The acute effects of methamphetamine, amphetamine and p-chloroamphetamine on the cortical serotonergic system of the rat brain: Evidence for differences in the effects of methamphetamine and amphetamine

European Journal of Pharmacology, 116 (1985) 11-16

ll

Elsevier

T H E ACUTE E F F E C T S OF M E T H A M P H E T A M I N E , A M P H E T A M I N E A N D p-

C H L O R O A M P H E T A M I N E O N T H E C O R T I C A L S E R O T O N E R G I C S Y S T E M OF T H E RAT BRAIN: E V I D E N C E F O R D I F F E R E N C E S IN T H E E F F E C T S OF M E T H A M P H E T A M I N E A N D AMPHETAMINE

*

M I C H A E L A. P E A T

1,2,**,

P A U L A F. W A R R E N

1.2,

C H A R L E S B A K H I T a and JAMES W. GIBB 1

1 Department of Biochemical Pharmacology and Toxicology and -' Center for Human Toxicology, Unwersity of Utah, Salt Lake Ci(v, UT 84112, U.S.A. Received 30 May 1985, accepted 5 July 1985

M.A. PEAT, P.F. WARREN, C. BAKHIT and J.W. GIBB, The acute effects of methamphetamine, amphetamine and p-chloroamphetamine on the cortical serotonergic system of the rat brain: evidence for differences in the effects of methamphetamine and amphetamine, European J. Pharmacol. 116 (1985) 11-16. Cortical tryptophan hydroxylase (TPH) activity was reduced 3 h after a 10 or 15 mg/kg i.p. dose of either amphetamine (AMP), methamphetamine (METH), or p-chloroamphetamine (PCA). These injections of METH or PCA also decreased cortical serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations; none of the four doses of AMP decreased indoleamine concentrations. The time course of the effects following a 15 mg/kg dose of each amine was also different. Cortical TPH activity was reduced by all three amines for periods up to 24 h, whereas only METH and PCA significantly decreased 5-HT and 5-HIAA concentrations for long periods. These data suggest that each of the amphetamines may inhibit TPH activity, whereas only METH and PCA produced long-lasting decreases in indoleamine concentrations, reflecting either varying degrees of toxicity or differential effects of AMP on enzyme activity and neurotransmitter concentrations. Amphetamine

Tryptophan hydroxylase

5-HT

1. Introduction

Recently, increasing attention has been focused on the effects of amphetamines on the brain serotonergic system. Chronic or sub-acute administration of amphetamine (AMP) or methamphetamine (METH) produces prolonged decreases in regional tryptophan hydroxylase (TPH) activity and concentrations of serotonin (5-HT) and 5-hydroxyindoleactic acid (5-HIAA) (Trulson and Jacobs, 1979; 1980; Hotchkiss and Gibb, 1980; Ricaurte et al., 1980; Bakhit et al., 1981; Trulson

* Supported by the USPHS Grant DA-00869 and the Thrasher Research Fund. ** To whom all correspondence should be addressed: Chemical Toxicology Institute, P.O. Box 8209, 1167 Chess Drive, Suite E, Foster City, CA 94404, U.S.A. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.

and Trulson, 1982). Brain T P H activity is also reduced by acute administration of A M P (Knapp et al., 1974) or M E T H (Bakhit and Gibb, 1981). There are, however, few reports on the acute effects of these amphetamines on the regional concentrations of indoleamines. Morgan et al. (1972) reported a marked reduction in brain 5-HT after M E T H administration but not after an equimolar dose of AMP. More recently Ricaurte et al. (1983) reported similar data following administration of repeated doses of M E T H or A M P to fluoxetine pretreated rats. Recent reports on the combined effects of iprindole, an inhibitor of the p-hydroxylation of A M P or METH, with either AMP (Fuller and Hemrick-Luecke, 1980; 1982a; Steranka, 1982; Warren et al., 1984) or M E T H (Peat et al., 1983) have suggested differing effects of A M P or M E T H

12 oll the serotonergic system. Following the administration of iprindole and AMP, no long-term decreases were seen in regional 5-HT or 5-HIAA concentrations, whereas consecutive injections of iprindole and M E T H caused prolonged decreases in cortical and striatal concentrations of these indoleamines (Peat et al., 1983). Although there are few reports comparing A M P and METH, researchers in the area have been concerned about differences between the two amines; this study was therefore designed to determined the effects of the acute administration of M E T H , A M P or p-chloroamphetamine (PCA) on the cortical and striatal serotonergic system. A single injection of PCA has previously been shown to cause long-term decreases in indoleamine concentrations and T P H activity (Sanders-Bush et al., 1975) and was included in this study to evaluate the possibility that the amphetamines have differing degrees of toxicity with PCA being the most and AMP the least toxic.

2. Materials and methods

Male Sprague-Dawley rats, 200-300 g, were housed in a 12-h light-dark cycle and given free access to water and standard laboratory feed. All drugs were administered in 0.9% saline by i.p. injection. Doses of the amphetamines are indicated in the appropriate figure legends. Treated and control (saline) rats were killed between 9:00 a.m. and 2:00 p.m. to avoid complicating the interpretation of the data because of diurnal rhythms in enzyme activities and neurotransmitter concentrations. For the dose-response experiments rats were killed by decapitation 3 h after injection and for the time-course studies at the time points shown in the appropriate figures. The whole brain was rapidly removed and neostriatal tissue and whole cerebral cortex dissected out and frozen separately on dry ice. Tissues were stored at - 85°C until analysis. Tissues were homogenized (1:3.5) in 50 mM 4-(2-hydroxyethyl)-l-piperazine ethane-ethanesulfonic and acid buffer (Sigma Chemical Co., St. Louis, MO), pH 7.4, containing 0.2% Triton X-100 and 5 mM dithiothreitol. Homogenates were

centrFuged for 15 rain at 19000 x~f. Duplicate 7.5-ffl aliquots were taken from the supernatant for the T P H assay. T P H was measured by a modified ~4CO2 trapping procedure (Ichiyama et al., 1970; Sitaram and Lees, 1978). The details of the T P H assay h a v e been described previously (Hotchkiss et al., 1979). High-performance liquid chromatography with fluorescence detection was used to measure tissue concentrations of 5-HT, 5-HIAA and tryptophan (TRP) (Peat and Gibb, 1983). Briefly, tissues were homogenized (1 : 5) in 0.1 M perchloric acid containing sodium metabisulfite (4 mM). After centrifuging at 1 5 0 0 0 x g for 15 min, a 50-~1 aliquot was injected onto a 15 cm x 4.6 mm Ultrasphere-ODS (5/*) column (Rainin Instrument Co., Inc., Woburn, MA). The eluent consisted of phosphate buffer (pH 3.3) containing sodium heptane sulfonic acid (1 g / l ) and methanol-water (3:2) as organic modifier. Indoles were detected using an excitation and emission wavelength of 290 and 330 nm, respectively. Quantitation was performed by comparison with known standard solutions. ( _+) M E T H hydrochloride, ( _+)AMP sulfate and PCA were supplied by the National Institute on Drug Abuse; Smith, Kline and French Laboratories, Philadelphia, PA and Sigma Chemical Co., St. Louis, MO, respectively.

3. Results

Figures 1 to 3 illustrate the effects of AMP, M E T H or PCA on T P H activity (fig. 1), 5-HT and 5-HIAA concentrations (fig. 2) and T R P concentrations (fig. 3) in the cerebral cortex 3 h after a single dose of 1.0, 2.5, 10 or 15 m g / k g i.p. Enzyme activity was significantly depressed by the 10 and 15 m g / k g doses of all three drugs (fig. 1); it was also significantly decreased by the lower doses (1.0 and 2.5 m g / k g ) of PCA. The effects of these three amines on cortical indoleamine concentrations were different (fig. 2). In contrast to M E T H and PCA, none of the four doses of A M P significantly altered either cortical 5-HT or 5-HIAA concentrations. Similar results were observed when the effects of the three amines on cortical T R P concentrations were compared (fig. 3). Of note PCA

13

-~

_, . . . .

,2o

AMP

0

AMP

140q

80 40 t ~_

180t ~

/~

'''~' ~q:

i

l

....

I

[

I

u m 40

~

/ 180 cn 140 ioo,-.

0

I

i

i 120t .

I

PCA

i

I

METH

.....

. .......

.--,--

,co

I

1.0

I

I

80 t o. 40 i~Xt~0

.....

I /

2.5 IOD 15.0 DOSE(mg/kg)

140 J ,oo3~ _.~_-" . . . . . . . . . . . "~ I I I I 1.0 2.5 I0.0 1,5.0 DOSE(mg/kg)

Fig. 1. Effect of varying doses of AMP, METH or PCA on cortical TPH activity 3 h after injection. Control enzyme activity was 15.8+0.94 nmol of TRP oxidized/g of tissue per h (n =15). * P < 0.01 and ** P < 0.001 compared to saline control.

Fig. 3. Effect of varying doses of AMP, METH or PCA on cortical TRP concentrations 3 h after injection. Control concentration was 3.3+0.19 n g / m g of tissue (n =15). * P < 0.01 and ** P < 0.001 compared to saline controls.

produced by far the most dramatic increases in cortical TRP concentrations. The changes observed in neostriatal TPH activ-

ity, 5-HT and 5-HIAA concentrations, following administration of the three amines, were similar to those observed in the cerebral cortex (data not shown). However, the effect of AMP on neostriatal TRP concentration was different, with AMP significantly increasing the concentration of the precursor after 10 m g / k g and 15 m g / k g (133 _+ 8% and 158 _+ 13% of control respectively). Quantitative differences between the three amphetamines were also observed when the time course of the effects were monitored. Cortical TPH activity was reduced for up to 24 h by a 15 m g / k g dose of each drug; however, for AMP and METH some recovery in enzyme activity was observed at 24 h whereas it was still significantly depressed (to 7% of control) by PCA(fig. 4). The most dramatic differences between the effects of AMP and METH were observed when cortical indoleamine concentrations were considered, with METH producing a much longer decrease in 5-HT concentration (fig. 5). Both PCA and METH also had an effect to increase 5-HIAA concentrations after 30 min; this was not observed with AMP. Figure 6 shows the time Course of the effects of the three amines on cortical TRP concentrations; again differences

120-I

AMP

80_1-T . . . . --~--_-~-~- _-~.~_..~--.J 40-1 0 O[

'

-- 5-HT --;- 5-HIAA

§ ,204

0

I

I

I~

-.~-. --

i

........

.::_

. . . . . . . .

8o- i

/ , . . . . . . ~__ / "~'~ j

.o4.;

0

,

.E..

8 r7_i

:_.

,

l

1.0

,

2.5

** . . . . .

_.

",~-~ "k~

[

,

I0.0 15.0

DOSE (m9 / kg)

Fig. 2. Effect of varying doses of AMP, METH or PCA on cortical 5-HT and 5-HIAA concentrations 3 h after injection. Control concentrations were: 5-HT, 0.36 n g / m g of tissue (n = 15) and 5-HIAA, 0.19+0.01 (n =15). * P < 0 . 0 1 and * * P < 0.001 compared to saline controls.

14

120

o.

AMP

220 ~ 14O

AMP i

~" ~l &

--

*

J

l u

/

g J-

g

220q

u 120-

METH ~-~-

?

METH

w Z p . -, 8 0 ,~ .

.

.

.

.

.

.

.

.

.

.

.

z

,

t40

~

~ 40-1

T~__~y>~ ~

S ~8o l

pc~

22ol

i:z-

8°t %

,

4o~

,~o1

--\~t

02505

I

3

6 TIME (hrs)

Fig. 4. Effect of a 15 m g / k g dose cortical T P H activity 0.25, 0.5, injection. Control e n z y m e activity o x i d i z e d / g of tissue per (n - 30). compared to saline controls.

12

24

of A M P , M E T H or P C A on 1, 3, 6, 12 and 24 h after was 23.8 ± 0.47 nmol of T R P * P < 0.01 and ** P < 0.001

AMP

~2°1

'k ~,

.4 .......

:: ?'"

I0

,,~; 3

6 TIME (hrs)

12

24

Fig. 6. Effect of a 15 m g / k g dose of A M P , M E T H or PCA on cortical T R P concentrations 0.25, 0.5, 1, 3, 6, 12 and 24 h after injection. Control concentration was 3.9 ± 0.1 n g / m g of tissue (n = 30). * P < 0.01 and ** P < 0.001 compared to saline controls.

between the three agents were observed. PCA was by far the most effective in increasing cortical TRP concentrations. Similar results (data not shown) were observed when neostriatal parameters were compared, with PCA causing the most pronounced and long-lasting effects.

:::_~.

8o1

120

/" - ,

1 0 2 5 05

METH ",,

4. Discussion s

**

t

_/--

80- ~,~

**

. . . . e\',

/ \,~.~

",

4O-

~[':

0 25 Q5

I

3

6 TIME (hrs)

12

24

Fig. 5. Effect of a 15 m g / k g dose of A M P , M E T H or PCA on cortical 5 - H T and 5 - H I A A concentrations 0.25, 0.5, 1, 3, 6, 12 and 24 h after injection. Control concentrations were 5-HT, 0.27 ± 0.01 n g / m g of tissue (n = 30) and 5 - H I A A , 0.17 + 0.004 n g / m g of tissue (n = 30). N o 5 - H T or 5 - H I A A were detected at 12 and 24 h in the PCA treated animals. * P < 0.01 and • * P < 0.001 compared to saline controls.

Investigators studying the effects of amphetamines on the dopaminergic and serotonergic systems have been concerned about the apparent differences between METH and AMP on the serotonergic system. This study demonstrates that AMP, METH and PCA do indeed have different effects on the cortical and neostriatal serotonergic systems following a single injection. Three hours after the administration of 10 and 15 m g / k g all three amines significantly decreased TPH activity, whereas only PCA and METH reduced 5-HT and 5-HIAA concentrations. Data from the time-course studies are also indicative of differing effects. Although all three amines significantly depressed

15 cortical enzyme activity for periods up to 24 h, both METH and PCA reduced cortical indoleamine concentrations for similar periods; AMP reduced 5-HT and 5-HIAA concentrations only at 1 .h. These results may indicate that the three amines have a similar mechanism of action to reduce TPH activity and varying effects on transmitter and metabolite concentrations. The exact mechanism by which the three amines depress the enzyme is not yet apparent. Knapp et al. (1974) have suggested that AMP acts directly on TPH. However, they did not find an effect of AMP on the enzyme in vitro or in synaptosomes, and they ruled out the possibility that the drug was acting through a metabolic product. The time period of depression of TPH activity caused by the three agents was different. This may suggest a gradation in the toxicity caused by the three amines with AMP being the least and PCA the most toxic to the enzyme. This is further supported by studies using consecutive injections of iprindole and either AMP or METH. Peat et al. (1983) demonstrated that cortical TPH activity was significantly reduced 7 days after a single injection of iprindole and METH, whereas enzyme activity had recovered following injection of iprindole and AMP (Warren et al., 1984). METH and PCA have similar effects in reducing cortical 5-HT concentrations for at least 12 h (fig. 5). Long-term decreases in this neurotransmitter concentration have been observed by others following a single injection of PCA (Sanders-Bush et al., 1975) or sub-acute administration of M E T H (Hotchkiss and Gibb, 1980; Bakhit et al., 1981). Initially, PCA and METH caused an increase in cortical 5-HIAA concentrations possibly reflecting an increased release of serotonin a n d / o r inhibition of uptake. PCA has previously been shown to inhibit strongly the uptake and stimulate the release of synaptosomal 5-HT (Wong et al., 1973). Increased release and turnover of 5-HT by METH has also been reported by Leonard (1972). Numerous reports have shown that AMP also increases neurotransmitter release and inhibits reuptake (Homan and Ziance, 1981; Azzaro and Rutledge, 1973; Raiteri et al., 1975; Leonard, 1972); from our data cortical 5HIAA concentrations are significantly increased at

30 min when compared to those at 15 min. However at the earlier time-point, cortical 5-HIAA concentrations were significantly reduced by AMP, reflecting inhibition of monoamine oxidase (MAO)-catalyzed oxidation of 5-HT (a substrate of type A MAO). The ability of AMP to inhibit MAO in vivo has been demonstrated (Green and E1 Hait, 1980; Miller et al., 1980). It is possible that AMP, therefore, is more potent as an inhibitor of MAO-A than as a releaser of 5-HT. Fuller and Hemrick-Luecke (1982b) have also shown that the p-halogenated derivatives of AMP are more potent inhibitors of MAO-A in vitro than AMP itself, thus the inability of PCA to cause a reduction of 5-HIAA concentrations in the cortex after 15 min is suggestive that this amine is a more effective releaser or inhibitor of reuptake of 5-HT than inhibitor of MAO-A. The reduction in cortical 5-HT concentrations caused by METH and PCA occurred at approximately the same time as the reduction in TPH activity. A similar pattern was seen in the recovery of neurotransmitter concentrations and enzyme activity over 24 h. However, the reduction in cortical and neostriatal TPH activity caused by AMP was observed earlier and persisted for longer periods than the decrease in 5-HT concentrations (figs. 4 and 5). These data are suggestive of either a differential effect of AMP on in vivo enzyme activity and neurotransmitter concentrations or varying degrees of toxicity of the three amines on these parameters. Morgan et al. (1972) and Ricaurte et al. (1983) have previously reported a marked reduction in 5-HT concentrations following METH administration which was not observed after AMP. This study shows that administration of AMP, M E T H and PCA produces long-lasting and dosedependent decreases in cortical and neostriatal T P H activity, reflecting a possible in vivo inhibition of the enzyme. The order of potency for this effect was PCA > METH > AMP. However, only METH and PCA produced long-lasting and dosedependent decreases in 5-HT and 5-HIAA. This may indicate varying degrees of toxicity of the three amines or a differential effect of AMP on enzyme activity and neurotransmitter concentrations.

16

References Azzaro. A.J. and C.O. Rutledge, 1973, Selectivity of release of norepinephrine, dopamine and 5-hydroxytryptamine by amphetamine in various regions of rat brain, Biochem. Pharmacol. 24, 2801. Bakhit, C. and J.W. Gibb, 1981, Methamphetamine-induced depression of tryptophan hydroxylase: Recovery following acute treatment, European J. Pharmaco[. 76, 229. Bakhit, C., M.E. Morgan, M.A. Peat and J.W. Gibb, 1981, Long-term effects of methamphetamine on synthesis and metabolism of 5-hydroxytryptamine in various regions of the rat brain, Neuropharmacology 20, 1135. Fuller, R.W. and S. Hemrick-Luecke, 1980, Long-lasting depletion of striatal dopamine by a single injection of amphetamine on iprindole-treated rats, Science (Wash. D.C.) 209, 395. Fuller, R.W. and S. Hemrick-Luecke, 1982a, Further studies on the long-term depletion of striatal dopamine in iprindoletreated rats by amphetamine, Neuropharmacology 21, 433. Fuller, R.W. and S. Hemrick-Luecke, 1982b, Influence of ring and side chain substituents on the selectivity of amphetamine as a monoamine oxidase inhibilor, Res. C o m m u n . Subst. Abuse 3, 159. Green, A.L. and M.A.S. El Hait, 1980, A new approach to the assessment of the potency of reversible monoamine oxidase inhibitors in vivo, and its application to ( + ) a m p h e t a m i n e , p-methoxyamphetamine and harmaline, Biochem. Pharmacol. 29, 2781. Homan, H.D. and R.J. Ziance, 1981, The effects of damphetamine and potassium on serotonin release and metabolism in rat cerebral cortex tissue, Res. C o m m u n . ('hem. Pathol. Pharmacol. 31, 223. Hotchkiss, A.J. and J.W. Gibb, 1980, Long-term effects of muhiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain, J. Pharmacol. Exp. Ther. 214, 257. Hotchkiss, A.J., M.E. Morgan and J.W. Gibb, 1979, The longlasting effects of multiple doses of methamphetamine on neostriatal tryptophan hydroxylase, tyrosine hydroxylase, choline acetyl transferase and glutamate decarboxylase activities, Life Sci. 25, 1373. Ichiyama, A., N. Shigenobu, N. Yasotomi and O. Hayaishi, 1970, Enzymic studies on the biosynthesis of serotonin in mammalian brain, J. Biol. Chem. 245, 1699. Knapp, S., A.J. Mandell and M.A. Geyer, 1974, Effects of amphetamines on regional tryptophan hydroxylase activity and synaptosomal conversion of tryptophan to 5-hydroxytryptamine in rat brain, J. Pharmacol. Exp. Ther. 185, 43. Leonard, B.E., 1972, Effect of four amphetamines on brain biogenic amines and their metabolites, Biochem. Pharmacol. 21, 1289. Miller, H.H., P.A. Shore and D.E. Clarke, 1980, In vivo monoamine oxidase inhibition by d-amphetamine, Biochem. Pharmacol. 29, 1347.

M o r g a n , C.I)., F. C a t t a b e n i and E. ('osta, 1972, Methamphetamine, fenfluramine and their N-dealkylated metabolites: Effects on monoamine concentrations in rat tissues, J. Pharmacol. Exp. Ther. 180, 127. Peat, M.A. and J.W. Gibb, 1983, High-performance liquid chromatographic determination of indoleamines, dopamine and norepinephrine in rat brain with fluorometric detection, Anal. Biochem. 128, 275. Peat, M.A., P.F. Warren and J.W. Gibb, 1983, The effects of a single dose of methamphetamine and iprindole on the serotonergic and dopaminergic system of the rat brain, J. Pharmacol. Exp. Ther. 255, 126. Raiteri, M., A. Bertallini, F. Angelini and G. Levi, 1975, d-Amphetamine as a releaser or reuptake inhibitor of biogenie amines in synaptosomes, European J. Pharmacol. 34, 189. Ricaurte, G.A., R.W. Fuller, K.W. Perry, L.S. Seiden and C.R. Schuster, 1983, Fluoxetine increases long-lasting neostriatal dopamine depletion after administration of d-methamphetamine and d-amphetamine, Neuropharmacology 22, 1165. Ricaurte, G.A., C.R. Schuster and L.A. Selden, 1980, Long-term effects of repeated methylamphetamine administration on dopamine and serotonin neurons in the rat brain: A regional study, Brain Res. 193, 153. Sanders-Bush, E., J.A. Bushing and F. Sulser, 1975, Long-term effects of p-chloroamphetamine and related drugs on central serotonergic mechanisms, J. Pharmacol. Exp. Ther. 192, 33. Sitaram, B.R. and G.J. Lees, 1978, Diurnal rhythm and turnover of tryptophan hydroxylase in pineal gland of the rat, J. Neurochem. 31, 1021. Steranka, L.R., 1982, Long-term decreases in striatal dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid after a single injection of amphetamine in iprindole-treated rats: Time-course and time-dependent interactions with amfonelic acid, Brain Res. 234, 123. Trulson, M.E. and B.L. Jacobs, 1979, Chronic amphetamine administration to cats: Behavioral and neurochemical evidence for decreased serotonergic function, J. Pharmacol. Exp. Ther. 211,375. Trulson, M.E. and B i . Jacobs, 1980, Chronic amphetamine administration decreases brain tryptophan hydroxylase activity in cats, Life Sci. 26, 229. Trulson, M.E. and V.M. Trulson, 1982, Reduction in brain serotonin synthesis following chronic methamphetamine administration in rats, European J. Pharmacol. 83, 97. Warren, P.F., M.A. Peat and J.W. Gibb, 1984, The effects of a single dose of amphetamine and iprindole on the serotonergic system of the rat brain, Neuropharmacology 23, 803. Wong, D.T., J. Horng and R.W. Fuller, 1973, Kinetics of serotonin accumulation into synaptosomes of rat brain-effects of amphetamine and chloroamphetamine, Biochem. Pharmacol. 22, 311.