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BEHAVIORAL PHARMACOLOGY

The -Opioid Receptor Agonist SNC80 [(+)-4-[(R)--[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide] Synergistically Enhances the Locomotor-Activating Effects of Some Psychomotor Stimulants, but Not Direct Dopamine Agonists, in Rats

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan (E.M.J., J.H.W.); Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas (M.G.B.); and Chemical Biology Research Branch, National Institute on Drug Abuse, National Institutes of Health, Bethesda, Maryland (J.E.F., K.C.R.)

Received April 2, 2007; accepted November 5, 2007.

	Abstract

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The nonpeptidic -opioid agonist SNC80 [(+)-4-[(R)--[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide] produces many stimulant-like behavioral effects in rodents and monkeys, such as locomotor stimulation, generalization to cocaine in discrimination procedures, and antiparkinsonian effects. Tolerance to the locomotor-stimulating effects of SNC80 develops after a single administration of SNC80 in rats; it is not known whether cross-tolerance develops to the effects of other stimulant compounds. In the initial studies to determine whether SNC80 produced cross-tolerance to other stimulant compounds, it was discovered that amphetamine-stimulated locomotor activity was greatly enhanced in SNC80-pretreated rats. This study evaluated acute cross-tolerance between -opioid agonists and other locomotor-stimulating drugs. Locomotor activity was measured in male Sprague-Dawley rats implanted with radiotransmitters, and activity levels were recorded in the home cage environment. Three-hour SNC80 pretreatment produced tolerance to further -opioid receptor stimulation but also augmented greatly amphetamine-stimulated locomotor activity in a dose-dependent manner. Pretreatments with other -opioid agonists, (+)BW373U86 [(+)-4-[(R)--[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide] and oxymorphindole (17-methyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2',3'-indolomorphinan), also modified amphetamine-induced activity levels. SNC80 pretreatment enhanced the stimulatory effects of the dopamine/norepinephrine transporter ligands cocaine and nomifensine (1,2,3,4-tetrahydro-2-methyl-4-phenyl-8-isoquinolinanmine maleate salt), but not the direct dopamine receptor agonists SKF81297 [R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide] and quinpirole [trans-(-)-(4R)-4,4a, 5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g] quinoline monohydrochloride]. In conclusion, SNC80 enhanced the locomotor-stimulating effects of monoamine transporter ligands suggesting that -opioid receptor activation might alter the functional activity of monoamine transporters or presynaptic monoamine terminals.

In general, drugs with reinforcing properties, such as cocaine, amphetamine, morphine, and heroin, increase dopamine release in the striatum either by acting at the dopamine terminals directly in the striatum or by acting directly or indirectly on dopaminergic cell bodies in the substantia nigra or ventral tegmental area. Similarly, administration of peptidic -opioid agonists into the nucleus accumbens, ventral tegmental area, or the interpeduncular nucleus stimulated dopamine release as measured by in vivo microdialysis (Spanagel et al., 1990; Longoni et al., 1991; Dourmap et al., 1992; Devine et al., 1993; Billet et al., 2004; Fusa et al., 2005; Hirose et al., 2005). However, one study reported that DP-DPE did not alter basal dopamine release but did increase potassium-stimulated dopamine release in the striatum as measured by in vivo voltammetry (Pentney and Gratton, 1991). Conversely, systemic administration of the non-peptidic -opioid agonists SNC80 and BW373U86 did not increase dopamine release as measured by in vivo microdialysis at doses that stimulated locomotor activity and produced conditioned place preference (Longoni et al., 1998). These studies suggest that there may be differences between the actions of peptidic and nonpeptidic -opioid agonists, possibly due to the route of administration (i.c.v. for peptides and peripheral for nonpeptides) or to the pharmacological differences between -opioid agonists. For example, the non-peptide SNC80 has a lower K_i (approximately 8-fold) for the -opioid receptor and is more efficacious than the peptide DPDPE, even though these compounds have similar selectivities for the -over the µ-opioid receptor (Calderon et al., 1997; Clark et al., 1997). However, no studies have directly explored these differences in vivo. The effects of -opioid receptor activation on the dopamine system and dopamine-related behaviors are complex and not well understood.

One major difference in behavior between -opioid agonists and psychomotor stimulants can be observed following repeated drug administration. For example, a single administration of the -opioid agonist SNC80 produced tolerance to its locomotor-stimulating effects, such that subsequent SNC80 doses did not produce similar increases in activity levels (Jutkiewicz et al., 2005b). It is unknown whether tolerance to the locomotor-stimulating effects of -opioid agonists is related to changes in the dopaminergic system. This is seldom the case with repeated amphetamine or co-caine treatments because repeated administration regimens most often produce locomotor sensitization and enhanced dopamine responses to amphetamine and cocaine (Robinson et al., 1998; Vanderschuren et al., 1999; Kuczenski and Segal, 2001).

In the present study, experiments were conducted to determine whether SNC80 administration produces cross-tolerance to other compounds with stimulatory activity. Surprisingly, the initial experiment showed that rats tolerant to further SNC80 stimulation demonstrated large increases in amphetamine-stimulated locomotor activity. The present study further investigated these findings by evaluating the effects of SNC80 pretreatments on locomotor activity stimulated by dopamine/norepinephrine transporter ligands (amphetamine, cocaine, and nomifensine) and direct dopamine agonists (SKF81297 and quinpirole). It was expected that 3-h SNC80 pretreatment would enhance the locomotor-stimulating effects of all drugs tested. Unexpectedly, these data demonstrated that 3-h SNC80 pretreatments only enhanced the locomotor-stimulating effects of monoamine transporter ligands but not direct dopamine agonists.

	Materials and Methods

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Subjects
Male Sprague-Dawley rats (250–350 g) were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and housed in groups of three animals per cage upon arrival. Food and water were freely available for all rats, and the housing and experimental rooms were maintained on a 12 h light/dark cycle with lights on at 7:00 AM at an average temperature of 21°C. The experimental protocols were approved by the University of Michigan University Committee on the Use and Care of Animals and conformed to the guidelines established by the NIH Guide for the Use of Laboratory Animals.

Surgical Implant and Telemetry System
To measure changes in locomotor activity, singly housed rats were implanted with telemetry devices (model ER-4000 E-Mitter; Mini Mitter Co., Bend, OR). Under ketamine (90 mg/kg i.p.) and xylazine (10 mg/kg i.p.) anesthesia, the abdomen was prepared for surgery using Betadine and alcohol. A 1- to 2-cm-long rostral-caudal incision was made in the skin and muscle to expose the peritoneal cavity. The radiotransmitter was placed inside the peritoneal cavity, and the muscle and skin were closed separately with absorbable gastrointestinal 4-0-coated Vicryl suture. Implant surgeries were conducted at least 6 days prior to conducting an experiment. The implanted transmitters produced locomotor activity signals that were sent to a receiver (model ER-4000 Receiver; Mini Mitter Co.) placed directly under the home cage of each rat. Data were collected and processed simultaneously by the Vital View data acquisition system (Mini Mitter Co.).

Testing Regimens
Each rat was exposed to only one treatment condition or one treatment regimen and then euthanized. Rats in their home cages were placed on the receivers at least 15 h prior to data acquisition. Baseline locomotor activity was collected for at least 40 min prior to injections without disturbing the rats' home cages. Baseline activity counts are shown in Fig. 1 only, but similar basal levels were observed for all experiments. For injections, a cage was lifted off its receiver, and the rat was removed from its cage for the injection. Each treatment group represents data averaged from six to eight rats. Procedures specific for each experiment are described below. Data are represented as mean locomotor activity counts for each treatment group ± S.E.M.

View larger version (26K): [in this window] [in a new window]   Fig. 1. SNC80 (a) and amphetamine (b) dose-dependently increased locomotor activity in the home cage environment of male Sprague-Dawley rats. Injections of SNC80 or amphetamine occurred at time 0 with baseline activity counts displayed prior to injection. c, effects of vehicle or various doses of NTI administered s.c. 30 min prior to 10 mg/kg SNC80, and NTI dose-dependently attenuated SNC80-induced locomotor activity. Activity counts following SNC80 injections are shown. d, effects of vehicle or 10 mg/kg NTI administered 30 min prior to 1.0 mg/kg amphetamine injection, and NTI did not block amphetamine-induced locomotor activity. The graph illustrates activity counts following the amphetamine injection.

Experiment 1. In the first experiment, SNC80-stimulated locomotor activity was compared with amphetamine-stimulated activity levels under the following experimental design. Vehicle or different doses of SNC80 (1.0, 3.2, 10, or 32 mg/kg) or amphetamine (0.32, 1.0, or 3.2 mg/kg) were administered s.c. at time 0, and locomotor activity counts were recorded for at least 200 min after injection. In addition, the -opioid antagonist naltrindole (NTI) was used as a pretreatment to attenuate SNC80- and amphetamine-stimulated activity to determine whether the -opioid receptor was involved in these locomotor effects. The NTI pretreatment was administered s.c. 30 min prior to SNC80 or amphetamine.

Experiment 2. In the second experiment, SNC80 and/or amphetamine were given in 3-h intervals to determine whether rapid tolerance or cross-tolerance develops following drug administration. Pretreatments were administered 3 h prior to a challenge injection (expressed as "pretreatment + challenge" in graph legends). The pretreatment injection occurred at time 0, and the challenge injection occurred at 180 min. Pretreatments were vehicle solutions, different doses of SNC80 (1, 3.2, 10, 32 mg/kg), or 1.0 mg/kg amphetamine. The challenge drug was either SNC80 (10 mg/kg) or amphetamine (1.0 mg/kg). These doses were chosen because they produce similar locomotor responses, as demonstrated in Fig. 1.

Experiment 3. The time course of the SNC80-induced enhancement of amphetamine was evaluated by pretreating rats with SNC80 for various lengths of time prior to amphetamine challenge. Vehicle or 10 mg/kg SNC80 was administered 1, 4, 6, or 24 h prior to an amphetamine challenge (1.0 mg/kg).

Experiment 4. In experiment 4, two different -opioid agonists [(+)BW373U86 or OMI] were administered as pretreatments to amphetamine challenge to demonstrate that the amphetamine enhancement was not SNC80-specific. (+)BW373U86 is more potent and efficacious than SNC80 but is less selective for the -opioid receptor; OMI is a partial agonist at the -opioid receptor (Clark et al., 1997; Jutkiewicz et al., 2005c). Vehicle or different doses of the full -opioid agonist (+)BW373U86 (1, 3.2, or 10 mg/kg) or the partial -opioid agonist OMI (10 or 32 mg/kg) were administered s.c. as a pretreatment 3 h prior to an amphetamine challenge (1.0 mg/kg). The pretreatment injection occurred at time 0, and the challenge injection occurred at 180 min.

Experiment 5. This experiment investigated whether the effect of SNC80 pretreatment on amphetamine challenge was -opioid receptor-mediated by administering naltrindole soon after SNC80. In addition, naltrindole was administered just before the amphetamine challenge (when the pretreatment effects dissipated) to determine whether -opioid receptor activation continued at the time of amphetamine challenge. SNC80 (10 mg/kg) was administered as a 3-h pretreatment to the amphetamine challenge. NTI was administered s.c. 30 min after the SNC80 pretreatment or 30 min before the amphetamine challenge.

Experiment 6. Experiment 6 investigated the effects of simultaneous administration of amphetamine with SNC80 to determine whether coadministration of these compounds can produce similar enhanced amphetamine responses. Amphetamine (1.0 mg/kg) was administered simultaneously with 0.1 or 1.0 mg/kg SNC80, and activity counts were displayed for 200 min following this combination injection. For comparison, the locomotor effects of 1.0 mg/kg amphetamine alone or 1.0 mg/kg SNC80 alone were also shown as well as activity counts from the amphetamine challenge following 10 mg/kg SNC80 pretreatment.

Experiment 7. This experiment tested whether SNC80 could enhance the locomotor effects of dopamine-related drugs, such as the monoamine transporter reuptake inhibitors, cocaine and nomifensine, or the direct dopamine D₁ agonist, SKF81297, or the direct dopamine D₂-like agonist, quinpirole. Vehicle or 10 mg/kg SNC80 was administered as a 3-h pretreatment to different challenge drugs (s.c.): 5.6 mg/kg cocaine, 3.2 mg/kg nomifensine, 3.2 mg/kg SKF81297, or 3.2 mg/kg quinpirole. The pretreatment injection occurred at time 0, and the challenge injection occurred at 180 min.

Drugs
All drugs were administered s.c. and injected in a volume of 1 ml/kg. D-Amphetamine sulfate [(+)--methylphenethylamine sulfate], cocaine (ecgonine methyl ester benzoate hydrochloride), R(+)-SKF81297, (-)-quinpirole, oxymorphindole (Portoghese et al., 1990), and NTI were dissolved in sterile water. Nomifensine was dissolved in water with a few drops of lactic acid. Amphetamine, cocaine, and NTI were obtained from the National Institute on Drug Abuse, and SKF81297 and quinpirole were purchased from Sigma-Aldrich (St. Louis, MO). (+)BW373U86 and SNC80 were synthesized as previously described (Calderon et al., 1994) and dissolved in 8% 1 M HCl. Solutions ranged from pH 3 to 7, and the pH of the vehicle solutions was adjusted to match the drug solutions.

Data Analysis
Locomotor activity counts were summed over 5-min intervals for the duration of an experiment. Data collected began at least 40 min prior to drug injection or pretreatment. Mean locomotor activity counts were calculated for each experimental group and graphed as activity at time after injection (±2 min). Statistical analyses were performed on the locomotor activity over time for the challenge injection only using two-way analyses of variance with Tukey's post hoc test (GraphPad Prism Software, GraphPad Software Inc., San Diego, CA; or SigmaStat; Systat Software, Inc., San Diego, CA). For all tests, significance was set at p < 0.05.

	Results

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Experiment 1. SNC80 produced dose-dependent increases in locomotor activity (Fig. 1a) [pretreatment: F(4,1131) = 147.1, p < 0.0001; interaction: F(152,1131) = 2.9, p < 0.0001]. In this experimental design, vehicle injections stimulated some activity from the handling and injecting procedures, which returned to baseline levels within 30 to 40 min. The low dose of 1.0 mg/kg SNC80 stimulated small, but significant (p < 0.0001), increases in activity above vehicle levels, and the higher doses of 3.2 to 32 mg/kg SNC80 produced robust and significant increases in locomotor activity (all p < 0.0001) lasting approximately 2 h. All doses were significantly different from each other except for 10 and 32 mg/kg SNC80 as determined by Tukey's post hoc test.

Amphetamine also produced dose-dependent increases in locomotor activity (Fig. 1b) [pretreatment: F(3,456) = 22.9, p < 0.0001; interaction: F(114,456) = 3.3, p < 0.0001]. The low dose of amphetamine (0.32 mg/kg amphetamine) produced small, but nonsignificant, increases in activity as compared with vehicle (p = 0.7). High doses of amphetamine stimulated locomotor activity producing large increases in activity lasting 2.5 to 3 h (Fig. 1b). The high doses of amphetamine (1 and 3.2 mg/kg) were not different from each other but produced significant increases in activity as compared with vehicle (p = 0.002 and 0.0002, respectively). SNC80 (10 mg/kg) and 1.0 mg/kg amphetamine produced similar activity responses as demonstrated in Fig. 1; therefore, these doses were used for further experiments in this study. The locomotor-stimulating effects of SNC80 were attenuated by the selective -opioid antagonist NTI in a dose-dependent manner (Fig. 1c), but the highest dose of NTI (10 mg/kg) did not alter the locomotor-stimulating effects of amphetamine (Fig. 1d).

Experiment 2. As previously demonstrated (Jutkiewicz et al., 2005b), repeated SNC80 injections produced tolerance to the locomotor-stimulating effects [statistical analyses for second injection, 180–350 min only; pretreatment: F(2,510) = 370.5, p < 0.0001; interaction: F(66,510) = 7.4, p < 0.0001]. An initial injection of SNC80 (10 mg/kg at time 0) produced a large increase in locomotor activity counts that returned to baseline levels in approximately 2 to 2.5 h (Figs. 1a and 2a). However, the second injection of SNC80 administered 3 h later stimulated a limited amount of locomotor activity relative to the first injection of SNC80. Locomotor activity produced by the second injection of SNC80 was larger than that produced by a vehicle injection alone administered at 180 min (p < 0.0001) and significantly less activity than rats receiving SNC80 for the first time at the 180-min time point (p < 0.0001) (Fig. 2a). These data demonstrated that acute tolerance developed to the stimulant activity of SNC80; however, at this time, rats were also sensitized to the stimulant effects of amphetamine. For example, a 3-h SNC80 pretreatment greatly enhanced the stimulatory effects of amphetamine challenge (1.0 mg/kg) [interaction: F(33,340) = 2.34, p < 0.0001], producing a greater than 2-fold increase in the total locomotor activity counts stimulated by amphetamine challenge (Fig. 2c). Post-hoc tests revealed a significant enhancement of amphetamine-stimulated locomotor activity in SNC80-pretreated rats at 198 to 293 min (p < 0.05). In comparison, a 3-h amphetamine pretreatment (1.0 mg/kg) produced a small, but significant, increase in activity stimulated by a 1.0 mg/kg amphetamine challenge [interaction: F(33,340) = 1.90, p = 0.003; post-hoc tests, p < 0.05 at 198 and 208 min only] (Fig. 2b). In addition, 3-h amphetamine pretreatment (1.0 mg/kg) did not alter the locomotor activity stimulated by 10 mg/kg SNC80 challenge [interaction: F(33,340) = 1.38, p = 0.09] (Fig. 2d).

View larger version (25K): [in this window] [in a new window]   Fig. 2. The effects of repeated injections of SNC80 or amphetamine on locomotor activity in rats. SNC80 pretreatment enhanced amphetamine-induced locomotor activity at a time when rats were tolerant to further SNC80 stimulation. Rats were administered one injection at time 0 (pretreatment) and a second injection at 180 min (challenge) as indicated by a break in the x-axis. a, effects of 3-h vehicle or SNC80 pretreatment (10 mg/kg) on 10 mg/kg SNC80 challenge. b, effects of 3-h vehicle or amphetamine pretreatment (1.0 mg/kg) on activity stimulated by 1.0 mg/kg amphetamine challenge. c, effects of 3-h SNC80 (10 mg/kg) pretreatment on amphetamine (1.0 mg/kg)-stimulated locomotor activity. d, effects of 3-h amphetamine (1.0 mg/kg) pretreatment on SNC80 (10 mg/kg)-stimulated locomotor activity.

View larger version (37K): [in this window] [in a new window]   Fig. 3. SNC80 pretreatments dose-dependently increased amphetamine-stimulated locomotor activity. To simplify visually these data, SNC80 pretreatment doses are displayed on separate graphs. Locomotor activity was recorded throughout the pretreatment and amphetamine challenge. a, vehicle or 1 or 3.2 mg/kg SNC80 was administered 180 min prior to 1.0 mg/kg amphetamine challenge. b, effect of vehicle or 10 or 32 mg/kg SNC80 pretreatment on amphetamine-stimulated locomotor activity. The data from 10 mg/kg SNC80 pretreatment are repeated from Fig. 2 for reference.

Experiment 3. The time course of SNC80-stimulated enhancement of amphetamine-induced locomotor activity was evaluated (Fig. 4). A 1-h pretreatment with SNC80 produced a small increase in amphetamine-stimulated locomotor activity (Fig. 4a). With these data, there was no significant interaction because the lines are parallel [F(41,420) = 1.32, p = 0.1], but there was a significant main effect for pretreatment [F(1,420) = 11.17, p < 0.0001], demonstrating that SNC80 pretreatment elevated activity levels stimulated by amphetamine challenge. SNC80 administered as a 4-h pretreatment to amphetamine challenge significantly increased amphetamine-stimulated activity [interaction: F(33,340) = 1.68, p = 0.013] (Fig. 4b) but to a lesser extent than observed with a 3-h SNC80 pretreatment (see Figs. 2c and 3a for comparison). Four-hour pretreatment with SNC80 significantly enhanced amphetamine-stimulated activity from 253 to 268 min (p < 0.05). Following a 6-h pretreatment, SNC80 pretreatment did not significantly increase amphetamine-stimulated activity, but there was a trend for SNC80-induced enhancement of amphetamine [interaction: F(61,620) = 1.30, p = 0.07] (Fig. 4c). The SNC80-induced enhancement of amphetamine dissipated following a 24-h SNC80 pretreatment (Fig. 4d), as demonstrated by no statistical interaction [F(40,369) = 0.74, p = 0.88] and no pretreatment effect [F(1,369) = 0.23, p = 0.63].

View larger version (29K): [in this window] [in a new window]   Fig. 4. The effects of different SNC80 pretreatment times on amphetamine-stimulated locomotor activity. SNC80 enhanced amphetamine-induced locomotor activity up to 4 h after its administration, and the effect dissipated after a 6-h pretreatment. Vehicle or 10 mg/kg SNC80 was administered 1 (a), 4 (b), 6 (c), or 24 (d) h prior to amphetamine challenge (1.0 mg/kg). Activity counts following the amphetamine challenge are displayed on the graphs.

View larger version (30K): [in this window] [in a new window]   Fig. 5. The effects of the -opioid agonists (a) (+)BW373U86 (BW) and (b) OMI on locomotor activity stimulated by 1.0 mg/kg amphetamine challenge. Only the high-efficacy agonist BW greatly enhanced the locomotor-stimulating effects of amphetamine, in a manner similar to SNC80 pretreatment. Vehicle or increasing doses of (+)BW373U86 or OMI were administered 180 min prior to the amphetamine challenge (1.0 mg/kg). The graphs display activity counts recorded during the pretreatment and amphetamine challenge.

On the other hand, the low efficacy -opioid agonist OMI did not increase the magnitude of the amphetamine effect in terms of total activity counts but altered the time course of the stimulatory effects of amphetamine [interaction: F(106,810) = 3.53, p < 0.0001] (Fig. 5b). For example, the high dose of OMI pretreatment lengthened the duration of amphetamine-induced locomotor activity but also suppressed the initial increase in activity following the amphetamine challenge. As compared with vehicle pretreatment in post hoc tests (p < 0.05), 10 mg/kg OMI pretreatment increased amphetamine-stimulated activity from 313 to 403 min, and 32 mg/kg OMI pretreatment decreased amphetamine-stimulated activity from 203 to 233 min and increased activity from 308 to 438 min.

Experiment 5. To determine whether the SNC80-induced enhancement of amphetamine-stimulated locomotor activity was -opioid receptor-mediated, NTI (1.0 mg/kg) was administered 30 min after SNC80 pretreatment (Fig. 6a) or 30 min prior to amphetamine challenge (Fig. 6b). NTI administered shortly after SNC80 pretreatment attenuated the locomotor-stimulating effects of SNC80 (data not shown) and also blocked the enhanced amphetamine effect in SNC80-pretreated rats (Fig. 6a). Similarly, NTI administered 30 min prior to amphetamine challenge partially blocked the SNC80-induced enhancement of amphetamine-stimulated locomotor activity (Fig. 6b), demonstrating this effect was also mediated through the -opioid receptor.

View larger version (35K): [in this window] [in a new window]   Fig. 6. The effects of the selective -opioid antagonist NTI on SNC80 pretreatment-induced enhancement of amphetamine-stimulated locomotor activity. NTI attenuated the SNC80-induced enhancement of amphetamine, suggesting that the -opioid receptor mediated this effect. The graphs display activity counts recorded during the amphetamine challenge. a, vehicle or 1.0 mg/kg NTI was administered 30 min after the 10 mg/kg SNC80 pretreatment, and the 1.0 mg/kg amphetamine challenge was administered 180 min after the SNC80 pretreatment. b, vehicle or 1.0 mg/kg NTI was administered 30 min before the 1.0 mg/kg amphetamine challenge, which was given 180 min after SNC80 pretreatment (10 mg/kg).

View larger version (20K): [in this window] [in a new window]   Fig. 7. The effects of simultaneous administration of SNC80 and amphetamine on activity levels. Simultaneous administration of low doses of SNC80 with amphetamine mimicked the enhanced amphetamine responses induced by SNC80 pretreatment. SNC80 (either 0.1 or 1.0 mg/kg) was administered simultaneously with 1.0 mg/kg amphetamine and compared with the effects of 1.0 mg/kg amphetamine alone or 1.0 mg/kg SNC80 alone. For comparison, amphetamine challenge (1.0 mg/kg) following 10 mg/kg SNC80 pretreatment is shown.

View larger version (26K): [in this window] [in a new window]   Fig. 8. The effects of SNC80 pretreatments on locomotor stimulation produced by various compounds. SNC80 pretreatment also enhanced the locomotor-stimulating effects of cocaine and nomifensine, but not direct dopamine agonists SKF81297 and quinpirole. Rats were administered one injection at time 0 (pretreatment) and a second injection at 180 min (challenge) as indicated by a break in the x-axis. Vehicle or 10 mg/kg SNC80 were administered as 180-min pretreatments to cocaine (a), nomifensine (b), the direct dopamine D1 agonist SKF81297 (c), or the direct dopamine D2/D3 agonist quinpirole (d). The graphs display activity counts recorded during the pretreatment and amphetamine challenge.

	Discussion

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Pretreatment with the -opioid agonist SNC80 also increased the locomotor-stimulating effects of other monoamine transporter ligands, such as the reuptake blockers cocaine and nomifensine. SNC80 did not significantly enhance the locomotor-stimulating effects of the direct dopamine D1 agonist SKF18297 and the dopamine D2/D3 agonist quinpirole. In summary, SNC80 was able to enhance the locomotor effects of ligands that act at monoamine transporters, such as amphetamine, cocaine, and nomifensine, but did not increase the effects of direct dopamine receptor agonists.

Similarly, one previous study demonstrated that simultaneous administration of the peptidic -opioid agonist DPDPE with cocaine or the selective dopamine reuptake inhibitor GBR12909, but not the direct dopamine agonist apomorphine, potentiated locomotor activity (Waddell and Holtzman, 1998). However, these effects of DPDPE were not specific for the -opioid receptor because selective µ-and -opioid antagonists blocked the additive effects of DPDPE with cocaine. Since DPDPE and GBR12909 were only administered simultaneously, it is not known if these effects are simply additive or if they are longer lasting, as observed in the present study.

One interesting aspect of the current study is that the SNC80-induced enhancement of amphetamine-stimulated locomotor activity occurred when the stimulant effects of SNC80 had dissipated and when rats were tolerant to further -opioid receptor activation. The initial locomotor-stimulating effects of SNC80 dissipated after approximately 3 h, which may be due to the elimination of compound from circulation or to acute tolerance development. Although one previous report with a radiolabeled SNC80 analog ([³H]SNC121) demonstrated that drug levels in the brain were negligible 60 min after an i.p. injection (Schetz et al., 1996), another study suggested that SNC80 levels remained elevated for at least 3 h following an s.c. injection (Jutkiewicz et al., 2005a). Three hours after the initial SNC80 administration, rats were tolerant to further -opioid agonist stimulation, suggesting that tolerance developed during the first SNC80 exposure. These data would suggest that -opioid receptors were rapidly desensitized and/or down-regulated during SNC80 exposure. Although -opioid receptors contributing to locomotor activity were tolerant to further -opioid agonist stimulation, the effects of amphetamine were enhanced through a -opioid receptor-mediated mechanism. Naltrindole had no effect on amphetamine-induced stimulation alone (as shown in Fig. 1); however, it almost completely eliminated the SNC80-induced enhancement of amphetamine. In conclusion, these data suggested that some SNC80 remained in the circulation without stimulating locomotor activity alone but enhanced the effects of amphetamine through a -opioid receptor-mediated mechanism. In confirmation of this, simultaneous administration of a low dose of SNC80 (1.0 mg/kg) with amphetamine mimicked the enhancement of amphetamine produced by 3-h pretreatment with 10 mg/kg SNC80. These data suggest that low, inactive levels of circulating SNC80 bind to the -opioid receptor and alter the effects of monoaminergic transporter ligands; however, the mechanism through which this occurs is unknown.

One possible mechanism is that -opioid receptor activation transiently enhances activity of monoaminergic neurons alone, and the amphetamine challenge further increases these elevated monoamine levels. SNC80 might produce prolonged elevations in monoamine levels without persistent increases in locomotor activity, considering SNC80 pretreatment has no observable effects at the time of amphetamine challenge (3 h after injection). In support of this, the majority of -opioid receptors in the striatum are located on terminals or axons of GABAergic and cholinergic neurons apposed to dopaminergic neurons in frontal brain regions (Svingos et al., 1999). Inhibition of GABA neurons could relieve the constant inhibitory influence on monoaminergic neurons, thus increasing monoaminergic activity overall. These behaviorally inactive increases in dopamine/norepinephrine activity could potentiate the effects of amphetamine to promote exaggerated locomotor responses. However, one previous study demonstrated that peripheral administration of SNC80 did not increase dopamine release in the nucleus accumbens or the caudate putamen as measured by in vivo microdialysis (Longoni et al., 1998), suggesting that -opioid receptor activation does not enhance monoaminergic activity.

Another possible mechanism is that -opioid receptors on monoamine terminals could modulate directly transporter activity. Previous studies reported that -opioid receptors were actually located on dopamine terminals, as suggested by decreased enkephalin and [³H]naloxone binding in the striatum following 6-hydroxydopamine lesions in the substantia nigra (Pollard et al., 1977). This was supported by more recent observations demonstrating that small populations of -opioid receptors were colocalized with dopamine transporters on dopaminergic terminals (Svingos et al., 1999). Furthermore, it was shown that -opioid peptides enhanced dopaminergic activity in rat striatal slices in the presence of tetrodotoxin and in microdialysis experiments following intrastriatal kainic acid lesions to destroy interneurons, suggesting that -opioid receptors localized on dopamine terminals alter the dopaminergic system (Lubetzki et al., 1982; Dourmap et al., 1992). Consequently, this might imply that -opioid receptors directly on dopamine/norepinephrine terminals could alter the activity of the transporters in the presence of amphetamine.

In conclusion, these findings demonstrate that -opioid agonists have some stimulant-like behaviors, although these behaviors may not necessarily be correlated with an increase in dopamine release. A single 3-h pretreatment with SNC80 produced tolerance to further -opioid receptor activation but also greatly enhanced the locomotor-stimulating effects of amphetamine. The -opioid agonist-induced enhancement of amphetamine-stimulated activity occurred when SNC80 was administered simultaneously and as a pretreatment. This enhancement of amphetamine was attenuated by naltrindole demonstrating -opioid receptor mediation. SNC80 also enhanced the effects of other dopamine/monoamine transporter ligands such as cocaine and nomifensine. The present study expanded on previous literature to demonstrate that the nonpeptidic -opioid agonists, SNC80 and (+)BW373U86, enhance the locomotor effects of cocaine and other transporter ligands but also that this potentiation is prolonged and outlasts the actions of the -opioid agonist itself. In future studies, different classes of -opioid agonists should be investigated to determine whether agonists other than benzhydrylpiperazines can enhance the activity of monoamine transporter ligands. Overall, these data suggest that -opioid agonists might alter the actions of monoamine transporters or the activity of presynaptic terminals containing these transporters. At this point, it is unknown whether the -opioid agonists are indirectly or directly altering the action of monoamine transporter ligands. Future in vitro studies will evaluate the neurobiological and cellular mechanisms involved in the SNC80-induced potentiation of transporter activity.

	Footnotes

 
This study was supported by U.S. Public Health Service Grants DA00254, DA020669, T32 GM07767, T32 DA07267, and T32 DA07268. This study was also supported in part by the Intramural Research Program of the National Institutes of Health, by the National Institute of Diabetes and Digestive and Kidney Diseases, and by the National Institute on Drug Abuse.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.123844.

ABBREVIATIONS: DPDPE, [D-Pen²,D-Pen⁵]-enkephalin; SNC80, (+)-4-[(R)--[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-(methoxybenzyl)-N,N-diethylbenzamide; (+)BW373U86, (+)-4-[(R)--[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide; nomifensine, 1,2,3,4-tetrahydro-2-methyl-4-phenyl-8-isoquinolinanmine maleate salt; R(+)-SKF81297, R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide; quinpirole, trans-(-)-(4R)-4,4a, 5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g] quinoline monohydrochloride; NTI, naltrindole, 17-cyclopropylmethyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2',3'-indolomorphinan; oxymorphindole, 17-methyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2',3'-indolomorphinan; GBR12909, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine; TAN-67, (±)2-methyl-4-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a-octahydroquinolino[2,3,3-g]isoquinoline; OMI, oxymorphindole.

Address correspondence to: Dr. Emily M. Jutkiewicz, Department of Pharmacology, University of Michigan Medical School, 1301 Medical Science Research Building III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0632. E-mail: ejutkiew{at}umich.edu

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