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Influence of methamphetamine on nigral and striatal tyrosine hydroxylase activity and on striatal dopamine levels
European Journal of Pharmacology, 36 (1976) 363--371
363
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
INFLUENCE OF METHAMPHETAMINE ON NIGRAL A N D STRIATAL TYROSINE H Y D R O X Y L A S E ACTIVITY A N D ON STRIATAL DOPAMINE LEVELS * FREDERICK J. KOGAN **, WILLIAM K. NICHOLS and JAMES W. GIBB
Department of Pharmacology, Colleges of Pharmacy and Medicine, University of Utah, Salt Lake City, Utah, U.S.A. Received 10 July 1975, revised MS received 12 November 1975, accepted 15 December 1975
F.J. KOGAN, W.K. NICHOLS and J.W. GIBB, Influence of methamphetamine on nigral and striatal tyrosine hydroxylase activity and on striatal dopamine levels, European J. Pharmacol. 36 (1976) 363--371. In previous reports, methamphetamine was shown to depress tyrosine hydroxylase (TH) activity in the rat corpus striatum. To evaluate further the mechanism of this decrease in TH activity, enzyme activity was measured in the rat corpus striatum and substantia nigra after repetitive and single-dose methamphetamine administration. Following repeated doses of methamphetamine, nigral TH activity decreased and reached 45% of controls at 12 hr and returned to normal at 60 hr. Striatal TH activity decreased to 40% of control at 36 hr and returned toward normal at 60 hr. When methamphetamine was administered every 6 hr for 30 hr and then discontinued, nigral TH activity returned toward control levels 4 days prior to recovery of striatal TH activity. Methamphetamine initially increased striatal dopamine levels at 6 hr (170% of control). Dopamine levels then decreased in parallel with striatal TH activity but failed to increase as the enzyme recovered. Concurrent administration of chlorpromazine with methamphetamine prevented the methamphetamine-induced decrease in nigral and striatal TH activity and striatal dopamine levels. The results indicate that the methamphetamine-induced depression of striatal and nigral TH activity may be related to increased stimulation of dopamine receptors in the striatum. Tyrosine hydroxylase Dopamine
Corpus striatum
Substantia nigra
1. Introduction Methamphetamine is one of many drugs which affect tyrosine hydroxylase (TH) activity. Mandel and Morgan (1970) reported that methamphetamine markedly increased TH activity in chick adrenal glands. Koda and Gibb (1971, 1973), Fibiger and McGeer (1971) and Buening and Gibb {1974) found that chronic treatment with methamphetamine produced an increase in TH activity in the adrenal gland * Part of this study was presented at the Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, U.S.A., in April 1975. ** Recipient, Hoffmann--LaRoche Fellowship.
Chlorpromazine
Methamphetamine
and a decrease in enzyme activity in the corpus striatum. The central effects of methamphetamine appear to be dependent upon intact dopamine (DA) receptor activity. Chlorpromazine is believed to block DA receptors and has been shown to antagonize many of the effects of amphetamines in animals, including aggregation toxicity (Lasagna and McCann, 1957), stereotyped behavior (Randrup et al., 1963), and hyperthermia (Morpurgo and Theobald, 1967). Chlorpromazine is also effective in the clinical management of amphetamine poisoning (Espelin and Done, 1968). The amphetamine-induced depression of the firing rate of the DA-containing cells in the substantia nigra can be prevented or reversed by chlorproma-
364 zine (Bunney and Aghajanian, 1973; Bunney et al., 1973b). Buening and Gibb (1974) observed that chlorpromazine, administered concurrently with methamphetamine prevented the methamphetamine-induced depression of striatal TH activity. The mechanism by which methamphetamine depresses striatal TH activity is still unclear. It has been suggested that amphetamine, by increasing the concentration of DA postsynaptically in the striatum, might initiate a feedback inhibition of the nigro-striatal neurons (Bunney and Aghajanian, 1973; Bunney et al., 1973a, 1973b). Electrophysiological evidence supporting this hypothesis has been presented by Yoshida and Precht (1971) who found that stimulation of the caudate nucleus had an inhibitory effect on the neurons of the substantia nigra and that this inhibition was probably produced by a striatonigral neuronal pathway. The objective of this study was to elucidate further the mechanism by which methamphetamine depresses TH activity in the rat brain nigro-striatal dopaminergic pathway. Experimental evidence suggests that the methamphetamine-induced depression of nigral and striatal TH activity may be related to increased stimulation of dopaminergic receptors in the corpus striatum and that this depression in enzyme activity is initiated by a striato-nigral neuronal feedback circuit.
F.J. KOGAN ET AL. chlorpromazine, as the base, 15 mg/kg, intraperitoneally (i.p.). Groups of animals were sacrificed by decapitation at various intervals after the initiation of treatment. In order to minimize possible variability due to diurnal cycles, all animals were sacrificed in the morning between 8.00 a.m. and 10.00 a.m. The corpus striatum and substantia nigra were immediately removed, wrapped in parafilm, frozen on dry ice, and stored a t - - 2 0 ° C . All tissues were assayed within 5 days after sacrifice. No significant change in TH activity or DA levels occurred in this period of time. During the preparation of tissues for assay, tissue homogenates and other incubation reagents were kept on ice. L-Tyrosine-l-14C (40--60 mCi/mmol) was obtained from New England Nuclear; NCS tissue solubilizer from Amersham/Searle; Ltyrosine, 2-mercaptoethanol and pyridoxal phosphate from Calbiochem; and 2-amino-4h y d r o x y -6,7 -dimethyl- 5,6,7,8-tetrahydropteridine • HC1 ( D M P H 4 ) f r o m Aldrich Chemical Co. The plastic wells and rubber stoppers used with the reaction vessels for the TH assay were purchased from the Kontes Glass Co. Bio-Rex 70, 200--400 mesh, sodium form, was obtained from Bio-Rad Laboratories. Dopamine • HC1 was obtained from the Regis Chemical Co. Reagent grade chemicals were used in all experiments.
2.2. Tyrosine hydroxylase assay 2. Materials and methods
2.1. General procedures Male Sprague--Dawley rats weighing between 180--240 g were housed three per cage (16 in X 9 in X 8 in) in a room with controlled lighting (12 hr of light, 12 hr of darkness) and temperature (26°C). Food and water were offered ad libitum. Rats were injected every 6 hr (9.00 a.m., 3.00 p.m., 9.00 p.m. and 3.00 a.m.) with saline, 1 ml/kg, subcutaneously (s.c.); methamphetamine, as the base, 15 mg/kg, s.c.;
The substantia nigra and corpus striatum were weighed, homogenized in 0.4 and 1.0 ml, respectively, of glass-distilled water in ground-glass homogenizer tubes. The homogenates were centrifuged for 15 min at 27,000 X g at 4°C. Aliquots of the supernatant fraction were assayed for TH activity by the coupled decarboxylase assay of Waymire et al. (1971). In this assay, L-dopa-1-14 C formed by hydroxylation of L-tyrosine-l-14C was converted to DA and carbon dioxide by a crude preparation of aromatic L-amino acid decarboxylase prepared from hog kidney. The evolved 14 CO2 was counted by liquid scintil-
NIGRAL AND $TRIATAL TH ACTIVITY AFTER METHAMPHETAMINE lation spectroscopy. The coupled decarboxylase assay was linear from 5 to 35 min. In a total volume of 0.5 ml the assay medium contained: 0.1 ml of the supernatant fraction from tissue homogenates; 0.2 ml of crude decarboxylase enzyme preparation; 500 mmol sodium acetate buffer, pH 6.1; 0.01 mmol ferrous sulfate; 2.0 mmol DMPH4; 20 mmol 2mercaptoethanol; 0.1 pmol pyridoxal phosphate; and L-tyrosine-l-' 4 C, approximately 1.1 X l 0 s dpm (9.0 X 10 -1° mol) with 0.05 pmol L-tyrosine for determining TH activity in the corpus striatum or L-tyrosine-1 -~4 C, approximately 3.3 X l 0 s dpm (2.7 X 10:9 mol) with 0.047 pmol L-tyrosine for determining the TH activity in the substantia nigra. Small test tubes (15 mm X 85 mm) were used as the reaction vessels. Plastic wells containing a small folded filter paper wick plus 0.2 ml NCS tissue solubilizer were suspended from rubber injection vial caps sealing the test tubes. The reaction vessels were incubated in a New Brunswick metabolic shaker at 250 r.p.m, and 37°C for 20 min (corpus striatum) or 30 min (substantia nigra). 5-min tissue blanks were measured for each tissue sample. Blanks containing the complete reaction mixture, except t h a t glass-distilled water was substituted for tissue supernatant, were also incubated as described above. The incubation was stopped by the addition of 0.5 ml 10% trichloroacetic acid at the times indicated. The acidified media were incubated an additional 45 min to trap ' 4 CO2 in the NCS solubilizer. At the end of this period, the plastic wells were removed, wiped with absorbent paper and placed in vials with 15 ml scintillation fluid. The scintillation fluid contained 0.5 g p-bis-[2-(4-methyl-5-phenyloxazolyl)]-benzene (dimethyl-POPOP) and 4.0 g 2,5-diphenyloxazoline (PPO) per liter of toluene. Radioactivity was counted in a Packard Liquid Scintillation Spectrometer with an efficiency of 89%.
2. 3. Dopamine determination Striatal dopamine levels were determined by the method of Barchas et al. (1972). Fro-
365
zen striata were homogenized in 0.5 ml icecold 75% ethanol containing 0.2% ethylenediamine tetra-acetic acid disodium salt (EDTA). The samples were then washed with 1.5 ml 75% ethanol containing 0.2% EDTA and centrifuged (30,000 × g at 4°C) for 15 min. The supernatants were decanted and diluted with an equal volume of ice-cold glass-distilled water and then passed through a Bio-Rex 70, 200--400 mesh, column (3 cm X 0.6 cm). All columns were first washed with 10 ml of 0.02 M sodium phosphate buffer, pH 6.5, containing 2% EDTA and then with 6 ml glass-distilled water. The DA was eluted with 3.0 ml of 0.5 N acetic acid. Column blanks and known amounts of dopamine were determined along with the samples. To a 0.6 ml aliquot of the DA eluate, 0.3 ml of a 0.5 M potassium phosphate buffer, pH 7.0 was added. The sample was then adjusted to pH 6.8 by the addition of 1 N potassium carbonate. The dopamine was oxidized by the addition of 0.06 ml 0.5% sodium periodate to the sample. After 60 sec, 0.3 ml alkaline sulfite solution (1.25 g Na2 SO3 initially dissolved in 5 ml glass-distilled water and 45 ml 5 N NaOH added) was added and followed immediately by 0.3 ml of 0.5 M citrate buffer, pH 4.0, and 0.5 ml 3 M phosphoric acid. 5 min after the addition of the oxidizing agent, the samples and standards were assayed fluorometrically at an excitation wavelength of 316 mp and an emission wavelength of 370 mp.
3. Results
3.1. Effect o f methamphetamine on nigral tyrosine hydroxylase activity The cell bodies of the nigro-striatal dopamine system are located in the substantia nigra of the midbrain and the axons terminate in the corpus striatum. In order to elucidate further the mechanism by which methamphetamine depresses TH activity in the corpus striatum, enzyme activity was measured in the dopaminergic cell bodies of the nigro-striatal dopamine system.
366
F.J. K O G A N E T AL.
Methamphetamine (15 mg/kg) was administered s.c. every 6 hr for periods as long as 54 hr, and nigral TH activity was determined at various times, in each instance, 6 hr after the last injection. In the 30-min and 3-hr determinations, only one dose was given. Following chronic methamphetamine administration, nigral TH activity returned to normal levels (fig. 1), even though administration of methamphetamine was continued. Enzyme activity decreased initially at 6 hr (p < 0.05) and fell to 45% of control at 12 hr. TH activity generally remained depressed for 48 hr (50% of control) and returned to control values at 60 hr.
3.2. Effect o f methamphetamine on striatal tyrosine hydroxylase activity As in the substantia nigra, methamphetamine, administered as described above in
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Fig. 1. Effect o f m e t h a m p h e t a m i n e o n striatal a n d nigral t y r o s i n e h y d r o x y l a s e activity. M e t h a m p h e t a m i n e ( 1 5 mg/kg, s.c.) was a d m i n i s t e r e d every 6 hr for 54 hr. Rats were sacrificed 6 hr a f t e r t h e last injection, e x c e p t for t h e 30 rain a n d 3 hr d e t e r m i n a tions. T h e first p o i n t o f each line r e p r e s e n t s t h e m e a n of 2 8 - - 3 6 c o n t r o l o b s e r v a t i o n s . All o t h e r p o i n t s represent the mean of 10--24 observations. Brackets indicate +- S.E.M. T h e h a t c h e d areas w i t h i n t h e figure d e p i c t t h e p o o l e d c o n t r o l values _+ S.E.M. for t h e stria t u m a n d nigra at each t i m e p o i n t over t h e t r e a t m e n t period, a, Significantly d i f f e r e n t f r o m c o n t r o l (p < 0.01). b, D i f f e r e n t f r o m c o n t r o l at p < 0.01. c,d, D i f f e r e n t f r o m 36 h r c o r p u s striatal value at p < 0.05 a n d p < 0.001, respectively, e, Significantly d i f f e r e n t f r o m c o n t r o l (p < 0.05). f, Significantly d i f f e r e n t f r o m 36 a n d 48 hr nigral value (p < 0.001).
3.1., produced a decrease in TH activity followed by a return toward control levels (fig. 1). The first significant depression was observed 6 hr after administration of the drug. Enzyme activity then decreased linearly until it reached 40% of control values at 36 hr. Upon the continued administration of methamphetamine, TH activity progressively returned, at 48 and 60 hr, toward normal. 3.3. Recovery of striatal and nigral tyrosine hydroxylase activity after 30 hr o f chronic methamphetamine administration Chronic methamphetamine administration has been shown to produce a post-amphetamine depression (Kosman and Unna, 1968; Griffith, 1966; Griffith et al., 1970). This state may be related to decreased transmitter stores after amphetamine administration which may be due to a decline in TH activity. To evaluate the time necessary for striatal and nigral TH activity to return toward control levels after chronic methamphetamine administration, rats were injected every 6 hr for 30 hr with methamphetamine and sacrificed on succeeding days after the last injection of drug. At 36 hr, nigral TH activity was 50% of control (fig. 2). Nigral TH activity continued to decrease for 3 days after cessation of treatment (day 3, 20% of control). By the following day (day 4), enzyme activity returned rapidly toward control levels, to a value of 70% of control. On the other hand, at 36 hr striatal TH activity was 40% of control and remained depressed for seven days after repetitive treatment with methamphetamine had been stopped. In contrast to nigral TH activity, enzyme activity in the striatum did not begin to recover significantly until eight days after discontinuing the drug, at which time it had returned to 60% of the control activity.
3.4. Effect o f a single dose o f methamphetamine (15 mg/kg) on nigral and striatal tyrosine hydroxylase activity Further information on the effect of methamphetamine on the hypothesized striato-
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3.5. Effect o f chlorpromazine on the methamphetamine-induced depression of nigral and striatal tyrosine hydroxylase activity C h l o r p r o m a z i n e , an a g e n t w h i c h is r e p o r t e d to b l o c k D A r e c e p t o r s (Carlsson and Lind-
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Fig. 3. Effect of a single dose-of methamphetamine on striatal and nigral tyrosine hydroxylase activity. Rats were injected with methamphetamine (15 mg/kg, s.c.) and were sacrificed at various times after injection. The first point represents the mean of 13-24 observations. All other points represent the mean of 9--21 observations. Brackets indicate ± S.E.M. The hatched areas within the figure depict the pooled control values -+ S.E.M. for the striatum and nigra at each time point over the treatment period, p values as compared to saline control: a, p < 0.01; b,p < 0.05; c, p < 0.001; d, differs from 24 hr value (p < 0.01).
qvist, 1 9 6 3 ; Nyb~ick et al., 1 9 6 8 ; A n d d n et al., 1 9 7 0 ) .was used t o evaluate t h e role o f D A in m e d i a t i n g t h e decrease o f nigral and striatal T H activity. C h l o r p r o m a z i n e (15 m g / k g ) was a d m i n i s t e r e d s.c. every 6 hr f o r 30 hr. Rats were sacrificed 6 hr after t h e last injection. C h l o r p r o m a z i n e alone did n o t alter TH activity in either t h e s u b s t a n t i a nigra or c o r p u s striat u m (fig. 4). C h l o r p r o m a z i n e , w h e n administ e r e d c o n c u r r e n t l y with m e t h a m p h e t a m i n e , p r e v e n t e d the m e t h a m p h e t a m i n e - i n d u c e d decrease in T H activity in the substantia nigra and c o r p u s s t r i a t u m {fig. 4).
3.6. Effect o f chlorpromazine on methamphetamine-induced changes in striatal dopamine content M e t h a m p h e t a m i n e initially p r o d u c e d a significant increase in D A in the c o r p u s s t r i a t u m at 6 hr (fig. 5). D A levels r e t u r n e d to n o r m a l at 12 hr, decreased significantly at 24 hr and r e m a i n e d depressed for 60 hr. Repetitive
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Fig. 5. Effect of methamphetamine on striatal dopamine levels. Rats were injected every 6 hr for 30 hr with methamphetamine (15 mg/kg, s.c.). Rats were sacrificed 6 hr after the last injection, except for the 30 min and 3 hr determinations. The first point represents the mean of 18 observations. All other points represent the mean of 4--6 observations. Brackets indicate ± S.E.M. p values as compared to saline control: a , p < 0.001.
TABLE 1 Effect of chlorpromazine on the methamphetamineinduced decrease of striatal dopamine levels. Rats were injected every 6 hr for 30 hr with saline, 1 ml/kg, s.c.; methamphetamine, 15 mg/kg, s.c. and chlorpromazine, 15 mg/kg, i.p. Rats were sacrificed 6 hr after the last injection. Values in the table are expressed as the mean ± S.E.M. Numbers in parentheses indicate the number of animals in each group.
chlorpromazine treatment alone had no effect o n s t r i a t a l D A levels ( t a b l e 1). H o w e v e r , w h e n administered concurrently with methamphetamine, chlorpromazine prevented the methamphetamine-induced d e c r e a s e in s t r i a t a l D A levels.
Treatment
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T h e r e s u l t s p r e s e n t e d d e m o n s t r a t e t h a t repetitive high doses of methamphetamine prod u c e a t i m e - d e p e n d e n t d e c r e a s e in s t r i a t a l T H activity during the first 36 hr of treatment (fig. 1). C h r o n i c m e t h a m p h e t a m i n e t r e a t m e n t p r o d u c e d a m a r k e d d e c r e a s e in n i g r a l T H act i v i t y a t 6 h r (fig. 1). I n c o n t r a s t t o n i g r a l T H activity, striatal enzyme activity decreased
NIGRAL AND STRIATAL TH ACTIVITY AFTER METHAMPHETAMINE slowly and steadily over the first 36 hr. The dramatic decline in nigral TH, as opposed to the more gradual decline in striatal enzyme activity, implies that changes in the enzyme activity occur first in the dopaminergic cell bodies located in the substantia nigra and subsequently in the dopaminergic terminals in the corpus striatum. In order to separate more clearly the effects of methamphetamine on nigral and striatal TH activity during repetitive methamphetamine administration, the drug was administered at 6-hr intervals for 30 hr after which the animals were then allowed to recover (fig. 2). Tyrosine hydroxylase activity initially returned toward control values in the substantia nigra within four days after chronic methamphetamine treatment was discontinued. Enzyme activity in the striatum did not begin to recover until eight days after discontinuing the drug. These findings indicate that after chronic methamphetamine treatment, TH activity returns initially in the cell bodies located in the substantia nigra and then progressively returns in the axon projections located in the corpus striatum. These results are consistent with the theory that enzymes involved in catecholamine biosynthesis are transported from their site of synthesis, the perikaryon, to the main site of their action, the nerve terminals (Thoenen et al., 1973; Singh et al., 1974). Further evidence in support of this hypothesis is demonstrated by the effect of a single dose of methamphetamine (fig. 3). The decrease in nigral TH activity preceded that in the striatum by 18 hr and recovery also occurred at least 12 hr earlier. This evidence further substantiates the hypothesis that changes in TH activity in the nigro-striatal dopaminergic pathway after methamphetamine treatment occur initially in the cell bodies rather than in the axon terminals. After an initial significant increase (at 6 hr) striatal DA levels decreased in parallel with the decline in striatal TH activity for the first 36 hr (fig. 5). The increase in striatal DA at 6 hr may be the result of: (1) a decreased endproduct inhibition of TH, caused by the re-
369
lease of DA from the nerve terminal (Neff and Costa, 1966; Spector et al., 1967); and (2) an increased affinity of TH for the pteridine cofactor (Zivkovic et al., 1974). The decrease in striatal DA levels from 6 to 60 hr after initiation of repetitive methamphetamine treatment may be explained by the decrease in TH activity, the rate-limiting step in catecholamine biosynthesis (Levitt et al., 1965). The continued suppression of striatal DA levels at 48 and 60 hr, even as striatal TH activity was returning toward normal, may be explained by a number of experimental observations. Striatal DA appears to be stored in two compartments, a small functional compartment and a larger main storage compartment (Javoy and Glowinski, 1971). The functional compartment appears to contain newly synthesized DA and plays an important role in the release process {Besson et al., 1969; Javoy and Glowinski, 1971). The main storage c o m p a r t m e n t appears to contain amines stored for longer times and may be considered as a reservoir (Javoy and Glowinski, 1971; Glowinski, 1973). As suggested by the experiments of H~iggendal and Dahlstr6m (1971), amines localized in the functional compartments may be stored in newly formed vesicles while those found in the main storage compartments could represent amines stored in older vesicles. Amphetamines release newly synthesized DA from striatal slices (Besson et al., 1969), reduce the accumulation of newly synthesized DA in the striatum (Javoy et al., 1970), and inhibit DA uptake into striatal synaptosomes (Coyle and Snyder, 1969; Horn et al., 1971). These findings may account for the continued depression of striatal DA levels seen after chronic methamphetamine treatment. Even though striatal TH activity is recovering toward normal at 48 hr, DA levels are not immediately replenished because: (1) newly synthesized DA is released preferentially; (2) methamphetamine appears to augment the release of newly synthesized DA; and {3) methamphetamine may inhibit the reuptake of DA into axon terminals. The blockade by chlorpromazine of the
370
methamphetamine-induced decrease in nigral and striatal TH activity is of great interest (fig. 4). Chlorpromazine is presumed to block striatal DA receptors. It would appear that the decrease in TH activity in the striatum and nigra after chronic treatment with methamphetamine is dependent upon intact dopaminergic receptor activity. Bunney and Aghajanian (1973) and Bunney et al. (1973a, 1973b) have reported that amphetamine administered to rats i.v. inhibited the firing of the dopaminergic neurons in the zona compacta of the substantia nigra and that chlorpromazine prevented or reversed the amphetamine-induced depression of the firing rate. These investigators suggest that amphetamine may be exerting its effects on the firing rate of the dopaminergic cells through a postsynaptic inhibitory feedback circuit. Further electrophysiological evidence demonstrating the existence of an inhibitory pathway had been presented by Yoshida and Precht (1971). These workers found that stimulation of the caudate nucleus produced an inhibitory effect on the neurons of the substantia nigra and that this inhibition was probably produced by a striato-nigral neuronal pathway. The present results may be interpreted in a similar manner. Methamphetamine, by facilitating the release of striatal DA, which in turn stimulates the postsynaptic dopaminergic receptors, may produce a decrease in the firing rate of the nigral dopaminergic cells by activating a hypothetical postsynaptic feedback circuit. This decrease in firing rate may be responsible for the depression in TH activity in the substantia nigra and, subsequently, in the corpus striatum after chronic methamphetamine administration. Chlorpromazine blocked the methamphetamine-induced decrease in striatal DA levels (table 1). By blocking postsynaptic DA receptors in the corpus striatum, chlorpromazine may attenuate the influence of the hypothesized striato-nigral neuronal feedback circuit in decreasing TH activity in the cell bodies of the substantia nigra and, subsequently, in the axon terminals, of the nigro-
F.J. KOGAN ET AL.
striatal dopaminergic pathway after methamphetamine administration. The results from the present investigation suggest that the methamphetamine-induced depression of nigral and striatal TH activity is related to increased stimulation of DA receptors in the corpus striatum. Activation of a hypothesized striato-nigral neuronal feedback inhibitory circuit by methamphetamine could explain the depression in enzyme activity. Acknowledgements The authors wish to thank Abbott Laboratories (North Chicago, Illinois) and Smith, Kline and French Laboratories (Philadelphia, Pennsylvania) for their generous supply of methamphetamine and chlorpromazine, respectively. We are also grateful to Ms. Carlene Dowe!l for her skillful technical assistance. This investigation was supported by NIH Grant DA00869.
References And~n, N.E., S.G. Butcher, H. Corrodi, K. Fuxe and U. Ungerstedt, 1970, Receptor ac*Avity and turnover of dopamine and noradrenaline after neuroleptics, European J. Pharmacol. 11,303. Barchas, J., E. Erdelyi and P. Angwin, 1972, Simultaneous determination of indole and catecholamines in tissues using a weak cation-exchange resin, Anal. Biochem. 50, 1. Besson, M.J., A. Cheramy, P. Feltz and J. Glowinski, 1969, Release of newly synthesized dopamine from dopamine-containing terminals in the striatum of the rat, Proc. Nat. Acad. Sci. U.S.A. 62, 741. Buening, M.K. and J.W. Gibb, 1974, Influence of methamphetamine and neuroleptic drugs on tyrosine hydroxylase activity, European J. Pharmacol. 26, 30. Bunney, B.S. and G.K. Aghajanian, 1973, Electrophysiological effects of amphetamine and dopaminergic neurons, in: International Catecholamine Symposium, 3rd, University of Strasbourg, Frontiers in Catecholamine Research, eds. E. Usdin and S.H. Snyder (Pergamon Press, New York) p. 957. Bunney, B.S., G.K. Aghajanian and R.H. Roth, 1973a, Amphetamine, L-dopa and apomorphine: Effects on activity of rat dopaminergic neurons, Federation Proc. 32, 247.
NIGRAL AND STRIATAL TH ACTIVITY A F T E R METHAMPHETAMINE Bunney, B.S., J.R. Walters, R.H. Roth and G.K. Aghajanian, 1973b, Dopaminergic neurons: Effect of antipsychotic drugs and amphetamine on single cell activity, J. Pharmacol. Exptl. Therap. 185, 560. Carlsson, A. and M. Lindqvist, 1963, Effect of chlorpromazine or haloperidol on formation of 3methoxytyramine and normetanephrine in mouse brain, Acta Pharmacol. Toxicol. 20, 140. Coyle, J.T. and S.H. Snyder, 1969, Catecholamine uptake by synaptosomes in homogenates of rat brain: Stereospecificity in different areas, J. Pharmacol. Exptl. Therap. 170, 221. Espelin, D.E. and A.K. Done, 1968, Amphetamine poisoning, N. Eng. J. Med. 278, 3. Fibiger, H.C. and E.G. McGeer, 1971, Effect of acute and chronic methamphetamine treatment on tyrosine hydroxylase activity in brain and adrenal medulla, European J. Pharmacol. 16,176. Glowinski, J., 1973, Some characteristics of the 'functional' and 'main storage' compartments in central catecholamine neurons, Brain Res. 62,489. Griffith, J.D., 1966, A study of illicit amphetamine drug traffic in Oklahoma City, Amer. J. Psychiat. 125, 560. Griffith, J.D., J.H. Cavanaugh, J. Held and J.A. Oates, 1970, Experimental psychosis induced by the administration of d-amphetamine, in: Amphetamines and Related Compounds; Proceedings of the Mario Negri Institute for Pharmacological Research, Milan, Italy, eds. E. Costa and S. Garattifli (Raven Press, New York) p. 897. I-t~ggendal, J. and A. DahlstrSm, 1971, The recovery of noradrenaline in adrenergic nerve terminals of the rat after reserpine treatment, J. Pharm. Pharmacol. 23, 81. Horn, A.S., J.T. Coyle and S.H. Snyder, 1971, Catecholamine uptake by synaptosomes from rat brain, Mol. Pharmacol. 7, 66. Javoy, F. and J. Glowinski, 1971, Dynamic characteristics of the 'functional c o m p a r t m e n t ' of dopamine in dopaminergic terminals of the rat striatum, J. Neurochem. 18, 1305. Javoy, F., M. Hamon and J. Glowinski, 1970, Disposition of newly synthesized amines in cell bodies and terminals of central catecholaminergic neurons. (I) Effects of amphetamine and thioproperazinc on the metabolism of CA in the caudate nucleus, the substantia nigra, and the ventromedial nucleus of the hypothalamus, European J. Pharmacol. 10, 178. Koda, L.Y. and J.W. Gibb, 1971, The effect of repeated large doses of methamphetamine on adrenal and brain tyrosine hydroxylase, Pharmacologist 13, 253. Koda, L.Y. and J.W. Gibb, 1973, Adrenal and striatal tyrosine hydroxylase activity after methamphet-
371
amine, J. Pharmacol. Exptl. Therap. 185, 42. Kosman, M.E. and K.R. Unna, 1968, Long-term effects of amphetamine and methamphetamine in mice and rats-, Clin. Pharmacol. Therap. 9 , 2 4 0 . Lasagna, L. and W.P. McCann, 1957, Effect of 'tranquilizing' drugs on amphetamine toxicity in aggregated mice, Science 125, 1241. Levitt, M., S. Spector, A. Sjoerdsma and S. Udenfriend, 1965, Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmacol. Exptl. Therap. 148, 1. Mandell, A.J. and M. Morgan, 1970, Amphetamine-induced increase in tyrosine hydroxylase activity, Nature (London) 227, 75. Morpurgo, C. and W. Theobald, 1967, Pharmacological modifications of the amphetamine-induced hyperthermia in rats, European J. Pharmacol. 2, 287. Neff, N.H. and E. Costa, 1966, The influence of monoamine oxidase inhibition on catecholamine synthesis, Life Sci. 5, 951. Nyb~/ck, H., Z. Borzecki and G. Sedvall, 1968, Accumulation and disappearance of catecholamines formed from tyrosine-14C in mouse brain; effect of some psychotropic drugs, European J. Pharmacol. 4, 395. Randrup, A., I. Munkvad and P. Udsen, 1963, Adrenergic mechanisms and amphetamine-induced abnormal behavior, Acta Pharmacol. Toxicol. 20, 145. Singh, V.K., H.C. Fibiger, E.G. McGeer and P.L. McGeer, 1974, Analysis of proteins undergoing axonal transport in nigro-striatal neurons in the rat, J. Neuroehem. 22, 119. Spector, S., R. Gordon, A. Sjoerdsma and S. Udenfriend, 1967, End-product inhibition of tyrosine hydroxylase as a possible mechanism for regulation of norepinephrine synthesis, Mol. Pharmacol. 3, 549. Thoenen, H., U. Otten and F. Oesch, 1973, Axoplasmic transport of enzymes involved in the synthesis of noradrenaline: Relationship between the rate of transport and subcellular distribution, Brain Res. 62, 471. Waymire, J.C., R. Bjur and N. Weiner, 1971, Assay of tyrosine hydroxylase by coupled deearboxylation of dopa formed from 1-14 C-L-tyrosine, Anal. Biochem. 43, 588. Yoshida, M. and W. Precht, 1971, Monosynaptic inhibition of neurons of the substantia nigra by caudato-nigral fibers, Brain Res. 32, 225. Zivkovic, B., A. Guidotti and E. Costa, 1974, Effects of neuroleptics on striatal tyrosine hydroxylase: changes in affinity for the pteridine cofactor, Mol. Pharmacol. 10, 727.
363
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
INFLUENCE OF METHAMPHETAMINE ON NIGRAL A N D STRIATAL TYROSINE H Y D R O X Y L A S E ACTIVITY A N D ON STRIATAL DOPAMINE LEVELS * FREDERICK J. KOGAN **, WILLIAM K. NICHOLS and JAMES W. GIBB
Department of Pharmacology, Colleges of Pharmacy and Medicine, University of Utah, Salt Lake City, Utah, U.S.A. Received 10 July 1975, revised MS received 12 November 1975, accepted 15 December 1975
F.J. KOGAN, W.K. NICHOLS and J.W. GIBB, Influence of methamphetamine on nigral and striatal tyrosine hydroxylase activity and on striatal dopamine levels, European J. Pharmacol. 36 (1976) 363--371. In previous reports, methamphetamine was shown to depress tyrosine hydroxylase (TH) activity in the rat corpus striatum. To evaluate further the mechanism of this decrease in TH activity, enzyme activity was measured in the rat corpus striatum and substantia nigra after repetitive and single-dose methamphetamine administration. Following repeated doses of methamphetamine, nigral TH activity decreased and reached 45% of controls at 12 hr and returned to normal at 60 hr. Striatal TH activity decreased to 40% of control at 36 hr and returned toward normal at 60 hr. When methamphetamine was administered every 6 hr for 30 hr and then discontinued, nigral TH activity returned toward control levels 4 days prior to recovery of striatal TH activity. Methamphetamine initially increased striatal dopamine levels at 6 hr (170% of control). Dopamine levels then decreased in parallel with striatal TH activity but failed to increase as the enzyme recovered. Concurrent administration of chlorpromazine with methamphetamine prevented the methamphetamine-induced decrease in nigral and striatal TH activity and striatal dopamine levels. The results indicate that the methamphetamine-induced depression of striatal and nigral TH activity may be related to increased stimulation of dopamine receptors in the striatum. Tyrosine hydroxylase Dopamine
Corpus striatum
Substantia nigra
1. Introduction Methamphetamine is one of many drugs which affect tyrosine hydroxylase (TH) activity. Mandel and Morgan (1970) reported that methamphetamine markedly increased TH activity in chick adrenal glands. Koda and Gibb (1971, 1973), Fibiger and McGeer (1971) and Buening and Gibb {1974) found that chronic treatment with methamphetamine produced an increase in TH activity in the adrenal gland * Part of this study was presented at the Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, U.S.A., in April 1975. ** Recipient, Hoffmann--LaRoche Fellowship.
Chlorpromazine
Methamphetamine
and a decrease in enzyme activity in the corpus striatum. The central effects of methamphetamine appear to be dependent upon intact dopamine (DA) receptor activity. Chlorpromazine is believed to block DA receptors and has been shown to antagonize many of the effects of amphetamines in animals, including aggregation toxicity (Lasagna and McCann, 1957), stereotyped behavior (Randrup et al., 1963), and hyperthermia (Morpurgo and Theobald, 1967). Chlorpromazine is also effective in the clinical management of amphetamine poisoning (Espelin and Done, 1968). The amphetamine-induced depression of the firing rate of the DA-containing cells in the substantia nigra can be prevented or reversed by chlorproma-
364 zine (Bunney and Aghajanian, 1973; Bunney et al., 1973b). Buening and Gibb (1974) observed that chlorpromazine, administered concurrently with methamphetamine prevented the methamphetamine-induced depression of striatal TH activity. The mechanism by which methamphetamine depresses striatal TH activity is still unclear. It has been suggested that amphetamine, by increasing the concentration of DA postsynaptically in the striatum, might initiate a feedback inhibition of the nigro-striatal neurons (Bunney and Aghajanian, 1973; Bunney et al., 1973a, 1973b). Electrophysiological evidence supporting this hypothesis has been presented by Yoshida and Precht (1971) who found that stimulation of the caudate nucleus had an inhibitory effect on the neurons of the substantia nigra and that this inhibition was probably produced by a striatonigral neuronal pathway. The objective of this study was to elucidate further the mechanism by which methamphetamine depresses TH activity in the rat brain nigro-striatal dopaminergic pathway. Experimental evidence suggests that the methamphetamine-induced depression of nigral and striatal TH activity may be related to increased stimulation of dopaminergic receptors in the corpus striatum and that this depression in enzyme activity is initiated by a striato-nigral neuronal feedback circuit.
F.J. KOGAN ET AL. chlorpromazine, as the base, 15 mg/kg, intraperitoneally (i.p.). Groups of animals were sacrificed by decapitation at various intervals after the initiation of treatment. In order to minimize possible variability due to diurnal cycles, all animals were sacrificed in the morning between 8.00 a.m. and 10.00 a.m. The corpus striatum and substantia nigra were immediately removed, wrapped in parafilm, frozen on dry ice, and stored a t - - 2 0 ° C . All tissues were assayed within 5 days after sacrifice. No significant change in TH activity or DA levels occurred in this period of time. During the preparation of tissues for assay, tissue homogenates and other incubation reagents were kept on ice. L-Tyrosine-l-14C (40--60 mCi/mmol) was obtained from New England Nuclear; NCS tissue solubilizer from Amersham/Searle; Ltyrosine, 2-mercaptoethanol and pyridoxal phosphate from Calbiochem; and 2-amino-4h y d r o x y -6,7 -dimethyl- 5,6,7,8-tetrahydropteridine • HC1 ( D M P H 4 ) f r o m Aldrich Chemical Co. The plastic wells and rubber stoppers used with the reaction vessels for the TH assay were purchased from the Kontes Glass Co. Bio-Rex 70, 200--400 mesh, sodium form, was obtained from Bio-Rad Laboratories. Dopamine • HC1 was obtained from the Regis Chemical Co. Reagent grade chemicals were used in all experiments.
2.2. Tyrosine hydroxylase assay 2. Materials and methods
2.1. General procedures Male Sprague--Dawley rats weighing between 180--240 g were housed three per cage (16 in X 9 in X 8 in) in a room with controlled lighting (12 hr of light, 12 hr of darkness) and temperature (26°C). Food and water were offered ad libitum. Rats were injected every 6 hr (9.00 a.m., 3.00 p.m., 9.00 p.m. and 3.00 a.m.) with saline, 1 ml/kg, subcutaneously (s.c.); methamphetamine, as the base, 15 mg/kg, s.c.;
The substantia nigra and corpus striatum were weighed, homogenized in 0.4 and 1.0 ml, respectively, of glass-distilled water in ground-glass homogenizer tubes. The homogenates were centrifuged for 15 min at 27,000 X g at 4°C. Aliquots of the supernatant fraction were assayed for TH activity by the coupled decarboxylase assay of Waymire et al. (1971). In this assay, L-dopa-1-14 C formed by hydroxylation of L-tyrosine-l-14C was converted to DA and carbon dioxide by a crude preparation of aromatic L-amino acid decarboxylase prepared from hog kidney. The evolved 14 CO2 was counted by liquid scintil-
NIGRAL AND $TRIATAL TH ACTIVITY AFTER METHAMPHETAMINE lation spectroscopy. The coupled decarboxylase assay was linear from 5 to 35 min. In a total volume of 0.5 ml the assay medium contained: 0.1 ml of the supernatant fraction from tissue homogenates; 0.2 ml of crude decarboxylase enzyme preparation; 500 mmol sodium acetate buffer, pH 6.1; 0.01 mmol ferrous sulfate; 2.0 mmol DMPH4; 20 mmol 2mercaptoethanol; 0.1 pmol pyridoxal phosphate; and L-tyrosine-l-' 4 C, approximately 1.1 X l 0 s dpm (9.0 X 10 -1° mol) with 0.05 pmol L-tyrosine for determining TH activity in the corpus striatum or L-tyrosine-1 -~4 C, approximately 3.3 X l 0 s dpm (2.7 X 10:9 mol) with 0.047 pmol L-tyrosine for determining the TH activity in the substantia nigra. Small test tubes (15 mm X 85 mm) were used as the reaction vessels. Plastic wells containing a small folded filter paper wick plus 0.2 ml NCS tissue solubilizer were suspended from rubber injection vial caps sealing the test tubes. The reaction vessels were incubated in a New Brunswick metabolic shaker at 250 r.p.m, and 37°C for 20 min (corpus striatum) or 30 min (substantia nigra). 5-min tissue blanks were measured for each tissue sample. Blanks containing the complete reaction mixture, except t h a t glass-distilled water was substituted for tissue supernatant, were also incubated as described above. The incubation was stopped by the addition of 0.5 ml 10% trichloroacetic acid at the times indicated. The acidified media were incubated an additional 45 min to trap ' 4 CO2 in the NCS solubilizer. At the end of this period, the plastic wells were removed, wiped with absorbent paper and placed in vials with 15 ml scintillation fluid. The scintillation fluid contained 0.5 g p-bis-[2-(4-methyl-5-phenyloxazolyl)]-benzene (dimethyl-POPOP) and 4.0 g 2,5-diphenyloxazoline (PPO) per liter of toluene. Radioactivity was counted in a Packard Liquid Scintillation Spectrometer with an efficiency of 89%.
2. 3. Dopamine determination Striatal dopamine levels were determined by the method of Barchas et al. (1972). Fro-
365
zen striata were homogenized in 0.5 ml icecold 75% ethanol containing 0.2% ethylenediamine tetra-acetic acid disodium salt (EDTA). The samples were then washed with 1.5 ml 75% ethanol containing 0.2% EDTA and centrifuged (30,000 × g at 4°C) for 15 min. The supernatants were decanted and diluted with an equal volume of ice-cold glass-distilled water and then passed through a Bio-Rex 70, 200--400 mesh, column (3 cm X 0.6 cm). All columns were first washed with 10 ml of 0.02 M sodium phosphate buffer, pH 6.5, containing 2% EDTA and then with 6 ml glass-distilled water. The DA was eluted with 3.0 ml of 0.5 N acetic acid. Column blanks and known amounts of dopamine were determined along with the samples. To a 0.6 ml aliquot of the DA eluate, 0.3 ml of a 0.5 M potassium phosphate buffer, pH 7.0 was added. The sample was then adjusted to pH 6.8 by the addition of 1 N potassium carbonate. The dopamine was oxidized by the addition of 0.06 ml 0.5% sodium periodate to the sample. After 60 sec, 0.3 ml alkaline sulfite solution (1.25 g Na2 SO3 initially dissolved in 5 ml glass-distilled water and 45 ml 5 N NaOH added) was added and followed immediately by 0.3 ml of 0.5 M citrate buffer, pH 4.0, and 0.5 ml 3 M phosphoric acid. 5 min after the addition of the oxidizing agent, the samples and standards were assayed fluorometrically at an excitation wavelength of 316 mp and an emission wavelength of 370 mp.
3. Results
3.1. Effect o f methamphetamine on nigral tyrosine hydroxylase activity The cell bodies of the nigro-striatal dopamine system are located in the substantia nigra of the midbrain and the axons terminate in the corpus striatum. In order to elucidate further the mechanism by which methamphetamine depresses TH activity in the corpus striatum, enzyme activity was measured in the dopaminergic cell bodies of the nigro-striatal dopamine system.
366
F.J. K O G A N E T AL.
Methamphetamine (15 mg/kg) was administered s.c. every 6 hr for periods as long as 54 hr, and nigral TH activity was determined at various times, in each instance, 6 hr after the last injection. In the 30-min and 3-hr determinations, only one dose was given. Following chronic methamphetamine administration, nigral TH activity returned to normal levels (fig. 1), even though administration of methamphetamine was continued. Enzyme activity decreased initially at 6 hr (p < 0.05) and fell to 45% of control at 12 hr. TH activity generally remained depressed for 48 hr (50% of control) and returned to control values at 60 hr.
3.2. Effect o f methamphetamine on striatal tyrosine hydroxylase activity As in the substantia nigra, methamphetamine, administered as described above in
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Fig. 1. Effect o f m e t h a m p h e t a m i n e o n striatal a n d nigral t y r o s i n e h y d r o x y l a s e activity. M e t h a m p h e t a m i n e ( 1 5 mg/kg, s.c.) was a d m i n i s t e r e d every 6 hr for 54 hr. Rats were sacrificed 6 hr a f t e r t h e last injection, e x c e p t for t h e 30 rain a n d 3 hr d e t e r m i n a tions. T h e first p o i n t o f each line r e p r e s e n t s t h e m e a n of 2 8 - - 3 6 c o n t r o l o b s e r v a t i o n s . All o t h e r p o i n t s represent the mean of 10--24 observations. Brackets indicate +- S.E.M. T h e h a t c h e d areas w i t h i n t h e figure d e p i c t t h e p o o l e d c o n t r o l values _+ S.E.M. for t h e stria t u m a n d nigra at each t i m e p o i n t over t h e t r e a t m e n t period, a, Significantly d i f f e r e n t f r o m c o n t r o l (p < 0.01). b, D i f f e r e n t f r o m c o n t r o l at p < 0.01. c,d, D i f f e r e n t f r o m 36 h r c o r p u s striatal value at p < 0.05 a n d p < 0.001, respectively, e, Significantly d i f f e r e n t f r o m c o n t r o l (p < 0.05). f, Significantly d i f f e r e n t f r o m 36 a n d 48 hr nigral value (p < 0.001).
3.1., produced a decrease in TH activity followed by a return toward control levels (fig. 1). The first significant depression was observed 6 hr after administration of the drug. Enzyme activity then decreased linearly until it reached 40% of control values at 36 hr. Upon the continued administration of methamphetamine, TH activity progressively returned, at 48 and 60 hr, toward normal. 3.3. Recovery of striatal and nigral tyrosine hydroxylase activity after 30 hr o f chronic methamphetamine administration Chronic methamphetamine administration has been shown to produce a post-amphetamine depression (Kosman and Unna, 1968; Griffith, 1966; Griffith et al., 1970). This state may be related to decreased transmitter stores after amphetamine administration which may be due to a decline in TH activity. To evaluate the time necessary for striatal and nigral TH activity to return toward control levels after chronic methamphetamine administration, rats were injected every 6 hr for 30 hr with methamphetamine and sacrificed on succeeding days after the last injection of drug. At 36 hr, nigral TH activity was 50% of control (fig. 2). Nigral TH activity continued to decrease for 3 days after cessation of treatment (day 3, 20% of control). By the following day (day 4), enzyme activity returned rapidly toward control levels, to a value of 70% of control. On the other hand, at 36 hr striatal TH activity was 40% of control and remained depressed for seven days after repetitive treatment with methamphetamine had been stopped. In contrast to nigral TH activity, enzyme activity in the striatum did not begin to recover significantly until eight days after discontinuing the drug, at which time it had returned to 60% of the control activity.
3.4. Effect o f a single dose o f methamphetamine (15 mg/kg) on nigral and striatal tyrosine hydroxylase activity Further information on the effect of methamphetamine on the hypothesized striato-
NIGRAL AND STRIATAL TH ACTIVITY AFTER METHAMPHETAMINE
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3.5. Effect o f chlorpromazine on the methamphetamine-induced depression of nigral and striatal tyrosine hydroxylase activity C h l o r p r o m a z i n e , an a g e n t w h i c h is r e p o r t e d to b l o c k D A r e c e p t o r s (Carlsson and Lind-
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qvist, 1 9 6 3 ; Nyb~ick et al., 1 9 6 8 ; A n d d n et al., 1 9 7 0 ) .was used t o evaluate t h e role o f D A in m e d i a t i n g t h e decrease o f nigral and striatal T H activity. C h l o r p r o m a z i n e (15 m g / k g ) was a d m i n i s t e r e d s.c. every 6 hr f o r 30 hr. Rats were sacrificed 6 hr after t h e last injection. C h l o r p r o m a z i n e alone did n o t alter TH activity in either t h e s u b s t a n t i a nigra or c o r p u s striat u m (fig. 4). C h l o r p r o m a z i n e , w h e n administ e r e d c o n c u r r e n t l y with m e t h a m p h e t a m i n e , p r e v e n t e d the m e t h a m p h e t a m i n e - i n d u c e d decrease in T H activity in the substantia nigra and c o r p u s s t r i a t u m {fig. 4).
3.6. Effect o f chlorpromazine on methamphetamine-induced changes in striatal dopamine content M e t h a m p h e t a m i n e initially p r o d u c e d a significant increase in D A in the c o r p u s s t r i a t u m at 6 hr (fig. 5). D A levels r e t u r n e d to n o r m a l at 12 hr, decreased significantly at 24 hr and r e m a i n e d depressed for 60 hr. Repetitive
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TABLE 1 Effect of chlorpromazine on the methamphetamineinduced decrease of striatal dopamine levels. Rats were injected every 6 hr for 30 hr with saline, 1 ml/kg, s.c.; methamphetamine, 15 mg/kg, s.c. and chlorpromazine, 15 mg/kg, i.p. Rats were sacrificed 6 hr after the last injection. Values in the table are expressed as the mean ± S.E.M. Numbers in parentheses indicate the number of animals in each group.
chlorpromazine treatment alone had no effect o n s t r i a t a l D A levels ( t a b l e 1). H o w e v e r , w h e n administered concurrently with methamphetamine, chlorpromazine prevented the methamphetamine-induced d e c r e a s e in s t r i a t a l D A levels.
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5.8 +- 0.4 ** (10)
* Significantly different from saline treatment (p < o.OOD. ** Different from methamphetamine treatment (p < 0.001).
T h e r e s u l t s p r e s e n t e d d e m o n s t r a t e t h a t repetitive high doses of methamphetamine prod u c e a t i m e - d e p e n d e n t d e c r e a s e in s t r i a t a l T H activity during the first 36 hr of treatment (fig. 1). C h r o n i c m e t h a m p h e t a m i n e t r e a t m e n t p r o d u c e d a m a r k e d d e c r e a s e in n i g r a l T H act i v i t y a t 6 h r (fig. 1). I n c o n t r a s t t o n i g r a l T H activity, striatal enzyme activity decreased
NIGRAL AND STRIATAL TH ACTIVITY AFTER METHAMPHETAMINE slowly and steadily over the first 36 hr. The dramatic decline in nigral TH, as opposed to the more gradual decline in striatal enzyme activity, implies that changes in the enzyme activity occur first in the dopaminergic cell bodies located in the substantia nigra and subsequently in the dopaminergic terminals in the corpus striatum. In order to separate more clearly the effects of methamphetamine on nigral and striatal TH activity during repetitive methamphetamine administration, the drug was administered at 6-hr intervals for 30 hr after which the animals were then allowed to recover (fig. 2). Tyrosine hydroxylase activity initially returned toward control values in the substantia nigra within four days after chronic methamphetamine treatment was discontinued. Enzyme activity in the striatum did not begin to recover until eight days after discontinuing the drug. These findings indicate that after chronic methamphetamine treatment, TH activity returns initially in the cell bodies located in the substantia nigra and then progressively returns in the axon projections located in the corpus striatum. These results are consistent with the theory that enzymes involved in catecholamine biosynthesis are transported from their site of synthesis, the perikaryon, to the main site of their action, the nerve terminals (Thoenen et al., 1973; Singh et al., 1974). Further evidence in support of this hypothesis is demonstrated by the effect of a single dose of methamphetamine (fig. 3). The decrease in nigral TH activity preceded that in the striatum by 18 hr and recovery also occurred at least 12 hr earlier. This evidence further substantiates the hypothesis that changes in TH activity in the nigro-striatal dopaminergic pathway after methamphetamine treatment occur initially in the cell bodies rather than in the axon terminals. After an initial significant increase (at 6 hr) striatal DA levels decreased in parallel with the decline in striatal TH activity for the first 36 hr (fig. 5). The increase in striatal DA at 6 hr may be the result of: (1) a decreased endproduct inhibition of TH, caused by the re-
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lease of DA from the nerve terminal (Neff and Costa, 1966; Spector et al., 1967); and (2) an increased affinity of TH for the pteridine cofactor (Zivkovic et al., 1974). The decrease in striatal DA levels from 6 to 60 hr after initiation of repetitive methamphetamine treatment may be explained by the decrease in TH activity, the rate-limiting step in catecholamine biosynthesis (Levitt et al., 1965). The continued suppression of striatal DA levels at 48 and 60 hr, even as striatal TH activity was returning toward normal, may be explained by a number of experimental observations. Striatal DA appears to be stored in two compartments, a small functional compartment and a larger main storage compartment (Javoy and Glowinski, 1971). The functional compartment appears to contain newly synthesized DA and plays an important role in the release process {Besson et al., 1969; Javoy and Glowinski, 1971). The main storage c o m p a r t m e n t appears to contain amines stored for longer times and may be considered as a reservoir (Javoy and Glowinski, 1971; Glowinski, 1973). As suggested by the experiments of H~iggendal and Dahlstr6m (1971), amines localized in the functional compartments may be stored in newly formed vesicles while those found in the main storage compartments could represent amines stored in older vesicles. Amphetamines release newly synthesized DA from striatal slices (Besson et al., 1969), reduce the accumulation of newly synthesized DA in the striatum (Javoy et al., 1970), and inhibit DA uptake into striatal synaptosomes (Coyle and Snyder, 1969; Horn et al., 1971). These findings may account for the continued depression of striatal DA levels seen after chronic methamphetamine treatment. Even though striatal TH activity is recovering toward normal at 48 hr, DA levels are not immediately replenished because: (1) newly synthesized DA is released preferentially; (2) methamphetamine appears to augment the release of newly synthesized DA; and {3) methamphetamine may inhibit the reuptake of DA into axon terminals. The blockade by chlorpromazine of the
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methamphetamine-induced decrease in nigral and striatal TH activity is of great interest (fig. 4). Chlorpromazine is presumed to block striatal DA receptors. It would appear that the decrease in TH activity in the striatum and nigra after chronic treatment with methamphetamine is dependent upon intact dopaminergic receptor activity. Bunney and Aghajanian (1973) and Bunney et al. (1973a, 1973b) have reported that amphetamine administered to rats i.v. inhibited the firing of the dopaminergic neurons in the zona compacta of the substantia nigra and that chlorpromazine prevented or reversed the amphetamine-induced depression of the firing rate. These investigators suggest that amphetamine may be exerting its effects on the firing rate of the dopaminergic cells through a postsynaptic inhibitory feedback circuit. Further electrophysiological evidence demonstrating the existence of an inhibitory pathway had been presented by Yoshida and Precht (1971). These workers found that stimulation of the caudate nucleus produced an inhibitory effect on the neurons of the substantia nigra and that this inhibition was probably produced by a striato-nigral neuronal pathway. The present results may be interpreted in a similar manner. Methamphetamine, by facilitating the release of striatal DA, which in turn stimulates the postsynaptic dopaminergic receptors, may produce a decrease in the firing rate of the nigral dopaminergic cells by activating a hypothetical postsynaptic feedback circuit. This decrease in firing rate may be responsible for the depression in TH activity in the substantia nigra and, subsequently, in the corpus striatum after chronic methamphetamine administration. Chlorpromazine blocked the methamphetamine-induced decrease in striatal DA levels (table 1). By blocking postsynaptic DA receptors in the corpus striatum, chlorpromazine may attenuate the influence of the hypothesized striato-nigral neuronal feedback circuit in decreasing TH activity in the cell bodies of the substantia nigra and, subsequently, in the axon terminals, of the nigro-
F.J. KOGAN ET AL.
striatal dopaminergic pathway after methamphetamine administration. The results from the present investigation suggest that the methamphetamine-induced depression of nigral and striatal TH activity is related to increased stimulation of DA receptors in the corpus striatum. Activation of a hypothesized striato-nigral neuronal feedback inhibitory circuit by methamphetamine could explain the depression in enzyme activity. Acknowledgements The authors wish to thank Abbott Laboratories (North Chicago, Illinois) and Smith, Kline and French Laboratories (Philadelphia, Pennsylvania) for their generous supply of methamphetamine and chlorpromazine, respectively. We are also grateful to Ms. Carlene Dowe!l for her skillful technical assistance. This investigation was supported by NIH Grant DA00869.
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