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Methamphetamine pretreatment and the vulnerability of the striatum to methamphetamine neurotoxicity
Neuroscience Vol. 72, No. 3, pp. 593-600, 1996
~ ) Pergamon
0306-4522(95)0058 7-0
Elsevier Science Ltd Copyright © 1996 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00
M E T H A M P H E T A M I N E P R E T R E A T M E N T A N D THE V U L N E R A B I L I T Y OF THE S T R I A T U M TO METHAMPHETAMINE NEUROTOXICITY S. S T E P H A N S and B. Y A M A M O T O * Departments of Psychiatry and Neuroscience, Case Western Reserve University School of Medicine, Cleveland, OH 44106, U.S.A. A~traet--Pretreatment with intermittent low-dose administrations of stimulants increases mesostriatal dopamine transmission upon administration of a challenge dose. This occurs without evidence of a long-term dopamine or serotonin depletion. The purpose was to examine whether pretreatment with low doses of methamphetamine enhances dopamine and/or glutamate efflux and the subsequent depletion of dopamine and serotonin produced by neurotoxic challenge doses of methamphetamine. Microdialysis was used to measure simultaneously extracellular concentrations of dopamine and glutamate in the striatum and prefrontal cortex of awake rats. Basal extracellular concentrations of dopamine and glutamate were unaltered following pretreatment with methamphetamine. The increase in methamphetamine-induced striatal dopamine effiux was not significantly different between methamphetamine and saline pretreated groups. In contrast, after high challenge doses of methamphetamine, dopamine efltux in prefrontal cortex was enhanced to a greater extent in methamphetamine pretreated rats as compared to saline pretreated controls. Acute methamphetamine did not enhance glutamate efflux in prefrontal cortex after pretreatment with saline or methamphetamine. The increase in striatal glutamate effiux was blunted in rats pretreated with methamphetamine. When measured 4 days later, dopamine and serotonin content in striatum was depleted in all rats acutely challenged with methamphetamine. However, these depletions were attenuated in rats pretreated with methamphetamine. An acute methamphetamine challenge did not affect dopamine tissue content in the prefrontal cortex of any rats. Serotonin content in cortex was depleted in all groups following the methamphetamine challenge administration, but these depletions were diminished in methamphetamine-pretreated rats. These results are the first evidence that an intermittent pretreatment regimen with low doses of methamphetamine, followed by a 1 week withdrawal, reduces the vulnerability of striatal dopamine and serotonin terminals and cortical serotonin terminals to methamphetamine neurotoxicity. These findings provide evidence for the mechanism leading to methamphetamine neurotoxicity. Key words: microdialysis, prefrontal cortex, dopamine, serotonin.
Pretreatment with intermittent and repeated low doses of psychostimulants causes an increase in stimulant-induced behaviors. The characteristic augmentations are typically seen after a withdrawal period and upon a challenge administration o f the drug. The behavioral consequences o f amphetamine or cocaine pretreatment are manifested as enhanced locomotor activity and behavioral stereotypy '2'j8 and collectively referred to as behavioral sensitization. Other consequences that result from stimulant pretreatment include increased dopaminergic transmission in vitro 5"18"33 and in t)iuo, j0"29'34 Although increased behavior and augmented dopamine (DA) transmission have both been reported to occur, there is evidence o f a dissociation between these two variables? 1'3°'3s'47"48 Therefore, a pretreatment regimen that produces behavioral sensitization may or *To whom correspondence should be addressed. Abbreviations: DA, dopamine; EDTA, ethylenediaminete-
tra-acetate; HPLC, high-pressure liquid chromatography; NMDA, N-methyl-D-aspartate.
may not be associated with an increase in D A neurotransmission. While most studies of the effects of psychostimulant pretreatment have focused on cocaine and D-amphetamine, fewer studies have examined the effects of pretreatment with methamphetamine on subsequent augmentations in behavior and neurotransmitter efflux. Nevertheless, repeated intermittent low doses of methamphetamine also produce behavioral sensitization s'43 and enhanced dopamine transmission in striatum following a challenge administration, s'LS'49 These augmented effects following low doses are in marked contrast to the effects observed following chronic administration or multiple acute injections of high doses o f methamphetamine. High-dose administration results in decreased tissue concentrations of dopamine and serotonin, 32'37'44 a reduction in D A high affinity uptake sites ~ and inhibition of tyrosine hydroxylase and tryptophan hydroxylase activities) C o m m o n mechanisms seem to mediate the consequences of repeated low doses of methamphetamine 593
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S. Stephans and B. Yamamoto
(6 mg/kg i.m.) and ketamine (70 mg/kg i.m.). The skull was a n d high neurotoxic doses o f m e t h a m p h e t a m i n e . exposed and a 1 mm diameter hole was drilled through the Both methamphetamine pretreatment and neurotoxic bone above either the striatum (4-1.2 mm AP; _+3.2 mm doses of m e t h a m p h e t a m i n e cause a n increase in lateral to the midline suture) or the medial prefrontal cortex dopamine efnux. 8,15,27,49 C o a d m i n i s t r a t i o n o f d o p a ( + 3 . 3 m m AP; +_0.7mm lateral to the midline suture). 3. Dura was carefully removed and a 21-gauge stainless steel mine a n t a g o n i s t s prevents the n e u r o t o x i c effects o f guide cannula was then stereotaxically placed into the hole m e t h a m p h e t a m i n e , 4'9'z8'39 a n d w h e n s i m u l t a n e o u s l y and on to the surface of the cortex overlying the intended administered with repeated low doses o f m e t h probe implantation site. The guide cannula with a stylet a m p h e t a m i n e , also blocks the s u b s e q u e n t e n h a n c e d obturator was fixed to the skull with cranioplastic cement behavioral a n d n e u r o c h e m i c a l responses to challenge and three set screws. The vertical placement of the microdialysis probe in striatum or prefrontal cortex was predeteradministrations, s,43 mined by gluing a ring of PE 20 tubing at a measured T h e glutamatergic system h a s also b e e n implicated distance along the length of the probe. The PE 20 served as in the a u g m e n t e d responses d u r i n g a m p h e t a m i n e a n d a mechanical "stop" when the probe was inserted through m e t h a m p h e t a m i n e p r e t r e a t m e n t as well as in toxicity the guide cannula and into the striatum or prefrontal cortex. following a high dose o f m e t h a m p h e t a m i n e . N Microdialysis probes and perfusion Methyl-D-aspartate ( N M D A ) a n d n o n - N M D A anA concentric-shaped dialysis probe was constructed as tagonists block the d e v e l o p m e n t o f b e h a v i o r a l previously described, s° The dialysis membrane (13,000 mol. sensitization w h e n c o a d m i n i s t e r e d w i t h the l o w wt cut-off) was 4.0 mm in length for both striatum and i n t e r m i t t e n t injections o f D - a m p h e t a m i n e . 13'~4'46 Coprefrontal cortex probes. The average relative recovery for DA and metabolites was approximately 10%. The relative a d m i n i s t r a t i o n o f n o n - N M D A a n t a g o n i s t s with the recovery for glutamate was 19%. challenge a d m i n i s t r a t i o n also p r e v e n t s the expression Perfusion flow was 2.0 #l/min and was controlled by a o f the a u g m e n t e d behavioral responses. 14 Similarly, microinjection pump (Harvard Instruments, South Natick, a d m i n i s t r a t i o n o f toxic doses o f m e t h a m p h e t a m i n e or MA, U.S.A.). A stainless steel spring tether was used to D - a m p h e t a m i n e increases g l u t a m a t e efflux, 1'22'25'26A1 connect the swivel to the animal. The perfusion medium was a modified Dulbecco's phosphate-buffered saline containing a n d a n t a g o n i s m o f N M D A receptors p r e v e n t s the 138mM NaCl, 2.7mM KC1, 0.SmM MgC12, 1.5mM neurotoxicity to m e t h a m p h e t a m i n e . 3,4°,45 KH2PO4, 8.1 mM Na2HPO4, 1.2 mM CaC12, and 10.0 mM A l t h o u g h m e t h a m p h e t a m i n e - i n d u c e d sensitization D-glucose; pH 7.4. a n d neurotoxicity are associated with a n increase in Three days following surgery (day 8 of the withdrawal period) the dialysis experiment was performed. Rats were d o p a m i n e r g i c a n d glutamatergic t r a n s m i s s i o n , n o placed into the same pre-assigned chamber in which they studies to date have a t t e m p t e d to relate these two received the daily injections. The stylet was removed from p h e n o m e n a . In particular, n o studies h a v e directly the guide cannula and replaced with the dialysis probe. The examined whether a methamphetamine pretreatment probe was inserted into and through the cannula to the regimen followed by a w i t h d r a w a l p e r i o d e n h a n c e s point where the guide cannula abutted a pre-positioned ring of PE 20 tubing on the probe. This positioning permitted the d o p a m i n e a n d / o r g l u t a m a t e efflux a n d s u b s e q u e n t l y exposed portion of the dialysis membrane (4.0 mm for both alters the vulnerability o f n e u r o n s to the n e u r o t r a n s striatum and prefrontal cortex) to extend beyond the end of mitter depleting effects p r o d u c e d by high doses of the guide cannula and precisely into the brain area of m e t h a m p h e t a m i n e . Therefore, the p u r p o s e o f this interest. The tip of the probe was located 7 mm from the cortical surface for striatum and 4 mm from the cortical study was to investigate w h e t h e r p r i o r exposure to surface for prefrontal cortex. A 3-h equilibration period was low doses o f m e t h a m p h e t a m i n e e x a c e r b a t e s the neuallowed before samples were collected. Following the 3-h rotoxic effects o f acute high m e t h a m p h e t a m i n e doses equilibration period, three baseline samples and all subo n d o p a m i n e a n d serotonin n e u r o n s in s t r i a t u m a n d sequent dialysate samples were collected every 30 min. After cortex. the baseline samples, challenge injections of saline vehicle or methampbetamine hydrochloride (7.5 mg/kg) were administered subcutaneous once every 2 h over a period of 6 h for EXPERIMENTAL PROCEDURES a total of three injections. Four days after the dialysis experiment, rats were killed Drug treatment by rapid decapitation, and the brain was quickly removed Male Sprague-Dawley rats (Zivic Miller) weighing befrom the cranium and immediately frozen on dry ice. Probe tween 175 and 300 g were housed three per cage. The drug placements were verified from frozen coronal sections. The pretreatment regimen consisted of the following: rats were prefrontal cortex and anteroventrolateral striatum were removed from the animal colony at 10.30-11.00 a.m. each then dissected from a 250- and 400-#m slice respectively, on day, weighed, placed into a round plastic experimental the side contralateral to the probe implantation site. The chamber and immediately injected i.p. with 2mg/kg dissections were immediately frozen and stored at - 8 0 ° C (+)methamphetamine hydrochloride or 0.15 M NaC1 vefor subsequent tissue content analysis. hicle. Each rat was put in the same designated chamber with Biochemical measurements fresh bedding each day. After 3 h, the rats were returned to the home cage in the vivarium. This procedure was repeated Each dialysate sample (20 #1) was assayed for dopamine, daily for 7 days. All rats were then withdrawn from drug serotonin, and metabolites by high-pressure liquid chromatreatment for 7 days. During this period of withdrawal, tography (HPLC) with electrochemical detection as prerats were implanted with probes for the microdialysis viously described. 4t Separation was achieved with a 3-pm, experiments. C18 column (100 x 2.0 mm) and a mobile phase consisting of 32 mM citric acid, 54.3 mM sodium acetate, 0.074 mM Surgery Na:EDTA, 0.215raM octyl sodium sulfate and 3% On the fourth day of withdrawal from the drug treatment, methanol (pH 4.2). Detection was with a Princeton Applied rats were anesthetized with a combination of xylazine Research Instrument Model 400 Electrochemical Detector
Methamphetamine toxicity and striatal vulnerability and a glassy carbon electrode maintained at a potential of 0.65 V. Another 20-/~1 aliquot of each dialysate sample was assayed for amino acids by HPLC with electrochemical detection following precolumn derivatization with o-pthaldialdehyde. 7 The derivatization reagent was prepared by dissolving 27mg of o-pthaldialdehyde in l ml of 100% methanol, 9 ml of 0.I M sodium tetraborate (pH 9.4) and 5/~1 of ~-mercaptoethanol. This stock solution was then diluted 1:3 with 0.1 M sodium tetraborate. This reagent mixture (10#1) was then automatically added to each dialysate sample by an autosampler, mixed and allowed to react for exactly 2 min before injection. Amino acids were separated on a 3-gm C18 reverse phase column and eluted with 0.1 M sodium phosphate buffer (pH 6.4) containing 28% methanol and 50mg/l EDTA. Detection was with a glassy carbon electrode maintained at 0.7 V by an LC4B amperometric detector, The flow rate was 0.25 ml/min. Tissue from striatum and medial prefrontal cortex was sonicated in ice-cold 0.1 M HCIO4, centrifuged at 13,000 g for 5min, and the supernatant assayed for dopamine, serotonin and metabolites by HPLC with electrochemical detection. Protein determinations were performed using Bradford's Protein Assay.
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an acute methamphetamine challenge (P < 0 . 0 5 , F = 5.9, d.f. = 1,12) (Fig. IB). Following the first m e t h a m p h e t a m i n e challenge injection, cortical d o p a mine in methamphetamine-pretreated rats increased by 30-fold, whereas saline-pretreated rats only increased by 12-fold over baseline. Cortical d o p a m i n e in methamphetamine-pretreated rats remained elevated over that o f saline-pretreated rats during the course o f the experiment.
Glutamate dialysis data The baseline concentrations of glutamate in dialysate samples were 3.4 _+ 2.0 ng/20/~l (N = for the striatum and 5.9 + 2.4 n g / 2 0 p l (N = 22) the medial prefrontal cortex. The acute doses m e t h a m p h e t a m i n e caused a significant increase in
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(+)Methamphetamine hydrochloride, o-pthaldialdehyde and #-mercaptoethanol were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Xylazine was purchased from Miles Inc. (Shawnee Mission, KS, U.S.A.) and ketamine from Fort Dodge Laboratories, Inc. (Fort Dodge, IA, U.S.A.). HPLC columns were purchased from Phenomenex (Torrance, CA, U.S.A.). Dialysis membrane was purchased from Spectrum (Houston, TX, U.S.A.).
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The baseline concentrations of dopamine in the dialysate samples from striatum and medial prefrontal cortex were 6 . 1 1 + 0 . 3 1 p g / 2 0 / ~ 1 ( N = 2 8 ) and 0.46 __+0.06 pg/20/tl (N = 25), respectively. The acute challenge injections of methamphetamine significantly increased the extracellular concentrations of d o p a m i n e in both the striatum (P < 0.02, F = 79.4, d.f. = 1,24) and medial prefrontal cortex over time (P < 0.02, F = 52.8, d.f. = 1,21) as shown in Fig. IA and B. There was no significant effect o f m e t h a m p h e t amine pretreatment on the increases in extracellular d o p a m i n e in the striatum following the acute challenges with methamphetamine (Fig. IA). In contrast, there was a significant simple main effect of methamphetamine pretreatment on the increase in extracellular dopamine concentrations in cortex following
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Time (h) Fig. 1. The effect of drug pretreatment on the eiflux of dopamine in (A) striatum and (B) medial prefrontal cortex after the acute administration of methamphetamine or saline. Meth/Meth, methamphetamine pretreatment/acute methamphetamine challenge; Sal/Meth, Saline pretreatment/acute methamphetamine challenge; Meth/Sal, methamphetamine pretreatment/acute saline challenge; Sal/Sal, saline pretreatment/acute saline challenge. The acute challenge injections of methamphetamine significantly increased dopamine efflux over time (P <0.02) in both striatum (F = 79.4) and cortex (F = 52.8). There was a significant simple main effect of pretreatment on extraeellular dopamine in medial prefrontal cortex in acute methamphetamine challenged rats (Meth/Meth vs Sal/Meth, P < 0 . 0 5 , F = 5.9). Extracellular cortical dopamine was higher in methamphetamine-pretreated rats. The times of the acute injections are denoted by the arrows. N = 5-9/group.
S. Stephans and B. Yamamoto
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Time (h) Fig. 2. The effect of drug pretreatment on the eftlux of glutamate in (A) striatum and (B) medial prefrontal cortex after the acute administration of methamphetamine or saline. Meth/Meth, methamphetamine pretreatment/acute methamphetamine challenge; Sal/Meth, Saline pretreatment/acute methamphetamine challenge; Meth/Sal, methamphetamine pretreatment/acute saline challenge; Sal/Sal saline pretreatment/acute saline challenge. In the striatum, the methamphetamine challenge administration significantly increased glutamate efflux (P < 0.02, F = 6.9). *Significantly different from Meth/Meth (P < 0.05). The times of the acute injections are denoted by the arrows. N = 4-6/group. extracellular concentration of glutamate in striatum following the third m e t h a m p h e t a m i n e challenge injection (P < 0.02, F = 6.9, d.f. = 1,19) (Fig. 2A). The methamphetamine-induced glutamate increase was significantly blunted in rats pretreated with methamphetamine compared with saline-pretreated rats (P < 0.05). Striatal glutamate in saline pretreated rats increased 5-fold over baseline c o m p a r e d with a 3-fold increase in methamphetamine-pretreated rats. In con-
Table 1 illustrates the tissue concentrations of dopamine and serotonin in the striatum and medial prefrontal cortex when assayed 4 days after the acute treatments with methamphetamine or saline. Additional animals not used for the dialysis experiments were also pretreated and then challenged with saline or the high doses o f methamphetamine. Pretreatment with m e t h a m p h e t a m i n e did not alter the tissue concentrations of d o p a m i n e in the striatum or prefrontal cortex in the saline challenged groups (Sal/Sal vs Meth/Sal). Multiple acute methamphetamine challenges, however, decreased striatal dopamine content in both saline-pretreated and methamphetamine-pretreated rats (P < 0.02, F = 63.1, d.f. = 1,78). There was also a significant effect of methamphetamine pretreatment on the striatal dopamine depletions produced by the high doses of methamphetamine (Pretreatment x Challenge, P < 0.02, F = 7.4, d.f. = 1,78). The acute high doses of methamphetamine caused a greater depletion in striatal tissue dopamine content in saline-pretreated rats (62% decrease) than in rats pretreated with methamphetamine (41% decrease). In contrast, dopamine tissue content was not significantly depleted in the prefrontal cortex following administration of toxic doses of methamphetamine, regardless o f pretreatment. Pretreatment with methamphetamine did not alter the tissue concentrations of serotonin in the striatum or prefrontal cortex when administered a saline challenge (Sal/Sal vs Meth/Sal), (Table 1). A significant depletion of serotonin was observed in striatum of rats administered methamphetamine, regardless of drug pretreatment (P < 0.05, F = 4 . 2 , d.f. = 1,62), (methamphetamine-pretreated rats, 19% decrease; saline-pretreated, 33% decrease). In prefrontal cortex serotonin tissue content was also significantly depleted in both methamphetamine- and saline-pretreated rats following the challenge administration of m e t h a m p h e t a m i n e (P < 0,02, F = 16.5, d.f. = 1,54). In addition, there was a significant effect of drug
Table 1. The effect of methamphetamine pretreatment on the dopamine and serotonin content in the striatum and prefrontal cortex Pretreatment/challenge Saline/Saline Methamphetamine/Saline Saline/Methamphetamine Methamphetamine/Methamphetamine
Striatum Dopamine Serotonin 207. I + 13.4 185.5 __+11.5 79.0 __+10.2" 122.8 + 13. I*
4.58 + 3.81 _ 3.05 _ 3.73 +
0.30 0.23 0.49* 0.42
Prefrontal cortex Dopamine Serotonin 2.25 -t- 0.29 2.50 _ 0.45 2.22 _ 0.35 1.66 + 0.24
6.50 +__0.33 6.03 + 0.42 3.44 + 0.44* 5.04 + 0.61
Dopamine and serotonin content were measured 4 days after the acute administration of methamphetamine (methamphetamine, 7.5 mg/kg, s.c. once every 2 h for a total of three injections) or saline. The results are reported in pg/gg of protein. *Significantly different from saline/saline group post-hoc Tukey-test (P < 0.05). N = 11-24/group.
Methamphetamine toxicity and striatal vulnerability pretreatment on the depletions in cortical serotonin content produced by the challenge administration of methamphetamine. Methamphetamine produced a 47% decrease in serotonin content in the salinepretreated group and a 22% decrease in serotonin content in the methamphetamine-pretreated group (Pretreatment x Challenge, P < 0.05, F = 4.3, d.f. = 1,54). A post-hoe comparison revealed that drug pretreatment differentially affected methamphetamine-induced serotonin depletions in both striaturn and prefrontal cortex, such that saline- but not methamphetamine-pretreated rats administered a methamphetamine challenge had significantly lower serotonin concentrations than saline-pretreated controls administered a saline challenge (P < 0.05). DISCUSSION The present results illustrate that pretreatment with intermittent low doses of methamphetamine differentially alters the subsequent increases in glutamate and dopamine efflux in striatum and prefrontal cortex after neurotoxic doses of methamphetamine. The magnitude of these effects was paralleled by the subsequent differential depletions of dopamine and serotonin content in the striatum and cortex.
Methamphetamine and striatal glutamate effiux The observed attenuation of the methamphetamine-induced increase in extracellular glutamate concentrations in rats pretreatcd with methamphetamine may account for the finding that methamphetamine pretreatment decreased the dopamine depleting effects of methamphetamine in striatum. Although methamphetamine increased the extracellular concentrations of glutamate in striatum as reported previously, L25'4~ (Fig. 2B) this increase was significantly blunted at the later time points in rats pretreated with methamphetamine. These effects were paralleled by a diminished depletion of tissue dopamine in striatum 4 days after the challenge administrations. Antagonism of methamphetamine-induced striatal glutamate efflux has been shown to coincide with protection against methamphetamine-induced dopamine depletions in this same brain area. 41 It has been hypothesized that following a methamphetamine-induced energy impairment in striatum, 6 dopamine neurons are more vulnerable to the neurotoxic effects of glutamate. 25 A blunted increase in glutamate concentrations may circumvent calcium-mediated damage or, alternatively, may suppress the transporter-mediated DA release stimulated by glutamate 2~ and the subsequent generation of cytotoxic quinones and free radicals. 2°
Methamphetamine and striatal dopamine effiux The lack of a significant enhancement of striatal dopamine efflux to a methamphetamine challenge in
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rats pretreated with methamphetamine differs from previous studies illustrating augmented efflux of striatal dopamine in rats behaviorally sensitized to methamphetamine.8'~5"49 However, more recent reports show that a dissociation exists between behavioral sensitization and increased neurochemical responses. Therefore, certain paradigms that cause behavioral sensitization do not necessarily produce corresponding augmentations in extracellular dopamine concentrations upon challenge administration.H'a°'as'47'4g Moreover, it has recently been shown that following stimulant pretreatment, a withdrawal period exceeding 1 week is required to produce an augmented dopaminergic response. 3° Therefore, the relatively short withdi'awal period of 1 week used in the present study may account for the lack of an augmented increase in extracellular dopamine in the striatum.
Methamphetamine and cortical dopamine effiux The finding that methamphetamine pretreatment enhanced dopamine efflux during the high-dose methamphetamine challenge in prefrontal cortex is consistent with that of Robinson et al. 33 They reported that repeated intermittent administration of amphetamine increased mesocortical dopamine activity after a single low-dose challenge of D-amphetamine. The exact mechanisms responsible for the augmented methamphetamine-induced cortical dopamine efflux are not known. It has been reported that there is an increase in brain tissue amphetamine after chronic amphetamine treatment? 9 Therefore, it is possible that methamphetamine pretreatment resulted in the accumulation of methamphetamine in tissue. In contrast, chronic methamphetamine treatment has been shown to produce lower brain tissue concentrations of methamphetamine. 36 Therefore, methamphetamine accumulation in tissue is not a likely explanation for the enhanced cortical dopamine efflux observed upon the challenge administration in rats pretreated with methamphetamine. Furthermore, the basal extracellular concentrations of dopamine prior to the challenges were not significantly different between saline and methamphetamine-pretreated groups. Another possible explanation to account for the augmentation in methamphetamine-induced dopamine efflux in cortex is that prior methamphetamine exposure enhances the releasability of dopamine. Castaneda et al. reported that repeated amphetamine administrations can enhance both carrier-mediated and impulse-mediated dopamine release, perhaps by increasing both the cytoplasmic and vesicular pools of dopamine) In addition, changes in the density of the dopamine transporter following methamphetamine pretreatment may reduce uptake of dopamine 23 or enhance the transporter dependent release of dopamine. 49 Taken together, these possibilities are suggestive that a resultant decrease in the uptake of dopamine or increased methamphetamine-induced
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dopamine efflux, along with enhanced impulse-mediated release may culminate in the augmentation of the extracellular concentrations of dopamine.
Methamphetamine toxicity, dopamine and glutamate A depletion of dopamine tissue content was not observed in cortex 4 days after acute high-dose challenges with methamphetamine in methamphetamine-pretreated rats, despite the increase in extracellular dopamine concentration. However, methamphetamine did not increase the extraceilular concentration of glutamate in this region. An increase in both dopamine and glutamate efttux may be necessary to produce a long-term depletion of dopamine content. Therefore, analogous to the effects in the striatum, glutamate efflux appears to play an important role in mediating the long-term neurotoxic effects of methamphetamine to dopamine neurons. 41 The acute challenge of methamphetamine depleted the serotonin content in striatum and prefrontal cortex in both saline- and methamphetaminepretreated groups. This is consistent with previous findings illustrating that methamphetamine is toxic to serotonin neurons. 32'37 The mechanism leading to methamphetamine-induced serotonin depletions is not known. However, because methamphetaminepretreated rats, compared with saline-pretreated controls, exhibit relative resistance to serotonin toxicity in both brain areas following high doses, it seems that the toxicity to serotonin neurons is not exclusively dependent on dopamine efflux. Methamphetamine-induced depletions in serotonin tissue content may be analogous to the toxicity observed in striatum after M D M A , in that an increase in the activities of both the dopamine and the serotonin systems are required to observe the toxic effects to serotonin neurons. ~4'42 Methamphetamineinduced toxicity to serotonin neurons may rely on non-vesicular efflux of serotonin in addition to increased dopamine release. Although the extracellular concentrations of serotonin were not measured during the challenge administrations, it can be speculated that methamphetamine-induced serotonin effiux was blunted in methamphetamine-pretreated rats as compared with saline-pretreated controls. Reduced serotonin transporter activity in cortex may have diminished the uptake of dopamine into serotonin neurons, thereby minimizing cytotoxicity.35 The finding that methamphetamine pretreatment resulted in relative resistance to methamphetamine toxicity is compatible with the tolerance of the striatal dopamine and serotonin neurons to the toxic effects of acute methamphetamine administration following chronic treatment. 36 The present results indicate that in addition to striatal dopamine and serotonin neurons, serotonin neurons in cortex previously exposed to low doses of methamphetamine are resistant to the
subsequent effects of high doses of methamphetamine. The finding that chronic methamphetamine followed by neurotoxic doses leads to reduced brain tissue concentrations of methamphetamine a6 may explain the mechanism by which methamphetamine pretreatment confers resistance to the neurotransmitter-depleting effects of methamphetamine. However, this explanation is not applicable to the finding that methamphetamine-induced cortical dopamine efflux is enhanced in rats pretreated with methamphetamine. None the less, possible alterations in transporter protein density or changes in the distribution of dopamine within the nerve terminal produced by methamphetamine pretreatment, as previously described, could account for the augmented cortical dopamine efflux despite lower concentrations of methamphetamine in the brain. The tissue content data may reflect the transient changes in the regulation of tyrosine hydroxylase and tryptophan hydroxylase activities in a manner such that alterations in the biosynthesis of dopamine and serotonin produced by an acute challenge of methamphetamine may be affected by the pretreatment with methamphetamine. Previous studies have shown that a single acute injection of methamphetamine produces an initial decrease in enzyme activity during the first 1-3 days after administration. This is followed by a partial recovery towards control activity levels.2'9,16'17 In the present study, tissue dopamine and serotonin were assayed 4 days after the challenge administration. The depleted tissue concentrations of these neurotransmitters in rats challenged with methamphetamine could be due to a transient decrease in biosynthesis rather than a longlasting neurotoxic depletion. Thus the tissue content data measured 4 days after the acute challenge with methamphetamine may be reflective of an altered regulation of biosynthesis produced by the pretreatment with methamphetamine rather than due to neurotoxicity per se. However, this explanation is unlikely, based on the findings that the recovery in tyrosine hydroxylase activity after methamphetamine is not paralleled by a recovery in the tissue dopamine content. 16'~7 In addition, tryptophan hydroxylase activity remains depressed after multiple high doses 9 similar to those administered in this study. Moreover, our previous studies indicate that a similar acute challenge regimen of methamphetamine produced a depletion of dopamine content when observed even after a more prolonged period of 7 days. 25,41 CONCLUSIONS
In summary, the present results illustrate that both striatal and cortical dopaminergic and glutamatergic systems are differentially affected by a low-dose intermittent pretreatment regimen of methamphetamine and subsequent high acute doses of methamphetamine. It can be concluded that a low-dose
Methamphetamine toxicity and striatal vulnerability p r e t r e a t m e n t regimen o f m e t h a m p h e t a m i n e reduces the vulnerability of striatal d o p a m i n e a n d s e r o t o n i n terminals a n d cortical serotonin terminals to the toxic effects o f high m e t h a m p h e t a m i n e doses. These findings also provide further s u p p o r t for the role o f
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g l u t a m a t e in mediating m e t h a m p h e t a m i n e - i n d u c e d neurotoxicity. Acknowledgements--This work was supported by grants
DA07606, MH41684 and a gift from Hitachi Ltd.
REFERENCES
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(1988) The long-term effects of repeated amphetamine treatment in vivo on amphetamine, KCI and electrical stimulation evoked striatal dopamine release /n vitro. Life Sci. 42, 2447-2456. 6. Chan P., Di Monte D. A., Luo J., DeLanney L. E., Irwin I. and Langston J. W. (1994) Rapid ATP loss caused by methamphetamine in the mouse striatum: relationship between energy impairment and dopaminergic neurotoxicity. J. Neurochem. 62, 2484-2487. 7. Donzanti B. A. and Yamamoto B. K. (1988) An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters from brain tissue and microdialysis perfusates. Life Sci. 43, 913-922. 8. Hamamura T., Akiyama K., Akimoto K., Kashihara K., Okumura K., Ujike H. and Otsuki S. (1991) Co-administration of either a selective Dj or D 2 dopamine antagonist with methamphetamine prevents methamphetamine-induced behavioral sensitization and neurochemical change, studied by in vivo intracerebral dialysis. Brain Res. 546, 40-46. 9. Hotchkiss A. J. and Gibb J. W. (1980) Long-term effects of multiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain. J. Pharmac. exp. Ther. 214, 257-262. 10. Kalivas P. W. and Duffy P. (1990) Effect of acute and daily cocaine treatment on extracellular dopamine in the nucleus accumbens. Synapse 5, 48-58: 11. Kalivas P. W. and Duffy P. (1993) Time course of extraceUular dopamine and behavioral sensitization to cocaine. I. Dopamine axon terminals. J. Neurosci. 13, 266-275. 12. Kalivas P. W., Duffy P., DuMars L. A. and Skinner C. (1988) Behavioral and neurochemical effects of acute and daily cocaine administration in rats. J. Pharmac. exp. Ther. 245, 485-492. 13. Karler R., Calder L. D., Chaudhry I. A. and Turkanis S. A. (1989) Blockade of "reverse tolerance" to cocaine and amphetamine by MK-801. Life Sci. 45, 599-606. 14. Karler R., Calder L. D. and Turkanis S. A. (1991) DNQX blockade of amphetamine behavioral sensitization. Brain Res. 552, 295-300. 15. Kazahaya Y., Akimoto K. and Otsuki S. (1989) Subehronic methamphetamine treatment enhances methamphetamineor cocaine-induced dopamine efliux in vivo. Biol. Psychiatry 25, 903-912. 16. Koda L. Y. and Gibb, J. W. (1973) Adrenal and striatal tyrosine hydroxylase activity after methamphetamine. J. Pharmac. exp. Ther. 185, 42-48. 17. Kogan F. J., Nichols W. K. and Gibb J. W. (1976) Influence of methamphetamine on nigral and striatal tyrosine hydroxylase activity and on striatal dopamine levels. Eur. J. Pharmac. 36, 363-371. 18. Kolta M. G., Shreve P., De Souza V. and Uretsky N. J. (1985) Time course of the development of the enhanced behavioral and biochemical responses to amphetamine after pretreatment with amphetamine. Neuropharmacology 24, 823-829. 19. Kuhn C. M. and Schanberg S. M. (1978) Metabolism of amphetamine after acute and chronic administration to the rat. J. Pharmac. exp. Ther. 2,07, 544-554. 20. Lafon-Cazal M., Pietri S., Culcasi M. and Bockaert J. (1993) NMDA-dependent superoxide production and neurotoxicity. Nature 364, 535-537. 21. Lonart G. and Zigmond M. J. (1991) High glutamate concentrations evoke Ca + ÷-independent dopamine release from striatal slices: a possible role of reverse dopamine transport. J. Pharmac. exp. Ther. 256, 1132-1138. 22. Mora F. and Porras A. (1993) Effects of amphetamine on the release of excitatory amino acid neurotransmitters in the basal ganglia of the conscious rat. Can. J. Physiol. Pharmac. 71, 348-351. 23. Nakayama M., Koyama T. and Yamashita I. (1993) Long-lasting decrease in dopamine uptake sites following repeated administration of methamphetamine in the rat striatum. Brain Res. 601, 209-212. 24. Nash J. F., Meltzer H. Y. and Yamamoto B. K. (1990) Effect of 3,4-methylenedioxymethamphetamine on 3,4-dihydroxyphenylalanine accumulation in the striatum and nucleus accumbens. J. Neurochem. 54, 1062-1067. 25. Nash J. F. and Yamamoto B. K. (1992) Methamphetamine neurotoxicity and striatal glutamate release: comparison to 3,4-methylenedioxymethamphetamine. Brain Res. 581, 237-243. 26. Nash J. F. and Yamamoto B. K. (1993) Effect of D-amphetamine on the extracellular concentrations of glutamate and dopamine in iprindole-treated rats. Brain Res. 627, 1-8. 27. O'Dell S. J., Weihmuller F. B. and Marshall J. F. (1991) Multiple methamphetamine injections induce marked increases in extracellular striatal dopamine which correlate with subsequent neurotoxicity. Brain Res. 564, 256-260. 28. O'Dell S. J., Weihmuller F. B. and Marshall J. F. (1993) Methamphetamineoinduced dopamine overflow and injury to striatal dopamine terminals: attenuation by dopamine D~ or D 2 antagonists. J. Neurochem. 60, 1792-1799. 29. Patrick S. L., Thompson T. L., Walker J. M. and Patrick R. L. (1991) Concomitant sensitization of amphetamineinduced behavioral stimulation and in vivo dopamine release from rat caudate nucleus. Brain Res. 538, 343-346.
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30. Paulson P. E. and Robinson.T.E. (1995) Amphetamine-induced time-dependent sensitization of dopamine neurotransmission in the dorsal and ventral striatum: a microdialysis study in behaving rats. Synapse 19, 56~5. 31. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. 32. Ricaurte G. A., Schuster C. R. and Seiden L. S. (1980) Long-term effects of repeated methylamphetamine administration on dopamine and serotonin neurons in the rat brain: a regional study. Brain Res. 193, 153-163. 33. Robinson T. E., Becker J. B., Moore C. J., Castaneda E. and Mittleman G. (1985) Enduring enhancement in frontal cortex dopamine utilization in an animal model of amphetamine psychosis. Brain Res. 343, 374-377. 34. Robinson T. E., Jurson P. A., Bennet J. A. and Bentgen K. M. (1988) Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by prior experience with (+)-amphetamine: a microdialysis study in freely moving rats. Brain Res. 462, 211-222. 35. Schmidt C. J. (1987) Neurotoxicity of the psychedelic amphetamine, methylenedioxymethamphetamine. J. Pharmac. exp. Ther. 240, 1-7. 36. Schmidt C. J., Gehlhert D. R., Peat M. A., Sonsalla P. K. Hanson G. R., Wamsley J. K. and Gibb J. W. (1985) Studies on the mechanism of tolerance to methamphetamine. Brain Res. 343, 305-313. 37. Schmidt C. J. and Gibb J. W. (1985) Role of the dopamine uptake carrier in the neurochemical response to methamphetamine: effects of amfonelic acid. Eur. J. Pharmac. 109, 73-80. 38. Segal D. S. and Kuczenski R. (1992) In vivo microdialysis reveals a diminished amphetamine-induced DA response corresponding to behavioral sensitization produced by repeated amphetamine pretreatment. Brain Res. 571,330-337. 39. Sonsalla P. K., Gibb J. W. and Hanson G. R. (1986) Roles of D~ and D 2 dopamine receptor subtypes in mediating the methamphetamine-induced changes in monoamine systems. J. Pharmac. exp. Ther. 238, 932-937. 40. Sonsalla P. K., Nicklas W. J. and Heikkila R. E. (1989) Role for excitatory amino acids in methamphetamine-induced nigrostriatal dopaminergic toxicity. Science 243, 398-400. 41. Stephans S. E, and Yamamoto B. K. (1994) Methamphetamine-induced neurotoxicity: roles for glutamate and dopamine effiux. Synapse 17, 203-209. 42. Stone D. M., Johnson M., Hanson G. R. and Gibb J. W. (1988) Role for endogenous dopamine in the central serotonergic deficits induced by 3,4-methylenedioxymethamphetamine. J. Pharmac. exp. Ther. 247, 79-87. 43. Ujike H., Onoue T., Akiyama K., Hamamura T. and Otsuki S. (1989) Effects of selective D-1 and D-2 dopamine antagonists on development of methamphetamine-induced behavioral sensitization. Psychopharmacology 98, 89-92. 44. Wagner G. C., Ricaurte G. A., Seiden L. S., Schuster C. R., Miller R. J. and Westley J. (1980) Long-lasting depletions of striatal dopamine and loss of dopamine uptake sites following repeated administration of methamphetamine. Brain Res. 181, 151-160. 45. Weihmuller F. B., O'Dell S. J. and Marshall J. F. (1992) MK-801 protection against methamphetamine-induced striatal dopamine terminal injury is associated with attenuated dopamine overflow. Synapse 11, 155-163. 46. Wolf M. E. and Jeziorski M. (1993) Coadministration of MK-801 with amphetamine, cocaine or morphine prevents rather than transiently masks the development of behavioral sensitization. Brain Res. 613, 291-294. 47. Wolf M. E., White F. J. and Hu X. (1994) MK-801 prevents alterations in the mesoaceumbens dopamine system associated with behavioral sensitization to amphetamine. J. Neurosci. 14, 1735-1745. 48. Wolf M. E., White F. J., Nassar R., Brooderson R. J. and Khansa M. R. (1993) Differential development of autoreceptor subsensitivity and enhanced dopamine release during amphetamine sensitization. J. Pharmac. exp. Ther. 264, 249-255. 49. Yamada S., Kojima H., Yokoo H., Tsutsumi T., Takamuki K., Anraku S., Nishi S. and Inanaga K. (1988) Enhancement of dopamine release from striatal slices of rats that were subchronically treated with methamphetamine. Biol. Psychiatry 24, 399-408. 50. Yamamoto B. K. and Davy S. (1992) Dopaminergic modulation of glutamate release in striatum as measured by microdialysis. J. Neurochem. 58, 1736-1742. (Accepted 4 December 1995)
~ ) Pergamon
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Elsevier Science Ltd Copyright © 1996 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00
M E T H A M P H E T A M I N E P R E T R E A T M E N T A N D THE V U L N E R A B I L I T Y OF THE S T R I A T U M TO METHAMPHETAMINE NEUROTOXICITY S. S T E P H A N S and B. Y A M A M O T O * Departments of Psychiatry and Neuroscience, Case Western Reserve University School of Medicine, Cleveland, OH 44106, U.S.A. A~traet--Pretreatment with intermittent low-dose administrations of stimulants increases mesostriatal dopamine transmission upon administration of a challenge dose. This occurs without evidence of a long-term dopamine or serotonin depletion. The purpose was to examine whether pretreatment with low doses of methamphetamine enhances dopamine and/or glutamate efflux and the subsequent depletion of dopamine and serotonin produced by neurotoxic challenge doses of methamphetamine. Microdialysis was used to measure simultaneously extracellular concentrations of dopamine and glutamate in the striatum and prefrontal cortex of awake rats. Basal extracellular concentrations of dopamine and glutamate were unaltered following pretreatment with methamphetamine. The increase in methamphetamine-induced striatal dopamine effiux was not significantly different between methamphetamine and saline pretreated groups. In contrast, after high challenge doses of methamphetamine, dopamine efltux in prefrontal cortex was enhanced to a greater extent in methamphetamine pretreated rats as compared to saline pretreated controls. Acute methamphetamine did not enhance glutamate efflux in prefrontal cortex after pretreatment with saline or methamphetamine. The increase in striatal glutamate effiux was blunted in rats pretreated with methamphetamine. When measured 4 days later, dopamine and serotonin content in striatum was depleted in all rats acutely challenged with methamphetamine. However, these depletions were attenuated in rats pretreated with methamphetamine. An acute methamphetamine challenge did not affect dopamine tissue content in the prefrontal cortex of any rats. Serotonin content in cortex was depleted in all groups following the methamphetamine challenge administration, but these depletions were diminished in methamphetamine-pretreated rats. These results are the first evidence that an intermittent pretreatment regimen with low doses of methamphetamine, followed by a 1 week withdrawal, reduces the vulnerability of striatal dopamine and serotonin terminals and cortical serotonin terminals to methamphetamine neurotoxicity. These findings provide evidence for the mechanism leading to methamphetamine neurotoxicity. Key words: microdialysis, prefrontal cortex, dopamine, serotonin.
Pretreatment with intermittent and repeated low doses of psychostimulants causes an increase in stimulant-induced behaviors. The characteristic augmentations are typically seen after a withdrawal period and upon a challenge administration o f the drug. The behavioral consequences o f amphetamine or cocaine pretreatment are manifested as enhanced locomotor activity and behavioral stereotypy '2'j8 and collectively referred to as behavioral sensitization. Other consequences that result from stimulant pretreatment include increased dopaminergic transmission in vitro 5"18"33 and in t)iuo, j0"29'34 Although increased behavior and augmented dopamine (DA) transmission have both been reported to occur, there is evidence o f a dissociation between these two variables? 1'3°'3s'47"48 Therefore, a pretreatment regimen that produces behavioral sensitization may or *To whom correspondence should be addressed. Abbreviations: DA, dopamine; EDTA, ethylenediaminete-
tra-acetate; HPLC, high-pressure liquid chromatography; NMDA, N-methyl-D-aspartate.
may not be associated with an increase in D A neurotransmission. While most studies of the effects of psychostimulant pretreatment have focused on cocaine and D-amphetamine, fewer studies have examined the effects of pretreatment with methamphetamine on subsequent augmentations in behavior and neurotransmitter efflux. Nevertheless, repeated intermittent low doses of methamphetamine also produce behavioral sensitization s'43 and enhanced dopamine transmission in striatum following a challenge administration, s'LS'49 These augmented effects following low doses are in marked contrast to the effects observed following chronic administration or multiple acute injections of high doses o f methamphetamine. High-dose administration results in decreased tissue concentrations of dopamine and serotonin, 32'37'44 a reduction in D A high affinity uptake sites ~ and inhibition of tyrosine hydroxylase and tryptophan hydroxylase activities) C o m m o n mechanisms seem to mediate the consequences of repeated low doses of methamphetamine 593
594
S. Stephans and B. Yamamoto
(6 mg/kg i.m.) and ketamine (70 mg/kg i.m.). The skull was a n d high neurotoxic doses o f m e t h a m p h e t a m i n e . exposed and a 1 mm diameter hole was drilled through the Both methamphetamine pretreatment and neurotoxic bone above either the striatum (4-1.2 mm AP; _+3.2 mm doses of m e t h a m p h e t a m i n e cause a n increase in lateral to the midline suture) or the medial prefrontal cortex dopamine efnux. 8,15,27,49 C o a d m i n i s t r a t i o n o f d o p a ( + 3 . 3 m m AP; +_0.7mm lateral to the midline suture). 3. Dura was carefully removed and a 21-gauge stainless steel mine a n t a g o n i s t s prevents the n e u r o t o x i c effects o f guide cannula was then stereotaxically placed into the hole m e t h a m p h e t a m i n e , 4'9'z8'39 a n d w h e n s i m u l t a n e o u s l y and on to the surface of the cortex overlying the intended administered with repeated low doses o f m e t h probe implantation site. The guide cannula with a stylet a m p h e t a m i n e , also blocks the s u b s e q u e n t e n h a n c e d obturator was fixed to the skull with cranioplastic cement behavioral a n d n e u r o c h e m i c a l responses to challenge and three set screws. The vertical placement of the microdialysis probe in striatum or prefrontal cortex was predeteradministrations, s,43 mined by gluing a ring of PE 20 tubing at a measured T h e glutamatergic system h a s also b e e n implicated distance along the length of the probe. The PE 20 served as in the a u g m e n t e d responses d u r i n g a m p h e t a m i n e a n d a mechanical "stop" when the probe was inserted through m e t h a m p h e t a m i n e p r e t r e a t m e n t as well as in toxicity the guide cannula and into the striatum or prefrontal cortex. following a high dose o f m e t h a m p h e t a m i n e . N Microdialysis probes and perfusion Methyl-D-aspartate ( N M D A ) a n d n o n - N M D A anA concentric-shaped dialysis probe was constructed as tagonists block the d e v e l o p m e n t o f b e h a v i o r a l previously described, s° The dialysis membrane (13,000 mol. sensitization w h e n c o a d m i n i s t e r e d w i t h the l o w wt cut-off) was 4.0 mm in length for both striatum and i n t e r m i t t e n t injections o f D - a m p h e t a m i n e . 13'~4'46 Coprefrontal cortex probes. The average relative recovery for DA and metabolites was approximately 10%. The relative a d m i n i s t r a t i o n o f n o n - N M D A a n t a g o n i s t s with the recovery for glutamate was 19%. challenge a d m i n i s t r a t i o n also p r e v e n t s the expression Perfusion flow was 2.0 #l/min and was controlled by a o f the a u g m e n t e d behavioral responses. 14 Similarly, microinjection pump (Harvard Instruments, South Natick, a d m i n i s t r a t i o n o f toxic doses o f m e t h a m p h e t a m i n e or MA, U.S.A.). A stainless steel spring tether was used to D - a m p h e t a m i n e increases g l u t a m a t e efflux, 1'22'25'26A1 connect the swivel to the animal. The perfusion medium was a modified Dulbecco's phosphate-buffered saline containing a n d a n t a g o n i s m o f N M D A receptors p r e v e n t s the 138mM NaCl, 2.7mM KC1, 0.SmM MgC12, 1.5mM neurotoxicity to m e t h a m p h e t a m i n e . 3,4°,45 KH2PO4, 8.1 mM Na2HPO4, 1.2 mM CaC12, and 10.0 mM A l t h o u g h m e t h a m p h e t a m i n e - i n d u c e d sensitization D-glucose; pH 7.4. a n d neurotoxicity are associated with a n increase in Three days following surgery (day 8 of the withdrawal period) the dialysis experiment was performed. Rats were d o p a m i n e r g i c a n d glutamatergic t r a n s m i s s i o n , n o placed into the same pre-assigned chamber in which they studies to date have a t t e m p t e d to relate these two received the daily injections. The stylet was removed from p h e n o m e n a . In particular, n o studies h a v e directly the guide cannula and replaced with the dialysis probe. The examined whether a methamphetamine pretreatment probe was inserted into and through the cannula to the regimen followed by a w i t h d r a w a l p e r i o d e n h a n c e s point where the guide cannula abutted a pre-positioned ring of PE 20 tubing on the probe. This positioning permitted the d o p a m i n e a n d / o r g l u t a m a t e efflux a n d s u b s e q u e n t l y exposed portion of the dialysis membrane (4.0 mm for both alters the vulnerability o f n e u r o n s to the n e u r o t r a n s striatum and prefrontal cortex) to extend beyond the end of mitter depleting effects p r o d u c e d by high doses of the guide cannula and precisely into the brain area of m e t h a m p h e t a m i n e . Therefore, the p u r p o s e o f this interest. The tip of the probe was located 7 mm from the cortical surface for striatum and 4 mm from the cortical study was to investigate w h e t h e r p r i o r exposure to surface for prefrontal cortex. A 3-h equilibration period was low doses o f m e t h a m p h e t a m i n e e x a c e r b a t e s the neuallowed before samples were collected. Following the 3-h rotoxic effects o f acute high m e t h a m p h e t a m i n e doses equilibration period, three baseline samples and all subo n d o p a m i n e a n d serotonin n e u r o n s in s t r i a t u m a n d sequent dialysate samples were collected every 30 min. After cortex. the baseline samples, challenge injections of saline vehicle or methampbetamine hydrochloride (7.5 mg/kg) were administered subcutaneous once every 2 h over a period of 6 h for EXPERIMENTAL PROCEDURES a total of three injections. Four days after the dialysis experiment, rats were killed Drug treatment by rapid decapitation, and the brain was quickly removed Male Sprague-Dawley rats (Zivic Miller) weighing befrom the cranium and immediately frozen on dry ice. Probe tween 175 and 300 g were housed three per cage. The drug placements were verified from frozen coronal sections. The pretreatment regimen consisted of the following: rats were prefrontal cortex and anteroventrolateral striatum were removed from the animal colony at 10.30-11.00 a.m. each then dissected from a 250- and 400-#m slice respectively, on day, weighed, placed into a round plastic experimental the side contralateral to the probe implantation site. The chamber and immediately injected i.p. with 2mg/kg dissections were immediately frozen and stored at - 8 0 ° C (+)methamphetamine hydrochloride or 0.15 M NaC1 vefor subsequent tissue content analysis. hicle. Each rat was put in the same designated chamber with Biochemical measurements fresh bedding each day. After 3 h, the rats were returned to the home cage in the vivarium. This procedure was repeated Each dialysate sample (20 #1) was assayed for dopamine, daily for 7 days. All rats were then withdrawn from drug serotonin, and metabolites by high-pressure liquid chromatreatment for 7 days. During this period of withdrawal, tography (HPLC) with electrochemical detection as prerats were implanted with probes for the microdialysis viously described. 4t Separation was achieved with a 3-pm, experiments. C18 column (100 x 2.0 mm) and a mobile phase consisting of 32 mM citric acid, 54.3 mM sodium acetate, 0.074 mM Surgery Na:EDTA, 0.215raM octyl sodium sulfate and 3% On the fourth day of withdrawal from the drug treatment, methanol (pH 4.2). Detection was with a Princeton Applied rats were anesthetized with a combination of xylazine Research Instrument Model 400 Electrochemical Detector
Methamphetamine toxicity and striatal vulnerability and a glassy carbon electrode maintained at a potential of 0.65 V. Another 20-/~1 aliquot of each dialysate sample was assayed for amino acids by HPLC with electrochemical detection following precolumn derivatization with o-pthaldialdehyde. 7 The derivatization reagent was prepared by dissolving 27mg of o-pthaldialdehyde in l ml of 100% methanol, 9 ml of 0.I M sodium tetraborate (pH 9.4) and 5/~1 of ~-mercaptoethanol. This stock solution was then diluted 1:3 with 0.1 M sodium tetraborate. This reagent mixture (10#1) was then automatically added to each dialysate sample by an autosampler, mixed and allowed to react for exactly 2 min before injection. Amino acids were separated on a 3-gm C18 reverse phase column and eluted with 0.1 M sodium phosphate buffer (pH 6.4) containing 28% methanol and 50mg/l EDTA. Detection was with a glassy carbon electrode maintained at 0.7 V by an LC4B amperometric detector, The flow rate was 0.25 ml/min. Tissue from striatum and medial prefrontal cortex was sonicated in ice-cold 0.1 M HCIO4, centrifuged at 13,000 g for 5min, and the supernatant assayed for dopamine, serotonin and metabolites by HPLC with electrochemical detection. Protein determinations were performed using Bradford's Protein Assay.
595
an acute methamphetamine challenge (P < 0 . 0 5 , F = 5.9, d.f. = 1,12) (Fig. IB). Following the first m e t h a m p h e t a m i n e challenge injection, cortical d o p a mine in methamphetamine-pretreated rats increased by 30-fold, whereas saline-pretreated rats only increased by 12-fold over baseline. Cortical d o p a m i n e in methamphetamine-pretreated rats remained elevated over that o f saline-pretreated rats during the course o f the experiment.
Glutamate dialysis data The baseline concentrations of glutamate in dialysate samples were 3.4 _+ 2.0 ng/20/~l (N = for the striatum and 5.9 + 2.4 n g / 2 0 p l (N = 22) the medial prefrontal cortex. The acute doses m e t h a m p h e t a m i n e caused a significant increase in
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(+)Methamphetamine hydrochloride, o-pthaldialdehyde and #-mercaptoethanol were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Xylazine was purchased from Miles Inc. (Shawnee Mission, KS, U.S.A.) and ketamine from Fort Dodge Laboratories, Inc. (Fort Dodge, IA, U.S.A.). HPLC columns were purchased from Phenomenex (Torrance, CA, U.S.A.). Dialysis membrane was purchased from Spectrum (Houston, TX, U.S.A.).
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The baseline concentrations of dopamine in the dialysate samples from striatum and medial prefrontal cortex were 6 . 1 1 + 0 . 3 1 p g / 2 0 / ~ 1 ( N = 2 8 ) and 0.46 __+0.06 pg/20/tl (N = 25), respectively. The acute challenge injections of methamphetamine significantly increased the extracellular concentrations of d o p a m i n e in both the striatum (P < 0.02, F = 79.4, d.f. = 1,24) and medial prefrontal cortex over time (P < 0.02, F = 52.8, d.f. = 1,21) as shown in Fig. IA and B. There was no significant effect o f m e t h a m p h e t amine pretreatment on the increases in extracellular d o p a m i n e in the striatum following the acute challenges with methamphetamine (Fig. IA). In contrast, there was a significant simple main effect of methamphetamine pretreatment on the increase in extracellular dopamine concentrations in cortex following
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Dopamine data are presented in pg/20/11. Glutamate data are presented in ng/20 #1. Tissue content data are expressed as pg//~g of protein. All dialysis data (mean _ S.E.M.) were analysed by a two-way ANOVA with repeated measures incorporating the factors pretreatment (methamphetamine or saline) x challenge (methamphetamine or saline) x time. Post-hoe Tukey tests were performed to compare glutamate dialysate concentrations at specific time points. Tissue content data were analysed by a two-way ANOVA with post-hoe Tukey tests where appropriate.
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Time (h) Fig. 1. The effect of drug pretreatment on the eiflux of dopamine in (A) striatum and (B) medial prefrontal cortex after the acute administration of methamphetamine or saline. Meth/Meth, methamphetamine pretreatment/acute methamphetamine challenge; Sal/Meth, Saline pretreatment/acute methamphetamine challenge; Meth/Sal, methamphetamine pretreatment/acute saline challenge; Sal/Sal, saline pretreatment/acute saline challenge. The acute challenge injections of methamphetamine significantly increased dopamine efflux over time (P <0.02) in both striatum (F = 79.4) and cortex (F = 52.8). There was a significant simple main effect of pretreatment on extraeellular dopamine in medial prefrontal cortex in acute methamphetamine challenged rats (Meth/Meth vs Sal/Meth, P < 0 . 0 5 , F = 5.9). Extracellular cortical dopamine was higher in methamphetamine-pretreated rats. The times of the acute injections are denoted by the arrows. N = 5-9/group.
S. Stephans and B. Yamamoto
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trast, there was no effect of the methamphetamine challenge injections on extracellular cortical glutamate, indicating that the neurotoxic regimen of methamphetamine did not significantly alter glutamate effiux in prefrontal cortex in either methamphetamine- or saline-pretreated rats, as shown in Fig. 2B.
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I
I
Time (h) Fig. 2. The effect of drug pretreatment on the eftlux of glutamate in (A) striatum and (B) medial prefrontal cortex after the acute administration of methamphetamine or saline. Meth/Meth, methamphetamine pretreatment/acute methamphetamine challenge; Sal/Meth, Saline pretreatment/acute methamphetamine challenge; Meth/Sal, methamphetamine pretreatment/acute saline challenge; Sal/Sal saline pretreatment/acute saline challenge. In the striatum, the methamphetamine challenge administration significantly increased glutamate efflux (P < 0.02, F = 6.9). *Significantly different from Meth/Meth (P < 0.05). The times of the acute injections are denoted by the arrows. N = 4-6/group. extracellular concentration of glutamate in striatum following the third m e t h a m p h e t a m i n e challenge injection (P < 0.02, F = 6.9, d.f. = 1,19) (Fig. 2A). The methamphetamine-induced glutamate increase was significantly blunted in rats pretreated with methamphetamine compared with saline-pretreated rats (P < 0.05). Striatal glutamate in saline pretreated rats increased 5-fold over baseline c o m p a r e d with a 3-fold increase in methamphetamine-pretreated rats. In con-
Table 1 illustrates the tissue concentrations of dopamine and serotonin in the striatum and medial prefrontal cortex when assayed 4 days after the acute treatments with methamphetamine or saline. Additional animals not used for the dialysis experiments were also pretreated and then challenged with saline or the high doses o f methamphetamine. Pretreatment with m e t h a m p h e t a m i n e did not alter the tissue concentrations of d o p a m i n e in the striatum or prefrontal cortex in the saline challenged groups (Sal/Sal vs Meth/Sal). Multiple acute methamphetamine challenges, however, decreased striatal dopamine content in both saline-pretreated and methamphetamine-pretreated rats (P < 0.02, F = 63.1, d.f. = 1,78). There was also a significant effect of methamphetamine pretreatment on the striatal dopamine depletions produced by the high doses of methamphetamine (Pretreatment x Challenge, P < 0.02, F = 7.4, d.f. = 1,78). The acute high doses of methamphetamine caused a greater depletion in striatal tissue dopamine content in saline-pretreated rats (62% decrease) than in rats pretreated with methamphetamine (41% decrease). In contrast, dopamine tissue content was not significantly depleted in the prefrontal cortex following administration of toxic doses of methamphetamine, regardless o f pretreatment. Pretreatment with methamphetamine did not alter the tissue concentrations of serotonin in the striatum or prefrontal cortex when administered a saline challenge (Sal/Sal vs Meth/Sal), (Table 1). A significant depletion of serotonin was observed in striatum of rats administered methamphetamine, regardless of drug pretreatment (P < 0.05, F = 4 . 2 , d.f. = 1,62), (methamphetamine-pretreated rats, 19% decrease; saline-pretreated, 33% decrease). In prefrontal cortex serotonin tissue content was also significantly depleted in both methamphetamine- and saline-pretreated rats following the challenge administration of m e t h a m p h e t a m i n e (P < 0,02, F = 16.5, d.f. = 1,54). In addition, there was a significant effect of drug
Table 1. The effect of methamphetamine pretreatment on the dopamine and serotonin content in the striatum and prefrontal cortex Pretreatment/challenge Saline/Saline Methamphetamine/Saline Saline/Methamphetamine Methamphetamine/Methamphetamine
Striatum Dopamine Serotonin 207. I + 13.4 185.5 __+11.5 79.0 __+10.2" 122.8 + 13. I*
4.58 + 3.81 _ 3.05 _ 3.73 +
0.30 0.23 0.49* 0.42
Prefrontal cortex Dopamine Serotonin 2.25 -t- 0.29 2.50 _ 0.45 2.22 _ 0.35 1.66 + 0.24
6.50 +__0.33 6.03 + 0.42 3.44 + 0.44* 5.04 + 0.61
Dopamine and serotonin content were measured 4 days after the acute administration of methamphetamine (methamphetamine, 7.5 mg/kg, s.c. once every 2 h for a total of three injections) or saline. The results are reported in pg/gg of protein. *Significantly different from saline/saline group post-hoc Tukey-test (P < 0.05). N = 11-24/group.
Methamphetamine toxicity and striatal vulnerability pretreatment on the depletions in cortical serotonin content produced by the challenge administration of methamphetamine. Methamphetamine produced a 47% decrease in serotonin content in the salinepretreated group and a 22% decrease in serotonin content in the methamphetamine-pretreated group (Pretreatment x Challenge, P < 0.05, F = 4.3, d.f. = 1,54). A post-hoe comparison revealed that drug pretreatment differentially affected methamphetamine-induced serotonin depletions in both striaturn and prefrontal cortex, such that saline- but not methamphetamine-pretreated rats administered a methamphetamine challenge had significantly lower serotonin concentrations than saline-pretreated controls administered a saline challenge (P < 0.05). DISCUSSION The present results illustrate that pretreatment with intermittent low doses of methamphetamine differentially alters the subsequent increases in glutamate and dopamine efflux in striatum and prefrontal cortex after neurotoxic doses of methamphetamine. The magnitude of these effects was paralleled by the subsequent differential depletions of dopamine and serotonin content in the striatum and cortex.
Methamphetamine and striatal glutamate effiux The observed attenuation of the methamphetamine-induced increase in extracellular glutamate concentrations in rats pretreatcd with methamphetamine may account for the finding that methamphetamine pretreatment decreased the dopamine depleting effects of methamphetamine in striatum. Although methamphetamine increased the extracellular concentrations of glutamate in striatum as reported previously, L25'4~ (Fig. 2B) this increase was significantly blunted at the later time points in rats pretreated with methamphetamine. These effects were paralleled by a diminished depletion of tissue dopamine in striatum 4 days after the challenge administrations. Antagonism of methamphetamine-induced striatal glutamate efflux has been shown to coincide with protection against methamphetamine-induced dopamine depletions in this same brain area. 41 It has been hypothesized that following a methamphetamine-induced energy impairment in striatum, 6 dopamine neurons are more vulnerable to the neurotoxic effects of glutamate. 25 A blunted increase in glutamate concentrations may circumvent calcium-mediated damage or, alternatively, may suppress the transporter-mediated DA release stimulated by glutamate 2~ and the subsequent generation of cytotoxic quinones and free radicals. 2°
Methamphetamine and striatal dopamine effiux The lack of a significant enhancement of striatal dopamine efflux to a methamphetamine challenge in
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rats pretreated with methamphetamine differs from previous studies illustrating augmented efflux of striatal dopamine in rats behaviorally sensitized to methamphetamine.8'~5"49 However, more recent reports show that a dissociation exists between behavioral sensitization and increased neurochemical responses. Therefore, certain paradigms that cause behavioral sensitization do not necessarily produce corresponding augmentations in extracellular dopamine concentrations upon challenge administration.H'a°'as'47'4g Moreover, it has recently been shown that following stimulant pretreatment, a withdrawal period exceeding 1 week is required to produce an augmented dopaminergic response. 3° Therefore, the relatively short withdi'awal period of 1 week used in the present study may account for the lack of an augmented increase in extracellular dopamine in the striatum.
Methamphetamine and cortical dopamine effiux The finding that methamphetamine pretreatment enhanced dopamine efflux during the high-dose methamphetamine challenge in prefrontal cortex is consistent with that of Robinson et al. 33 They reported that repeated intermittent administration of amphetamine increased mesocortical dopamine activity after a single low-dose challenge of D-amphetamine. The exact mechanisms responsible for the augmented methamphetamine-induced cortical dopamine efflux are not known. It has been reported that there is an increase in brain tissue amphetamine after chronic amphetamine treatment? 9 Therefore, it is possible that methamphetamine pretreatment resulted in the accumulation of methamphetamine in tissue. In contrast, chronic methamphetamine treatment has been shown to produce lower brain tissue concentrations of methamphetamine. 36 Therefore, methamphetamine accumulation in tissue is not a likely explanation for the enhanced cortical dopamine efflux observed upon the challenge administration in rats pretreated with methamphetamine. Furthermore, the basal extracellular concentrations of dopamine prior to the challenges were not significantly different between saline and methamphetamine-pretreated groups. Another possible explanation to account for the augmentation in methamphetamine-induced dopamine efflux in cortex is that prior methamphetamine exposure enhances the releasability of dopamine. Castaneda et al. reported that repeated amphetamine administrations can enhance both carrier-mediated and impulse-mediated dopamine release, perhaps by increasing both the cytoplasmic and vesicular pools of dopamine) In addition, changes in the density of the dopamine transporter following methamphetamine pretreatment may reduce uptake of dopamine 23 or enhance the transporter dependent release of dopamine. 49 Taken together, these possibilities are suggestive that a resultant decrease in the uptake of dopamine or increased methamphetamine-induced
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dopamine efflux, along with enhanced impulse-mediated release may culminate in the augmentation of the extracellular concentrations of dopamine.
Methamphetamine toxicity, dopamine and glutamate A depletion of dopamine tissue content was not observed in cortex 4 days after acute high-dose challenges with methamphetamine in methamphetamine-pretreated rats, despite the increase in extracellular dopamine concentration. However, methamphetamine did not increase the extraceilular concentration of glutamate in this region. An increase in both dopamine and glutamate efttux may be necessary to produce a long-term depletion of dopamine content. Therefore, analogous to the effects in the striatum, glutamate efflux appears to play an important role in mediating the long-term neurotoxic effects of methamphetamine to dopamine neurons. 41 The acute challenge of methamphetamine depleted the serotonin content in striatum and prefrontal cortex in both saline- and methamphetaminepretreated groups. This is consistent with previous findings illustrating that methamphetamine is toxic to serotonin neurons. 32'37 The mechanism leading to methamphetamine-induced serotonin depletions is not known. However, because methamphetaminepretreated rats, compared with saline-pretreated controls, exhibit relative resistance to serotonin toxicity in both brain areas following high doses, it seems that the toxicity to serotonin neurons is not exclusively dependent on dopamine efflux. Methamphetamine-induced depletions in serotonin tissue content may be analogous to the toxicity observed in striatum after M D M A , in that an increase in the activities of both the dopamine and the serotonin systems are required to observe the toxic effects to serotonin neurons. ~4'42 Methamphetamineinduced toxicity to serotonin neurons may rely on non-vesicular efflux of serotonin in addition to increased dopamine release. Although the extracellular concentrations of serotonin were not measured during the challenge administrations, it can be speculated that methamphetamine-induced serotonin effiux was blunted in methamphetamine-pretreated rats as compared with saline-pretreated controls. Reduced serotonin transporter activity in cortex may have diminished the uptake of dopamine into serotonin neurons, thereby minimizing cytotoxicity.35 The finding that methamphetamine pretreatment resulted in relative resistance to methamphetamine toxicity is compatible with the tolerance of the striatal dopamine and serotonin neurons to the toxic effects of acute methamphetamine administration following chronic treatment. 36 The present results indicate that in addition to striatal dopamine and serotonin neurons, serotonin neurons in cortex previously exposed to low doses of methamphetamine are resistant to the
subsequent effects of high doses of methamphetamine. The finding that chronic methamphetamine followed by neurotoxic doses leads to reduced brain tissue concentrations of methamphetamine a6 may explain the mechanism by which methamphetamine pretreatment confers resistance to the neurotransmitter-depleting effects of methamphetamine. However, this explanation is not applicable to the finding that methamphetamine-induced cortical dopamine efflux is enhanced in rats pretreated with methamphetamine. None the less, possible alterations in transporter protein density or changes in the distribution of dopamine within the nerve terminal produced by methamphetamine pretreatment, as previously described, could account for the augmented cortical dopamine efflux despite lower concentrations of methamphetamine in the brain. The tissue content data may reflect the transient changes in the regulation of tyrosine hydroxylase and tryptophan hydroxylase activities in a manner such that alterations in the biosynthesis of dopamine and serotonin produced by an acute challenge of methamphetamine may be affected by the pretreatment with methamphetamine. Previous studies have shown that a single acute injection of methamphetamine produces an initial decrease in enzyme activity during the first 1-3 days after administration. This is followed by a partial recovery towards control activity levels.2'9,16'17 In the present study, tissue dopamine and serotonin were assayed 4 days after the challenge administration. The depleted tissue concentrations of these neurotransmitters in rats challenged with methamphetamine could be due to a transient decrease in biosynthesis rather than a longlasting neurotoxic depletion. Thus the tissue content data measured 4 days after the acute challenge with methamphetamine may be reflective of an altered regulation of biosynthesis produced by the pretreatment with methamphetamine rather than due to neurotoxicity per se. However, this explanation is unlikely, based on the findings that the recovery in tyrosine hydroxylase activity after methamphetamine is not paralleled by a recovery in the tissue dopamine content. 16'~7 In addition, tryptophan hydroxylase activity remains depressed after multiple high doses 9 similar to those administered in this study. Moreover, our previous studies indicate that a similar acute challenge regimen of methamphetamine produced a depletion of dopamine content when observed even after a more prolonged period of 7 days. 25,41 CONCLUSIONS
In summary, the present results illustrate that both striatal and cortical dopaminergic and glutamatergic systems are differentially affected by a low-dose intermittent pretreatment regimen of methamphetamine and subsequent high acute doses of methamphetamine. It can be concluded that a low-dose
Methamphetamine toxicity and striatal vulnerability p r e t r e a t m e n t regimen o f m e t h a m p h e t a m i n e reduces the vulnerability of striatal d o p a m i n e a n d s e r o t o n i n terminals a n d cortical serotonin terminals to the toxic effects o f high m e t h a m p h e t a m i n e doses. These findings also provide further s u p p o r t for the role o f
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g l u t a m a t e in mediating m e t h a m p h e t a m i n e - i n d u c e d neurotoxicity. Acknowledgements--This work was supported by grants
DA07606, MH41684 and a gift from Hitachi Ltd.
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