or saline for 4 consecutive days and sacrificed on the afternoon of the 4th day. TPH activity was measured in the dorsal medulla by enzymatic assay and HPLC quantification of the end-product, 5-HTP. There was a significant decrease in TPH activity in antisense oligo-infused and PCPA-treated male mice compared to saline and serambled-oligo infused animals (ANOVA; *P < 0.05).
167
M.M. McCarthy et aL /Regulatory Peptides 59 (1995) 163-170 200
300
t, ,.¢
'
2oo,
-~ _= 100
100
o
0 # of BEAMS
# of HEAD DIPS
Fig. 2. Effect of oligo infusions on behavior in the hole-board apparatus. A separate set of animals were either injected i.p. with PCPA (300 mg/kg) once or were infused daily with antisense or scrambled oligo for 4 consecutive days and tested on the afternoon of the 4th day. There was a significant decrease in the time spent head-dipping in aniraals treated with PCPA as compared to the antisense and scrambled oligo-infused'animals (ANOVA; *P < 0.05).
s c r a m b l e d o l i g o - i n f u s e d and a n t i s e n s e o l i g o - i n f u s e d a n i m a l s s i g n i f i c a n t l y i m p r o v e d their p e r f o r m a n c e (paired t-test; P < 0.05 and P < 0.01, r e s p e c t i v e l y ) , w h e r e a s the p e r f o r m a n c e o f the P C P A - t r e a t e d animals w a s not i m p r o v e d , s u g g e s t i n g a l o c o m o t o r impairment ( s e e Fig. 4). 100
R O T O R O D T E S T #1
TIME DIPPING
R O T O R O D T E S T #2
Fig. 4. Effect of oligo infusions on locomotor behavior. At the completion of the hole-board apparatus and elevated plus-maze behavioral tests, the same animals were tested twice on a rotorod apparatus. The two tests were separated by 1-3 h. Animals infused with scrambled or antisense oligo exhibited a significant improvement in locomotor performance on the second test as indicated by the increased number of revolutions achieved (paired t-test; *P <0.05, * *P <0.01), whereas the PCPA-treated animals showed no improvement.
4. D i s c u s s i o n T h e u s e o f s y n t h e t i c a n t i s e n s e o l i g o n u c l e o t i d e s to s e l e c t i v e l y target m R N A and b l o c k its translation
-
• X
80,
PCPA (n=4)
[]
SCRAMBLED OLIGO (n--4)
•
ANTISENSE OLIGO (n=4)
60 ¸
Z o
40.
20'
0 O P E N ARMS ENTRANCES C L O S E D A R M E N T R A N C E S
TIME O N O P E N A R M S
Fig. 3. Effect of oligo infusions on behavior in the elevated plus-maze. The same animals as in Fig. 2 were tested on the elevated plus-maze immediately after the hole-board apparatus. There was a significant decrease in the number of entrances onto the open arms in both the antisense oligo-infused and the PCPA-treated animals compared to the scrambled-oligo infused animals (ANOVA; *P < 0.05).
168
M.M. McCarthy et al. / Regulatory Peptides 59 (1995) 163-170
into protein is becoming increasingly widespread both in vitro and in vivo. We report here that intracerebroventricular administration of antisense oligos to the mRNA coding for tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, successfully reduced TPH activity and related behavioral parameters in adult male mice. Two major mechanisms, which are not mutually exclusive, have been proposed for antisense oligo blockade of translation in the cell cytoplasm. The first involves the hybridization of the oligo in the upstream region of the mRNA where it either prevents the ribosomal subunit from attaching to the mRNA or blocks movement of the ribosome down the mRNA after it has attached. These mechanisms are frequently referred to as hybridization arrest. The second mechanism involves the activation of the nuclease RNase H which selectively degrades the RNA portion of an RNA-DNA duplex and can greatly increase the efficacy of antisense oligo action (see for review [16]). The oligo can hybridize at any region along the mRNA and still be effective if the RNase H mechanism is acting. The question of whether RNase H is a contributing factor to antisense oligo action in the brain remains unresolved (see [8]). A potential limiting factor in antisense oligo efficacy is the cellular content of the targeted mRNA. For example, highly abundant mRNAs such as those for the neuropeptides oxytocin and vasopressin, have proven problematic to reliably and continuously block for extended periods by antisense oligos [5,11]. Alternatively, relatively low abundance mRNAs, such as those for neurotransmitter or steroid receptors have been very effective targets for antisense oligo blockade [9,17]. The TPH mRNA content may have been a critical factor in determining antisense oligo effectiveness in the current experiments. The level of TPH mRNA has been characterized in different brain areas and cells lines. TPH mRNA is found in the pineal gland at 940 copies per cell whereas only 11 copies per cell are found in neurons of the raphe [7] and may make this gene product extremely sensitive to antisense oligo-mediated inhibition. Other variables that can alter the effectiveness of antisense oligos include the rate of translation of the mRNA and the half-life of the mRNA and the synthesized protein. These parameters can differ for
the same protein in different cells. For example, in raphe neurons, significantly more enzyme is translated per mRNA than in the pineal cells [3]. Furthermore, activity is regulated by the stability of the TPH enzyme. In pineal gland~ the enzyme has a half-life of less than 90 min [12,15], while in raphe neurons, it has a 2 - 3 day half-life [10]. The fact that more protein per unit of TPH mRNA is produced in the raphe than the pineal would make this a more desirable target since each mRNA blocked by antisense oligo would result in a comparatively greater reduction in protein synthesis. Alternatively, the greater stability of the protein in the raphe compared to the pineal would require that the antisense oligos be administered for a longer length of time to be effective. The number of cells synthesizing the targeted mRNA could also be a variable in determining antisense effectiveness. In the rat, a population of approximately 4000 TPH synthesizing cells was estimated for the dorsal raphe nucleus and when analyzed along the posteroanterior axis, the TPH distribution within the raphe subdivisions was quite homogeneous [18]. A half-life in the range of 1.6 days for TPH protein was also calculated in this study. Therefore it is apparent that in one system, the TPH synthesizing neurons of the raphe nuclei, there is a constellation of attributes that make it a desirable target for antisense oligo blockade. These include: (1) a relatively small number of cells, (2) with a fairy short half-life for the protein, and (3) a low abundance of mRNA, (4) that is translated at a high rate of protein per unit of TPH mRNA. All of these factors together would suggest that the observed reduction in TPH activity after antisense oligo administration reflects a decrease in TPH protein. However, it must be pointed out that the current experiments did not measure actual TPH protein and the possibility exists for an alternative mechanism of inhibition of TPH activity. An interesting aspect of the current findings is that antisense oligos for TPH appear to be anxiogenic as indicated by a decrease in entrances onto the open arms of an elevated plus-maze when compared to scrambled oligo infusion. Treatment with PCPA had the same effect, as well as a decrease in time spent head-dipping on the hole-board apparatus, also suggestive of increased anxiety. However, tests of anxiety are often difficult to interpret due to
M.M. McCarthy et al. /Regulatory Peptides 59 (1995) 163-170
confounding locomotor effects. There is substantial evidence implicating 5-HT in control of motor behavior. Clearly the dorsal raphe projections to the nigro-striatal system would be expected to influence locomotion and it has been suggested that this projection inhibits general motor output of the basal ganglia. Alternatively, an excitatory 5-HT projection to spinal cord is known to mediate complex patterns of motor behavior and behavioral evidence suggests opposing actions of 5-HT in these two CNS regions (see [6]). Based on these observations it has been proposed that any involvement of 5-HT in anxiety merely reflects a general disruption of locomotor responses that manifests as an apparent change in anxiety, but in fact alters other behavioral responses as well. The current data address this issue. In addition to measures of anxiety, we also measured locomotor coordination on a rotorod apparatus. This behavioral test requires more then generalized movement since the animals must stay balanced with good hind and forelimb strength to stay upright on the continuously accelerating rotating rods. Animals traditionally improve their performance during a second session on the rotorod apparatus and this was observed to be the case for both the antisense oligo and scrambled oligo-infased animals. However, the PCPA-treated animals did not show any improvement on the second test, suggesting that their balance and coordination were severely impaired. Therefore, one side-effect of PCPA treatment may be increased ataxia or other motor impairments and these confounding effects were not observed when TPH activity was reduced by antisense oligonucleotides. However, the possibility that limitations of sample size or procedural factors prevented the accurate assessment of PCPA treatment on locomotor function, cannot be ruled out. Interest in the therapeutic uses of antisense oligonucleotides is continuing to grow. The data presented here demonstrate how target parameters can influence the effectiveness of antisense oligonucleotides in particular systems. The observation that TPH antisense oligo treatment apparently produced less undesirable side.-effects than the TPH inhibitor PCPA, supports the notion that this approach offers additional advantages over those pharmacological agents already available.
169
References [1] Boadle-Biber, M.C., Codey, K.C., Graves, L., Phan, T.H. and Rosecrans, J., Increase in the activity of tryptophan hydroxylase from cortex and midbrain of male Fischer 344 rats in response to acute or repeated sound stress, Brain Res., 482 (1989) 306-316. [2] Cooper, R.R. and Melcer, I., The enzymatic oxidation of tryptophan to 5-hydroxytryptophan in the biosynthesis of serotonin, J. Pharmacol. Exp. Ther., 132 (1961) 265-268. [3] 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 (1980) 537-547. [4] Durcan, M.J., Lister, R.G. and Linnoila, M., Behavioral effects of the inhibitors of phenylethanolamine-N-methyltransferase, LY 78335 and LY 134046, and their interactions with ethanol, Psychopharmacology, 101 (1990) 196-202. [5] Flanagan, L.M., McCarthy, M.M., Brooks, P.J., Pfaff, D.W. and McEwen, B.S., Arginine vasopressin levels (AVP) after daily infusions of antisense oligonucleotides into the supraoptic nucleus (SON), Soc. Neurosci. Abstr., 18 (1992) 205-207. [6] Iversen, S.D., 5-HT and anxiety, Neuropharmacology, 23 (1984) 1553-1560. [7] Kim, K.S., Wesse, 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. [8] McCarthy, M.M., Use of antisense oligonucleotides to block gene expression in the central nervous system. In E.R. de Kloet and W. Sutano (Eds.), Methods in Neurosciences: Neurobiology of Steroids, Vol 22, Academic Press, New York, NY, 1994, pp. 342-356. [9] McCarthy, M.M., Masters, D.B., Rimvall, K., SchwartzGiblin, S. and Pfaff, D.W., Intracerebral administration of antisense oligodeoxynucleotides to GAD65 and GAD67 mRNAs modulate reproductive behavior in the female rat, Brain Res., 636 (1994) 209-220. [10] Meek, J.L. and Neff, N.H., Tryptophan 5-hydroxylase: approximation of half-life and rate of axonal transport, J. Neurochem., 19 (1972) 1519-1525. [11] Neuman, I., Porter, D.W., Landgraf, R. and Pittman, Q.J., Rapid effect on suckling of an oxytocin antisense oligonucleotide administered into rat supraoptic nucleus, Am. J. Physiol., (1994) R842-R858. [12] Sitaram, B.R. and Lees, G.J., Diurnal rhythm and the turnover of tryptophan hydroxylase in the pineal gland of the rat, J. Neurochem., 31 (1978) 1021-1026. [13] Stoll J. and Goldman D., Isolation and structural characterization of the murine tryptophan hydroxylase gene, J, Neurosci. Res., 28 (1991) 457-465. [14] Stoll, J., Kozak, C.A. and Goldman, D., Characterization and chromosomal mapping of a cDNA encoding tryptophan hydroxylase from a mouse mastocytoma cell line, Genomics, 7 (1990) 88-96.
170
M.M. McCarthy et al. / Regulatory Peptides 59 (1995) 163-170
[15] Sugden, D., Grady, R., Jr. and Mefford, I.N., Measurement of tryptophan hydroxylase activity in rat pineal glands and pinealocytes using an HPLC assay with electrochemical detection, J. Pineal Res., 6 (1989) 285-292. [16] Uhlmann, E. and Peyman, A., Antisense oligonucleotides: A new therapeutic principle, Chem, Rev., 90 (1990) 544-584. [17] Wahlestedt, C., Golanov, E., Yamamoto, S. Yee, F., Ericson, H., Yoo, H., Inturrisi, C.E. and Reis, D.J., Antisense oligodeoxynucleotides to NMDA-Ra receptor channel protect
cortical neurons from excitotoxicity and reduce focal ischaemic infarctions, Nature, 363 (1993) 260-263. [18] Weissman, 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-chlorophenylalanine administration. II. Anatomical distribution of the tryptophan-5-hydroxylase protein and regional variation of its turnover rate, Brain Res., 536 (1990) 46-55.
Regulatory Peptides 59 (1995) 163-170
Antisense oligonucleotide inhibition of tryptophan hydroxylase activity in mouse brain M.M. McCarthy a,*, D.A. Nielsen b, D. Goldman b a Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD, USA b Laboratory ofNeurogenetics, NIAAA, NIH, Rockville, MD, USA
Received 10 March 1995; accepted 27 June 1995
Abstract To examine in vivo effectiveness of antisense oligonucleotides against tryptophan hydroxylase (TPH) mRNA, adult male swiss-NIH mice were implanted with in-dwelling cannula into the 4th ventricle and after recovery infused with either antisense oligonucleotide to TPH, scrambled control oligo or saline vehicle for four consecutive days. An additional group of animals bearing cannu'~la were injected a single time i.p. with the TPH inhibitor para-chlorophenylalanine (PCPA; 300 mg/kg). All animals were sacrificed on the afternoon of the 4th day of treatment. TPH activity was measured by enzymatic assay and HPLC quantification of end-product synthesis. There was a significant decrease ( > 50%) in TPH activity in both the PCPA-treated and antisense-oligo infused animals compared to either scrambled-oligo or saline-infused subjects (ANOVA; P < 0.05). There was no difference between saline and scrambled oligo-infusion. In a separate group of animals treated in the same way, behavioral tests were conducted on the afternoon of the 4th day. Two tests of anxiety, the hole-board apparatus and the elevated plus-maze, indicated some significant effects of PCPA treatment and/or antisense oligo-infusion but confounding effects due to alterations in locomotion could not be ruled out. However, tests on a rotorod apparatus indicated that antisense oligo-infused animals retained good balance and coordination in that their performance significantly improved ,an the second test, as did that of scrambled-oligo infused animals. In contrast, PCPA-treated animals did not improve, suggesting that locomotor performance had been impaired. These data support the notion that antisense oligo blockade may offer advantages over pharmacological manipulations of enzyme activity. Keywords: Tryptophan hydroxylase;Antisense oligonucleotide;Mouse brain; Inhibition
1. Introduction Tryptophan hydroxylase (TPH; EC 1.14.16.4) is the rate-limiting enzyme in the biosynthesis of serotonin [2]. It catalyzes the biopterin-dependent, monooxygenation of tryptophan to 5-hydroxytryptophan (5-HTP), which is subsequently decarboxyl-
* Corresponding author. Fax: + 1 (410) 7068341.
ated to form the neurotransmitter serotonin, also called 5-HT. The substrate for TPH, tryptophan, is present in brain at close to saturating concentrations so it is the amount of TPH and its level of activity that regulates 5-HT levels. Serotonergic neurons are predominantly found in the seven raphe nuclei of the brain stem and coalesce into three major ascending projections to the forebrain. A clear distinction is made between two pathways projecting to the nigro-striatal system, and
0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0167-0115(95)00102-6
164
M.M. McCarthy et al./ RegulatoryPeptides59 (1995) 163-170
arising exclusively from the dorsal raphe nucleus, and a ventral pathway arising from the ventral raphe and projecting to limbic and cortical systems (see [6]). A full length, murine TPH cDNA has been cloned [13,14] and the sequence encodes a 447 amino acid protein. Sequence analysis of the 5' upstream region indicates the presence of many putative regulatory elements. In addition to the conserved TATA and CCAAT boxes, there are sequences related to the consensus binding sequences of AP-1, AP-2 and AP-3. Regulatory sites are present for cyclic AMP regulation, Spl, NF-1, metals, glucocorticoid and octomer binding sites (Nielsen, unpublished observations). Thus there is considerable potential for regulation of TPH gene expression. The role of 5-HT in various behaviors has been investigated pharmacologically as well as by inhibition of 5-HT by para-chloroamphetamine (PCPA) and lesioning of serotonergic neurons by the toxin, 5,7-dihydroxytryptamine (5,7-DHT). In the rat, TPH concentration has been found to decrease between 44 and 63% two days after administration of PCPA [18]. We report here the use of antisense oligonucleotides against TPH mRNA administered directly into the mouse brain to alter TPH activity and serotonin synthesis, resulting in modulation of anxiety-related behaviors. The antisense oligo treatment was equally effective at reducing TPH activity to that seen with PCPA but possibly with fewer side effects.
2. M e t h o d s
Animals Adult male NIH-Swiss mice were cannulated into tlae 4th ventricle using PE-10 tubing pre-cut to the appropriate length and secured in place by an additional piece of plastic tubing around the cannula that was super-glued to the skull. Cannula were kept sealed until the time of infusion with a small piece of stainless steel wire. Surgery was performed under pentobarbital anesthesia (50 mg/kg/i.p.), after which animals were individually housed on a 12/12 h light/dark cycle with food and water available ad lib. The mice weighed between 22 and 28 g at the time of testing and were naive to the testing apparatuses.
Drug treatments Oligonucleotides. The oligonucleotides were obtained from a commercial source (Oligos Etc., Wilsonville, OR) and were HPLC purified. The sequence of the antisense oligo was: 5'-ATCATGGTGAATCTG-3' and was in the region of the translation start codon. The control oligo was a scrambled sequence, 5 ' - T C G A A T C G T T A A T C G - ' 3 with roughly the same nucleotide content as the antisense oligo and with no obvious homology to cDNA sequences available in the Genbank database. Oligos were dissolved in saline at a concentration of 1 /xg//.*l and infusion volume was 4 /zl, for a total dose of 4/xg. Control infusions consisted of an equal volume of saline. Para-chlorophenylalanine (PCPA). As an additional comparison, animals with in-dwelling cannula received a single i.p. injection of PCPA (Sigma) at a dose of 300 m g / k g BW coincident with the first infusion of oligonucleotides. Intraventicular infusions All infusions were performed in the morning during the light portion of the light/dark cycle. Animals were lightly anesthetized with the inhalant anesthetic Flurothane, the stainless steel wire removed, and infused with 4 / z l of saline using a 10-/~1 Hamilton syringe connected to PE-10 tubing and a 33-g syringe needle. Infusions lasted approximately 2 min with the needle left in place an additional 30 s before removal and replacement of the stainless steel wire. Animals were infused on four consecutive days and either sacrificed or behaviorally tested on the afternoon of the 4th day. Behavioral tests Holeboard testing. The holeboard consisted of a Plexiglas box (40 × 40 × 30 cm) the floor of which had four equally spaced holes, 3 cm in diameter. In two opposite walls, 2 cm above the floor, were four equally spaced infra-red photobeams to measure movement in the box. There were also photobeams beneath each hole to measure the number and duration of head-dips. The apparatus was interfaced with a PDP- 11 computer running SKED-11 software (State Systems Inc., Kalamazoo, MI). The test was conducted in a dimly lit quiet room and involved placing a mouse in the center of the floor and allowing it to
M.M. McCarthyet al./Regulatory Peptides59 (1995) 163-170
explore for 5 min. This test was always conducted prior to the elevated plus-maze test. Elevated plus-maze. The plus-maze was made of black Plexiglas and consisted of two opposite facing open arms (30 x 5 cra) and two closed arms (30 × 5 cm) with a 5 × 5 cm central area. The closed arms had clear 15 cm high Plexiglas walls which were open at the ends. The whole plus-maze apparatus was mounted on a base which raised it 38.5 cm above the floor. The testing involved placing the mouse in the center of the maze and allowing it to move around the apparatus for 5 min. The number and duration of entries into either the open or closed arms were recorded using an event recorder interfaced with a PDP-11 computer running SKED-11 software. The principal measures taken were: (1) the percentage of the total number of entries which were into open arms; (2) the percentage of the total time spent on the arms that was spent on the open arms; and (3) the total number of arm-entries. Testing was conducted in a dimly lit quiet room immediately after the holeboard procedure and was based on previous procedures used in this laboratory [4]. Rotorod. Animals were tested on an automated rotorod apparatus (Harvard Apparatus) for two trials separated by 1-3 h. These were conducted on the same day and after the hole-board and elevated plus-maze behavioral tests. The animal was placed on the immobile rod and the apparatus turned on. The speed of rotation accelerated at a constant rate during the 5 min te:st. The number of revolutions successfully completed by the animal was counted automatically with a cutoff of 300 revolutions. Tissue On the afternoon of the 4th consecutive day of oligo infusions, some animals were sacrificed by cervical dislocation, tire brains removed and the midbrain region containing the raphe nuclei rapidly dissected and frozen on dry ice. Tissue was weighed and then homogenized in 1:2 weight/volume of buffer. Tryptophan hydroxylr, tse activity assay Supematant extracts of frozen tissue were prepared and the tryptophan hydroxylase activity was
165
measured the same day. The enzyme assay is based on that of [1] and involved conversion of tryptophan to 5-HTP measured in the presence of a decarboxylase inhibitor to prevent further metabolism to 5-HT. Tissue samples were homogenized in 2 volumes Tris-HC1 buffer (50 /~M, pH 7.4) with a Tissue Tearer (45 s at setting 5; Biospec Product Inc., Racine, WI). The homogenate was centrifuged at 33,000 g for 30 rain at 4°C. Dithiotriotol, in a final concentration of 2 mM was added to the resulting supematant fraction which was then run through a Sephadex G-25M column (Pharmacia) equilibrated with 0.05 M Tris-HC1 (pH 7.4). This filtration step removed small molecules such as endogenous tryptophan. The pink-colored protein fraction which appeared in the void volume of the column was collected and 50/.d aliquots were assayed. The reaction was carried out in a total volume of 300/~1 in buffer containing Tris-HC1 (20 ~M), NADPH, 6MPH4 (50 /zM), D T r (2raM) brocresine (40 nmoles) and catalase (1000 U). After a 10 min preincubation at 37°C, the reaction was initiated by addition of 0.06/~moles L-tryptophan (D-tryptophan was added to the blanks and standards) and incubated at 37°C for 20 min. The final concentration of L-tryptophan was 200 /.~M. The enzyme reaction was terminated with 30 /.d of 70% perchloric acid and the precipitated protein removed by centrifugation for 30 s at 12,000 g in an Eppendorf microfuge. The 5-HTP formed was separated from the other assay constituents by high-pressure liquid chromatography (HPLC) with a mobile phase of 0.075 M sodium phosphate buffer, 0.038 mM EDTA, 0.646 mM octyl sodium sulfate and 0.01% triethylamine. The pH was adjusted to 3.0 with phosphoric acid and acetonitrile added to a final concentration of 13%. The 5-HTP was then quantified in a 20 /zl injection volume by electrochemical detection using a Coulochem detector (Model 5100A) set at a 0.5 ml/min flow rate. A set of standards in the range of 220 to 1400 picograms of 5-HTP were run with each assay. Protein was quantified using the DC Protein Assay kit (Bio-Rad). Data are expressed as pmol 5 - H T P / m g protein/min. Statistics Data were analyzed by one-way ANOVA with a post-hoc Neuman-Keuls to establish a value of P <
M.M. McCarthyet al. /Regulatory Peptides59 (1995) 163-170
166
Experiment two: effect of antisense oligos, scrambled oligo and P C P A on anxiety and locomotor behavior.
0.05. In the case of the rotorod apparatus, the same animals were tested twice and so data were analyzed by paired t-test to compare performance on test # 1 and test # 2 .
Behavioral tests of anxiety There were no effects of antisense or scrambled oligos on behavior in the hole-board apparatus. However, animals treated with P C P A exhibited a significantly decreased amount of time spent head-dipping compared to the other two groups ( A N O V A ; F[2,9] = 6.27; P < 0.05; see Fig. 2). There was no effect of P C P A treatment on the number o f beams crossed or the number of head dips. On the elevated plus-maze there were significantly fewer entrances onto the open arms by antisense oligo-infused and PCPA-treated animals compared to scrambled oligo-infused mice ( A N O V A ; F[2,9] = 5.7; P < 0.05). There were no other significant differences but a notably high level of variability (S.E.M. was greater than the mean) in the time spent on the open arm in animals treated with" P C P A (see Fig. 3).
3. Results
3.1. Administration of antisense and scrambled oligos in vivo Experiment one: effect of antisense oligos, scrambled oligo and P C P A on tryptophan hydroxylase activity in brain tissue. There was a significant decrease in tryptophan hydroxylase activity as indicated by a decreased rate of synthesis of 5-HTP in brain tissue microdissected from the region of the dorsal raphe in animals infused daily with antisense oligonucleotide or given an i.p. injection of P C P A compared to either animals infused with saline or scrambled oligo ( A N O V A ; F[3,14] = 3.90; P < 0.05; see Fig. 1). There was no difference in the mean mg P / s a m p l e across groups.
Locomotor behavior A l l animals performed equally well on the first rotorod test. However, on the second test both the
30-
E 20
¸
10
¸
T
T
[] [] P'] []
SALINE(n=6) PCPA(n--4) SCRAMBLEDOLIGO(n=5) ANTISENSEOLIGO(n=5)
r~ [...
Fig. 1. Tryptophan hydroxylase activity in dorsal medulla. Animals were either injected i.p. with PCPA (300 mg/kg) once or were infused daily with antisense oligo, scrambled oligo or saline for 4 consecutive days and sacrificed on the afternoon of the 4th day. TPH activity was measured in the dorsal medulla by enzymatic assay and HPLC quantification of the end-product, 5-HTP. There was a significant decrease in TPH activity in antisense oligo-infused and PCPA-treated male mice compared to saline and serambled-oligo infused animals (ANOVA; *P < 0.05).
167
M.M. McCarthy et aL /Regulatory Peptides 59 (1995) 163-170 200
300
t, ,.¢
'
2oo,
-~ _= 100
100
o
0 # of BEAMS
# of HEAD DIPS
Fig. 2. Effect of oligo infusions on behavior in the hole-board apparatus. A separate set of animals were either injected i.p. with PCPA (300 mg/kg) once or were infused daily with antisense or scrambled oligo for 4 consecutive days and tested on the afternoon of the 4th day. There was a significant decrease in the time spent head-dipping in aniraals treated with PCPA as compared to the antisense and scrambled oligo-infused'animals (ANOVA; *P < 0.05).
s c r a m b l e d o l i g o - i n f u s e d and a n t i s e n s e o l i g o - i n f u s e d a n i m a l s s i g n i f i c a n t l y i m p r o v e d their p e r f o r m a n c e (paired t-test; P < 0.05 and P < 0.01, r e s p e c t i v e l y ) , w h e r e a s the p e r f o r m a n c e o f the P C P A - t r e a t e d animals w a s not i m p r o v e d , s u g g e s t i n g a l o c o m o t o r impairment ( s e e Fig. 4). 100
R O T O R O D T E S T #1
TIME DIPPING
R O T O R O D T E S T #2
Fig. 4. Effect of oligo infusions on locomotor behavior. At the completion of the hole-board apparatus and elevated plus-maze behavioral tests, the same animals were tested twice on a rotorod apparatus. The two tests were separated by 1-3 h. Animals infused with scrambled or antisense oligo exhibited a significant improvement in locomotor performance on the second test as indicated by the increased number of revolutions achieved (paired t-test; *P <0.05, * *P <0.01), whereas the PCPA-treated animals showed no improvement.
4. D i s c u s s i o n T h e u s e o f s y n t h e t i c a n t i s e n s e o l i g o n u c l e o t i d e s to s e l e c t i v e l y target m R N A and b l o c k its translation
-
• X
80,
PCPA (n=4)
[]
SCRAMBLED OLIGO (n--4)
•
ANTISENSE OLIGO (n=4)
60 ¸
Z o
40.
20'
0 O P E N ARMS ENTRANCES C L O S E D A R M E N T R A N C E S
TIME O N O P E N A R M S
Fig. 3. Effect of oligo infusions on behavior in the elevated plus-maze. The same animals as in Fig. 2 were tested on the elevated plus-maze immediately after the hole-board apparatus. There was a significant decrease in the number of entrances onto the open arms in both the antisense oligo-infused and the PCPA-treated animals compared to the scrambled-oligo infused animals (ANOVA; *P < 0.05).
168
M.M. McCarthy et al. / Regulatory Peptides 59 (1995) 163-170
into protein is becoming increasingly widespread both in vitro and in vivo. We report here that intracerebroventricular administration of antisense oligos to the mRNA coding for tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, successfully reduced TPH activity and related behavioral parameters in adult male mice. Two major mechanisms, which are not mutually exclusive, have been proposed for antisense oligo blockade of translation in the cell cytoplasm. The first involves the hybridization of the oligo in the upstream region of the mRNA where it either prevents the ribosomal subunit from attaching to the mRNA or blocks movement of the ribosome down the mRNA after it has attached. These mechanisms are frequently referred to as hybridization arrest. The second mechanism involves the activation of the nuclease RNase H which selectively degrades the RNA portion of an RNA-DNA duplex and can greatly increase the efficacy of antisense oligo action (see for review [16]). The oligo can hybridize at any region along the mRNA and still be effective if the RNase H mechanism is acting. The question of whether RNase H is a contributing factor to antisense oligo action in the brain remains unresolved (see [8]). A potential limiting factor in antisense oligo efficacy is the cellular content of the targeted mRNA. For example, highly abundant mRNAs such as those for the neuropeptides oxytocin and vasopressin, have proven problematic to reliably and continuously block for extended periods by antisense oligos [5,11]. Alternatively, relatively low abundance mRNAs, such as those for neurotransmitter or steroid receptors have been very effective targets for antisense oligo blockade [9,17]. The TPH mRNA content may have been a critical factor in determining antisense oligo effectiveness in the current experiments. The level of TPH mRNA has been characterized in different brain areas and cells lines. TPH mRNA is found in the pineal gland at 940 copies per cell whereas only 11 copies per cell are found in neurons of the raphe [7] and may make this gene product extremely sensitive to antisense oligo-mediated inhibition. Other variables that can alter the effectiveness of antisense oligos include the rate of translation of the mRNA and the half-life of the mRNA and the synthesized protein. These parameters can differ for
the same protein in different cells. For example, in raphe neurons, significantly more enzyme is translated per mRNA than in the pineal cells [3]. Furthermore, activity is regulated by the stability of the TPH enzyme. In pineal gland~ the enzyme has a half-life of less than 90 min [12,15], while in raphe neurons, it has a 2 - 3 day half-life [10]. The fact that more protein per unit of TPH mRNA is produced in the raphe than the pineal would make this a more desirable target since each mRNA blocked by antisense oligo would result in a comparatively greater reduction in protein synthesis. Alternatively, the greater stability of the protein in the raphe compared to the pineal would require that the antisense oligos be administered for a longer length of time to be effective. The number of cells synthesizing the targeted mRNA could also be a variable in determining antisense effectiveness. In the rat, a population of approximately 4000 TPH synthesizing cells was estimated for the dorsal raphe nucleus and when analyzed along the posteroanterior axis, the TPH distribution within the raphe subdivisions was quite homogeneous [18]. A half-life in the range of 1.6 days for TPH protein was also calculated in this study. Therefore it is apparent that in one system, the TPH synthesizing neurons of the raphe nuclei, there is a constellation of attributes that make it a desirable target for antisense oligo blockade. These include: (1) a relatively small number of cells, (2) with a fairy short half-life for the protein, and (3) a low abundance of mRNA, (4) that is translated at a high rate of protein per unit of TPH mRNA. All of these factors together would suggest that the observed reduction in TPH activity after antisense oligo administration reflects a decrease in TPH protein. However, it must be pointed out that the current experiments did not measure actual TPH protein and the possibility exists for an alternative mechanism of inhibition of TPH activity. An interesting aspect of the current findings is that antisense oligos for TPH appear to be anxiogenic as indicated by a decrease in entrances onto the open arms of an elevated plus-maze when compared to scrambled oligo infusion. Treatment with PCPA had the same effect, as well as a decrease in time spent head-dipping on the hole-board apparatus, also suggestive of increased anxiety. However, tests of anxiety are often difficult to interpret due to
M.M. McCarthy et al. /Regulatory Peptides 59 (1995) 163-170
confounding locomotor effects. There is substantial evidence implicating 5-HT in control of motor behavior. Clearly the dorsal raphe projections to the nigro-striatal system would be expected to influence locomotion and it has been suggested that this projection inhibits general motor output of the basal ganglia. Alternatively, an excitatory 5-HT projection to spinal cord is known to mediate complex patterns of motor behavior and behavioral evidence suggests opposing actions of 5-HT in these two CNS regions (see [6]). Based on these observations it has been proposed that any involvement of 5-HT in anxiety merely reflects a general disruption of locomotor responses that manifests as an apparent change in anxiety, but in fact alters other behavioral responses as well. The current data address this issue. In addition to measures of anxiety, we also measured locomotor coordination on a rotorod apparatus. This behavioral test requires more then generalized movement since the animals must stay balanced with good hind and forelimb strength to stay upright on the continuously accelerating rotating rods. Animals traditionally improve their performance during a second session on the rotorod apparatus and this was observed to be the case for both the antisense oligo and scrambled oligo-infased animals. However, the PCPA-treated animals did not show any improvement on the second test, suggesting that their balance and coordination were severely impaired. Therefore, one side-effect of PCPA treatment may be increased ataxia or other motor impairments and these confounding effects were not observed when TPH activity was reduced by antisense oligonucleotides. However, the possibility that limitations of sample size or procedural factors prevented the accurate assessment of PCPA treatment on locomotor function, cannot be ruled out. Interest in the therapeutic uses of antisense oligonucleotides is continuing to grow. The data presented here demonstrate how target parameters can influence the effectiveness of antisense oligonucleotides in particular systems. The observation that TPH antisense oligo treatment apparently produced less undesirable side.-effects than the TPH inhibitor PCPA, supports the notion that this approach offers additional advantages over those pharmacological agents already available.
169
References [1] Boadle-Biber, M.C., Codey, K.C., Graves, L., Phan, T.H. and Rosecrans, J., Increase in the activity of tryptophan hydroxylase from cortex and midbrain of male Fischer 344 rats in response to acute or repeated sound stress, Brain Res., 482 (1989) 306-316. [2] Cooper, R.R. and Melcer, I., The enzymatic oxidation of tryptophan to 5-hydroxytryptophan in the biosynthesis of serotonin, J. Pharmacol. Exp. Ther., 132 (1961) 265-268. [3] 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 (1980) 537-547. [4] Durcan, M.J., Lister, R.G. and Linnoila, M., Behavioral effects of the inhibitors of phenylethanolamine-N-methyltransferase, LY 78335 and LY 134046, and their interactions with ethanol, Psychopharmacology, 101 (1990) 196-202. [5] Flanagan, L.M., McCarthy, M.M., Brooks, P.J., Pfaff, D.W. and McEwen, B.S., Arginine vasopressin levels (AVP) after daily infusions of antisense oligonucleotides into the supraoptic nucleus (SON), Soc. Neurosci. Abstr., 18 (1992) 205-207. [6] Iversen, S.D., 5-HT and anxiety, Neuropharmacology, 23 (1984) 1553-1560. [7] Kim, K.S., Wesse, 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. [8] McCarthy, M.M., Use of antisense oligonucleotides to block gene expression in the central nervous system. In E.R. de Kloet and W. Sutano (Eds.), Methods in Neurosciences: Neurobiology of Steroids, Vol 22, Academic Press, New York, NY, 1994, pp. 342-356. [9] McCarthy, M.M., Masters, D.B., Rimvall, K., SchwartzGiblin, S. and Pfaff, D.W., Intracerebral administration of antisense oligodeoxynucleotides to GAD65 and GAD67 mRNAs modulate reproductive behavior in the female rat, Brain Res., 636 (1994) 209-220. [10] Meek, J.L. and Neff, N.H., Tryptophan 5-hydroxylase: approximation of half-life and rate of axonal transport, J. Neurochem., 19 (1972) 1519-1525. [11] Neuman, I., Porter, D.W., Landgraf, R. and Pittman, Q.J., Rapid effect on suckling of an oxytocin antisense oligonucleotide administered into rat supraoptic nucleus, Am. J. Physiol., (1994) R842-R858. [12] Sitaram, B.R. and Lees, G.J., Diurnal rhythm and the turnover of tryptophan hydroxylase in the pineal gland of the rat, J. Neurochem., 31 (1978) 1021-1026. [13] Stoll J. and Goldman D., Isolation and structural characterization of the murine tryptophan hydroxylase gene, J, Neurosci. Res., 28 (1991) 457-465. [14] Stoll, J., Kozak, C.A. and Goldman, D., Characterization and chromosomal mapping of a cDNA encoding tryptophan hydroxylase from a mouse mastocytoma cell line, Genomics, 7 (1990) 88-96.
170
M.M. McCarthy et al. / Regulatory Peptides 59 (1995) 163-170
[15] Sugden, D., Grady, R., Jr. and Mefford, I.N., Measurement of tryptophan hydroxylase activity in rat pineal glands and pinealocytes using an HPLC assay with electrochemical detection, J. Pineal Res., 6 (1989) 285-292. [16] Uhlmann, E. and Peyman, A., Antisense oligonucleotides: A new therapeutic principle, Chem, Rev., 90 (1990) 544-584. [17] Wahlestedt, C., Golanov, E., Yamamoto, S. Yee, F., Ericson, H., Yoo, H., Inturrisi, C.E. and Reis, D.J., Antisense oligodeoxynucleotides to NMDA-Ra receptor channel protect
cortical neurons from excitotoxicity and reduce focal ischaemic infarctions, Nature, 363 (1993) 260-263. [18] Weissman, 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-chlorophenylalanine administration. II. Anatomical distribution of the tryptophan-5-hydroxylase protein and regional variation of its turnover rate, Brain Res., 536 (1990) 46-55.