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Long-term effects of methamphetamine on the synthesis and metabolism of 5-hydroxytryptamine in various regions of the rat brain
0028.3908/8l/l21135-06)02.00/0 Pergamon Press Lid
LONG-TERM EFFECTS OF METHAMPHETAMINE ON THE SYNTHESIS AND METABOLISM OF 5HYDROXYTRYPTAMINE IN VARIOUS REGIONS OF THE RAT BRAIN C. BAKHIT. M. E. MORGAN.M. A. PEAT and J. W. GIBB Departments of Biochemical Pharmacology and Toxicology and Pharmacology, University of Utah, Salt Lake City, UT 84112, U.S.A. (Accepted 26 June 1981) Summary-Repeated administration of methamphetamine for 30 hr produced large decreases in the activity of tryptophan hydroxyfase and in the levels of S-hydroxytryptamine (5-HT) and 5hydroxyindoleacetic acid (5-HIAA) in several serotonergic nerve terminal regions of the rat brain. Six sequential doses of methamphetamine (15 mg/kg, s.c.), given every 6 hr, produced a differential regional reduction in the levels of 5-HT. 5-HIAA and in tryptophan hydroxylase activity. Among the regions tested, the neostriaturn and cerebral cortex were most affected and the hypothalamus was least affected. A significant recovery in the levels of 5-HT, 5-HIAA and tryptophan hydroxylase activity had occurred by 10 days following drug treatment. Recovery of enzyme activity in the hypothalamus, spinal cord and olfactory tubercle was complete, whereas enzyme activity was still significantly depressed in the neostriatum, nucleus accumbens, cerebral cortex and hippocampus. Similar trends were obtained for 5-HT and 5-HIAA. Tryptophan levels in some regions were increased at 36 hr and remained elevated 110 days after methamphetamine treatment.
Since the report by Knapp, Mandell and Geyer (1974)
that acute injection of amphetamine or methamphetamine to rats produced a decrease in brain tryptophan hydroxylase activity and synaptosomal conversion of tryptophan to 5-hydroxytryptamine (S-I-IT), increasing attention has been paid to the effects of amphetamine and meth~phet~ine on the serotonergic system. Hotchkiss, Morgan and Gibb (1979) showed that subacute administration of methamphetamine produced a profound depression of tryptophan hydroxylase activity in the neostriatum which persisted for 30 days. Hotchkiss and Gibb (1980) have also shown that the extent of the depression of neostriatal tryptophan hydroxylase by subacute administration of meth~phetamine was dependent on both the amount of drug and the number of doses administered. In addition, Trulson and Jacobs (1979) and Ricaurte, Schuster and Seiden (1980) observed a decrease in S-HT levels following chronic administration of amphetamine or methamphetamine. The present study was undertaken to determine the regional response of the serotonergic system to the effects of subacute administration of methamphetamine and also the regional recovery from these effects. Simultaneous determination in various brain regions of several neurochemical parameters of the serotonergic system were made following subacute administration of methamphetamine and also, at 110 days after cessation of drug treatment. METHODS
Male Sprague-Dawley rats weighing were housed 3 per cage in a temperature
200-300 g controlled
room 24°C with a 12-hr light-dark cycle. Food and water were offered ad libitumi Methamphetamine (15 mg/kg, s.c.) was administered first at 6:OOp.m. and then once every 6 hr for 6 doses. Groups of control and treated rats were sacrificed 6 hr after the last injection for the 36-hr group and another group of control and treated rats were maintained for 110 days before being sacrificed. The whole brain was removed rapidly after decapitation and chilled on ice. The spinal cord was dissected immediately after decapitation. The brain was placed on its dorsal surface on ice and a chilled razor blade was used to make coronal cuts. The optic chiasma was utilized as a landmark. Two cuts were made, 3 and 2 mm respectively, anterior to the optic chiasma. The nucleus accumbens was dissected from the above l-mm slice. Another cut was made at the optic chiasma and the slice formed from this cut and the one at 2-mm anterior were used to obtain the olfactory tubercle and neostriatum. The brain was then placed on its ventral surface and a cut was made at the intersection of the superior and inferior colliculi. A 1.5-mm coronal cut was made rostra1 to the last slice and the slice was used to obtain the raphe. The segment anterior to the last cut was used to obtain the cerebral cortex, hippocampus and hypothalamus. All dissected tissues were wrapped in parafilm and frozen at -70°C until assayed. At the time of assay, lO-15-mg portions from the neostriatum, hippocampus, hypothalamus, cerebral cortex and spinal cord were used to measure metabolite levels; the remaining tissue was used for the determination of tryptoph~ hydroxylase activity.
1135
C.
1136
BAKHIT
Because of an inadequate amount of tissue, the other regions were assayed only for enzyme activity. All tissue samples of the two groups of rats (36-hr and llO-day, as well as the control groups) from the same brain region were assayed at the same time. The activity of tryptophan hydroxylase was measured by a modified ~4CO~-trapping method (Ichiyama, Nakamura, Nishizuka and Hayaishi, 1970: Sitaram and Lees, t978), as described by Hotchkiss et al. (1979). Briefly, tissues were homogenized in 50mM HEPES buffer, pH 7.4, and centrifuged at 19,OOOgfor 15 min. A 7..5+1 aliquot was taken for the tryptophan hydroxylase assay and a similar aliquot was diluted to 50~1 and assayed for tyrosine hydroxylase as described elsewhere (Hotchkiss et al., 1979). The final reaction mixture contained: HEPES, 50mM, pH 7.4 at 23°C; L-[“C]tryptophan, 0.1 mM; L-[r4C]tryptophan, 0.02 mM (10 nCi); and hog kidney L-aromatic amino acid decarboxyIase. The trapping of i4C02 was performed on a piece of filter paper moistened with 40 ~1 of hyamine hydroxide. Metabolite levels were determined simultaneously by high pressure liquid chromatography (HPLC) with fluorescence detection as described by Peat and Gibb (unpublished). Individual tissues were homogenized in lOO$ of 0.1 M perchloric acid, 0.04 mM sodium metabisulfite and 100 ~1 of 0.1 ng/bl N-acetylserotonin (internal standard). The homogenate was centrifuged for 15 min at 1085 g and samples of supernatant were assayed using the following HPLC conditions: a 4.6 x 150 mm Ultrasphere ODS (5 w) column; the eluting solvent contained 34% methanol: water (3:2) and 0.02 M potassium phosphate (PH 3.6) monobasic plus 1 g/I sodium heptane sulfonic acid. The flow rate was I .8 ml/min. The spectrofluorometer was equipped with an 18-~1 flow cell and the excitation and emission wavelengths were 300 and 346 nm respectively. Peak height ratios were used to quantiate the amounts of indoleamines in the various regions examined by HPLC. Standards were made up in the perchloric acid solution and mixed in a 1: 1 ratio with the internal standard solution. RESULTS
Thirty-six hours after initiating treatment repeated doses of methamphet~ine produced large decreases in tryptophan hydroxylase activity in all sefotonergic nerve terminal regions examined (Figs l-7). There was a significant quantitative variation in the decrease of tryptophan hydroxylase activity from one region to another. In the cerebral cortex (Fig. 1) and neostriaturn (Fig. 2). enzyme activity was profoundly depressed so that no activity was detected in treated rats at this time. Enzyme activity in the nucleus accumbens (Fig. 3), olfactory tubercle (Fig. 4) and hippocampus (Fig. 5) was less than 20% of control values, whereas in the h~othal~us (Fig. 6) and spinal cord (Fig. 7) tryptophan hydroxylase activity was decreased to about 60”/, that of control; this suggests
et trl.
that the latter regions are less sensitive to the effect of methamphetamine. As suggested in an earlier report by Hotchkiss and Gibb (1980), no significant difference in tryptophan hydroxylase activity between control and treated rats was observed in the cell body region of the raphe (data not shown). Also in agreement with what has been found previously (Bakhit, Morgan and Gibb, 1981) was the variation in basal CEREBRAL
CORTEX
I I....I
3;
CONTROL
36 hr
Yk
r-i
fi0 days
M~T~AMPHETAMINE
Fig. 1. Effect of repeated doses of methamphetamine (IS mg/kg, s.c.) on tryptophan hydroxylase activity in the cerebral cortex at 36 hr and 110 days following drug treatment. (For more details, see Methods section). *Statistically different from control (P < 0.05). The number of animals in each group was 5 or more:
NEOSTRIATUM 60
1
1 40
1i
T
CONTROL
36 hr
llOdoy8
METHAMPHETAMINE
Fig. 2. Etfects of repeated doses of methamphetamine on tryptophan hydroxylase activity in the neostriatum. For more detail see Figure 1 and Methods section.
NUCLEUS
~CCUM~ENS
u 4
-x
1
..
CONTROL
36 hr
IlO&y*
Fig. 3. Effects of repeated doses of methamphet~ine on tryptophan hydroxyiase activity in the nucleus accumbens. For more detail see Figure t and Methods section.
Methamphetamine
enzyme activity from one region to another; the h~othalamus had the highest activity and the mesolimbic areas the lowest basal tryptophan hydroxyia~ activity. The activity of tryptophan hydroxylase in brains of rats that were allowed a long period (110 days) of recovery following drug treatment is shown in Figures OLFACTORY
1137
on S-HT metabolism
50
SPINAL CORD
1
TUEERCLE
CONTROL
110&y
36 hr
METHAMPWETAMINE
Fig. 7. Effects of repeated doses of methamphetamine on tryptophan hydroxyiase activity in the spinal cord. For more detail see Figure 1 and Methods section.
4
~ :ONTROL
36 hr
METHAMPHETAYINE
Fig. 4. Effects of repeated doses of methamphetamine on tryptophan hydroxylase activity in the olfactory tubercle. For more detail see Figure 1 and Methods section.
HIPPOCAMPUS
50 -I
METHAMPHETAYINE
Fig. 5. Effects of repeated doses of methamphetamine on tryptophan hydroxylase activity in the hippocampus. For more detail see Figure 1 and Methods section.
l-7. Significant recovery occurred in most regions but the quantitative recovery varied with the region; for exampie, almost complete recovery of enzyme activity occurred in the mesolimbic areas, hypothalamus and spinal cord, whereas only partial recovery was observed in the neostriatum, cerebral cortex and hippocampus. However, in certain regions like the mesolimbit areas and neostriatum, the greater recovery was observed at 110 days relative to the values at 36 hr. In the cerebral cortex and hip~mpu~ it is interesting to note, however, that tryptophan hydroxylase ao tivity was still depressed by 70% or more after 110 days. As a control parameter, tyrosine hydroxylase activity was measured in the neostriatum at 36 hr and 110 days. The activity of tyrosine hydroxylase was depressed to 45% of control at 36 hr and, at 110 days, enzyme activity was still significantly depressed with only partial recovery (72% of control, Fig. 8). These results are in agreement with earlier reports which showed that neostriatal tyrosine hydroxylase activity was depressed at 36 hr (Koda and Gibb, 1973; Kogan, Nichols and Gibb, 1976) and remained depressed 115
HYPOTHALAMUS
L
NEOSTRIATUM
GUNTROL
36lw
110 doy8
YETHAYPWETAYINE MEWAMPHETAMINE
Fig. 6. Effects of repeated doses of methamphetamine on tryptophan hydroxylase activity in the hypothalamus. For more detail see Figure 1 and Methods section.
Fig. 8. Effect of repeated doses of me&hetamine (15 mg/kg, s.c.) on neostriatal tyrosine hydroxylase activity at 36 hr and 110 days following drug treatment. *Statistically different from control (P < 0.05). The number of animals in each group was 5 or more.
C. BAKHIT
1138
days following methamphetamine treatment (Ellison. Eison, Huberman and Daniel, 1978). Simultaneous determinations of Shydroxytryptamine (SHT), S-hydroxyindoleacetic acid (5-HIAA) and tryptophan levels were made in various serotonergic nerve terminal regions. The decrease in the levels of S-HT and 5-HIAA correlated well with the reduced activity of tr~tophan hydroxylase in each region (Table 1). At 36 hr, the levels of S-NT and 5-HIAA were markedly depressed. No detectable amounts of these substances were found in the neostriatum and cerebral cortex following metamphetamine treatment (compare with tryptophan hydroxylase activity in Figs 1 and 2). In other regions tested, 5-HT and SHIAA levels were depressed by 50% or more and there was a close correlation between the depression in tryptophan hydroxylase !I-HIAA and 5-HT. Also, the regional depression of these substances by methamphetamine was similar to that of tryptophan hydroxylase. Tryptophan levels were increased only in the cerebral cortex at 36 hr but no change was observed in the other regions. At 110 days following methamphetamine treatment, significant recovery of 5-HT and 5-HIAA levels had occurred (Table 1). which again correlated with the activity of tryptophan hydroxylase at 110 days in the various regions. Partial recovery of 5-HT and 5-HIAA levels occurred in all regions tested except the hypothalamus which showed complete recovery in 5-HT levels but 5-HIAA recovered to only 80% of control. Tryptophan levels were significantly higher in the neostriatum and hy~thalamus but did not change in the cerebra1 cortex, hipp~mpus and spinal cord (Table 1).
et
al. DISCUSSION
In the present study the effects of methamphetamine on tryptophan hydroxylase activity and on 5-HT. 5-HIAA and tryptophan levels in various brain regions were examined. Repeated administration of methamphetamine produced large decreases in tryptophan hydroxylase activity, and in 5-HT and 5-HIAA levels. The response varied quantitatively in each brain region; some serotonergic nerve terminal regions were much more sensitive to the eh’ects of methamphetamine than other regions. For instance, tryptophan hydroxylase activity and 5-HT levels were virtually absent in the cerebral cortex and neostriaturn of the treated groups at 36 hr. whereas they were reduced to only about 50% in the hypothalamus. This indicates that methamphetamine has a selective effect, not only on the serotonergic system, as suggested by Hotchkiss er al. (1979) but also the effect of the drug on the serotonergic system is quantitatively different depending on the particular brain region.. However, it should be noted that the differential sensitivity of serotonergic nerve terminal regions observed in this study, may be due to selective disposition of the drug and/or differential responses of serotonergic nerve terminal regions to methamphetamine. The extent of recovery of tryptophan hydroxylase activity in the different regions of treated rat brains, that were maintained for 110 days following drug treatment, varied from region to region. Complete recovery was observed in the hypothalamus olfactory tubercle, and spinal cord, whereas tryptophan hydroxylase activity in the hipp~ampus and cerebral cortex was still depressed by 70y0 or more. Enzyme
Table 1. Effects of repeated doses of methampetamine (15 mg/kg se.) on the levels of 5-HT. 5-HIAA and tryptophan (TRP) in several serotonergic nerve terminal regions of the rat brain
Neostriatum
5-HT
Control 5-HIAA
TRP
5-HT
2.27 f 0.26 3.38 + 0.27
2.61 t 0.25 2.7 I: 0.07
12.2 + 1.52 14.2 f 1.28
ND 2.11 f 0.22*
2.31 + 0.42
1.60 ri; 0.33
12.1 + 2.1
110 days
2.86 f 0.37
2.85 f 0.16
13.1 i 1.04
36 hr
1.01 &-0.05
1.2 f 0.08
20.7 + 2.04
0.96 f 0.19* (30%) 3.02 + 0.26 (105%) ND
110 days
1.35 + 0.05
1.48 f 0.02
22.9 + 0.48
36 hr
1.24 + 0.09
1.59 1: 0.17
19.4 f 1.9
110 days
1.51 k 0.13
1.40 & 0.10 14.n7 + 0.66
36 hr
1.72 k 0.13
1.16 & 0.14
1i0 days
1.58 + 0.12
0.88 f 0.10 13.42 f 0.44
36 hr 110 days
(62%) Hypothalamus
C. Cortex
Hippocampus
Spinal cord
36 hr
14.8 + 1.99
0.41 * o.os* (30%) 0.24 f 0.02* (19%) 0.34 + 0.07* (34%) 0.86 4 0.08* (50%) 1.19 f 0.08* (75%)
Methamphetamine 5-HIAA ND 1.87 + 0.22* (69%) 0.79 * 0.15* (49%) 2.22 + 0.18* (79?9?) ND 0.23 + 0.02* (14%) 0.47 * 0.07’ (25%) 0.67 f 0.08’ (58%) 0.63 2 0.04* (72%)
TRP 14.5 + 1.16 18.0 f 1.02* (127%:) 13.8 + 0.89 16.3 f 1.03* (124%) 32.8 f 2.95* (158%) 23.6 + 1.13 18.8 f 1.6 15.5 f 1.5 14.8 f 1.17 13.0 + 0.93
The determinations were made in the same tissues as the tryptophan hydroxylase assay (for details see. text). Values are ND = not expressed as the mean i SEM ng/mg tissue weight. *Statistically significant compared to control P -c0.05. detectable.
Methamphetamine activity in the nucleus accumbens and neostriatum recovered considerably but was still significantly depressed after 110 days. The change in metabolite levels after administration of methamphetamine correlated well with the alteration of tryptophan hydroxylase activity. At 36 hr, 5-HT and 5-HIAA were not detected in the neostriatum and cerebral cortex of the treated rats and were markedly depressed in the other regions examined. The correlation between 5-HT depression in different brain regions and the depression of enzyme activity suggests that a primary effect of methamphetmaine on the synthesizing capability of the serotonergic neurons may be due to direct effects on the enzyme itself (Knapp rt al., 1974). or to destruction of the serotonergic nerve terminals. Unlike the effects on 5-HT and 5-HIAA, no significant changes in tryptophan levels were observed 36 hr after methamphetamine in any region except the cerebral cortex in which the level was increased. Tryptophan levels were also increased in the neostriatum and hypothalamus in the 1 lo-day treated group. Increases in brain tryptophan levels after acute administration of amphetamine have been reported previously (Tagliamonte, Tagliamonte Perez-Cruet, Stern and Gessa, 1971). An interesting observation in this study was the difference in the relative recovery of tryptophan hydroxylase activity in the different brain regions: the recovery of enzyme activity in the mesolimbic areas and neostriatum was much greater than in the other regions. This may be interpreted as an indication of the relative resilience of each region. For instance tryptophan hydroxylase activity in the olfactory tubercle was depressed by more than 90% at 36 hr, and yet recovered completely by 110 days. In contrast, enzyme activity in the cerebral cortex was not detected at 36 hr but had recovered to only 9% of control at 110 days. The changes observed in neurotransmitter levels correlate well with the enzyme activity and support the suggestion that each region responds differently to recovery from the effects of methamphetamine. The observation that 5-HT, 5-HIAA and tryptophan hydroxylase activity did not recover completely but actually remained depressed in certain regions such as the cerebral cortex and hippocampus suggest that an irreversible process such as nerve terminals destruction, impairment of the synthesizing machinery and/or disruption of axonal transport of the enzyme to the nerve terminal had occurred. Studies by other investigators with repeated administration of amphetamine or methamphetamine on the dopaminergic system have shown a long-lasting (115 days) depression of tyrosine hydroxylase in the striatum (Ellison et al., 1978) and damage of striatal dopamine nerve terminals (Lorez, 1981). However, this does not hold for regions where there was complete recovery of 5-HT, 5-HIAA levels and tryptophan hydroxylase activity such as the hypothalamus. olfactory tubercle
1139
on S-HT metabolism
and spinal cord. In these areas the effect of methamphetamine on the serotonergic system seemed to be reversible. Further work needs to be performed to elucidate the mechanism by which metamphetamine produces long-lasting effects on the serotonergic system. In contrast to the long-lasting, regionally selective effects seen with repeated doses of methamphetamine in this study, results from acute experiments (single injection) of metamphetamine show a marked depression of tryptophan hydroxylase activity 1 hr after drug administration, significant recovery of tryptophan hydroxylase activity 12 hr later and complete recovery of tryptophan hydroxylase activity when measured 2 weeks following drug treatment in all regions examined, including the cerebral cortex and neostriatum (Bakhit and Gibb, 1980). In addition, the time to complete recovery following acute treatment depended on the dose administered. The larger the These obserdose, the longer the time of recovery. vations with acute and chronic studies indicate that the long-lasting effects of metamphetamine depend not only on the dose administered, but also on the persistence of the drug at the site of action, which occurs with repeated administration. This is consistent with the report by Fuller and Hemrick (1980) who, in studies using iprindole to inhibit hepatic metabolism of amphetamine, found a decrease in striatal dopamine 7 days after a single dose (17.5 mg/kg) of amphetamine in iprindole-pretreated rats. In experiments with methamphetamine and iprindole, Peat and Gibb (unpublished) also found a similar effect on 5-HT levels. The long-lasting effects of repeated doses of methamphtamine on the serotonergic system are similar to the effects of a single injection of p-chloroamphetamine (PCA) reported by Sanders-Bush, Bushing and Sulser, (1972; 1975). Long-lasting (4 months) depletion of 5-HT levels and tryptophan hydroxylase adtivity were found following a single dose (10 mg/kg) of PCA. Thus. it appears that metamphetamine like PCA, can also be neurotoxic, but only if the levels of drug in the neuron are maintained at high concentrations for a significant period of time. In summary, methamphetamine produced marked differential regional inhibition of the neurotransmitter synthesizing capacity of the serotonergic system of the rat brain. The extent of the effects of methamphetamine and recovery from these effects varied differentially from region to region. Regional variation in blood flow, drug disposition. responsiveness and adaptability are possible factors which may operate to produce these differential effects.
REFERENCES
Bakhit, C. and Gibb. J. W. (1980). Metamphetamineinduced depression of brain tryptophan hydroxylase: Regional recovery following acute treatment. Sot. Neurosci. Abstr. 53: 32.
1140
C. BAKHITet al.
Bakhit, C., Morgan, M. E. and Gibb, J. W. (1981). Proprapranolol differentjally blocks the methamphe~mineinduced depression of tryptophan hydroxylase in various rat brain regions. Neurosci. Lett. 23: 99. Ellison, G., Eison, M. S., Huberman, H. S. and Daniel. F.. (1978). Long-term changes in dopaminergic innervation of caudate nucleus after continuous amphetamine administration. Science Wash. 201: 276278.Fuller. R. W. and Hemrick. S. K. (1980). Lone-lastine depletion of striatal dopamine by a single &jectio% of amphetamine in iprindole-treated rats. Science 209: 305-307. Hotchkiss, A. J. and Gibb, J. W. (1980). Long-term effects of multiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain. J. Pearce. exp. Ther. 214: 257-262. Hotchkiss, A. J., Morgan, M. E. and Gibb, J. W. (1979). The long-term effects of multiple doses of methamphetamine on neostriatal tryptophan hydroxylase, tyrosine hydroxylase, choline acetyltransferase and glutamate decarboxylase activities. Life Sci. 25: 1373-1378. Ichiyama, A., Nakamura, S., Nishizuka, Y. and Hayaishi, 0. (1970). Enzymatic studies on the biosynthesis of serotonin in mammalian brain. J. bid. Gem. 245: 1699-l 709. Knapp, S., Mandell, A. J. and Geyer, M. A. (1974). Effects of amphetamine in regional tryptophan hydroxylase activity and synaptosomal conversion of tryptophan to 5-hydroxytryptamine in rat brain. J. Pharmac. exp. Ther. 189: 676689. Koda, L. Y. and Gibb J. W. (1973). Adrenal and striatal tyrosine hydroxylase activity after methamphetam~ne. 1. Pharmac. exp. Ther. 185: 42-48.
Kogan, F. J., Nichols, W. K. and Gibb. J. W. (1976). Influence of methamphetamine on nigral and striatal tyrosine hydroxyltise activity and striatal dopamine levels. Eur. J. Pharmac. 36: 363-371. Lorez, H. (1981). Fluorescence histochemistry indicates damaged of striatal dopamine nerve terminals in rats after multiple doses of methamphetamine. Life. Sci. 28: 911. _ Ricaurte. G. A.. Schuster. C. R. and Seiden. L. S. (1980). Long-terms effects of repeated methylamphetamine administration on dopamine and serotonin neurons in the rat brain: A regional study. Brain Res. 193: 153163. Sanders-Bush, E., Bushing, J. and Sulser, F. (1972). Long term effects of p-chloro~mphetamine on tryptophan hydroxylase activity and on the levels of S-hydroxytryp tamine and 5-hydroxyindoleacetic acid in brain. Eur. J. Phwmac. 20: 385.
Sanders-Bush, G., Bushing, J. and Sulser, E. (1975). Longterm effects of p-chloroamphetamine and related drugs on central serotonergic mechanisms. J. Pharmac. exp. Ther. 192: 53.
Sitaram, 8. R. and Lees, G. J. (1978). Diurnal rhythm and trvptophan hvdroxvlase in the nineal eland of the rat. J. NGrochem. 3-l: IOil-1026. _ _ Taeliamonte. A.. Taaliamonte. P.. Perez-Cruet. J.. Stern. S. and Gessa, G. K. (1971). Effects of psychotropic drugs on tryptophan concentration in the rat brain. J. Pharmac. exp. Ther. 177: 475480. Trulson, M. E. and Jacobs, B. L. (1979). Long-term amphetamine treatment decreases brain serotonin metabolism: impli~t~ons for theories of schizophrenia. Science 205: 12951297.
LONG-TERM EFFECTS OF METHAMPHETAMINE ON THE SYNTHESIS AND METABOLISM OF 5HYDROXYTRYPTAMINE IN VARIOUS REGIONS OF THE RAT BRAIN C. BAKHIT. M. E. MORGAN.M. A. PEAT and J. W. GIBB Departments of Biochemical Pharmacology and Toxicology and Pharmacology, University of Utah, Salt Lake City, UT 84112, U.S.A. (Accepted 26 June 1981) Summary-Repeated administration of methamphetamine for 30 hr produced large decreases in the activity of tryptophan hydroxyfase and in the levels of S-hydroxytryptamine (5-HT) and 5hydroxyindoleacetic acid (5-HIAA) in several serotonergic nerve terminal regions of the rat brain. Six sequential doses of methamphetamine (15 mg/kg, s.c.), given every 6 hr, produced a differential regional reduction in the levels of 5-HT. 5-HIAA and in tryptophan hydroxylase activity. Among the regions tested, the neostriaturn and cerebral cortex were most affected and the hypothalamus was least affected. A significant recovery in the levels of 5-HT, 5-HIAA and tryptophan hydroxylase activity had occurred by 10 days following drug treatment. Recovery of enzyme activity in the hypothalamus, spinal cord and olfactory tubercle was complete, whereas enzyme activity was still significantly depressed in the neostriatum, nucleus accumbens, cerebral cortex and hippocampus. Similar trends were obtained for 5-HT and 5-HIAA. Tryptophan levels in some regions were increased at 36 hr and remained elevated 110 days after methamphetamine treatment.
Since the report by Knapp, Mandell and Geyer (1974)
that acute injection of amphetamine or methamphetamine to rats produced a decrease in brain tryptophan hydroxylase activity and synaptosomal conversion of tryptophan to 5-hydroxytryptamine (S-I-IT), increasing attention has been paid to the effects of amphetamine and meth~phet~ine on the serotonergic system. Hotchkiss, Morgan and Gibb (1979) showed that subacute administration of methamphetamine produced a profound depression of tryptophan hydroxylase activity in the neostriatum which persisted for 30 days. Hotchkiss and Gibb (1980) have also shown that the extent of the depression of neostriatal tryptophan hydroxylase by subacute administration of meth~phetamine was dependent on both the amount of drug and the number of doses administered. In addition, Trulson and Jacobs (1979) and Ricaurte, Schuster and Seiden (1980) observed a decrease in S-HT levels following chronic administration of amphetamine or methamphetamine. The present study was undertaken to determine the regional response of the serotonergic system to the effects of subacute administration of methamphetamine and also the regional recovery from these effects. Simultaneous determination in various brain regions of several neurochemical parameters of the serotonergic system were made following subacute administration of methamphetamine and also, at 110 days after cessation of drug treatment. METHODS
Male Sprague-Dawley rats weighing were housed 3 per cage in a temperature
200-300 g controlled
room 24°C with a 12-hr light-dark cycle. Food and water were offered ad libitumi Methamphetamine (15 mg/kg, s.c.) was administered first at 6:OOp.m. and then once every 6 hr for 6 doses. Groups of control and treated rats were sacrificed 6 hr after the last injection for the 36-hr group and another group of control and treated rats were maintained for 110 days before being sacrificed. The whole brain was removed rapidly after decapitation and chilled on ice. The spinal cord was dissected immediately after decapitation. The brain was placed on its dorsal surface on ice and a chilled razor blade was used to make coronal cuts. The optic chiasma was utilized as a landmark. Two cuts were made, 3 and 2 mm respectively, anterior to the optic chiasma. The nucleus accumbens was dissected from the above l-mm slice. Another cut was made at the optic chiasma and the slice formed from this cut and the one at 2-mm anterior were used to obtain the olfactory tubercle and neostriatum. The brain was then placed on its ventral surface and a cut was made at the intersection of the superior and inferior colliculi. A 1.5-mm coronal cut was made rostra1 to the last slice and the slice was used to obtain the raphe. The segment anterior to the last cut was used to obtain the cerebral cortex, hippocampus and hypothalamus. All dissected tissues were wrapped in parafilm and frozen at -70°C until assayed. At the time of assay, lO-15-mg portions from the neostriatum, hippocampus, hypothalamus, cerebral cortex and spinal cord were used to measure metabolite levels; the remaining tissue was used for the determination of tryptoph~ hydroxylase activity.
1135
C.
1136
BAKHIT
Because of an inadequate amount of tissue, the other regions were assayed only for enzyme activity. All tissue samples of the two groups of rats (36-hr and llO-day, as well as the control groups) from the same brain region were assayed at the same time. The activity of tryptophan hydroxylase was measured by a modified ~4CO~-trapping method (Ichiyama, Nakamura, Nishizuka and Hayaishi, 1970: Sitaram and Lees, t978), as described by Hotchkiss et al. (1979). Briefly, tissues were homogenized in 50mM HEPES buffer, pH 7.4, and centrifuged at 19,OOOgfor 15 min. A 7..5+1 aliquot was taken for the tryptophan hydroxylase assay and a similar aliquot was diluted to 50~1 and assayed for tyrosine hydroxylase as described elsewhere (Hotchkiss et al., 1979). The final reaction mixture contained: HEPES, 50mM, pH 7.4 at 23°C; L-[“C]tryptophan, 0.1 mM; L-[r4C]tryptophan, 0.02 mM (10 nCi); and hog kidney L-aromatic amino acid decarboxyIase. The trapping of i4C02 was performed on a piece of filter paper moistened with 40 ~1 of hyamine hydroxide. Metabolite levels were determined simultaneously by high pressure liquid chromatography (HPLC) with fluorescence detection as described by Peat and Gibb (unpublished). Individual tissues were homogenized in lOO$ of 0.1 M perchloric acid, 0.04 mM sodium metabisulfite and 100 ~1 of 0.1 ng/bl N-acetylserotonin (internal standard). The homogenate was centrifuged for 15 min at 1085 g and samples of supernatant were assayed using the following HPLC conditions: a 4.6 x 150 mm Ultrasphere ODS (5 w) column; the eluting solvent contained 34% methanol: water (3:2) and 0.02 M potassium phosphate (PH 3.6) monobasic plus 1 g/I sodium heptane sulfonic acid. The flow rate was I .8 ml/min. The spectrofluorometer was equipped with an 18-~1 flow cell and the excitation and emission wavelengths were 300 and 346 nm respectively. Peak height ratios were used to quantiate the amounts of indoleamines in the various regions examined by HPLC. Standards were made up in the perchloric acid solution and mixed in a 1: 1 ratio with the internal standard solution. RESULTS
Thirty-six hours after initiating treatment repeated doses of methamphet~ine produced large decreases in tryptophan hydroxylase activity in all sefotonergic nerve terminal regions examined (Figs l-7). There was a significant quantitative variation in the decrease of tryptophan hydroxylase activity from one region to another. In the cerebral cortex (Fig. 1) and neostriaturn (Fig. 2). enzyme activity was profoundly depressed so that no activity was detected in treated rats at this time. Enzyme activity in the nucleus accumbens (Fig. 3), olfactory tubercle (Fig. 4) and hippocampus (Fig. 5) was less than 20% of control values, whereas in the h~othal~us (Fig. 6) and spinal cord (Fig. 7) tryptophan hydroxylase activity was decreased to about 60”/, that of control; this suggests
et trl.
that the latter regions are less sensitive to the effect of methamphetamine. As suggested in an earlier report by Hotchkiss and Gibb (1980), no significant difference in tryptophan hydroxylase activity between control and treated rats was observed in the cell body region of the raphe (data not shown). Also in agreement with what has been found previously (Bakhit, Morgan and Gibb, 1981) was the variation in basal CEREBRAL
CORTEX
I I....I
3;
CONTROL
36 hr
Yk
r-i
fi0 days
M~T~AMPHETAMINE
Fig. 1. Effect of repeated doses of methamphetamine (IS mg/kg, s.c.) on tryptophan hydroxylase activity in the cerebral cortex at 36 hr and 110 days following drug treatment. (For more details, see Methods section). *Statistically different from control (P < 0.05). The number of animals in each group was 5 or more:
NEOSTRIATUM 60
1
1 40
1i
T
CONTROL
36 hr
llOdoy8
METHAMPHETAMINE
Fig. 2. Etfects of repeated doses of methamphetamine on tryptophan hydroxylase activity in the neostriatum. For more detail see Figure 1 and Methods section.
NUCLEUS
~CCUM~ENS
u 4
-x
1
..
CONTROL
36 hr
IlO&y*
Fig. 3. Effects of repeated doses of methamphet~ine on tryptophan hydroxyiase activity in the nucleus accumbens. For more detail see Figure t and Methods section.
Methamphetamine
enzyme activity from one region to another; the h~othalamus had the highest activity and the mesolimbic areas the lowest basal tryptophan hydroxyia~ activity. The activity of tryptophan hydroxylase in brains of rats that were allowed a long period (110 days) of recovery following drug treatment is shown in Figures OLFACTORY
1137
on S-HT metabolism
50
SPINAL CORD
1
TUEERCLE
CONTROL
110&y
36 hr
METHAMPWETAMINE
Fig. 7. Effects of repeated doses of methamphetamine on tryptophan hydroxyiase activity in the spinal cord. For more detail see Figure 1 and Methods section.
4
~ :ONTROL
36 hr
METHAMPHETAYINE
Fig. 4. Effects of repeated doses of methamphetamine on tryptophan hydroxylase activity in the olfactory tubercle. For more detail see Figure 1 and Methods section.
HIPPOCAMPUS
50 -I
METHAMPHETAYINE
Fig. 5. Effects of repeated doses of methamphetamine on tryptophan hydroxylase activity in the hippocampus. For more detail see Figure 1 and Methods section.
l-7. Significant recovery occurred in most regions but the quantitative recovery varied with the region; for exampie, almost complete recovery of enzyme activity occurred in the mesolimbic areas, hypothalamus and spinal cord, whereas only partial recovery was observed in the neostriatum, cerebral cortex and hippocampus. However, in certain regions like the mesolimbit areas and neostriatum, the greater recovery was observed at 110 days relative to the values at 36 hr. In the cerebral cortex and hip~mpu~ it is interesting to note, however, that tryptophan hydroxylase ao tivity was still depressed by 70% or more after 110 days. As a control parameter, tyrosine hydroxylase activity was measured in the neostriatum at 36 hr and 110 days. The activity of tyrosine hydroxylase was depressed to 45% of control at 36 hr and, at 110 days, enzyme activity was still significantly depressed with only partial recovery (72% of control, Fig. 8). These results are in agreement with earlier reports which showed that neostriatal tyrosine hydroxylase activity was depressed at 36 hr (Koda and Gibb, 1973; Kogan, Nichols and Gibb, 1976) and remained depressed 115
HYPOTHALAMUS
L
NEOSTRIATUM
GUNTROL
36lw
110 doy8
YETHAYPWETAYINE MEWAMPHETAMINE
Fig. 6. Effects of repeated doses of methamphetamine on tryptophan hydroxylase activity in the hypothalamus. For more detail see Figure 1 and Methods section.
Fig. 8. Effect of repeated doses of me&hetamine (15 mg/kg, s.c.) on neostriatal tyrosine hydroxylase activity at 36 hr and 110 days following drug treatment. *Statistically different from control (P < 0.05). The number of animals in each group was 5 or more.
C. BAKHIT
1138
days following methamphetamine treatment (Ellison. Eison, Huberman and Daniel, 1978). Simultaneous determinations of Shydroxytryptamine (SHT), S-hydroxyindoleacetic acid (5-HIAA) and tryptophan levels were made in various serotonergic nerve terminal regions. The decrease in the levels of S-HT and 5-HIAA correlated well with the reduced activity of tr~tophan hydroxylase in each region (Table 1). At 36 hr, the levels of S-NT and 5-HIAA were markedly depressed. No detectable amounts of these substances were found in the neostriatum and cerebral cortex following metamphetamine treatment (compare with tryptophan hydroxylase activity in Figs 1 and 2). In other regions tested, 5-HT and SHIAA levels were depressed by 50% or more and there was a close correlation between the depression in tryptophan hydroxylase !I-HIAA and 5-HT. Also, the regional depression of these substances by methamphetamine was similar to that of tryptophan hydroxylase. Tryptophan levels were increased only in the cerebral cortex at 36 hr but no change was observed in the other regions. At 110 days following methamphetamine treatment, significant recovery of 5-HT and 5-HIAA levels had occurred (Table 1). which again correlated with the activity of tryptophan hydroxylase at 110 days in the various regions. Partial recovery of 5-HT and 5-HIAA levels occurred in all regions tested except the hypothalamus which showed complete recovery in 5-HT levels but 5-HIAA recovered to only 80% of control. Tryptophan levels were significantly higher in the neostriatum and hy~thalamus but did not change in the cerebra1 cortex, hipp~mpus and spinal cord (Table 1).
et
al. DISCUSSION
In the present study the effects of methamphetamine on tryptophan hydroxylase activity and on 5-HT. 5-HIAA and tryptophan levels in various brain regions were examined. Repeated administration of methamphetamine produced large decreases in tryptophan hydroxylase activity, and in 5-HT and 5-HIAA levels. The response varied quantitatively in each brain region; some serotonergic nerve terminal regions were much more sensitive to the eh’ects of methamphetamine than other regions. For instance, tryptophan hydroxylase activity and 5-HT levels were virtually absent in the cerebral cortex and neostriaturn of the treated groups at 36 hr. whereas they were reduced to only about 50% in the hypothalamus. This indicates that methamphetamine has a selective effect, not only on the serotonergic system, as suggested by Hotchkiss er al. (1979) but also the effect of the drug on the serotonergic system is quantitatively different depending on the particular brain region.. However, it should be noted that the differential sensitivity of serotonergic nerve terminal regions observed in this study, may be due to selective disposition of the drug and/or differential responses of serotonergic nerve terminal regions to methamphetamine. The extent of recovery of tryptophan hydroxylase activity in the different regions of treated rat brains, that were maintained for 110 days following drug treatment, varied from region to region. Complete recovery was observed in the hypothalamus olfactory tubercle, and spinal cord, whereas tryptophan hydroxylase activity in the hipp~ampus and cerebral cortex was still depressed by 70y0 or more. Enzyme
Table 1. Effects of repeated doses of methampetamine (15 mg/kg se.) on the levels of 5-HT. 5-HIAA and tryptophan (TRP) in several serotonergic nerve terminal regions of the rat brain
Neostriatum
5-HT
Control 5-HIAA
TRP
5-HT
2.27 f 0.26 3.38 + 0.27
2.61 t 0.25 2.7 I: 0.07
12.2 + 1.52 14.2 f 1.28
ND 2.11 f 0.22*
2.31 + 0.42
1.60 ri; 0.33
12.1 + 2.1
110 days
2.86 f 0.37
2.85 f 0.16
13.1 i 1.04
36 hr
1.01 &-0.05
1.2 f 0.08
20.7 + 2.04
0.96 f 0.19* (30%) 3.02 + 0.26 (105%) ND
110 days
1.35 + 0.05
1.48 f 0.02
22.9 + 0.48
36 hr
1.24 + 0.09
1.59 1: 0.17
19.4 f 1.9
110 days
1.51 k 0.13
1.40 & 0.10 14.n7 + 0.66
36 hr
1.72 k 0.13
1.16 & 0.14
1i0 days
1.58 + 0.12
0.88 f 0.10 13.42 f 0.44
36 hr 110 days
(62%) Hypothalamus
C. Cortex
Hippocampus
Spinal cord
36 hr
14.8 + 1.99
0.41 * o.os* (30%) 0.24 f 0.02* (19%) 0.34 + 0.07* (34%) 0.86 4 0.08* (50%) 1.19 f 0.08* (75%)
Methamphetamine 5-HIAA ND 1.87 + 0.22* (69%) 0.79 * 0.15* (49%) 2.22 + 0.18* (79?9?) ND 0.23 + 0.02* (14%) 0.47 * 0.07’ (25%) 0.67 f 0.08’ (58%) 0.63 2 0.04* (72%)
TRP 14.5 + 1.16 18.0 f 1.02* (127%:) 13.8 + 0.89 16.3 f 1.03* (124%) 32.8 f 2.95* (158%) 23.6 + 1.13 18.8 f 1.6 15.5 f 1.5 14.8 f 1.17 13.0 + 0.93
The determinations were made in the same tissues as the tryptophan hydroxylase assay (for details see. text). Values are ND = not expressed as the mean i SEM ng/mg tissue weight. *Statistically significant compared to control P -c0.05. detectable.
Methamphetamine activity in the nucleus accumbens and neostriatum recovered considerably but was still significantly depressed after 110 days. The change in metabolite levels after administration of methamphetamine correlated well with the alteration of tryptophan hydroxylase activity. At 36 hr, 5-HT and 5-HIAA were not detected in the neostriatum and cerebral cortex of the treated rats and were markedly depressed in the other regions examined. The correlation between 5-HT depression in different brain regions and the depression of enzyme activity suggests that a primary effect of methamphetmaine on the synthesizing capability of the serotonergic neurons may be due to direct effects on the enzyme itself (Knapp rt al., 1974). or to destruction of the serotonergic nerve terminals. Unlike the effects on 5-HT and 5-HIAA, no significant changes in tryptophan levels were observed 36 hr after methamphetamine in any region except the cerebral cortex in which the level was increased. Tryptophan levels were also increased in the neostriatum and hypothalamus in the 1 lo-day treated group. Increases in brain tryptophan levels after acute administration of amphetamine have been reported previously (Tagliamonte, Tagliamonte Perez-Cruet, Stern and Gessa, 1971). An interesting observation in this study was the difference in the relative recovery of tryptophan hydroxylase activity in the different brain regions: the recovery of enzyme activity in the mesolimbic areas and neostriatum was much greater than in the other regions. This may be interpreted as an indication of the relative resilience of each region. For instance tryptophan hydroxylase activity in the olfactory tubercle was depressed by more than 90% at 36 hr, and yet recovered completely by 110 days. In contrast, enzyme activity in the cerebral cortex was not detected at 36 hr but had recovered to only 9% of control at 110 days. The changes observed in neurotransmitter levels correlate well with the enzyme activity and support the suggestion that each region responds differently to recovery from the effects of methamphetamine. The observation that 5-HT, 5-HIAA and tryptophan hydroxylase activity did not recover completely but actually remained depressed in certain regions such as the cerebral cortex and hippocampus suggest that an irreversible process such as nerve terminals destruction, impairment of the synthesizing machinery and/or disruption of axonal transport of the enzyme to the nerve terminal had occurred. Studies by other investigators with repeated administration of amphetamine or methamphetamine on the dopaminergic system have shown a long-lasting (115 days) depression of tyrosine hydroxylase in the striatum (Ellison et al., 1978) and damage of striatal dopamine nerve terminals (Lorez, 1981). However, this does not hold for regions where there was complete recovery of 5-HT, 5-HIAA levels and tryptophan hydroxylase activity such as the hypothalamus. olfactory tubercle
1139
on S-HT metabolism
and spinal cord. In these areas the effect of methamphetamine on the serotonergic system seemed to be reversible. Further work needs to be performed to elucidate the mechanism by which metamphetamine produces long-lasting effects on the serotonergic system. In contrast to the long-lasting, regionally selective effects seen with repeated doses of methamphetamine in this study, results from acute experiments (single injection) of metamphetamine show a marked depression of tryptophan hydroxylase activity 1 hr after drug administration, significant recovery of tryptophan hydroxylase activity 12 hr later and complete recovery of tryptophan hydroxylase activity when measured 2 weeks following drug treatment in all regions examined, including the cerebral cortex and neostriatum (Bakhit and Gibb, 1980). In addition, the time to complete recovery following acute treatment depended on the dose administered. The larger the These obserdose, the longer the time of recovery. vations with acute and chronic studies indicate that the long-lasting effects of metamphetamine depend not only on the dose administered, but also on the persistence of the drug at the site of action, which occurs with repeated administration. This is consistent with the report by Fuller and Hemrick (1980) who, in studies using iprindole to inhibit hepatic metabolism of amphetamine, found a decrease in striatal dopamine 7 days after a single dose (17.5 mg/kg) of amphetamine in iprindole-pretreated rats. In experiments with methamphetamine and iprindole, Peat and Gibb (unpublished) also found a similar effect on 5-HT levels. The long-lasting effects of repeated doses of methamphtamine on the serotonergic system are similar to the effects of a single injection of p-chloroamphetamine (PCA) reported by Sanders-Bush, Bushing and Sulser, (1972; 1975). Long-lasting (4 months) depletion of 5-HT levels and tryptophan hydroxylase adtivity were found following a single dose (10 mg/kg) of PCA. Thus. it appears that metamphetamine like PCA, can also be neurotoxic, but only if the levels of drug in the neuron are maintained at high concentrations for a significant period of time. In summary, methamphetamine produced marked differential regional inhibition of the neurotransmitter synthesizing capacity of the serotonergic system of the rat brain. The extent of the effects of methamphetamine and recovery from these effects varied differentially from region to region. Regional variation in blood flow, drug disposition. responsiveness and adaptability are possible factors which may operate to produce these differential effects.
REFERENCES
Bakhit, C. and Gibb. J. W. (1980). Metamphetamineinduced depression of brain tryptophan hydroxylase: Regional recovery following acute treatment. Sot. Neurosci. Abstr. 53: 32.
1140
C. BAKHITet al.
Bakhit, C., Morgan, M. E. and Gibb, J. W. (1981). Proprapranolol differentjally blocks the methamphe~mineinduced depression of tryptophan hydroxylase in various rat brain regions. Neurosci. Lett. 23: 99. Ellison, G., Eison, M. S., Huberman, H. S. and Daniel. F.. (1978). Long-term changes in dopaminergic innervation of caudate nucleus after continuous amphetamine administration. Science Wash. 201: 276278.Fuller. R. W. and Hemrick. S. K. (1980). Lone-lastine depletion of striatal dopamine by a single &jectio% of amphetamine in iprindole-treated rats. Science 209: 305-307. Hotchkiss, A. J. and Gibb, J. W. (1980). Long-term effects of multiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain. J. Pearce. exp. Ther. 214: 257-262. Hotchkiss, A. J., Morgan, M. E. and Gibb, J. W. (1979). The long-term effects of multiple doses of methamphetamine on neostriatal tryptophan hydroxylase, tyrosine hydroxylase, choline acetyltransferase and glutamate decarboxylase activities. Life Sci. 25: 1373-1378. Ichiyama, A., Nakamura, S., Nishizuka, Y. and Hayaishi, 0. (1970). Enzymatic studies on the biosynthesis of serotonin in mammalian brain. J. bid. Gem. 245: 1699-l 709. Knapp, S., Mandell, A. J. and Geyer, M. A. (1974). Effects of amphetamine in regional tryptophan hydroxylase activity and synaptosomal conversion of tryptophan to 5-hydroxytryptamine in rat brain. J. Pharmac. exp. Ther. 189: 676689. Koda, L. Y. and Gibb J. W. (1973). Adrenal and striatal tyrosine hydroxylase activity after methamphetam~ne. 1. Pharmac. exp. Ther. 185: 42-48.
Kogan, F. J., Nichols, W. K. and Gibb. J. W. (1976). Influence of methamphetamine on nigral and striatal tyrosine hydroxyltise activity and striatal dopamine levels. Eur. J. Pharmac. 36: 363-371. Lorez, H. (1981). Fluorescence histochemistry indicates damaged of striatal dopamine nerve terminals in rats after multiple doses of methamphetamine. Life. Sci. 28: 911. _ Ricaurte. G. A.. Schuster. C. R. and Seiden. L. S. (1980). Long-terms effects of repeated methylamphetamine administration on dopamine and serotonin neurons in the rat brain: A regional study. Brain Res. 193: 153163. Sanders-Bush, E., Bushing, J. and Sulser, F. (1972). Long term effects of p-chloro~mphetamine on tryptophan hydroxylase activity and on the levels of S-hydroxytryp tamine and 5-hydroxyindoleacetic acid in brain. Eur. J. Phwmac. 20: 385.
Sanders-Bush, G., Bushing, J. and Sulser, E. (1975). Longterm effects of p-chloroamphetamine and related drugs on central serotonergic mechanisms. J. Pharmac. exp. Ther. 192: 53.
Sitaram, 8. R. and Lees, G. J. (1978). Diurnal rhythm and trvptophan hvdroxvlase in the nineal eland of the rat. J. NGrochem. 3-l: IOil-1026. _ _ Taeliamonte. A.. Taaliamonte. P.. Perez-Cruet. J.. Stern. S. and Gessa, G. K. (1971). Effects of psychotropic drugs on tryptophan concentration in the rat brain. J. Pharmac. exp. Ther. 177: 475480. Trulson, M. E. and Jacobs, B. L. (1979). Long-term amphetamine treatment decreases brain serotonin metabolism: impli~t~ons for theories of schizophrenia. Science 205: 12951297.