I

'/_ 30_ Neuroscience Letters, 105 (1990) 300-306Elsevier Scientific Publishers Ireland Ltd.

tant role in the toxic m_

NSL06391 systemically active, non-

'.'dj we explored _he role ofreport that dextr0rphan

_,L in rat brain. This observ

The N-methyl-D-aspartate (NMDA) receptor MDMA-ind eaneurotMale Sprague-Dawle

antagonist, dextrorphan, prevents the neurotoxic mic MDMA was synthe

effects of 3,4-methylenedioxymethamphetamine t4j.Dextrorphanwas geGroups of rats (n= 6) w

¥, (MDMA) in rats trorphan alone (45 mgjincreasing doses of dext

'_3I Kevin T. Finnegan, John J. Skratt, lan Irwin and James W. Langston givensubcutaneously,e_'_ ' phanorsaline(givenin

t .,! Institute For Medical Research, San Jose, CA 95128 (U.S.A.) and The California Parkinson's Foundation,' San Jose, CA 95128 (U.S.A.) tion of MDMA. Animal

_-...2 (Received 2 May 1989; Accepted 16 June 1989) hippocampus, and Cort,obtained by making twoKey words.' Excitatory amino acid; Dextrorphan; N-Methyl-D-aspartate; Neurotoxicity; 3,4-Methylene- and the second anterior

dioxymethamphetamine; Serotonin

,; by dissecting away theUsing the systemically active, non-competitive N-methyl-o-aspartate (NMDA) receptor antagonist dex- sum, septum, and anter

trorphan, we explored the role of the NMDA receptor-calcium channel complex in the toxic mechanismlated from the remainin

of action of 3,4-methylenedioxymethamphetamine (MDMA). Rats were treated with MDMA, dextror-

_. phan, or the combination of MDMA and increasing doses of dextrorphan, and then killed 10 days later with a blunt scalpel an

for the assay of serotonin and dopamine in the striatum, hippocampus, and cortex. Dextrorphan totally underlying cerebral pedrprevented the serotonin-depleting effects of MDMA in the striatum, with a lessened but still significant by the horns of the lat_'

blockade noted in the hippocampus and cortex. These findings may provide a clue to the molecular events from the adjoining cort

underlying MDMA-induced neurotoxicity, pies were stored in liqui.'_'t acetic acid (5-HIAA), d

3,4-Methylenedioxymethamphetamine (MDMA) produces potent and selective homovanillic acid (HV__ neurotoxic effects on CNS serotonergic neurons in both rodents and primates [9, 12, detection, as described e

30, 32]. In humans, MDMA induces stimulant and psychedelic effects [33, 34],proba- MDMA (10 rog/kg)bly giving rise to its popularity as a drug of abuse among certain segments of the 52%.The hippocampuspopulation [26]. Because of increasing concerns that MDMA might cause serotoner- of MDMA, the concent_

gic damage in humans the drug has recently become the focus of intense scientific respectively (Table I). i%study. Although oxidative stress [37] or the generation of toxic compounds such as marker associated with

5,6-dihydroxytryptamine [9] have been suggested to play a role in the toxic effects the 3 brain areas (data ]of MDMA, its biochemical mechanism of action remains poorly understood, in the striatum were ur

? I Recently, Sonsalla et al. [36] have reported that the striatal dopamine-depleting el- rog/kg) prior to MDMAfects of methamphetamine, a compound structurally related to MDMA, are pre- inthe striatum. Lower d

" vented by systemically administered non-competitive N-methyl-D-aspar_ate in thisarea. The protect(NMDA) receptor antagonists. The authors suggest than NMDA receptor activation campus and cortex, witl

-'-_ and perhaps alterations in excitatory amino acid (EAA) transmission play an impor- dose ofdextrorphan. Theffects on 5-HT, with sm

(_ Correspondence.' K.T. Finnegan, Institute for Medical Research, 2260 Clove Drive, San Jose, CA 95128. or cortex, respectively (2U.S.A. Theseresultsshowth

' /0304-3940, 8%$ 03.50 ._ 1989 Elsevier Scientific Publishers Ireland Ltd.

06 301

td. ':_litant role in the toxic mechanism of action of methamphetamine. Using the potent,

systemically active, non-competitive NMDA receptor antagonist, dex.trorphan [6, 8], L I

we explored the rOle of NMDA receptors in the toxic actions of MDMA. We now _)report that dextrorphan prevents the serotonin (5-HT)-depleting effects of MDMA 4/.

in rat brain. This observation may provide a clue to the molecular events underlying %MDMA-inddced neurotoxicity, r-_

VMale Sprague-Dawley rats, weighing 200 g, were used for all experiments. Race-

mic MDMA was synthesized in our laboratory, according to the method of Braum ._

[4]. Dextrorphan was generously donated by Dr. Dennis Choi (Stanford University). _'Groups of rats (n = 6) were treated with saline, MDMA alone (10 mg/kg/dose), dex- .t._trorphan alone (45 rog/kg/dose), or the combination of MDMA (10 rog/kg) andincreasing doses of dextrorphan (7.5-45.0 mg/kg/dose). Five doses of MDMA were 7:3_l.7

given subcutaneously, each dose separated by a 6 h interval. A dose of either dextror-

phan or saline (given intraperitoneally) was administered 20 min before each injec-

tion of MDMA. Animals were killed 10 days later by decapitation, and the striatum, _,hippocampus, and cortex were rapidly dissected free over ice. Striatal tissue was %,

obtained by making two transverse sections: the first posterior to the olfactory bulband the second anterior to the optic chiasm. Anterior striatal tissue was then freed '0by dissecting away the cortical and septal parts of the slice, using the corpus callo-

sum, septum, and anterior commissure as landmarks. Hippocampal tissue was iso- , _fated from the remaining portion of the brain by first severing the corpus callosum ._

with a blunt scalpel and then dissecting the cerebral hemispheres away from the C'underlying cerebral peduncles. Taking advantage of the natural tissue plane created

by the horns of the lateral ventricle, hippocampal tissue was then gently separated ,[/[,from the adjoining cortical layer, a sample of which was also obtained. Tissue sam-

pies were stored in liquid nitrogen until assay. The levels of 5-HT, 5-hydroxyindole- L 1 Li

! acetic acid (5-HIAA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and _'_ homovanillic acid (HVA) were assayed by reverse-phase HPLC with electrochemical '%:.

detection,asdescribedelsewhere[13]. _"

MDMA (10 mg/kg) alone reduced the concentration of 5-HT in the striatum by

52%. The hippocampus and cortex were more sensitive to the amine-depleting effects.?'x

of MDMA, the concentrations of 5-HT in these areas being reduced by 73% and 86%, . ?respectively (Table I). MDMA-induced depletions of 5-HIAA, another biochemical ,_

marker associated with serotonergic neurons, were equivalent to those of 5-HT in .g._the 3 brain areas (data not shown). The concentrations of DA, DOPAC, and HVAin the striatum were unaffected (Table II). The administration of dextrorphan (45 J- _-,_rog/kg) prior to MDMA totally blocked the 5HT-<lepleting effects of this neurotoxin

in the striatum. Lower doses ofdextrorphan provided proportionately less protection

in this area. The protective effects of dextrorphan were less pronounced in the hippo- >>

campus and cortex, with significant protection being observed at only the 45 mg/kg %dose of dextrorphan. The administration of dextrorphan alone produced inconsistent :'

effects on 5-HT, with small but significant increases or decreases noted in the striatum '0

or cortex, respectively (Table I).

These results show that dextrorphan blocks the 5-HT-depleting effects of MDMA ,9:

rv

302

TABLE I TABLE II

CONCENTRATIONS OF SEROTONIN IN THE RAT STRIATUM, HIPPOCAMPUS, AND COR- CONCENTRATIONS OF DOPAI_

TEX : :' Values (ng/mg tissue) are expressedValues (ng/mg tissue) are expressed as the mean + S.E.M. for each treated group (n= 6). Values in paren- theses represent percent of control. (theses represent percent of control. Individual group means were compared using Student's t-tes_ cally significant dose-related effect c

(2-tailed), after a simple one-way ANOVA revealed a statistically significant Fvalue. DA, dopamine; DOPAC, dihydroxy

Striata Hippocampus Cortex DA ,

Saline control 0.340 + 0.026 0.305 + 0.026 0.233 _+0.023 Saline control 8.95+ 0.5Dextrorphan 0.415 + 0.024* 0.288 + 0.010 0.174 4-0.016* Dextrorphan l0.01 + 0.2

45 rog/kg i.p. (122.0%) (94.4%) (74.7%) 45 rog/kg J.p. ' (111.8%)''x 5 only x 5 only

MDMA 0.165 +0.015' 0.081 +0.015' 0.033 +_0.006* MDMA 9.21_+0.3(l0 rog/kg s.c. (48.5%) (26.6%) (14.2%) 10 rog/kg s.c. (102.9%)

x 5 only x 5 only

.._ Dextrorphan 0.168+ 0.019 0.080 + 0.004 0.032 + 0.002 Dextrorphan 9.074-0.227.5 mg/kg (49.4%) (26.2%) (13.7%) 7.5 rog/kg (101.3%)

andMDMA andMDMA

Dextrorphan 0.179+ 0.015 0.116 + 0.009 0.042 + 0.004 Dextrorphan 9_30_+0.4015mg/kg (52.6%) (38.0%) (18.0%) 15 rog/kg (103.9%)

andMDMA andMDMA

Dextrorphan 0.218+ 0.018** 0.118 _+0.012 0.051 _+0.004 Dextrorphan 10.60+ 0.3230 mg/kg (64.1%) (38.7%) (21.9%) 30 mg/kg (118.4%)

andMDMA andMDMA '

Dextrorphan 0.342_ 0.021'* 0.172_+_0.017'* 0.072_.+0.007** Dextrorphan 9.43+-0.2945 mg/kg (I00.6%) (56.4%) (30.9%) 45 mg/kg (105.4%)

andMDMA andMDMA

*P < 0.05, compared to saline; **P < 0.05, compared to MDMA alone. *P< 0.05, compared to saline.

in rat brain. Interestingly, dextrorphan and its methylated derivative, dextromethor- increased sensitivity of the corte

phan, also provide protection against the neuronal injury associated with several otb- density of specific [3H]glutam a

er pathogenic conditions. Dextrorphan blocks the neuronal damage induced by these two brain areas as compa:

hypoxemia in cell culture [16] and, in animal models of stroke, dextromethorphan regional distribution of recepto

prevents the cellular injury produced by transient cerebral ischemia [15, 28]. In vitro, of dextrorphan were less prono_

dextrorphan has also been shown to markedly attenuate the neurotoxicity associated the striatum, as larger doses ofd

with hypoglycemic states [25] as well as selectively preventing the neuroexcitation number of receptors. Further e:

produced by NMDA itself [6]. Current evidence indicates that the neuronal damage would help clarify the relationsh

observed under these conditions is caused in large part by an NMDA receptor-gated alterations in MDMA-induced t,

accumulation of calcium within target cells [7, 35]. The neuroprotective effects ofdcx- in the biodistribution of dextrorptrorphan appear primarily due to its capacity to inhibit calcium entry by binding to ations in the biodistribution of 1_a site on the NMDA receptor-channel complex [6, 8, 39]. Our data, showing that grees of protection.

dextrorphan protects against the 5-HT-depleting effects of MDMA, therefore raise The mechanism by which the b

the possibility that the NMDA receptor-calcium channel complex plays an important neuronal injury produced by MErole in the toxic mechanism of action of this widely abused drug, perhaps by gating et al. [22] report that application

intracellular influxes of calcium, depletion of 5-HT-like immunore_

Consistent with this proposal, we observed that MDMA produced larger deple- of NMDA receptors on 5-HT-co

tions of 5-HT in the hippocampus and cortex than in the striatum (Table I). This injury. One possibility then is that

e

C

303 _'r;4/,

TABLE II

CONCENTRATIONS OF DOPAMINE AND ITS METABOLITES IN THE RAT STRIATUM [.. I

Values (ng/mg tissue) are expressed as the mean ± S.E.M. for each treated group (n= 6). Values in paren- btheses represent percent of control. Given in combination with MDMA, dextrorphan produced no statisti- -r./_.

%cally significant dose-related effect on the concentration of DA, DOPAC, or HVA (one-way ANOVA).DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid. "%

DA DOPAC HVA ,_

Saline control 8.95± 0.55 1.94± 0.13 1.04+ 0.08 _

Dextrorphan 10.01± 0.29 1.33± 0.05* 0.95+ 0.03 ,k,_45 rog/kg i.p. (111.8%) (68.6%) (91.3%)

x 5 only _3.3_]3MDMA 9.21+0.30 1.79±0.11 1.07±0.04 '

10mg/kg s.c. (102.9_;) (92.3%) (102.8%)x 5 only

Dextrorphan 9.075:0.22 !.58 5:0.07 0.91+ 0.04 _,7.5mg/kg (l 01.3%) (81.4%) (87.5%) c_t,

%/.

and MDMA "_

Dextrorphan 9.305:0.40 1.79±0.12 1.01+0.05l 5 rog/kg (103.9%) (92.2%) (97.1%) '0

and MDMA

Dextrorphan 10.60+ 0.32 1.69± 0.06 1.01± 0.04 _x,x_'_30mg/kg (118.4%) (87.1%) (97.1%) ,,,

and MDMA

Dextrorphan 9.435:0.29 1.60± 0.09 1.08± 0.06 _'

45andmg/kgMDMA (105.4%) (82.5%) (103.8%) '._'('v[_

*P<0.05, compared to saline. L ] ,_

increased sensitivity of the cortex and hippocampus appears to parallel the increased

density of specific [3H]glutamate or [3H]phencyclidine ([3H]PCP) binding sites in ',%_

these two brain areas as compared to the striatum [24, 29]. These differences in the ,._regional distribution of receptors may also help explain why the protective effects _'of dextrorphan were less pronounced in the cortex and hippocampus compared to _:

the striatum, as larger doses of dextrorphan might be required to block the increased _*-_number of receptors. Further experiments employing larger doses of dextrorphan ×_

would help clarify the relationship between increased receptor density and regional .g._!

alterations in MDMA-induced toxicity. Alternatively, it is possible that differences L3__15_:in the biodistribution of dextrorphan within the brain, or dextrorphan-induced alter-

ations in the biodistribution of MDMA, may have contributed to the different de-

grees of protection.

The mechanism by which the NMDA receptor-channel complex is involved in the _'('_!,

neuronal injury produced by MDMA, if this indeed is the case, is uncertain. Millar _c_

et al. [22] report that application of NMDA itself produces a selective and lasting

depletion of 5-HT-like immunoreactivity in retinal tissue, suggesting that activation '0

of NMDA receptors on 5-HT-containing neurons is capable of producing cellular ._injury. One possibility then is that MDMA (or a metabolite) might directly activate . _':

' ;'v

i

3on{, NMDA receptors present on 5-HT nerve terminals, or alternatively, that MDMA in the biochemical chain

causes ia release of EAAs from endogenous stores which in turn activate NMDA amines, such_as para-cMo

tlc receptors. A third possibility is that MDMA may produce neuronal damage by bind- hal damage produced by 1lng to one of the other ligand binding sites present on the NMDA receptor-channel ogenic conditions whose r

f_ complex [1, 2, 14, 18, 21, 38]; for example, MDMA has been reported to bind with receptors, and calcium.a moderately high affinity (2.5 #M) to the PCP recognition site [20].

If any of these possibilities are true, it might be predicted that the direct injection The attthors wish to th_P of MDMA into brain would produce neuronal damage, analogous to that noted after and to gratefully acknowl

the injection of NMDA or other EAA receptor agonists. Molliver et al. [23], how'- the preparation of this maever, observed no immunocytochemical evidence of damage to cortical 5-HT-con-

_ taining fibers after the direct intracerebral administration of MDMA in rat brain· I Anis,N.A., Berry,S,C.,ButteInterestingly, a similar puzzle appears to exist for the structurally related neurotoxin, cyclidine,selectivelyreduce _

Pharmacol., 79 (1983) 565--55methamphetamine. Non-competitive NMDA antagonists are reported to block the 2 Atilt, B.,Evans,R.H., Franci

/(_ striatal dopamine-depleting effects of methamphetamine [36] but nigrostriatal dope- aminoacid induceddepolariminergic neurons are evidently not damaged by the direct infusion of NMDA [40] physiol.,32 (I980)424-442.

,_ or other EAA agonists [10] into the striatum. It is also unclear how a direct interne- 3 Boegman, R.J. and Parent,tion of MDMA with any binding site associated with the NMDA receptor-channel excitatory aminoacidagonisl

complex could give rise to the selective effects of this neurotoxin. NMDA receptors 4 Braum, U., Schulgin, A.T. a_dioxyPhenylisopropylamine

are also thought to exist on brain dopaminergic [17, 31] and noradrenergic neurons 192-196.[19] as well as on striatal intrinsic neurons [3, 27]. Yet there is little evidence that 5 Carpenter, C.L., Marks, S.SMDMA produces any significant damage to dopamine or norepinephrine-containi ng phan as calciumchannelant_

neurons [9, 12] (present studies). 6 Choi, D.W. and Peters,S., Dcity in cortical cell Culture, N

The above considerations suggest that the capacity of dextrorphan to prevent 7 Choi, D., Calcium-mediatedMDMA-induced toxicity may involve an action at sites other than, or in addition damage,TrendsNeurosci.,1

i to, its effects at the NMDA receptor-channel complex. It is noteworthy in this regard 8 Church,J., Lodge,D. and B_that EAAs produce calcium-dependent neurotoxic effects not only by activating tation ofratspinalneuronsl

7(, NMDA receptor-gated calcium channels, but also by producing neuronal depolari- 9 Commins,D.L., Vosmer,G..micai and histological evidelzation with a subsequent activation of voltage-dependent calcium channels [11]. Re-tons in therat brain, J, Phar,

;_' cent evidence indicates that dextrorphan also inhibits voltage-gated influxes of cai- l0 Coyle, J.T. and Schwarcz, 1;cium into neurons [5]. MDMA may therefore produce at least a portion of its damag- acid, Nature (Lond.),263(1_

3 lng effects by promoting calcium entry into 5-HT neurons through an activation of Il Dingledine, R., N-Methyl-a:

voltage-gated channels. For example, transient effects of MDMA on cellular energy campal pyramidalcells,J. PI

dynamics or cell membrane permeability could theoretically activate voltage-gated 12Finnegan, K.T., Ricaurte,(administered MDMA cause

_'_ calcium channels by producing alterations in the cell membrane potential. Thus dex- 141-144.

· trorphan may be particularly efficacious in preventing MDMA-induced neurotoxi- t3 Finnegan, K,T., DeLanney,city because of its ability to block multiple routes of calcium entry. Further experi- effectsof 5,7-dihydroxytrypl

· _ ments, employing selective NMDA receptor blocking drugs, will be required to a%ess 496(1989)251-256,44

_ _ the relative contribution of receptor-gated or voltage-gated calcium channels to the 14 Foster, A.C. and Fagg, G.Itheir characteristics and relatoxiceffectsof MDMA. f5George,C.P.,Goldberg,M..

In summary, our findings suggest that calcium entry, gated by either the NM DA cai ischemicneuronaldamarreceptor-channel complex or perhaps voltage-activated channels, may play an impor- 16 Goldberg,M.P., Pram, P.Ctant role in the toxic mechanism of action of MDMA. Together with the results of injuryin neuronalculture,b

_' Sonsalla and colleagues [36] regarding methamphetamine, the findings also raise the 17Jhamandas, K. and Marien,· byanexcitatoryaminoaci

possibility that intracellular influxes of calcium may represent the final common link 641--650.

c

305in the biochemical chain of events underlying the neurotoxicity of other phenethyl- _-amines, such as para-chloroamphetamine or fenfiuramine. It appears that the neuro- L inal damage produced by this class of drugs is to be added to the growing list of path- bogenic conditions whose neurotoxic effects appear to be mediated by the EAAs, their "' <5-

receptors,andcalcium.?

The authors wish to thank Dr. Dennis Choi for his generous gift of dextrorphan, ,-5.and to gratefully acknowledge Pamela Schmidt and David Rosner for their help in .:?thepreparationofthismanuscript. ?

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