Monoamine uptake inhibitors block the neurotoxic effects of methamphetamine (MA) upon dopaminergic and serotonergic neurons in the rat. The neurotoxic ...
Brain Research, 516 (1990) 1-7 Elsevier BRES 15427
1
Research Reports
The effects of monoamine uptake inhibitors and methamphetamine on neostriatal 6-hydroxydopamine (6-OHDA) formation, short-term monoamine depletions and locomotor activity in the rat Gerard J. Marek, Georgetta Vosmer and Lewis S. Seiden The University of Chicago, Department of Pharmacological and Physiological Sciences, Chicago, IL 60637 (U.S.A.) (Accepted 3 October 1989)
Key words: Methamphetamine; Neostriatum; Locomotor activity; Monoamine; 6-Hydroxydopamine
Monoamine uptake inhibitors block the neurotoxic effects of methamphetamine (MA) upon dopaminergic and serotonergic neurons in the rat. The neurotoxic effects of MA upon dopaminergic neurons have previously been suggested to be mediated via formation of 6-hydroxydopamine (6-OHDA) from endogenous stores of dopamine (DA). In the present experiments, administration of the DA uptake inhibitor amfonelic acid (AFA, 10 mg/kg, i.p.) did not block the formation of 6-OHDA in rats treated with a single s.c. 100 mg/kg dose of MA. Consistent with the lack of effect by AFA on MA-induced 6-OHDA formation, neither AFA (10 mg/kg, i.p.) nor the DA and 5-hydroxytryptamine (5-HT) uptake inhibitor mazindol (40 mg/kg, i.p., MAZ) blocked the depletion seen in neostriatal DA levels 1, 2 or 8 h following administration of a single 100 mg/kg dose of MA. In fact, AFA enhanced the DA depletions 2 and 8 h following MA administration. AFA also enhanced the MA-induced increase in locomotor activity in rats and this effect was blocked by lesions of dopaminergic neurons with i.v.t. (intraventricular) 6-OHDA in desipramine-pretreated rats. These results suggest that DA uptake inhibitors do not prevent the neurotoxic effect of MA on DA neurons by either preventing entry of MA into the cell or blocking the efflux of DA out of the cell. Instead, the DA uptake inhibitors appear to prevent the neurotoxic effect of MA upon dopaminergic neurons by blocking entry of 6-OHDA into the cell. INTRODUCTION Methamphetamine (MA) is known to exert neurotoxic effects on dopaminergic and/or serotonergic neurons in a n u m b e r of species including rats, cats, primates and guinea pigs 8'19'20'26'27'31'32. Experiments studying the effects of manipulation of intraneuronal D A pools with reserpine, a-methyl-p-tyrosine (AMT) and pargyline upon long-term MA-induced D A depletions suggest that either D A or a D A metabolite rather than a metabolite of M A is directly responsible for the neurotoxicity engendered by M A administration 9'33. Consistent with the above work, 6-hydroxydopamine ( 6 - O H D A ) has been detected in the rat brain following administration of a single, 100 mg/kg dose of M A 12'25. D A and 5-hydroxytryptamine (5-HT) uptake inhibitors block the long-term MA-induced decreases in brain D A and 5-HT, respectively 11'13'1a. With regard to the hypothesis of 6 - O H D A formation from D A , the uptake inhibitors could block M A neurotoxicity by preventing either the formation of 6 - O H D A or the effects of 6 - O H D A . The D A uptake inhibitors could block 6O H D A formation by either (1) preventing entry of M A
into the neuron or (2) preventing efflux of the neurotransmitter at the high-affinity m o n o a m i n e uptake site. The possibility that the D A uptake inhibitors block M A neurotoxicity by preventing the effects of 6 - O H D A is raised by the observation that amfonelic acid (AFA) did not block short-term depletions of neostriatal D A following M A 13. Thus, the D A uptake inhibitors could block entry of 6 - O H D A into the cell after its formation in the synaptic cleft. The present experiments were designed to differentiate between the hypotheses listed above concerning the mechanism by which the D A uptake inhibitors A F A and mazindo135 block the neurotoxic effects of M A upon dopaminergic neurons. First, formation of 6 - O H D A was measured in the neostriata of rats administered M A and the D A uptake inhibitor A F A . Second, neostriatal D A and hippocampal 5-HT were measured in the rat 30 min, 1, 2, 6 or 8 h following administration of M A and A F A or M A and mazindol ( M A Z : a D A and 5-HT uptake inhibitor7). Earlier results had suggested that A F A enhanced short-term MA-induced D A depletions 13. To assess whether this effect of A F A was an idiosyncratic one, M A Z was tested since it blocks both dopamine and
Correspondence: L.S. Seiden, The University of Chicago, Department of Pharmacological and Physiological Sciences, 947 East 58th Street, Chicago, IL 60637, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
s e r o t o n i n uptake. Third, we d e t e r m i n e d A F A ' s effect on D A release by measuring the behavioral and biochemical e f f e c t s o f M A a n d A F A in b o t h s h a m a n d 6 - O H D A t r e a t e d rats w h o s e l o c o m o t o r activity was m e a s u r e d f o r a 6 h period
in s t a b i l i m e t e r s b e f o r e
being
killed
for
n e u r o c h e m i c a l assay. A F A did n o t p r e v e n t t h e f o r m a t i o n of 6-OHDA
in M A - t r e a t e d rats. T h e r e s u l t s o f t h e s e
e x p e r i m e n t s s u g g e s t t h a t use o f t h e A F A (10 m g / k g , i . p . ) b l o c k s t h e n e u r o t o x i c a c t i o n o f a single s.c. 100 m g / k g dose of M A by preventing the entry of 6 - O H D A into the n e u r o n t h r o u g h t h e D A u p t a k e site a f t e r its f o r m a t i o n in t h e s y n a p t i c cleft. A l t e r n a t i v e l y , A F A m a y p r e v e n t t h e entry of 6-OHDA
into the n e u r o n after intracellularly
formed
is r e l e a s e d i n t o t h e
6-OHDA
s y n a p t i c cleft
concomitantly with the parent c o m p o u n d D A . MATERIALS AND METHODS
Animals Subjects for this study were male Sprague-Dawley rats (Harlan Sprague-Dawley, Terre Haute, IN) weighing 280 + 10 g when the drug treatments were initiated. Rats were housed singly in wiremesh cages with free access to laboratory chow (Teklad 4% Rat Diet) and water. Ambient temperature was maintained at 22 + 1 °C. Fluorescent lighting was automatically turned on at 06.00 h and off at 20.00 h. Drug treatment The neostriata of rats in the experiments studying acute effects of MA and DA uptake inhibitors on brain neurochemistry were assayed for the formation of 6-OHDA with reverse-phase ion-pair high-performance liquid chromatography with electrochemical detection. The results of this experiment were combined with additional experiments employing MA and AFA administration at 0.5, 1 and 2 h prior to the rats being decapitated for neurochemical assay. In these experiments, MA and a DA uptake inhibitor were
administered at the same time in order to measure the effects of MA and DA uptake inhibitors on DA and 5-HT. In Expt. 1 the groups of 4-6 rats were decapitated 0.5, 1, 2 and 8 h following drug administration for neurochemical assay. All rats were treated with either MA (100 mg/kg, s.c.) or saline in an equivalent volume of 4 ml/kg. All rats were also treated with either the appropriate vehicle or a DA uptake inhibitor: AFA (10 mg/kg, i.p.) or MAZ (40 mg/kg, i.p.). In this and all following studies, MA was dissolved in 0.9% NaCI. AFA was dissolved in a solution of 9:1 propylene glycol and 2.5 M K2CO3. MAZ was dissolved in saline acidified with several drops of lactic acid. Both DA uptake inhibitors were injected in a 1 ml/kg volume. In Expt. 2, groups of 4-10 rats were injected with either saline or 25, 50, 100 mg/kg MA. The rats were decapitated for neurochemical assay 8 h following the injection and their locomotor activity was measured during this 8 h period following drug administration in stabilimeter chambers. The 12 wire mesh stabilimeters (38 x 18 x 20 cm) were mounted on a pivoting axle, and the excursion of the tilt was carefully adjusted to be the minimum excursion necessary to operate a microswitch mounted at the edge of the cage 1°. The cages were enclosed in sound attenuating chambers which were ventilated by a fan and were illuminated by two 28 V houselights during the behavioral sessions. Test sessions were conducted between 08.00 and 16.00 h. The stabilimeter cages were connected to a PDP 11/73 microcomputer via a Coulbourn lablinc computer interface. The sessions were timed and data recorded using a Super SKED II Software System29. The test session consisting of drug treatment followed two days adaptation in which the rats were placed in the chambers and locomotor data was recorded. In Expt. 3, the effects of MA and AFA were assessed in groups of 3-5 rats that had been treated with an i.v.t. (intraventricular) infusion of either 6-OHDA or vehicle 2 weeks previously. The rats were anesthetized with Nembutal (40 mg/kg, i.p.). The rats were injected with desipramine (DMI, 30 mg/kg, i.p.) and pargyline (30 mg/kg, i.p. in a 15 mg/ml saline solution) 30 min prior to the i.v.t. 6-OHDA infusion. The rats were placed in a stereotaxis and received midline scalp incisions to expose the dorsal surface of the skull. Holes were bored at the coronal suture 1.5 mm on each side of the sagittal suture. The cannula was directed into the lateral ventricle at 0.2 mm posterior to bregma, 1.5 mm lateral to the midline, and 3.3 mm dorsal to the dura. 60 #g of 6-OHDA (free base) in 10 ~1 of 0.9% NaCI and 0.1% ascorbate was injected into
TABLE I
Neostriatal 6-OHDA formation in rats treated with MA (100 mg/kg) or saline and AFA (10 mg/kg) or vehicle (VEH) with 1'/2, I or 2 h pretreatment time Time l/2h No. of rats 6-OHDA detected/N x + S.E.M. Magnitude* + S.E.M. lh No. of rats 6-OHDA detected/N x _+ S.EIM. Magnitude* + S.E.M. 2h No. of rats 6-OHDA detected/N x + S.E.M. Magnitude* + S.E.M. Total no. of rats in which 6-OHDA was detected/N x + S.E.M. Magnitude* + S.E.M.
Saline-VEH
Saline-AFA (10 mg/kg)
M A - V E H (100 mg/kg)
MA-AFA
0/8 0.00 -
0/12 0.00 -
4/12 0.015 + 0.007 0.044 + 0.010
2/10 0.013 + 0.009 0.065 + 0.004
0/13 0.00 -
1/13 0.007 + 0.007 0.094
5/21 0.008 + 0.004 0.035 + 0.012
3/22 0.052 + 0.032 0.052 + 0.032
0/10 0.00 -
0/13 0.00 -
3/18 0.005 + 0.003 0.036 + 0.011
3/18 0.083 + 0.066 0.496 + 0.348
0/21 0.00 -
1/38 0.002 + 0.002 0.094
12/51 0.005 + 0.022 + 0.006
8/50 0.036 + 0.024 0.222 + 0.139
* The 'magnitude' of 6-OHDA levels was calculated by disregarding values of 0 in rats where 6-OHDA was not detected and taking the mean of only those rats in which 6-OHDA was detected.
TABLE II
Neostriatal DA levels in rats treated with MA (100 mg/kg) or saline (SAL ) and AFA (10 mg/kg), M A Z (40 mg/kg) or the appropriate vehicle (VEH) with a 1/2, 1, 2, 6 or 8 h pretreatment time Values = x + S.E.M. (N).
Condition
1/2h
1h
SAL-VEH MA-VEH (100 mg/kg) SAL-AFA (10 mg/kg) MA-AFA SAL-MAZ MA-MAZ
11.55 _+0.72 11.15 + 0.51 12.54 _ 0.78 12.22 + 1.13 -
(6) (6) (6) (5)
2h
12.37 _+0.81 10.42 + 0.50* 12.86 + 0.37 9.22 + 0.71" 7.59 +0.26"** 10.00_+ 0.92*
(4) (5) (5) (6) (5) (5)
11.00 + 0.69 5.31 + 0.24*** 8.56 + 0.40 2.81 + 0.25***** 6.28 +0.92"** 4.22 + 0.41"**
(5) (5) (5) (5) (5) (4)
6h
8h
10.29 + 0.69 3.86 _+ 0.65*** 9.93 + 0.76 4.81 _+ 0.76*** -
10.39 + 0.42 6.14+ 0.39"** 11.24 _+ 0.42 3.24 + 0.08***** 9.41 _+ 1.03 7.25 + 1.19"*
(5) (5) (5) (5) (5) (5)
Significantly different from SAL-VEH: *P < 0.05; **P < 0.01; ***P < 0.001. Significantly different from MA-VEH: **P < 0.01. each lateral ventricle over a 1 min period for a total infusion of 120 #g of 6-OHDA (free base). The cannula remained in situ for 5 min following the infusion. The incision was closed with wound clips. Control rats were treated identically except that no 6-OHDA was present in the injection vehicle. Two weeks following these i.v.t. infusions, these rats were adapted to the stabilimeter chambers during two 6 h behavioral sessions (between 08.00 and 14.30 h) on consecutive days. On the third day, rats received either saline or MA (100 mg/kg) and vehicle or A A (10 mg/kg). The rats were killed for neurochemical assay at the end of the 6 h behavioral session.
Regional brain dissection The neostriatum, nucleus accumbens, septum, frontal cortex, hypothalamus and hippocampus were obtained as described previously6 except that the neostriatal dissection was modified as described previously ~6. The rat tissue was wrapped in aluminum foil and stored in liquid nitrogen. DA and 5-HT level determinations Concentrations of 6-OHDA, DA, DOPAC and 5-HT were determined by reverse-phase ion-pair high-performance liquid chromatography with electrochemical detection as follows. RSIL C-18 HL columns (4.6 mm i.d. x 25 cm, Alitech) were eluted at a flow rate of 1 ml/min with a mobile phase containing 0.27 mM EDTA, 0.09 M citric acid, 0.005 M sodium phosphate, 0.09 mM octylsodium sulfate and 1% methanol. The potential of the working electrode was 0.8 V relative to the Ag/AgCI reference electrode. The neostriatal samples were sonicated in 400/A M PCA with 0.05% EDTA and centrifuged 10 min at 20,000 g immediately prior to assay by HPLC-EC. One hundred microliters of supernatant was injected onto the HPLC column. DA, DOPAC and 6-OHDA were quantified with an integrator (Shimadzu, CR5A chromatopac). Retention times were as follows: 6-OHDA, 16 min; DOPAC, 23
min; DA, 27 min. The sensitivity of the assay for 6-OHDA was 0.1 ng/100/A with the minimum amount of 6-OHDA detected in 100 #l of supernatant being 0.2 ng/100/A.
Statistical analysis The significance of differences between group means was assessed with Student's t-test34 or with either single or multiple factor ANOVAs followed by either the Dunnett test ~ or the Duncan multiple range test4, respectively, to allow for multiple comparisons between group means. Significance was accepted at the P < 0.05 level. Drugs and materials Methamphetamine was supplied by the National Institute of Drug Abuse (Bethesda, MD). Pargyline hydrochloride, 5-hydroxytryptamine creatinine sulfate, and dopamine hydrochloride, dihydroxyphenyl acetic acid (Sigma Chemical Co., St. Louis, MO); 6hydroxydopamine (Regis Chemical Co., Morton Grove, IL) were purchased from their suppliers. Amfonelic acid (Sterling-Winthrop Research Institute, Rensselaer, NY) and mazindol (Sandoz, East Hanover, N J) were generous gifts from their suppliers. RESULTS
Neostriatal 6 - O H D A
detected f o l l o w i n g M A
and A F A
treatment AFA
did not block formation
neostriatum of MA-treated
of 6-OHDA
in t h e
r a t s w h e n c o m b i n i n g all o f
t h e e x p e r i m e n t s in w h i c h r a t s w e r e k i l l e d f o r n e u r o c h e m i c a l a s s a y 0.5, 1 a n d 2 h f o l l o w i n g M A a d m i n i s t r a t i o n
TABLE III
Hippocampal 5-HT Values = x + S.E.M.
Condition
1h
2h
SAL-VEH MA-VEH (100 mg/kg) SAL-AFA (10 mg/kg) MA-AFA SAL-MAZ (40 mg/kg) MA-MAZ
0.283 + 0.005 0.076 + 0.005*** 0.308 ___0.018 0.097 + 0.009*** 0.291 + 0.014 0.280 + 0.036***
0.213 + 0.040 + 0.200 + 0.044 + 0.167 + 0.274 +
6h 0.027 0.002** 0.015 0.006** 0.045 0.060'**
Significantly different from SAL-VEH: *P < 0.05; **P < 0.01 ; ***P < 0.001. Significantly different from MA-VEH: ***P < 0.001.
0.254 0.030 0.218 0.109 -
8h + + + +
0.028 0.003*** 0.028 0.007***
0.267 0.047 0.440 0.061 0.352 0.150
+ + + + + +
0.016 0.016" 0.108 0.016" 0.090 0.013
(Table I). No 6 - O H D A was detected in any of the 21 rats in the saline-vehicle group. 6 - O H D A was detected in only 1 of 38 rats in the saline-AFA group. 6-OHDA was detected in 12 of 51 MA-vehicle-treated rats and 8 of 50 MA-AFA-treated rats. Considering the 12 MA-vehicle and 8 MA-AFA rats in whom 6 - O H D A was detected, 6 - O H D A levels were not decreased in the MA-AFA group compared to the 6 - O H D A levels observed in the MA-saline group.
Neostriatal DA after MA and DA uptake inhibitors The D A uptake inhibitors did not block short-term MA-induced D A depletions but instead enhanced shortterm MA-induced D A depletions (Table II). MA significantly decreased neostriatal D A levels 2, 6 and 8 h but not 0.5 and 1 h following MA administration. The D A uptake inhibitors also decreased neostriatal D A levels, although not greater than MA alone. AFA alone depleted D A levels only at the 2 h time point while M A Z alone depleted D A levels only at the 1 and 2 h time points. Neither AFA nor M A Z blocked short-term D A depletion induced by MA administration. The MAinduced neostriatal D A depletions were enhanced by AFA at the 2 and 8 h time points and by M A Z at the 2 h time point. Hippocampal 5-HT after MA and DA uptake inhibitors MA alone significantly depleted hippocampal 5-HT by approximately 80% at 1, 2, 6 and 8 h following drug administration (Table III). Neither AFA nor MAZ, by themselves, had any effect on hippocampal 5-HT levels. Coadministration of AFA with MA did not block the MA-induced 5-HT depletions, but coadministration of M A Z with MA completely blocked MA-induced 5-HT depletions seen 1 and 2 h following drug administration. M A Z partially blocked the 5-HT depletions seen 8 h following MA administration. Effects of MA on locomotor behavior and neurochemistry MA (25-100 mg/kg) increased locomotor activity in a dose-dependent manner as reflected by an increase of stabilimeter counts during an 8 h session (F3,23 = 3.32, P < 0.05, Fig. 1). MA also acutely decreased neostriatal D A (/73,23 = 30.14, P < 0.001, Fig. 1) and D O P A C (/73,23 = 9.07, P < 0.001) in a dose-dependent manner. MA and AFA effects on behavior and neurochemistry after 6-OHDA lesion MA increased locomotor activity (F1.22 = 32.45, P < 0.001, Fig. 2) in sham-lesioned rats treated in both the MA-vehicle and MA-AFA conditions (P < 0.01). AFA also increased locomotor activity (F1,22 = 13.79, P < 0.001) as evidenced by greater stabilimeter counts in
sham-lesioned rats treated with MA-AFA than with MA-vehicle (P < 0.001). 6 - O H D A lesions decreased locomotor activity (F1,22 = 26.80, P < 0.001) as revealed by the fact that the 6 - O H D A lesions blocked increased stabilimeter counts in both the MA-vehicle (P < 0.01) and the M A - A A (P < 0.001) groups compared to sham-lesioned rats with the same drug treatments. A significant MA x 6 - O H D A interaction (F1,22 = 24.49, P < 0.001) was observed. Administration of MA by itself depleted D A 6 h after drug administration by 62 + 6, 47 + 11, 64 + 3, 57 + 12, and 65 + 8% in the neostriatum (P < 0.001), nucleus accumbens (P < 0.01), frontal cortex (P < 0.05), hypothalamus (P < 0.01) and septum (P < 0.001), respectively. Administration of MA by itself depleted 5-HT 6 h after drug administration by 92 + 2, 88 + 3, 88 + 1, 71 + 8 and 64 + 10% in the frontal cortex, neostriatum, hippocampus, hypothalamus (P < 0.001) and septum (P < 0.01), respectively. MA, alone, depleted NE by 79 + 12, 74 + 3 and 65 + 8% in the
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