The effect of MDMA (3,4-methylenedioxymethamphetamine) on the 5-HT synthesis rate in the rat brain: an autoradiographic study

Brain Research 810 Ž1998. 76–86

Research report

The effect of MDMA Ž3,4-methylenedioxymethamphetamine. on the 5-HT synthesis rate in the rat brain: an autoradiographic study 1 Dorotea Muck-Seler , Sho Takahashi 2 , Mirko Diksic ¨ ˇ

)

Cone Laboratory for Neurosurgical Research, Department of Neurology and Neurosurgery, and Montreal Neurological Institute, McGill UniÕersity, 3801 UniÕersity St., Montreal, Quebec, Canada H3A 2B4 Accepted 25 August 1998

Abstract The effect of MDMA Ž3,4-methylenedioxymethamphetamine., a psychotropic amphetamine derivative, treatment on the rate of serotonin Ž5-hydroxytryptamine; 5-HT. synthesis in the rat brain was studied by autoradiography using a-w14Cx-methyl-L-tryptophan method. Three different treatment protocols were compared to the control Žsaline. treated rats: Ž1. rats treated twice with 10 mgrkg every 12 h Ž20 mgrkg total. and injected tracer for the synthesis measurements 15 h later; Ž2. rats treated with four injections of 5 mgrkg every 12 h Ž20 mgrkg total. and injected tracer for the synthesis measurement 17 h after the last dose; and Ž3. rats given eight injections of 5 mgrkg every 12 h for four days Ž40 mgrkg. and used in the synthesis study 14 days after the last dose. Results showed a significant decrease in the rate of synthesis in the majority of cerebral structures examined in the 10 mgrkg group. In contrast the group receiving the same total amount Ž20 mgrkg. of MDMA but over two days Ž4 = 5 mgrkg. showed a significant increase in 5-HT synthesis in comparison to controls. The 5-HT synthesis rates measured 14 days after the last dose Žfour days, 8 = 5 mgrkg. were significantly reduced. The findings suggest that MDMA can produce either an increase or a decrease in the 5-HT synthesis a short time after a total dose of 20 mgrkg depending on the dose fractionation. However, 14 days after total dose of 40 mgrkg given over four days the synthesis rate was significantly reduced in many brain structures. The latter suggests a possible effect of the MDMA neurotoxicity on the serotonergic neurons, in addition to a possible influence on 5-HT synthesis via a feedback mechanism. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Serotonin synthesis rate; MDMA; a-methyl-L-tryptophan; Ecstasy; Neurotoxicity

1. Introduction The psychotropic amphetamine derivatives 3,4-methylenedioxymethamphetamine ŽMDMA; ‘Ecstasy’. has been used for recreational w26x and therapeutic purposes in man w11x. It produces an euphoric action and unique effects on emotion. Lately there have been abundant immunocytochemical w24x, biochemical w13,16x and behavioural w16x investigations of the effect of MDMA on serotonergic neurons. MDMA acutely releases 5-HT w40x followed by degeneration of fine 5-HT axon terminals w11,24x, while

) Corresponding author. Fax: q 1-514-398-8195; E-mail: [email protected] 1 Permanent address: Laboratory for Molecular Neuropharmacology, Ruder Boskovic Institute, Zagreb, Croatia. 2 Permanent address: Department of Neurosurgery, Brain Research Institute, Niigata University, Niigata, Japan.

other 5-HT axons and raphe cell bodies are spared. In rats, this drug causes large reductions in brain levels of 5-HT by degeneratively affecting presynaptic 5-HT terminals. We have also reported differential influence on 5-HT synthesis in terminals and the cell bodies after treatment with D,L-fenfluramine w18x and fluoxetine w19x. The exact mode of the MDMA neurotoxic action has not been established as yet, but several possible mechanisms have been proposed. In vitro experiments have shown that methamphetamine can produce at physiological pH w41x 5,6-dihydroxytryptamine Ž5,6-DHT. a known serotonergic neuron toxin. After a single large dose of methamphetamine the formation of 5,6-DHT w7x and 6-hydroxydopamine w32x have been documented in the rat brain. It has been proposed that MDMA interacts with vesicular and plasma membrane transporters of 5-HT and dopamine w29,34,43x, and in addition of releasing 5-HT releases also dopamine w12,33,36x. Although, immunocytochemical staining for tyrosine hydroxylase demonstrates

0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 8 9 - 0

D. Muck-Seler ¨ ˇ et al.r Brain Research 810 (1998) 76–86

that catecholamine-containing axons are not damaged at short or long survival time after MDMA w22,24x, it has been suggested that dopamine ŽDA. somehow is involved in the neurotoxic effect of MDMA w39x. Since the total amount of 5-HT released and present in the tissue is also a function of an ongoing de novo synthesis of 5-HT and monoamine oxidase ŽMAO. activity, for a complete understanding of MDMA action it is important to know the influence of MDMA on 5-HT synthesis. In this respect, it has been reported that in in vitro MDMA is a competitive inhibitor of MAO-A Ž K i s 22 mmolrl. and to a lesser extent of MAO-B Ž K i s 370 mmolrl. w13x.

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3,4-Methylenedioxyamphetamine ŽMDA. is a main metabolite of MDMA w6x. Brain levels of MDA are higher than the concentration of Žq. MDMA after subcutaneously administration of lower doses Ž5–10 mgrkg. 3 h after Žq. MDMA injection. In this context it is interesting to note that a low dose Ž1.8 mgrkg. of MDA increases 5-HT but does not change 5-HIAA concentration in the frontal cortex of rabbit 2 h after MDMA injection w27x suggesting an increase in the 5-HT synthesis. Very little investigation has been done on the effect of MDMA on 5-HT synthesis rate in the rat brain. Schmidt and Taylor w31x reported that MDMA given to rats at dose

Fig. 1. A set of representative autoradiograms in control and MDMA-treated rats obtained at 1 h Žimages A–D and I–L. and 2.5 h Žimages E–H and M–P. after tracer injection Ž30 mCi of a-w14 Cxmethyl-L-tryptophan are shown for rats treated twice every 12 h with 10 mgrkg of MDMA ŽTreatment A. and injected tracer 15 h later. The sections shown were taken at different levels and there was no attempt made to get them at the same level, rather we wanted to exemplify tracer distribution through brain. Cross-sections in control rats are shown in inserts ŽA–H. and those from treated rats in inserts I to P. Structures identified by letters are as follows: pineal body ŽPB., dorsal raphe ŽDR., thalamus ŽTH., ventral hippocampus ŽHPV., dorsal hippocampus ŽHPD., visual cortex ŽOC., medial caudate ŽCAM., lateral caudate ŽCAL..

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of 20 mgrkg reduces in vitro measured activity of tryptophan hydroxylase ŽTPH. for up to seven days after a single dose. They concluded that TPH loses activity by an alteration in Vmax , possibly by released neurotransmitter. Since the release and subsequent blocking of vesicular uptake of 5-HT with MDMA could have effect on the enzymeŽs. synthesising 5-HT via feedback mechanism, we decided to investigate different dosing of MDMA on the rate of in vivo 5-HT synthesis Žequivalent to TPH activity.. The aim of the present study was to investigate the effect of a total doses of 20 mgrkg and 40 mgrkg of MDMA on the regional 5-HT synthesis rate in the rat brain. Serotonin synthesis was measured autoradiographically using a-w14 Cxmethyl-L-tryptophan w9,10,23x. Three groups of rats were studied: Ž1. a group injected 10 mgrkg

Žs.c. every 12 h twice Ž20 mgrkg total., and injected with tracer about 15 h after the last dose; Ž2. a group injected 5 mgrkg every 12 h for two days Ž20 mgrkg total., and injected with tracer about 17 h after the last dose; and 3. a group injected 5 mgrkg every 12 h for four days Ž40 mgrkg total. and injected with tracer 14 days after the last dose.

2. Material and methods 2.1. Animals and experimental procedure Male Wistar rats weighing 200–225 g were used in the group treated with 10 mgrkg while, Sprague–Dawley rats

Fig. 2. Similarly as in Fig. 1 only for rats treated with four injections of 5 mgrkg ŽTreatment B. every 12 h. Tracer was injected about 17 h after the last dose. Structures identification are as those in Fig. 1, in addition AC identifies nucleus accumbens.

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Žmale. between 200–230 g, were used in the groups treated with 5 mgrkg. The animals were kept under controlled temperature and 12 h lightrdark cycle Žlight on at 7 h. conditions for at least 3 days before being used in the experiments. All experiments were performed on animals deprived of food, with water given at libitum, over night before experiments; food depravation was more or less for the same length in all rats. For all groups the controls were subcutaneously Žs.c. injected with saline, while the treatment groups were injected s.c. with MDMA dissolved in saline. One group of rats was injected twice every 12 h with 10 mgrkg of MDMA ŽTreatment A. and tracer was injected about 15 h after the last dose. Second

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group was injected four times every 12 h with 5 mgrkg of MDMA ŽTreatment B. and tracer was injected about 17 h after the last dose. Third group was injected eight times every 12 h with 5 mgrkg of MDMA ŽTreatment C. and tracer was injected 14 days after the last dose. All animals, controls and treated ones, underwent the same surgical preparation as described before w9,10x. In short, under light halothane Ž0.5–1.5%. anaesthesia, plastic catheters were inserted in femoral artery Žfor blood sampling. and vein Žfor tracer injection.. Rats were placed in loose-fitting plaster casts and then allowed to wake up. About 30 mCi of a-w14 Cxmethyl-tryptophan Ž a-w14 CxMTrp. Žspecific activity of about 55 mCirmmol; synthesized by

Fig. 3. As in Fig. 1 but for rats treated eight times with 5 mgrkg of MDMA ŽTreatment C. and with tracer injected 14 days after the last dose. Structures identification are as those in Fig. 1, in addition AC identifies nucleus accumbens.

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the procedure described by Mzengeza et al. w21x. in 1 ml of saline was injected intravenously with an injection pump over 2 min. The specific activity of 55 mCirmmol should be sufficiently high to not disturb steady state of tryptophan transport andror hydroxylation catalysed by TPH Žsee discussion in Ref. w10x.. With the beginning of the tracer injection, arterial blood samples were taken at progressively increased time intervals up to killing time. Total 12 to 15 blood samples were taken. The blood samples were centrifuged for 3 min at 12,500 g. Twenty ml of plasma was taken for the radioactivity determination by a liquid scintillation counting to measure plasma radioactivity to obtain the input function. The HPLC analysis showed that plasma does not have any other radioactive metabolite of tracer w20x. The physiological state of the animals was assessed periodically and was within normal values for rats in the lab. Blood gases were determined with a micro blood gas analyser ŽModel 178, CIBA-CORNING, CANADA.. Total and free tryptophan concentrations were measured by the HPLC method described before w20x. All animals use procedures were in strict accordance with the Canadian Council on Animal Care guidelines, and were approved by the Animal Care Committee of McGill University.

Animals were killed by guillotine one or two and half hours after tracer injection. In general, one half of the total number of rats was killed at each experimental time. However, when an odd number of animal was used the larger number of rats was killed at two and half hours. The brains were removed, frozen in Freon and cut into 30 mm slices in a cryostat at about y208C. In order to obtained autoradiograms, brain sections mounted on glass slides were exposed to X-ray film along with 14 C-polymer standards. After 3 weeks films were developed and radioactivity concentrations in different brain structures determined. Using a microcomputer-based image analysing system ŽImage Calculator by Soquelec, Montreal consisting of a video camera, a frame grabber or MCIDrM4-Image Analysis System by Imaging Research, Canada. and appropriate software different brain structures were identified with the aid of the rat brain atlas w25x. Optical densities were converted into tissue tracer concentration Ž Ci) ŽT .; nCirg. utilizing a calibration curve made by plotting the optical density of 14 C-standards as a function of their tissue equivalent concentration. The tissue radioactivity concentrations ŽnCirg. were converted into distribution volumes ŽDV; mlrg. by dividing tissue concentrations with the plasma tracer concentration, Cp) ŽT . ŽnCirml., at the end of the

Fig. 4. A plot of the volume of distribution ŽDV. multiplied by the plasma free Trp Ž Cp ; nmolrml; y-axis. for control and treated rats in the 10 mgrkg group. Exposure time Ž Q: x-axis. calculated as the ratio of the plasma integral of the tracer radioactivity and the plasma radioactivity at the end of experiment w8x. Plots exemplify relationship in the dorsal raphe and hypothalamus. Note that the position of the points on the horizontal axis is not the same even if rats were killed at the same time after injection. The main reason for this is the difference in the plasma clearance of the tracer in different animals, which produce slightly different exposure times for different rats. The slope of these lines is equal to K ) Cp wnmolrgrminx which after division with the LC Ž0.42. gives the rate of serotonin synthesis.

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experiment. The 5-HT synthesis rates were calculated as described in Appendix A. The statistical significance of the results was evaluated by the two-tailed t-test or ANOVA comparing synthesis in the treatment to that in respective control group. This comparison was done after a significant main effect was found by one-sample t-test on the ratios of 5-HT synthesis in control to that in respective treatment group. The mean ratio with its standard deviation ŽS.D.. was compared to one with S.D. of zero Žthe null hypothesis., because if there is no difference in the 5-HT synthesis rates between two groups the mean ratio should not be significantly different from one. Differences with p - 0.05 were considered to be significant. There was no attempt made to make comparison between different treatment groups.

3. Results There was no difference in the body weight and the physiological parameters Že.g., PaO 2 , PaCO 2 , pH. among the animals in the treated and respective control groups at the time of tracer injection. The plasma free Trp concentration in control Ž9.4 " 1.1 nmolrml. and two days Ž4 = 5 mgrkg. MDMA treatment Ž11.3 " 1.5 nmolrml. groups were significantly different

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Ž p - 0.005; ANOVA.. There was no significant difference in the plasma free Trp concentration in rats two weeks after MDMA Ž8 = 5 mgrkg. treatment Ž8.8 " 3.9 nmolrml. and respective controls Ž9.4 " 3.9 nmolrml.. Similarly, there was no significant difference in the plasma free Trp in the group treated twice with 10 mgrkg of MDMA every 12 h Ž7.9 " 2.9 nmolrml. from that in the respective controls Ž9.0 " 1.9 nmolrml.. A set of representative autoradiograms obtained in the brains of control and treated rats for one, two, and four-day treatments Žshown in Figs. 1–3.. Controls are always shown on the left side of each figure. Even by visual examination it is easy to identify structures which contain a high concentration of serotonergic cell bodies Že.g., dorsal raphe, pineal body.. Examples of the regression plots used in the calculation of the unidirectional uptake constant Ž K ) ; mlrgrmin. in two brain structures of control and MDMA treated rats are shown in Fig. 4. In these figures the linear relationship w8x between the product of the plasma free Trp and DV Ž y-axis. are shown as a function of exposure time w Q s H0T Cp) Ž t .d trCp) ŽT .x. The rates of 5-HT synthesis measured in many brain structures are given in Tables 1–3. Standard deviations of the rates reported in Tables 1–3 were calculated from the standard deviations in K ) Cp reported by the variance–covariance matrix of the least-squares fit.

Table 1 Serotonin synthesis Ž R; pmolrgrmin. in representative discrete structures of the rat brain treated with Ž2 = 10 mgrkg; Treatment A. of MDMA every 12 h and tracer injected 15 h after the last dose Structure

Raphe

Cortex

Substantia nigra Striatum Thalamus Hippocampus

Geniculate body Hypothalamus Inferior olive Nucleus accumbens Pineal body Locus coeruleus

R Žpmolrgrmin.

Dorsal Median Magnus Visual Auditory Parietal Sensory Frontal Caudate lateral Caudate medial Ventral Dorsal Ventral CA3 Dorsal Medial Lateral

% Change from control

Control Ž n s 10.

MDMA Ž n s 12.

140 " 22 83 " 17 64 " 12 32 " 9 31 " 10 31 " 11 37 " 13 26 " 10 35 " 12 27 " 11 33 " 11 36 " 13 32 " 11 48 " 11 54 " 6 48 " 10 32 " 12 35 " 10 45 " 14 41 " 15 55 " 13 1462 " 490 43 " 11

50 " 9 43 " 10 33 " 9 18 " 5 13 " 5 15 " 6 19 " 5 11 " 5 9"5 13 " 6 16 " 8 15 " 6 12 " 6 23 " 6 26 " 6 24 " 6 9"6 9"6 24 " 5 23 " 9 26 " 8 711 " 416 10 " 6

y64 y48 y48 y42 y56 y53 y49 y56 y73 y53 y51 y59 y65 y54 y51 y51 y71 y73 y46 y45 y53 y52 y78

In the last column, the percentage of change in the treated group from the synthesis in the in the control group. The main effect was evaluated as described in Section 2. Rates are given as mean " standard deviation and decrease was significant in all structures Ž p - 0.05; ANOVA..

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Table 2 Serotonin synthesis rates Ž R; pmolrgrmin. measured in rat brain after MDMA injection Ž4 = 5 mgrkg; Treatment B. or saline Žcontrol. R Žpmolrgrmin. a

Structure

Raphe

Cortex

Substantia nigra Striatum

Thalamus Hippocampus Geniculate body Ventral tegmental area Medium forebrain bundle Hypothalamus Superior colliculus Inferior colliculus Nucleus accumbens Medial anterior olfactory n. Superior olive Pineal body

Dorsal Median Magnus Visual Auditory Parietal Sensory-motor Frontal Reticulata Compacta Globus pallidus Caudate lateral Caudate medial Ventral Dorsal Ventral Dorsal Medial Lateral

% Change from control

Control Ž n s 10.

MDMA Ž n s 11.

174 " 11 125 " 14 46 " 6 39 " 7 39 " 7 34 " 7 32 " 7 40 " 8 27 " 6 34 " 8 35 " 7 35 " 7 49 " 8 34 " 8 39 " 8 50 " 7 44 " 6 38 " 7 45 " 7 39 " 8 38 " 6 36 " 6 38 " 7 26 " 7 63 " 10 51 " 9 34 " 8 467 " 102

304 " 28 213 " 29 69 " 10 70 " 12 61 " 11 57 " 11 52 " 11 53 " 12 50 " 8 61 " 11 61 " 11 64 " 12 81 " 16 66 " 10 70 " 10 81 " 10 75 " 10 62 " 10 66 " 10 61 " 10 64 " 10 73 " 10 59 " 9 40 " 8 96 " 14 79 " 12 46 " 9 445 " 93

74 71 51 77 55 68 61 31 84 80 78 81 66 93 77 63 68 61 47 57 68 104 52 52 54 56 35 y5

Tracer was injected approximately 17 h after the last dose of MDMA. a Results are expressed as mean" S.D. n is the number of animals. The main effect was evaluated as described in Section 2. There was a significant increase Ž p - 0.05; ANOVA. in 5-HT synthesis rate in the MDMA group as comparison to that in the control group in all structures except pineal body.

In the one day treatment group Ž2 = 10 mgrkg; total 20 mgrkg; Treatment A. 5-HT synthesis rates were drastically reduced, in comparison to the respective control rats in all structures examined ŽTable 1.. The greatest decreases in the rate of 5-HT synthesis, expressed as % of control, were observed in the locus coeruleus Ž78%., substantia nigra Ž73%., lateral geniculate body Ž73%., and medial geniculate body Ž71%.. The cortical and subcortical structures showed a decrease of about 50%. A decrease of 64% was observed in the dorsal raphe, while the decrease of 48% was observed in median and magnus raphe nuclei, the brain areas containing serotonergic cell bodies. The two days MDMA treatment Ž4 = 5 mgrkg; total 20 mgrkg; Treatment B. induced increase in the 5-HT synthesis rate in all brain regions ŽTable 2.. The most pronounced raise was found in the hypothalamus Žq104%. and thalamus Žq93%. as well as in the regions that are the parts of nigro-striatal pathway like substantia nigra pars reticulata Žq84. and compacta Žq80%., caudate Žq81%. and globus pallidus Žq78%.. 5-HT synthesis was in-

creased in the raphe nuclei: dorsal Žq75%., median Žq70%., and magnus Žq50%.. Two weeks after multiple injections of MDMA Ž8 = 5 mgrkg; total 40 mgrkg; Treatment C. there was a decrease in 5-HT synthesis rate in some brain regions. The decrease was most marked in the substantia nigra pars reticulata Ž31%.. A moderate decrease in 5-HT synthesis rate was observed in substantia nigra pars compacta Ž17%., auditory cortex Ž17%., caudate Ž14%., hippocampus Ž14%.. A small but statistically significant Ž p - 0.05; two-tailed t-test. increase in 5-HT synthesis rate was still observed in the median raphe nuclei Ž13%.. No differences in 5-HT synthesis rate between saline controls and MDMA treated rats was found in all other brain regions after two weeks survival period ŽTable 3.. In the pineal body there was no effect of MDMA on the 5-HT synthesis in rats ŽTable 2. treated for two days with an accumulated dose of MDMA of 20 mgrkg Ž4 = 5 mgrkg.. However, one day treatment Ž2 = 10 mgrkg; Table 1. and a multiple s.c. injections of MDMA Ž8 = 5

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Table 3 Serotonin synthesis rates Ž R; pmolrgrmin. measured in rat brain after MDMA injection Ž8 = 5 mgrkg s.c.; every 12 h; Treatment C. or saline Žcontrol. R Žpmolrgrmin. a

Structure

Raphe

Cortex

Substantia nigra Striatum

Thalamus Hippocampus Geniculate body Ventral tegmental area Medium forebrain bundle Hypothalamus Superior colliculus Inferior colliculus Nucleus accumbens Medial anterior olfactory n. Superior olive Pineal body

Dorsal Median Magnus Visual Auditory Parietal Sensory-motor Frontal Reticulata Compacta Globus pallidus Caudate lateral Caudate medial Ventral Dorsal Ventral Dorsal Medial Lateral

% Change from Control and p

Control Ž n s 11.

MDMA Ž n s 11.

184 " 18 119 " 16 40 " 6 32 " 5 31 " 4 27 " 5 27 " 6 27 " 7 27 " 5 33 " 5 31 " 6 32 " 6 50 " 7 29 " 6 30 " 5 44 " 5 39 " 5 30 " 6 36 " 6 38 " 6 37 " 5 37 " 6 30 " 5 24 " 6 55 " 7 46 " 5 34 " 7 747 " 106

186 " 24 135 " 18 37 " 7 27 " 7 26 " 6 28 " 6 22 " 7 24 " 7 18 " 5 27 " 6 28 " 4 32 " 6 43 " 7 27 " 6 34 " 6 38 " 6 37 " 6 28 " 7 32 " 7 37 " 7 31 " 6 34 " 6 30 " 7 20 " 7 53 " 7 41 " 7 28 " 8 556 " 91

1; NS 13; p - 0.05 y8; NS y16; p - 0.05 y17; p - 0.05 3; NS y19; NS y12; NS y31; p - 0.01 y17; p - 0.03 y12; NS 0; NS y14; p - 0.02 y6; NS 10; NS y14; p - 0.02 y6; NS y5; NS y11; NS y13; NS y16; p - 0.03 y7; NS 0; NS y16; NS y4; NS y11; NS y18; NS y26; p - 0.01

Serotonin synthesis was measured 14 days after the last dose of MDMA. a Results are expressed as mean" S.D. n is the number of animals. Individual statistical comparison was done after the main effect was found to be significant Ž p - 0.001; one group t-test on the ratios; see Section 2 for details.. Percent change calculated relative to synthesis in control group. The significant difference in the synthesis rate by comparing treatment to corresponding control group Ž p - 0.05; ANOVA.. NS stand for not significant difference.

mgrkg; Table 3. produced a significant reduction of the 5-HT synthesis in the pineal body.

4. Discussion The dose of MDMA Ž5 mgrkg. used in present work is closer to the doses used by humans for recreational purpose w28x, but the multiple injections protocols applied here might not represent well the patterns used by humans. However, humans usually take a booster dose several hours after the first one which could be related to the multiple injections in the present protocol. In addition, interspecies differences in the MDMA metabolism were described w35x and should be kept in mind if an extrapolation of effects from rat to human. In the present work we have found the opposite, and the time-dependent effect of a low MDMA dose Ž20 mgrkg total; Treatments A and B. on 5-HT synthesis rate in the

rat brain ŽTables 1 and 2.. When rats were injected with the same total amount of MDMA Ž20 mgrkg total. but in two doses of 10 mgrkg ŽTreatment B., and the tracer injected about 15 h later after the last dose, the rates were reduced in the entire brain. In contrast to this reduction, the 5-HT synthesis rate was elevated in all structures when rats were treated with the same total dose of MDMA given in a dose of 5 mgrkg, and tracer injected about 17 h after the last dose. It is not expected that relatively small difference in the time of tracer injection Ž15 h; Treatment A, compared to 17 h; Treatment B. could have resulted in an opposite effect on the 5-HT synthesis ŽTables 1 and 2.. It is expected that rats treated with 2 = 10 mgrkg will have a higher concentration of 3,4-methylen edioxyamphetamine ŽMDA. present in the brain w6x, which could be involved in the overall effects on TPH activity. It should be noted that there was an increase of about 20% in the plasma free Trp in the two days treatment group, but this increase is substantially smaller than the

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increases in the 5-HT synthesis rates Ž35 to 104%.. Since it is known that the plasma free Trp correlates with the brain 5-HT synthesis w5,30x, the increases are probably in part related to the increase in the plasma Trp. Having the increase in 5-HT synthesis substantially greater than that of plasma Trp, the mechanism by which MDMA influences 5-HT synthesis is substantially more complex. An hypothesis of Schmidt and Taylor w31x, based on the in vitro measurements of the TPH activity, that MDMA produces probably indirectly reduction in the TPH activity, is not supported by data obtained after two days treatment, but would be supported by the data obtained after one day treatment. However, it should be noted that they used higher single dose Ž20 mgrkg. of MDMA. On the bases of data presented here and those of Schmidt and Taylor w31x it seems that a unit dose of the MDMA is the most important factor in producing inhibitory effect on TPH. TPH in the neurons damaged by MDMA could be activated, similarly to the activation of TPH observed after a local lesion of serotonergic terminals with 5,7-DHT w15,24x. This mechanism could be, at least in part, responsible for the increase in the 5-HT synthesis observed in rats given 4 = 5 mgrkg of MDMA over two days ŽTreatment B., as well as for not observing so great differences in rats in which synthesis was measured 14 days after the last dose ŽTreatment C.. Since the same total dose of the MDMA Ž20 mgrkg. given as two larger amounts Ž2 = 10 mgrkg; Treatment A. produced profound reduction in 5-HT synthesis it seems that MDMA might exert differential effect on serotonergic neurons when given more often at a lower doses. This reduction in 5-HT synthesis produced by MDMA is in line with the reported effect of MDMA on the ex vivo measured activity of TPH w31x. Two weeks after the final dose of MDMA Ž8 = 5 mgrkg; 4 days; Treatment C. a small but significant increase Ž13%. in 5-HT synthesis rate only in the median raphe with a significant decrease in the synthesis rate in several regions of the nerve terminals Žhippocampus, medial caudate, and substantia nigra. was observed. Battaglia et al. w3x reported that 20 mgrkg of MDMA given twice a day for 4 days, dose larger than one used in our work, produces profound reduction in the 5-HT uptake sites. This observation is also supported by others w17,24x who reported that there is a denervation of serotonergic terminals. It was also reported that the same dose as one used in our experiment produces two weeks after the last dose profound reduction in both 5-HT and 5-HIAA in most brain regions w2x. The reduction in the 5-HT synthesis observed by us two weeks after the last dose ŽTable 3. would support those earlier reports, and could be related to the loss of terminals. Since the reduction in the 5-HT synthesis is substantially less than one would expect from the reported terminal loss w2,3,24,28x, we would suggest that 5-HT synthesis was increased in the spared and damaged neuronal terminals by a compensatory mechanism. The latter would be similar to the observation reported for the

action of 5,7-DHT hypothalamic lesion on the 5-HT over entire ipsilateral brain w37x, and a substantial increase in the TPH mRNA in the dorsal raphe w4,14x. This compensatory increase in the 5-HT synthesis could in part be related to the observation in humans that there is no immediate neurological deficit seen in the abusers of this drug. Earlier reports that MDMA destroys serotonergic terminals but not cell bodies are supported by data presented here for the 5-HT synthesis showing that the rate of synthesis is at the control levels two weeks after the last MDMA dose ŽTable 3.. It is possible that the main reason for finding larger number of the brain structures in which 5-HT synthesis is below control levels is probably at least in part consequence of the denervation produced by MDMA w2,3,24,28x. The increase of synthesis in the median raphe could suggest existence of a compensatory mechanismŽs. which increased 5-HT synthesis in this raphe cell bodies as a part of compensation for the loss of some dorsal raphe projections to the structures receiving projections from both nuclei. This would also agree with the proposal that the projections from the dorsal raphe are more vulnerable to the MDMA toxicity w24x. Decrease in the 5-HT synthesis was significant in several structures Že.g., substantia nigra, neutral hippocampus. known to receive predominantly projection from dorsal raphe w1x. The reduction in the synthesis rate would be, probably even greater if there would not be compensatory mechanism, as well as partial recovery of damaged terminals w3,35x. Since there is an increase in 5-HT synthesis in the median raphe this increase could be a result of the compensatory mechanism in spared serotonergic neurons. This compensatory mechanism in median raphe neurons, and possibly those with projecting from dorsal raphe with damaged terminals w1x, probably contributes to the observation that the rate of 5-HT synthesis is less reduced ŽTable 3. than it would be expected on the bases of denervation based on the loss of uptake sites Že.g., parietal cortex, sensori motor cortex.. Interactions between serotonergic and dopaminergic systems of the brain have been described in numerous investigations w33,36,42x. There is evidence that MDMA increases release of DA w12,33,36x, probably, through carrierrtransporter-mediated process. 5-HT increases DA release via activation of 5-HT2 receptors. In this respect, the decrease in 5-HT synthesis rate, observed in the present work after administration of MDMA in a dose of 10 mgrkg ŽTreatment A. could be, at least in part, explained with the MDMA mediated increase in DA release, which would be expected to be greater w39x in 10 mgrkg than in 5 mgrkg ŽTreatment B. groups.

5. Conclusion In summary, we found a significant dose and time-dependent effect of MDMA on the 5-HT synthesis rate in the

D. Muck-Seler ¨ ˇ et al.r Brain Research 810 (1998) 76–86

rat brain regions. Its effect could be explained through direct or indirect effect on TPH activity and 17 h after the last dose. In addition, damaged axon terminals could stimulate synthesis of 5-HT specially at lower MDMA dose Ž5 mgrkg.. An activation of TPH, as part of compensatory mechanism, could also be in part responsible for finding a reduction in 5-HT synthesis only in few structures receiving majority innervation from dorsal raphe. Our findings would also be in line with reports suggesting that the damage to the brain serotonergic system is dose dependent w6x. Moreover, findings that the brain damage and recovery after MDMA is dependent on the dose and time through which this dose was administered, could have importance in the recreational use of this drug.

Acknowledgements Research was supported in part by the Medical Research Council of Canada ŽMT-13368. and National Institute of Health ŽRO1-NS-29629..

Appendix A The biological model and the way how to calculate rate of 5-HT synthesis has been described in details before w10,23x. After solving appropriate differential equations w10x total tissue tracer concentration Ž Ci )ŽT .; nCirg. at the end of an experiment can be expressed by the following equation w9,10,23x: Ci) Ž T . s K )

p

T

H0 eŽ

=

K 1) k 2)

T

H0 C ) Ž t . d t q Ž k k 2)6qk 3 ) . Ž tyT .

) 2 q k3

Cp) Ž t . d t

. Ž 1.

Here plasma to brain clearance K ) Žmlrgrmin. s Ž K 1 ) k 3 ).rŽ k 2 ) q k 3 )., with K 1 ) to k 3 ) being biological model rate constants discussed before w10x. ŽNote that K 1 ) incorporates free fraction of tracer.. After an apparent steady-state has been achieved, the exponential term becomes ‘small’ and approaches a constant value. After that time and division of Eq. Ž1. with the plasma tracer concentration Ž Cp )ŽT .; nCirml. there is a linear relationship between tissue distribution volume ŽDV s Ci) ŽT .rC p) ŽT .; mlrg. and the exposure time Ž Q s H0T Cp) Ž t .rCp) ŽT .; min. with a slope equal plasma to brain clearance K) and intercept equal apparent volume of precursor distribution Ž Vapp ; mlrg.. Since animals had somewhat different plasma free Trp Ž CpTrp ; pmolrml. equation for DV was multiplied by the plasma free Trp w8x ) resulting in Eq. Ž2.. Note that here K Trp represents a

85

product between K ) and ‘averaged’ plasma free Trp for a particular group of rats. DV Ž T . CpTrp s

Ci Ž T . Cp) Ž T .

) s K Trp

T

CpTrp

H0 C

) p

Ž t . d trCp) Ž T . q Vapp CpTrp . Ž 2 .

) . The regression lines of which slopes Ž K Trp were calculated from the linear relationship of the DVŽT. CpTrp Žpmolrg. and the exposure time Ž Q; min. are exemplified in Fig. 4. The rates of 5-HT synthesis Ž R; pmolrgrmin. ) were calculated by dividing the slope of those K Trp Žpmolrgrmin. with LC w8x measured in vivo in the rat ) brain w38x; Ž R s K Trp rLC; pmolrgrmin.. The LC is the ‘lumped constant’ found to be uniform throughout the rat brain and having an average value of 0.42 " 0.07 w38x. All products of the distribution volumes and CpTrp as a function of exposure time had a significant Ž p - 0.01; F-statistics. linear relation with a positive slope. To obtain more appropriate influences of different rat plasma concentration of free Trp on the calculation of the 5-HT synthesis rates, the product of K ) and CpTrp needed in the calculation of the synthesis rate Žsee above equation. was calculated from the linear relationship of DV and CpTrp product as a function of the exposure time as described before w8x.

References w1x E. Azmitia, M. Segal, An autoradiographic analysis of the differential ascending projections of the dorsal and medial raphe nuclei in the rat, Brain Res. 196 Ž1978. 405–415. w2x G. Battaglia, S.Y. Yeh, E. O’Hearn, M.E. Molliver, M.J. Kuhar, E.B. DeSouza, 3,4-Methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine destroy serotonin nerve terminals in rat brain: quantification of neurodegeneration by measurement of w3Hxparoxetine-labelled uptake sites, J. Pharmacol. Exper. Ther. 242 Ž1987. 911–916. w3x G. Battaglia, J. Sharkey, M.J. Kuhar, E.B. De Souza, Neuroanatomical specificity and time course of alterations in rat brain serotonergic pathways induced by MDMA Ž3,4-methylenedioxyamphetamine.: assessment using quantitative autoradiography, Synapse 8 Ž1991. 249–260. w4x C. Bendotti, A. Servadio, G. Forloni, H.A. Angere, R. Samanin, Increased tryptophan hydroxylase in RNA in raphe serotonergic neurons spared by 5,7-dihydroxytryptamine, Mol. Brain Res. 8 Ž1990. 343–348. w5x D.L. Bloxam, G. Curzon, A study of proposed determinants of brain tryptophan concentration in rats after portocaval anastomosis or sham operation, J. Neurochem. 31 Ž1978. 1255–1263. w6x T. Chu, Y. Kumagai, E.W. Distefano, A.K. Cho, Disposition of methylenedioxymethamphetamine and three metabolites in the brains of different rat strains and their possible roles in acute serotonin depletion, Biochem. Pharmacol. 51 Ž1996. 789–796. w7x D.L. Commins, K.J. Axt, G. Vosmor, L.S. Seiden, 5,6-Dihydroxytryptamine, a serotonergic neurotoxin, is formed endogenously in the rat brain, Brain Res. 403 Ž1987. 7–14. w8x M. Diksic, S. Nagahiro, M. Grdisa, ˇ The regional rate of serotonin

86

w9x w10x

w11x

w12x w13x

w14x

w15x

w16x

w17x

w18x

w19x

w20x

w21x

w22x

w23x

w24x

w25x w26x

D. Muck-Seler ¨ ˇ et al.r Brain Research 810 (1998) 76–86 synthesis estimated by the a-methyl-tryptophan method in rat brain from a single time point, J. Cereb. Blood Flow Metab. 15 Ž1995. 806–813. M. Diksic, Imaging of the serotoninergic neuronal system in the brain, Period. Biol. 93 Ž4. Ž1991. 525–532. M. Diksic, S. Nagahiro, T.L. Sourkes, Y. Yamamoto, A new method to measure brain serotonin synthesis in vivo: I. Theory and basic data for a biological model, Cereb. Blood Flow. Metab. 10 Ž1990. 1–12. A.R. Green, A.J. Cross, G.M. Goodwin, Review of the pharmacology and clinical pharmacology of 3,4-methylenedioxymethamphetamine ŽMDMA or Ecstasy., Psychopharmacology 119 Ž1995. 247–260. S. Koch, M.P. Galloway, induced dopamine release in vivo: role of endogenous serotonin, J. Neural Transm. 104 Ž2–3. Ž1997. 135–146. E.F. Kokotos-Leonardi, E.C. Azmitia, MDMA ŽEcstasy. inhibition of MAO type A and type B: comparison with fenfluramine and fluoxetine ŽProzac., Neuropsychopharmacology 10 Ž1996. 231–238. V. Ljubic-Thibal, A.J. Morin, M. Diksic, E. Hamel, Changes in tryptophan hydroxylase ŽTPH. mRNA expression in the dorsal raphe nucleus following lesion of the dorsolateral hypothalamus ŽDHL. in the rat. Soc. for Neuroscience, Ann Meeting, Washington, DC, November 16–21, 1996. V. Ljubic-Thibal, M. Diksic, E. Hamel, S. Raison, J.-F. Pujol, D. Weissman, Ipsilateral alterations in tryptophan hydroxylase activity in rat brain after hypothalamic 5,7-di-hydroxytryptamine lesion, Brain Res. 724 Ž1996. 222–231. M.G. McNamara, J.P. Kelly, B.E. Leonard, Some behavioral and neurochemical aspects of subacute Ž" .3,4-methylenedioxymethamphetamine administrations in rats, Pharmacol. Biochem. Behav. 52 Ž1995. 479–484. M.E. Molliver, Serotonergic neuronal systems: what their anatomic organization tells us about function, J. Clin. Psychopharmacol. 7 Ž1987. 3–23, wSuppl.x. D. Muck-Seler, M. Diksic, DL-Fenfluramine increases 5-HT synthe¨ ˇ sis rate in the terminals while decreasing it in the cell bodies of the rat brain, Brain Res. 737 Ž1996. 45–50. D. Muck-Seler, A. Jevric-Causevic, M. Diksic, Influence of fluoxe¨ ˇ tine on regional serotonin synthesis in the rat brain, J. Neurochem. 67 Ž1996. 2434–2442. D. Muck-Seler, M. Diksic, The acute effects of reserpine and ¨ ˇ NSD-1015 on the brain serotonin synthesis rate measured by an autoradiographic method, Neuropsychopharmacology 12 Ž1995. 251–262. S. Mzengeza, T.K. Venkatachalam, S. Rajagopal, M. Diksic, Synthesis of enantiomerically pure a-Ž14 C.methyl-L-tryptophan, Appl. Radiat. Isot. 44 Ž1993. 1167–1172. J.F. Nash, Ketanserin pretreatment attenuates MDMA-induced dopamine release in the striatum as measured by in vivo microdialysis, Life Sci. 47 Ž1990. 2401–2408. S. Nagahiro, A. Takada, M. Diksic, T.L. Sourkes, K. Missala, Y. Yamamoto, A new method to measure brain serotonin synthesis in vivo: II. A practical autoradiographic method tested in normal and lithium-treated rats, J. Cereb. Blood Flow Metab. 10 Ž1990. 13–21. E. O’Hearn, G. Battaglia, E.B. De Souza, M.J. Kuhar, M.E. Molliver, Methylenedioxyamphetamine ŽMDA . and methylenedioxymethamphetamine ŽMDMA. cause ablation of serotonergic axon terminals in forebrain: immunocytochemical evidence for neurotoxicity, J. Neurosurg. 8 Ž8. Ž1988. 2788–2803. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. S.J. Peroutka, Incidence of recreational use of 3,4-methylenedioxymethamphetamine ŽMDMA, Ecstasy. on undergraduate campus, N. Eng. J. Med. 317 Ž1987. 1542–1543.

w27x A.G. Romano, W. Du, J.A. Harvey, Methylenedioxymethamphetamine: a selective effect on cortical content and turnover of 5-HT, Pharmacol. Biochem. Behav. 49 Ž1994. 59–607. w28x G. Ricaurte, G. Bryan, L. Strauss, L. Seiden, C. Schuster, Hallucinogenic amphetamine selectively destroys brain serotonin nerve terminals, Science 229 Ž1985. 986–988. w29x G. Rudnick, S.C. Wall, The molecular mechanism of ecstasy w3,4methylenedioxymethamphetamine ŽMDMA. Serotonin transporters are targets for MDMA-induced serotonin release, Proc. Natl. Acad. Sci. 89 Ž1992. 1817–1821. w30x M. Salter, R.G. Knowles, C.I. Pogson, How does displacement of aluminium-bound tryptophan cause sustained increases in the free tryptophan concentration in plasma and 5-hydroxytryptamine synthesis in brain?, Biochem. J. 262 Ž1989. 365–368. w31x C.J. Schmidt, V.L. Taylor, Depression of rat brain tryptophan hydroxylase activity following the acute administration of methylenedioxymethamphetamine, Biochem. Pharmacol. 36 Ž1987. 4095–4102. w32x L.S. Seiden, G. Vosmerm, Formation of 6-hydroxydopamine in candate nucleus of the rat brain after a single large dose of methamphetamine, Pharmacol. Biochem. Behav. 21 Ž1984. 29–31. w33x R. Simontov, M. Tomhen, The abused drug MDMA ŽEcstasy. induces programmed death of human serotonergic cells, FASEB J. 11 Ž1997. 141–146. w34x T.D. Steel, D.E. Nicols, G.K.W. Yim, Stereochemical effects of 3,4-methylenedioxymethamphetamine ŽMDMA. and related amphetamine derivatives on inhibition of uptake of w3Hx anomalies into synoptosenses from different regions of rat brain, Biochem. Pharmacol. 36 Ž1987. 2297–2303. w35x D.M. Stone, G.R. Hanson, J.W. Gibb, Differences in the central serotonergic effects of methylenedioxymethamphetamine ŽMDMA. in mice and rats, Neuropharmacology 26 Ž1987. 1657–1661. w36x D.M. Stone, H. Johnson, G.R. Hanson, J.W. Gibb, Role of endogenous dopamine in the central serotonergic deficits induced by 3,4methylenedioxymethamphetamine, J. Pharmacol. Exp. Ther. 247 Ž1988. 79–87. w37x K. Tsuiki, D. Muck-Seler, M. Diksic, Autoradiographic evaluation ¨ ˇ of the influence of hypothalamic 5,7-dihydroxytryptamine lesion on brain serotonin synthesis rate, Biochem. Pharmacol. 49 Ž1995. 633– 642. w38x M. Vanier, K. Tsuiki, M. Grdisa, ˇ K. Worsley, M. Diksic, Determination of the lumped constant for the a-methytryptophan method of estimating the rate of serotonin synthesis, J. Neurochem. 64 Ž1995. 624–635. w39x S.R. White, T. Obradovic, K.M. Imel, M.J. Wheaton, The effects of methylenedioxymethamphetamine ŽMDMA, Ecstasy. on monoaminergic neurotransmission in the central neurons system, Prog. Neurobiol. 49 Ž1996. 455–479. w40x C.H. Wichems, C.K. Hollingsworth, B.A. Bennett, Modulation of methlenedioxymethamphetamine ŽMDMA. and other substituted amphetamines in cultured fetal raphe neurons: further evidence for calcium-independent mechanisms of release, Brain Res. 695 Ž1995. 10–18. w41x M.Z. Wrona, Z. Yang, M. McAdams, S. O’Connor-Coates, G. Dryhurst, Hydroxyl radical-mediated oxidation of serotonin: potential insights into the neurotoxicity of methamphetamine, J. Neurochem. 64 Ž1995. 1390–1400. w42x B.K. Yamamoto, J.F. Nash, G.A. Gudelsky, Modulation of methylenedioxymethamphetamine induced striatal dopamine release by the interaction between serotonin and g-aminobutiric acid in the substantia nigra, J. Pharmacol. Exp. Ther. 273 Ž1995. 1063–1070. w43x B.K. Yamamoto, L.J. Spanos, The acute effects of methylenedioxymethamphetamine in dopamine release in the awake-behaving rat, Eur. J. Pharmacol. 148 Ž1988. 195–203.