Endogenous neurotensin antagonizes methamphetamine-enhanced dopaminergic activity

BRAIN RESEARCH ELSEVIER

Brain Research 665 (1994) 237-244

Research report

Endogenous neurotensin antagonizes methamphetamine-enhanced dopaminergic activity John D. Wagstaff, Lloyd G. Bush, James W. Gibb, Glen R. Hanson * Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112, USA

Accepted 6 September 1994

Abstract

Neurotensin (NT) has been proposed to be an endogenous neuroleptic based on observations that i.c.v, administration of this peptide antagonizes dopamine-mediated behavior. Because NT influences dopamine activity, this peptide may contribute to the pathogenesis of psychotic disorders such as schizophrenia; however, the precise physiological effects of NT remain speculative. In order to elucidate the function of endogenous NT, a selective NT antiserum (NTAS) was administered i.c.v, through a push-pull cannula in unanesthetized, freely moving rats in combination with dopamine activation caused by methamphetamine (METH). Locomotor and rearing activities induced by a low dose of METH (0.5 mg/kg) were substantially enhanced (4-5-fold) in rats receiving NTAS compared to control animals receiving METH alone. Similarly raised antiserum to vasoactive intestinal polypeptide (VIP) did not alter METH-induced effects. To determine a possible mechanism for these observations, perfusate delivered into the cerebral ventricular space was collected by push-pull cannulae and assayed for dopamine release. METH-induced dopamine release was enhanced 4-5-fold by co-administration of NTAS but not VIP antiserum. To verify these observations, and to identify the site of dopamine release, this experiment was repeated utilizing microdialysis and the recently described NT antagonist, SR-48692. Results from this experiment were similar to those found using NTAS. Like NTAS, co-administration of the NT antagonist enhanced the behavioral responses to a low dose of METH. These studies with SR-48692 also revealed that blockade of NT receptors increased METH-induced release of dopamine from the nucleus accumbens. These findings are the first to demonstrate directly that endogenous NT antagonizes stimulated dopamine pathways and its inactivation substantially enhances METH-induced DA release and related behaviors. Keywords: Neurotensin; Dopamine; Methamphetamine; Schizophrenia; Locomotion; SR-48692; Nucleus accumbens

I. Introduction

Neurotensin (NT) is a neuroactive peptide which, when injected into the brain, exhibits neuroleptic properties such as hypothermia, decreased locomotor activity, muscle relaxation, and potentiation of barbiturateinduced sedation [23]. Because more than 90% of the N T receptors are located on dopamine (DA) terminals in the striatum and nucleus accumbens [3,29], it is likely that this peptide helps modulate D A activity associated with motor function and mental states. In order to elucidate its role, previous studies have examined the effects of exogenous N T injected into

* Corresponding author. 112 Skaggs Hall, University of Utah, Salt Lake City, UT 84112, USA. Fax: (1) (801) 585 5111. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01080-3

various brain regions. These studies have shown that D A systems are variably affected according to the site of administration. Thus, injections of N T into the substantia nigra or ventral tegmental area, primary regions of cell bodies associated with the nigrostriatal and corresponding mesolimbic D A pathways, enhance neuronal activity and increase terminal D A release from these pathways [4,7,15,16]. However, N T administered into the nucleus accumbens attenuates behavioral activities due to the D A agonist apomorphine [8], or microinjection of D A into the nucleus accumbens [17]. A possible explanation for these seemingly contradictory actions is a biphasic D A response to local administration of N T recently reported. Tanganelli et al. [31] showed that injections of a high concentration (1 /zM) of N T into the nucleus accumbens, a primary terminal region of the mesolimbic pathway, increases D A re-

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l e a s e in t h i s r e g i o n ; h o w e v e r a t l o w e r , m o r e p h y s i o l o g i cally r e l e v a n t c o n c e n t r a t i o n s (10 riM), N T r e d u c e s D A release through a GABA-mediated mechanism. P o t e n t C N S s t i m u l a n t s , s u c h as m e t h a m p h e t a m i n e (METH) and cocaine, produce a variety of behavioral effects, many of which are due to their ability to increase activity of extrapyramidal and mesolimbic DA p r o j e c t i o n s . A m o n g t h e s e D A - r e l a t e d e f f e c t s a r e inc r e a s e s in l o c o m o t o r activity, r e a r i n g a n d s t e r e o t y p i c a c t i v i t y [18,22,23]. I n t r a - a c c u m b e n s i n j e c t i o n s o f N T attenuate amphetamine-induced locomotor activity, w i t h o u t a l t e r i n g s t e r e o t y p i c b e h a v i o r [6,24]. T h e s e f i n d ings s u g g e s t t h a t a d m i n i s t e r e d N T a n t a g o n i z e s s o m e amphetamine-induced DA activities; however, the end o g e n o u s p h y s i o l o g i c a l r o l e o f c e n t r a l N T s y s t e m s in the response of DA pathways to the effects of CNS s t i m u l a n t s s u c h as t h e a m p h e t a m i n e s has not been determined. We administered a highly selective NT antiserum ( N T A S ) , i n t r a c e r e b r o v e n t r i c u l a r l y (i.c.v.) in c o m b i n a t i o n w i t h s y s t e m i c M E T H t r e a t m e n t t o e l u c i d a t e dir e c t l y t h e r o l e o f N T s y s t e m s in r e g u l a t i n g D A activity. Central administration of antiserum was employed previously to demonstrate the antinociceptive effects of e n d o g e n o u s N T s y s t e m s [2]. T o v e r i f y t h a t t h e e f f e c t s r e p o r t e d in t h i s s t u d y w e r e d u e t o i n a c t i v a t i o n o f N T by selective antibodies, a similar experiment was done substituting the recently described non-peptide NT ant a g o n i s t S R - 4 8 6 9 2 [9] f o r N T A S . R e s u l t s f r o m t h e present study demonstrate that an important physiological r o l e o f N T is t o m o d e r a t e a c t i v a t e d D A p a t h w a y s although endogenous NT systems appear to have little e f f e c t o n b a s a l D A activity.

2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (200-250 g, Simonson Laboratories, Gilroy, CA) were allowed to acclimate for two weeks. Animals were housed in a temperature-controlled room maintained on a 12-h light and dark cycle with free access to food and water.

2.2. Cannula implantation and perfusion Guide cannulae were constructed of 18-gauge thin-walled stainless steel hypodermic tubing. Push-pull cannulae consisted of 30gauge push and 22-gauge pull tubing joined by a short segment of polyethylene tubing. Each perfusion cannula was made to extend 1 mm beyond the tip of its corresponding guide cannula. Rats were anesthetized with chloral hydrate (375 mg/kg) and mounted in a stereotaxic apparatus. Unilateral guide cannulae were implanted into the right lateral ventricle (from bregma: A -0.8, L+l.5, and V -3.0) [26]. The guide cannulae were anchored to the skull with dental acrylic and stainless steel screws. To maintain patency, a stainless steel stylet was placed in the guide cannula and the animals were allowed to recover overnight. On the day of the experiment, the push-pull cannula was inserted into position and connected to an infusion pump (CMA/100,

Carnegie Medicin, Stockholm Swe.) through a dual-channel liquid swivel to allow free movement. The animal was perfused at a flow rate of 13 p,l/min with sterile filtered artificial cerebrospinal fluid (pH 7.4) containing NaCI, 122 mM; KCI, 3.0 mM; CaCI 2, 1.2 mM; MgCI2, 1.2 mM; KH2PO4, 0.4 mM; 0.03% bacitracin, and 0.08% bovine serum albumin. Rats were perfused for 2 h before samples were collected for analysis. Perfusate samples were collected every 15 min in 1.5-ml microcentrifuge tubes containing 50 ~1 of the high pressure liquid chromatographic (HPLC) mobile phase (0.15 M monochloroacetic acid, 0.2 mM ethylene-diamine-tetraacetic acid [EDTA], 0.003% sodium octyl sulfate, 12.5% methanol, pH 2.9). Samples were immediately frozen and stored at - 80°C until assayed for DA. Three consecutive samples were collected prior to the administration of antisera or METH to establish baseline DA levels. After 45 min of perfusion (time 0 on all figures) a bolus of 10 /zl NTAS (1 : 10 dilution) was initially infused into experimental animals. An air bubble was introduced just prior and immediately following this bolus to help determine its entrance into the brain. Administration of the NTAS bolus was followed by constant perfusion of NTAS (1:1,000) solution for the remainder of the experiment. Control animals were infused with artificial cerebral spinal fluid (CSF) with and without vasoactive intestinal polypeptide antiserum (VIPAS) following the same experimental protocol. After 15 min of perfusion with antiserum or antiserum vehicle, animals were injected with METH (0.5 mg/kg) or saline subcutaneously (s.c.). Following each experiment, rats were sacrificed with an overdose of chloral hydrate and the brains were removed and frozen. Ventricular placement of cannulae was verified visually from serial coronal sections.

2.3. Microdialysis probe construction, placement and perfusion Concentric microdialysis probes consisted of a length of fused silica tubing inside 23-guage stainless-steel hypodermic tubing measured to correspond with placement into the nucleus accumbens. The tubings were joined by polyethylene tubing and 2 mm of exposed dialysis membrane of 40,000 mw cutoff pore size (Hospal). Guide cannulae were constructed similarly to those described for push-pull cannulae, but designed to extend just dorsal to the nucleus accumbens (from bregma: A + 1.7, L _+1.2, V -6.0). Guide cannulae were implanted as described above. On the day of the experiment, the microdialysis probe was inserted through the guide cannula into the nucleus accumbens, connected to an infusion pump, and perfused at a flow rate of 2 / z l / m i n for at least 2 h prior to collection. After this period, dialysate was collected every 15 min into tubes containing 30 ~1 of the HPLC mobile phase, immediately frozen on dry ice, and stored at -80°C until assayed for DA. Following 3 baseline collections, SR-48692 (80 /~g/kg in 25% DMSO) or vehicle was administered i.p., and another 3 microdialysis samples were collected. This dosing protocol was employed to give maximal efficacy, and was verified by dose-response experiments (data not shown). Following collection of 3 samples, METH (0.5 mg/kg) or saline was given s.c., and dialysate collected for another hour. Probe placement was verified after the animals were sacrificed as described above.

2.4. Dopamine and metabolite analysis Levels of DA and 5-hydroxyindoleacetic acid (5-HIAA) were measured using HPLC-EC according to the method described by Johnson et al. [14]. Briefly, samples were thawed and filtered with a 0.2-tzm Microfilter system (Bioanalytical Systems, West Lafayette, IN), and 100/zl were injected onto a 10-cm Microsorb C-18 reversed phase column (3/zm, Rainin Instrument, Woburn, MA.) The mobile phase (0.15 M monochloroacetic acid, 0.2 mM ethylene-diamine-tetraacetic acid [EDTA], 0.003% sodium octyl sulfate, 12.5% methanol, pH 2.9) was pumped at a flow rate of 1.1 ml/min. The eluent was monitored using a glassy carbon electrode at a potential of+ 0.73 V

J.D. Wagstaff et al. / Brain Research 665 (1994) 237-244 vs. Ag/AgCI reference electrode using an amperometric electrochemical detector (model LC-4B, Bioanalytical Systems). Working standards were prepared fresh in the mobile phase from frozen stock solutions and standard curves used for quantification of each compound. External standards were injected intermittently during sample analysis to ensure consistency between injections. Data were analyzed using the Dynamax program (Rainin Instruments).

2.5. Behavior analysis Each experiment was conducted in a 2' × 2' observation box with a marked 6" square grid and located in an isolated room. Animals were monitored for the entire experiment. Locomotor activity was assessed by determining the number of complete squares the animal crossed during each 15-min period. Rearing was determined by recording the number of times an animal elevated up on the hind limbs with both forelimbs concurrently raised above body level, and is indicated graphically by the number of episodes in a 15-min time period.

2.6. Antisera Neurotensin antiserum was produced by a modification of standard techniques as previously described [19]. NT was conjugated to thyroglobulin, bovine serum albumin, ovalbumin, or hemocyanin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The mixture was allowed to sit at room temperature for 24 h, at which time an equal volume of 1 M hydroxylamine was added to the reaction mixture. After an additional 4 h of incubation, the conjugate was emulsified with complete or incomplete Freund's adjuvant and immediately injected intradermally into multiple sites on the backs of New Zealand White rabbits once a week for 4 weeks and subsequently at 6-week intervals. The antiserum was harvested via heart puncture 10 days after the final booster. This antiserum reliably detects 2 pg/sample of NT when used at a dilution of 1:50,000 for radioimmunoassays [19,30]. The antiserum is highly selective and has no cross-reactivity with 1,000-fold excess concentrations of other endogenous neuropeptides such as dynorphin A, metenkephalin, cholecystokinin or substance P. The antiserum has approximately 80% cross-reactivity with the 8 to 13 carboxyl-terminal fragment of NT. The VIP antiserum was produced in an identical manner, the only difference being the replacement of NT with VIP. This antiserum was chosen as a suitable control since VIP has not been shown to influence CNS DA systems. The VIP antiserum (VIPAS) detects 2 pg of VIP when used at a concentration of 1:160,000 in radioimmunoassays. Cross-reactivity for this antiserum was less than 1% for secretin, glucagon, and gastric inhibitory polypeptide [1] and not detectable with NT.

2. 7. Drugs METH (National Institute of Drug Abuse, Bethesda, MD) was dissolved in saline, and administered s.c. SR-48692 has been reported to be selective for the high-affinity, levocabastine insensitive NT receptor, with no activity at other tested receptor types [9]. This compound was dissolved in 25% DMSO, and administered i.p. SR-48692 was a generous gift of Dr. DanieUe Gully (Sanofe Recherche).

2.8. Statistics In order to facilitate the comparison of mean values_+S.E.M., Figs. 2 and 4 are expressed as percentage of control. Due to individual variability between each animal, DA and 5-HIAA levels were calculated as a percentage of each animal's baseline prior to

239

administration of antisera. Baseline levels between all groups were not significantly different and were pooled to determine basal DA release. The averages for control values are expressed in the figure legends. Analysis by ANOVA followed by Fisher's-PLSD (Protected Least Significant Difference) test for multiple comparisons was done to compare the means of each parameter against the values from corresponding control groups for each collection interval. Differences were considered significant when the probability that they were zero was less than 0.05.

3. Results

3.1. Effects of neurotensin antiserum on METH-induced behavior A low dose of M E T H (0.5 m g / k g ) was used for these studies since in dose-response experiments we found that this dose was close to threshold for eliciting significant behavioral changes in the rat. Perfusion of NTAS alone had no effect on locomotor activity (first time point of Fig. 1A); however, in combination with an injection of 0.5 m g / k g of METH, a dramatic increase in locomotor activity was immediately seen and persisted for the 45 min measured. To verify that this effect was not due to nonspecific antibodies, or other factors present in the antiserum, VIPAS was used in place of NTAS in a comparable experiment. This antiserum was raised in an identical manner as NTAS (see materials and methods), and thus would contain similar nonspecific constituents. The presence of VIPAS did not significantly alter the effects of a low dose of M E T H on locomotor activity. These results suggest that NTAS selectively enhanced METH-induced locomotion. Another behavior which has been attributed to enhanced DA-activity is rearing. In this study, rearing was defined as elevation of the rat onto its hind limbs with both forelimbs concurrently above body level. Neither M E T H (Fig. 1B) nor NTAS (see 15 min time point, Fig. 1B) alone significantly affected rearing activity. Like locomotion, the presence of NTAS induced a prominent METH-induced increase in rearing. Again using VIPAS as a control, the antiserum alone had no effect on rearing nor did it alter the effect of M E T H on this behavior.

3.2. Neurochemical effects of neurotensin antiserum Because the behaviors studied are associated with increases in D A activity, we assessed the effect of NTAS on dopamine release in the absence and presence of a low dose of M E T H . D A was measured in perfusate collected from the ventricle, and therefore represented a summation of D A release throughout the CNS. NTAS alone had no affect on total D A release (Fig. 2A; compare M E T H and M E T H + NTAS groups at 15 min time point with previous 3 time

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points). This result suggests that N T systems do not play a significant role in m o d u l a t i n g CNS basal D A activity since the inactivation of extracellular N T by N T A S did not alter baseline release of DA. M E T H alone also had no significant affect o n D A release m e a s u r e d from the ventricles (Fig. 2A) c o n f i r m i n g that 0.5 m g / k g was n e a r the threshold level n e e d e d to elicit D A - m e d i a t e d behavior changes. T h e c o m b i n a t i o n of M E T H a n d N T A S however, caused a n i m m e d i a t e rise (15 m i n after M E T H a d m i n i s t r a t i o n ) in D A release which was higher t h a n M E T H alone, but due to the large variability b e t w e e n animals did n o t reach significance c o m p a r e d to the M E T H + V I P A S group (Fig.

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NTAS METH Fig. 1. A: effects of i.c.v. NT antiserum (NTAS) on methamphetamine-induced locomotion. Rats were infused with NTAS, V|P antiserum (VIPAS) or artificial CSF and then injected with methamphetamine (0.5 mg/kg) or saline s.c. as explained in section 2. Rats were placed in a behavior observation box with a 6-ineh square grid and locomotor activity recorded. Locomotion is expressed as the number of complete squares crossed/15 min+_S.E.M, of 5-8 animals. No significant locomotor activity was observed during any time points prior to 15 min or in saline controls, therefore these values are not shown. B: effects of NT antiserum on methamphetamine-induced rearing. Rats were treated as described in (A), and evaluated for rearing behavior. Rearing is expressed as the number of times the animal elevated up on the hind limbs with both forelimbs concurrently raised above body level +_S.E.M. of 5-8 animals. No significant rearing activity was observed during any time periods prior to 15 rain or in saline controls, therefore these values are not shown. * P < 0.05 vs. corresponding values of METH and METH + VIPAS.

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Fig. 2. (A) Effects of NTAS on methamphetamine-induced dopamine release in the awake rat. Rats were treated as described in Fig. 1. Dopamine levels were analyzed in fluid perfused into and collected from the the lateral ventricle by push-pull cannulae. Data (means+_ S.E.M. of 5-8 animals) are expressed as a percentage of the mean of the controls (first 3 time points shown), prior to administration of antisera or drug. The average control values were 103.8_+16 pg/15 rain. Standard error bars for the first three time points for each group are so small that they do not extend past their respective symbols. (B) Effects of METH, NTAS and VIPAS alone and in combination on ventricular 5-HIAA levels. Rats were treated as described in Fig. 1. 5-HIAA levels were analyzed in fluid perfused into the lateral ventricle by push-pull cannulae. Data are expressed as a percentage of the mean of the controls (first 3 time points shown), prior to administration of antisera or drug. The average control values were 641.2+_112.3 pg/15 rain. **P < 0.05 vs. corresponding METH values * P < 0.05 vs. corresponding METH and METH + VIPAS values. 2A, see 30 m i n time point). However, 30 m i n following M E T H a d m i n i s t r a t i o n the D A release was significantly higher t h a n either M E T H or M E T H + V I P A S corres p o n d i n g groups (Fig. 2A, see 45 m i n time point). D A levels r e t u r n e d to control 45 m i n after M E T H administ r a t i o n (see 60 m i n time point). A g a i n V I P A S was used as a control for nonspecific effects that might be caused by the N T A S . Because V I P A S had n o significant effect o n D A release alone or in c o m b i n a t i o n with M E T H , the effect of N T A S on M E T H - i n d u c e d D A release likely was selective a n d d u e to the b i n d i n g of e n d o g e nous N T to specific N T a n t i b o d i e s in the a n t i s e r u m . 5-hydroxytryptamine (5-HT), or serotonin, release is

J.D. Wagstaffet aL /Brain Research 665 (1994) 237-244 also e n h a n c e d by M E T H [28], which m a y c o n t r i b u t e to its b e h a v i o r a l effects. 5 - H T t u r n o v e r was indirectly a s s e s s e d by m e a s u r i n g its m e t a b o l i t e , 5 - H I A A , since 5 - H T levels in the p e r f u s a t e w e r e b e l o w t h e sensitivity r a n g e o f o u r assay. 5 - H I A A levels w e r e not significantly a l t e r e d by M E T H a l o n e o r by e i t h e r a n t i s e r u m (Fig. 2B). W h e n M E T H was c o m b i n e d with N T A S o r V I P A S , b o t h g r o u p s s h o w e d similar, highly variable, i n c r e a s e s in 5 - H I A A levels.

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Fig. 4. Effects of METH and SR-48692 alone and in combination on METH-induced dopamine release in the nucleus accumbens. Animals were treated as described in Fig. 3A. Extracellular dopamine was analyzed from fluid collected by microdialysis. Data (means_+ S.E.M. of 9 animals) are expressed as a percentage of the mean of the controls (first 3 time points shown), prior to administration of SR-48692 or vehicle. The average control values were 6.6 _+4.2 pg/15 min. * P < 0.05 vs. corresponding METH group values.

nation, b o t h of t h e s e b e h a v i o r s w e r e significantly enh a n c e d . A s with the a n i m a l s given N T A S , SR-48692 c a u s e d m a x i m a l activity 30 min following the a d m i n i s t r a t i o n o f M E T H . L o c o m o t o r activity r e m a i n e d significantly e l e v a t e d for the e n t i r e o b s e r v a t i o n p e r i o d , while r e a r i n g r e t u r n e d to c o n t r o l levels at 45 min.

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Fig. 3. (A) Effects of METH and SR-48692 alone and in combination on METH-induced locomotor activity. Rats were injected with SR48692 (80 /xg/kg) i.p., followed by METH (0.5 mg/kg) s.c. as explained in section 2, and evaluated for locomotor activity as explained in Fig. 1A. Locomotion is expressed as the number of squares crossed/15 min-+ S.E.M. of 9 animals. (B) Effects of METH and SR-48692 alone and in combination on methamphetamine-induced rearing activity. Animals were treated as described in Fig. 3A and assessed for rearing activity as described in Fig. lB, None of the control animals exhibited any rearing activity. * P < 0.05 vs. corresponding METH group values.

In o r d e r to identify the site o f e n h a n c e d D A r e l e a s e c a u s e d by blocking N T transmission, microdialysis p r o b e s w e r e p l a c e d in the nucleus a c c u m b e n s . S R 48692, like N T A S , h a d no effect on basal e x t r a c e l l u l a r D A a l o n e (Fig. 4), c o n f i r m i n g the o b s e r v a t i o n that N T d o e s not play a significant role in m o d u l a t i n g basal D A r e l e a s e in this region. M E T H a l o n e also had no effect on e x t r a c e l l u l a r D A levels, which c o r r e s p o n d s to t h e lack o f activity seen in this g r o u p o f animals. H o w e v e r , w h e n SR-48692 was c o m b i n e d with M E T H , a r a p i d i n c r e a s e in e x t r a c e l l u l a r D A o f t h e nucleus a c c u m b e n s was s e e n 15 min following M E T H a d m i n i s t r a t i o n (60 min value), b u t r e t u r n e d to c o n t r o l levels by 30 min (75 min value).

4. Discussion Previous studies o f t h e effects o f N T on D A - m e d i a t e d b e h a v i o r s have r e p o r t e d t h e results of c e n t r a l adm i n i s t r a t i o n of e x o g e n o u s NT. T h e s e findings indirectly s u p p o r t the h y p o t h e s i s t h a t a m a j o r function o f

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NT systems in the CNS is to modulate DA systems. However, until recently, it has been difficult to demonstrate directly the endogenous physiological roles of NT pathways since no selective NT antagonist was available. By administering a highly specific NT antiserum we have shown, for the first time, that the inactivation of endogenous extracellular NT enhances the release of DA caused by exposure to low doses of M E T H and this in turn likely results in exaggerated locomotor and rearing activity. This role of NT was confirmed by substituting SR-48692, a new potent antagonist of the high-affinity NT receptor [9], for NTAS. Further, we observed with microdialysis that blockade of the NT receptor with this antagonist increased the METH-induced release of DA from the nucleus accumbens. The current study strongly suggests that a major role of central NT pathways is to stabilize excited DA systems since neutralization of N T activity with NTAS or SR-48692 alone had no effect on basal DA release or behavior (15 min time point in Figs. 1A,B and 2A) but dramatically enhanced METH-induced DA release and related behaviors. M E T H is also known to increase the release of 5-HT which suggests that this monoamine may contribute to the behavioral effects of this drug. However, since VIPAS had no significant effect on DA release or associated behaviors (Figs. 1A,B, and 2A), the fact that both antisera had similar effects on 5-HT (Fig. 2B) turnover suggests that 5-HT did not contribute to the effects of M E T H only augmented by NTAS. The high variability in the effects of these antisera on ventricular 5-HIAA may be due to nonspecific factors found in the antisera and not due to specific antibodies. Therefore, it appears that endogenous NT plays little if any role in the regulation of central 5-HT systems. The principal difference between NT antagonism by NTAS or SR-48692 was the more dramatic increase in behaviors seen with NTAS perfusion (compare Figs. 1A and B vs. Figs. 3A and B). Possible explanations for this difference are that NTAS was infused for the entire experiment, while SR-48692 was given as a single systemic dose, therefore, the antagonistic effect of NTAS would possibly be more sustained, and blockade of NT transmission more complete than blockade by SR-48692. Also, neutralizing released NT by NTAS would completely block all NT transmission, whereas SR-48692 only blocks NT activity at the high affinity NT receptor [9]. Thus, in those animals treated with this compound, extracellular NT would still be stimulating the low affinity NT receptor which may also play a role in modulating DA activity. Because blocking normal NT transmission enhanced the effects of a low dose of M E T H (0.5 mg/kg), it is likely that NT pathways are involved in physiological regulation and are clinically relevant. Alterations in

NT levels have been reported under conditions of stress [5], and i.c.v. NT administration has been shown to attenuate the response of rats to stress [32]. This suggests that NT systems are important regulators of enhanced DA activity that occurs in altered physiological conditions. If NT pathways temper DA activity during stress, a lack of NT activity might result in exaggerated DA responses that are associated with psychotic disorders, such as schizophrenia. In support of this hypothesis is the finding by Widerlov et al. [33] and others [20,21] that there is reduced NT levels in the cerebrospinal fluid of a subpopulation of schizophrenic patients. It is noteworthy that successful antipsychotic therapy with neuroleptic agents restores these N T levels to normal [33]. Other evidence of a NT role in some forms of psychosis are the reports that high doses of psychotomimetic drugs such as methamphetamine [11,19], cocaine [10,12], and phencyclidine [12], cause dramatic increases in NT levels in the nucleus accumbens and the neostriatum. A recent report by Hanson et al. [12] that high concentrations of M E T H block in vivo striatal release of NT, suggests that the increased NT tissue concentrations that are caused by psychotomimetic drugs are due to accumulation of this peptide in neuronal terminals from decreased activity of NT systems. The present findings suggest that this peptide does not have a significant role in the regulation of tonic DA activity, but rather helps to establish the physiological limits within which excited DA pathways can function. Thus, elimination of NT activity by psychotomimetic drugs would enhance the DA response to these potent stimulants and contribute to their psychotic effects. As mentioned, NT appears to have variable effects on central DA systems. When injected into regions with high densities of DA cell bodies, such as the substantia nigra or ventral tegmental area, NT increases electrical activity of DA neurons and increases DA turnover in terminal areas [4,7,15,16]. However, i.c.v., intracisternal or intra-accumbens N T administration antagonizes the locomotor effects of DA and indirect DA agonists such as d-amphetamine [6,8,17,25]. The present work supports the latter studies since NTAS given i.c.v., or SR-48692 given systemically potentiated the release of DA and corresponding behaviors induced by METH. This study also showed that the direct blockade of NT receptors by SR-48692 substantially enhanced the induced release of DA from the nucleus accumbens, and supports the hypothesis that the mesolimbic DA pathway has a major role in these NT sensitive METH-induced behaviors. The present studies did not specifically examine the role of extrapyramidal pathways in these behaviors. Currently there are conflicting data as to the location of NT receptors. Previous studies have reported

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that greater than 90% of NT receptors in the striatum, substantia nigra and ventral tegmental area are destroyed following 6-hydroxydopamine (6-OHDA) lesions while NT receptors in the nucleus accumbens are spared [13,27]. These findings imply that accumbens NT receptors are not located on DA terminals. However, more recent studies have shown 90-95% of NT receptors in the accumbens are destroyed following 6-OHDA treatment [3,29]. Because of these conflicting findings it is difficult to identify the site of D A / N T interaction within the nucleus accumbens. One possibility has been proposed recently by Tanganelli et al. [31] This group reported that NT at low concentrations (10 nM) increases the release of GABA while decreasing extracellular DA in the nucleus accumbens. Further, this effect on DA can be blocked by co-administering a GABA-A antagonist. This indicates that the antagonistic effect of NT on DA release in the nucleus accumbens is mediated by enhanced GABA release. If this is true, blocking NT receptors should decrease GABA release, causing a disinhibition of dopamine neurons resulting in enhanced DA release. This hypothesis is consistent with the present findings that blockade of NT receptors in combination with a low dose of METH enhances DA release from the nucleus accumbens. This study suggests that manipulation of central NT systems can have important clinical significance and possibly be targeted in the treatment of motor, psychiatric or neurological disorders involving DA pathway dysfunctions. Our findings suggest that the overall effects of specific NT antagonists is to potentiate activated DA systems. Such drugs may be useful in the treatment of Parkinson's disease as an adjunct to enhancing DA systems with L-DOPA therapy. NTAS or SR-48692 showed no effects when administered alone suggesting that NT antagonists may have few, or at least less severe side-effects than DA agonists. In contrast, NT-enhancing drugs may be useful in the treatment of disorders of exaggerated DA responses such as schizophrenia.

Acknowledgements This research was supported by USPHS Grants DA 00869 and DA 04221. Methamphetamine used in these studies was provided by the National Institutes on Drug Abuse. We are particularly grateful to Sanofe Recherche for the generous gift of SR-48692.

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