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Toluene inhalation produces regionally specific changes in extracellular dopamine
Drug and Alcohol Dependence 65 (2002) 243– 251 www.elsevier.com/locate/drugalcdep
Toluene inhalation produces regionally specific changes in extracellular dopamine Madina R. Gerasimov *, Wynne K. Schiffer, Douglas Marstellar, Richard Ferrieri, David Alexoff, Stephen L. Dewey Chemistry Department, Brookha6en National Laboratory, Upton, NY 11973, USA Received 20 March 2001; received in revised form 9 May 2001; accepted 11 May 2001
Abstract The aim of the present study was to investigate the effect of toluene inhalation on dopaminergic transmission in two distinct brain areas presumably involved in mediating the reward processes important for toluene abuse. Extracellular dopamine (DA) levels were measured in prefrontal cortex (PFC) and nucleus accumbens (NACC) of freely moving rats using in vivo microdialysis. Inhalation of a behaviorally relevant concentration of toluene (3000 ppm) produced a significant increase in the PFC but not in the NACC. However, the odorant isoamyl acetate, increased PFC DA levels by only 37%, significantly less than the 96% increase observed following toluene exposure. When toluene inhalation was combined with cocaine administration (20 mg/kg i.p.), the response to the combined challenge was not different from the response to toluene alone in the PFC. However, the combination of these two drugs produced a supradditive response of 802% in the NACC, compared with the 450% increase observed following cocaine alone. Recent reports indicate that toluene influences the function of several ionotropic receptors in a subunit specific manner. As further evidence of specific effects, our results indicate regionally specific changes in dopaminergic transmission following toluene exposure. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Toluene; Dopamine; Microdialysis; Nucleus accumbens; Prefrontal cortex
1. Introduction Solvent abuse, mainly among adolescents, has been reported to occur worldwide (Flanagan and Ives 1994; Kozel, 1995). In the US, for example, inhaled agents rank fourth behind alcohol, marijuana and tobacco in their incidence of abuse, while in Japan, organic solvents and amphetamine are two of the most commonly abused substances. Toluene is the principal intoxicant in many of the products involved in ‘sniffing’. Although inhalant abuse is now recognized as a worldwide problem, organic solvents are currently the least studied drugs of abuse. For example, relatively little is known about the underlying cellular mechanisms of action through which these substances produce their effects in the central nervous system (CNS). * Corresponding author. Tel.: + 1-631-3444395; fax: +1-6313447902. E-mail address: [email protected] (M.R. Gerasimov).
A review of the current literature clearly indicates the paucity of in vivo research designed to specifically address the acute affects of toluene on neurotransmitter concentration, release, or the response to pharmacological challenge (Balster, 1998). Exposure to toluene may produce behaviors consistent with many other drugs of abuse. Toluene, like benzodiazepines, alcohol and phencyclidine (PCP), has a demonstrated abuse liability in humans (Howard et al., 2001). Furthermore, its rewarding/reinforcing effects have been demonstrated in animal studies where it increased the response rate of intracranial self-stimulation (ICSS), induced conditioned place preference (CPP) (Yavich et al., 1994), and maintained self-administration in squirrel monkeys (Weiss et al., 1979). In view of the well established role of dopamine (DA) in underlying the reinforcing effects of many abused drugs, earlier ex vivo studies assessed changes in brain DA content and turnover following acute and chronic toluene exposure (von Euler, 1994). To our knowledge,
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however, only two studies assessed brain DA levels in freely moving animals during toluene inhalation. These studies measured DA levels in the corpus striatum and provided contradictory results. Stengard et al., 1994 found that exposure to toluene increased striatal DA 47% above baseline while Kondo et al., 1995 failed to observe any significant change. Due to its high lipid solubility, toluene has long been considered to act in a ‘non-specific’ manner, through the disordering of cell membrane lipids. In fact, behavioral evidence indicates that toluene shares many properties with other small lipophilic agents like inhaled anesthetics and ethanol (Rees et al., 1987a; Bowen et al., 1996). In addition, there exists behavioral similarities between toluene and classical non-volatile CNS depressants like benzodiazepines and barbiturates (Evans and Balster, 1991), compounds that produce their effects directly through the gamma-aminobutyric acid (GABA) receptor complex. For example, toluene, ethanol, midazolam and pentobarbital effectively decreased withdrawal symptoms produced by trichloroethane inhalation (Evans and Balster, 1993). Furthermore, mice trained to discriminate toluene from vehicle generalized to pentobarbital, but not morphine (Rees et al., 1987b). Toluene resembles benzodiazepines in its ability to produce anticonvulsant, antipunishment and antianxiety effects (Bowen et al., 1996; Wood et al., 1984). Finally, recent work by Bowen et al., 1999 demonstrated that toluene produced concentration-related partial substitution for phencyclidine (PCP) in a drug discrimination assay. Taken together, these findings suggest some specificity in the relevance of both NMDA receptor antagonism and GABA receptor stimulation in the mechanism of action of toluene. It appears that several neurotransmitter systems may mediate the complex symptomatology associated with toluene abuse. While nucleus accumbens (NACC) DA activity is considered to be the principal element in generating reward-related behaviors (Di Chiara, 1995; Gardner, 1997), recent studies utilizing ICSS, CPP and self-administration paradigms suggest that the prefrontal cortex (PFC) is also involved in the generation of reward (Tzschentke, 2000). Therefore, the present study was designed to test whether acute toluene inhalation altered extracellular DA levels in the NACC and PFC in freely moving animals. Furthermore, since earlier work (Gerasimov and Dewey, 1999; Gerasimov et al., 2000; Hemby et al., 1999) has demonstrated that addictive drugs often produce synergistic effects when co-administered, we also investigated possible interactions between cocaine and toluene. These data may provide insight into the apparent association between drug inhalation and other drugs of abuse where inhalant users were five times more likely than non-users to abuse illicit drugs (Schu¨ tz et al., 1994). Finally, in order to rule out possible effects of stress on extracellular DA
levels caused by a non-specific environmental stimuli (e.g. a pronounced odor) or a possible change in air composition, we exposed animals to the odorant isoamyl acetate under identical conditions to tolueneexposed animals.
2. Materials and methods
2.1. Animals and surgery All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local animal care committee. Experimentally naı¨ve, male Sprague– Dawley rats were used in all experiments (200–300 g, Taconic Farms, Germantown, NY) and were given food and water ad libitum. Each animal was housed individually on a 12-h light:12-h dark cycle. Animals were anesthetized with ketamine/xylazine mixture and placed in a stereotaxic frame. Siliconized guide cannulae (BAS, West Lafayette, INF) were implanted in the brain above the PFC (anterior-posterior to bregma 3.7, medial-lateral to bregma 1.0, dorsal-ventral to bregma 1.0) or NACC (anterior-posterior 1.5, medial-lateral 1.0, dorsal-ventral 5.6) 2 days before the microdialysis experiment. These coordinates were calculated according to the Paxinos and Watson atlas for the rat brain (Second edition, 1986). Dental acrylic and machine screws held the cannulae in place throughout the study. On the day of the experiment, animals were removed from their home cage and placed in the dialysis chamber. Concentric flow dialysis probes (4 mm for PFC, 2 mm for NACC) were lowered through the guide cannulae in awake animals, terminating in the structures of interest. The dialysis probes were perfused with Ringer’s solution (McGaw, Inc. Irvine, CA) at a flow rate of 2.0 ml/min. Dialysate samples were collected for 20 min and analyzed on-line. After all experiments, animals were euthanized with an overdose of chloral hydrate and brains dissected to verify probe placement.
2.2. Analytical procedure Dialysate samples were assayed for DA content by high-pressure liquid chromatography (HPLC) coupled with electrochemical detection. This HPLC system consists of a BAS reverse-phase microbore column (150×1 mm, 5 mm microbore column with C18 packing; BAS) directly attached to a conventional flow electrochemical detector equipped with a glassy carbon working electrode maintained at + 650 mV relative to a Ag/AgCl reference electrode (LC-4C, BAS). The microbore column was kept at a constant temperature of 27.6 °C. The range was set to 0.2 nA, and the electrical noise and output filter at 0.02 Hz. The mobile phase con-
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sisted of 8.0% acetonitrile, 1.0% tetrahydrofuran, 14.5 mM sodium phosphate monobasic, 30 mM sodium citrate, 27 mM disodium-EDTA, 10 mM diethylamine HCl, 1.95 mM decanesulfonic acid, sodium salt, and pH 4.0. An online degasser was used to ensure that the mobile phase was free of air. The net flow rate calibrated from the microbore column was 0.10 ml min − 1, obtained by using a flow splitting technique. Data was collected on-line using chronograph® software (BAS) as well as with a dual-pen strip chart recorder that records chemical activity, detection voltage on the chart recorder was set at 1 V.
2.3. Exposure to toluene and isoamyl acetate. Animals were exposed to toluene and isoamyl acetate for 40 min in a microdialysis chamber modified for this particular experiment (Fig. 1). The use of a rotating bowl with an optical monitoring device (Raturn®, BAS) eliminates the need for a liquid swivel, thus dramatically improving the recovery of DA in the dialysate. Rotation is prompted by rat’s movement, thus preventing the connecting lines from damage due to entanglement. An opening in the lid of the chamber allowed for free movement of the arm with the tether hooked to the animal collar, together with the tubing directly connecting the microdialysis probe with the on-line injector.
Fig. 1. Diagram demonstrating an animal undergoing simultaneous dialysis and toluene exposure. (A). Syringe pumping CSF at 2.0 ml/min, (B). On-line injector and LC analysis system, (C). Glass vestibule with sand and heated toluene, (D). Ceramic plate with holes allowed the heated vapor to rise into the microdialysis chamber
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After baseline measurements of dopamine were obtained (defined by at least four consecutive DA samples differing by less than 10%) the ceramic plate was lifted without disturbing the animal and a glass sand bath filled with toluene or isoamyl acetate (100 ml, Sigma– Aldrich, Milwaukee, WI) heated to boiling point was placed underneath the platform. The heating of the toluene-filled sand bath was performed in a ventilated hood using Thermolyne® heating plate. A grated floor allowed the heated vapor to rise into the microdialysis chamber. Air samples drawn every 10 min from the bowl at the level of the animal head indicated that initial levels of the vapors (immediately following introduction of the source into the bowl) were significantly lower (300–350 ppm) than stable concentrations of toluene and isoamyl acetate levels that were reached at 10 min. These vapor levels maintained at 3000950 ppm and 2500950 ppm, respectively, throughout the exposure period, and declined thereafter. Thus, the reported numbers actually represent the highest vapor levels that were achieved by this method. Air sampling for measurement of airborne toluene levels in the exposure chamber was conducted using a 10 ml glass, gas-tight syringe (Hamilton Valve Co.) equipped with a shut-off valve. The syringe was coupled to a 25× 0.16 cm Teflon tube. These syringes have Teflon barrels and stainless heads with a locking Teflon insert. Any other plastic material in the syringe could cause problems through solvent adsorption which will confuse a trace level analysis. The air sample withdrawn from the bowl was immediately locked inside the syringe to allow time to remove the Teflon sampling tube without complications of loss of sample from the syringe. The sample was then slowly dispelled through 0.5 ml of acetonitrile that was in a small Teflon sealed vial (crimp seal type). Head space volume was minimized by using these vials. The vial had to be vented while the 10 cm3 of air sample was dispelled through the solvent. We ran the outlet gas flow through a second volume of solvent to ensure that no toluene was passing through the first solvent trap and out. We used acetonitrile as a solvent because it does not carry a trace load of toluene. Toluene losses from the solvent were not a problem once it was entrained in the solvent. This was verified by standardizing the flame ionization detector response to amounts of toluene, by measuring standard stability over time (a few h). There was no noticeable toluene loss from the standards as long as the vials remained sealed. Actual samples were analyzed within 15 min of drawing the sample. Gas chromatographic analysis was carried out using a Hewlett–Packard 5890A capillary gas chromatograph equipped with a flame ionization detector whose output was interfaced with a Vision 4 chromatography acquisition system (Scientific Systems, State College, PA). One microliter
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samples was injected and split 1:100 onto a 60 m× 0.025 mm SE-30 column (J and W Capillary) initially maintained at 50 °C, and then temperature programmed to 100 °C at a 2 °C/min rate. The injector temperature was maintained at 225 °C and the flame ionization detector maintained at 350 °C throughout the analysis. Standard samples of toluene were prepared in two ways to effect an accurate range of amounts of substance from below 5 to 50 000 ppm. Standards were prepared for the detector response measurements from the below 5 to 5000 ppm range by pipetting 1– 10 ml volumes of toluene using calibrated analytical pipettes into 1–10 ml volumetric flasks containing HPLC grade acetonitrile solvent. Standards were made up in triplicate, as well as detector response measurements carried out in triplicate. Standards for higher concentrations of toluene ( \5000 ppm) were made up in HPLC grade hexane solvent using the same procedures described. In these instances, the slight impurity level of toluene in the hexane solvent was insignificant relative to the sample injected. Standards were typically kept refrigerated and maintained their integrity for several days without a loss of sample. A rigorous calibration was performed on detector response at the onset of the study and then spot checked daily within the critical range to ensure that response was within 2% of the expected valve. In the control (air exposure) studies, animals remained in the modified chambers with the lid in place. In the studies designed to examine a potential synergistic effect between toluene and cocaine, cocaine (20 mg/kg i.p.) was administered immediately prior to toluene exposure.
2.4. Statistics Baseline OA level was calculated for each animal separately as the average of three pre-drug samples not differing from each other by more than 10%. This average number was subsequently used for each animal to calculate individual drug response. Peak increases in extracellular DA, expressed as a percent change from these baseline values, were compared for every group with a one-way analysis of variance (ANOVA) followed by a post hoc Student’s Newman – Keuls test. Significance levels were set at P B0.05.
3. Results
3.1. Effect of 6apor exposure alone and in combination with cocaine on extracellular DA le6els in the PFC. Extracellular DA levels increased immediately following the onset of toluene inhalation and reached a
Fig. 2. Effect of toluene (3000 ppm) and isoamyl acetate (2500 ppm) vapors on extracellular DA levels in the prefrontal cortex of rats. Values are mean 9S.E.M. and expressed as percent of basal levels (n = 5 – 9 per group). Exposure started at time 0 and ended at time 40 min. *, PB 0.05 different from the peak effect in animals exposed to isoamyl acetate (ANOVA and post-hoc Student’s Newman–Keuls test).
maximum of 96% above baseline by the end of the 40 min exposure (n=9) (Fig. 2). DA levels returned to baseline 100 min after the source of toluene vapor was removed. This peak increase was significantly different (P B0.05) from the increase following isoamyl acetate exposure (37% above baseline, n= 6). The peak increase of 71% following combined cocaine and toluene administration (n= 9) was not significantly different from elevations observed in animals exposed to toluene alone (96% above baseline). In addition, the temporal parameters of this peak increase and subsequent return to baseline were not different between these two groups either. This increase, however, was significantly different from the cocaine-induced increase of 165% above baseline (n= 6) (P=0.01) (Fig. 3).
3.2. Effect of 6apor exposure alone and in combination with cocaine on extracellular DA le6els in the NACC. There was no significant difference between the DAergic response to air (n= 4), isoamyl acetate (n=4) or toluene (n=9) alone (data not shown). Conversely, cocaine administration produced an increase of 450% above baseline (n= 9). When combined with toluene, however, extracellular DA increased by 802% (n =9). This increase was significantly different from the response to cocaine or toluene alone (PB0.001). In both groups DA peaked 40 min post exposure and returned to baseline in 90 min (Fig. 4).
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4. Discussion Toluene increased extracellular PFC DA to a greater extent than did isoamyl acetate, while neither had any effect on extracellular NACC DA. In addition, inhalation of toluene immediately following cocaine administration, had no effect on increases in PFC DA but significantly potentiated the response observed in the NACC. The majority of studies reported in the literature were designed to address the effects of toluene on DA neurons by utilizing biochemical assays mainly reflective of intracellular concentrations (Fuxe et al., 1982; von Euler et al., 1988; von Euler, 1994). While these levels may be altered by both synthesis and exocytotic release of DA, they may not indicate whether DAergic transmission is increased or decreased. Additionally, these studies focused primarily on persistent and shortterm effects of chronic toluene exposure and are thus of limited relevance to understanding the potential acute effects. Mesocorticolimbic DA activity is regulated by both GABAergic and glutamatergic input. Beckstead et al., 2000 demonstrated that toluene significantly enhanced GABAA receptor function in a dose-dependent and reversible manner. Of particular significance, however, is their observation that the application of the highest concentration of toluene did not influence GABAA receptor activity in the absence of GABA. Toluene might also disrupt excitatory amino acid input by in-
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hibiting NMDA-mediated currents in vitro (Cruz et al., 1998) and in vivo (Cruz et al., 1999). Taken together, these data suggest that neurochemically specific as well as non-specific mechanisms of action need to be considered. The moderately high concentrations of toluene used in this study (3000 ppm) are self-administered (Weiss et al., 1979), produce discriminative stimulus effects (Rees et al., 1987a) and have a distinct antipunishment effect (Wood et al., 1984). In addition, this concentration exerts anxyolitic-like action on burying behavior and in elevated plus-maze test performance (Lopez-Rubaclava et al., 2000). In the present study, toluene exposure increased extracellular DA in the PFC (Fig. 1) while having no effect in the NACC (Fig. 3). To our knowledge, this is the first direct in vivo evidence that toluene inhalation alters extracellular DA in this cortical region. Furthermore, the unique finding of specific regional effects supports the hypothesis that, notwithstanding toluene’s reported distribution according to lipid content (Gospe and Calaban, 1988), some of its effects may be due to selective interactions with neurotransmitter receptor proteins. That is, even though the lowest brain/blood toluene concentration ratio was found in cerebral cortex (Ameno et al., 1992; Kiriu et al., 1990), we observed significant changes in extracellular DA levels following toluene inhalation in this brain region. DA cell bodies in the VTA receive reciprocal excitatory amino acid input from PFC, and in turn project
Fig. 3. Effect of combined toluene and cocaine administration on extracellular DA levels in the prefrontal cortex of rats. Values are means9S.E.M. expressed as percent of basal levels (n= 6–9/group). Exposure started at time 0 and ended at time 40 min. **, PB 0.01 different from the peak effect in animals exposed to either toluene alone or to combination
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Fig. 4. Effect of combined toluene and cocaine administration on extracellular DA levels in the nucleus accumbens of rats. Values are means of9 S.E.M. expressed as percent of basal levels (n= 9 per group). Exposure started at time 0 and ended at time 40 min. ***, P= 0.001 different from the peak effect in animals exposed to either toluene or cocaine alone.
back to the NACC. Recent evidence indicates that PFC afferents synapse on either mesocortical DA neurons or on mesolimbic GABA neurons (Carr and Sesack, 2000). Therefore, it is conceivable that the regionally specific changes observed in the present study represent indirect effects of toluene on these subpopulations of VTA cell bodies. In fact, PFC DA increased immediately following toluene inhalation, suggesting that transient activation of GABAA receptors and/or non-competitive inhibition of NMDA receptors might have indirectly contributed to this marked increase. It should be noted, however, that isoamyl acetate increased PFC DA levels as well, but to a much lesser degree. This difference addresses the argument that increases observed following toluene exposure are directly related to a non-specific olfactory mechanism or to a change in the molecular composition of breathed air. Beckstead et al., 2000 demonstrated that toluene, at concentrations of 0.2– 0.9 mM, or 20 – 90 mg/ml (which approximately corresponds to 30 mg/g in rats exposed to 1000–2000 ppm of toluene (Kishi et al., 1988), significantly increased ligand-gated currents in GABAA and glycine receptors. Accordingly, human plasma toluene concentrations in abusers have been reported to be in the 0.01–0.1 mM range (King, 1982). Interestingly, toluene IC50 values for inhibiting NMDA-induced currents is in the same range of 0.2–2 mM (Cruz et al., 1998). This is also close to the effective concentrations
of other non-competitive NMDA receptor antagonists that preferentially stimulate PFC DA (Hata et al., 1990; Nishijima et al., 1994) via GABA-mediated disinhibition. The notion that toluene produces neurobehavioral effects similar to those of non-competitive NMDA antagonists is further supported by evidence from Bowen et al., 1999 who demonstrated that toluene, at a concentration of 1000–4000 ppm, partially substituted for a pharmacologically relevant dose of PCP (2 mg/ kg). The clinical significance of the present findings is further emphasized by the work of (von Euler et al., 1993, 2000) who proposed that subchronic toluene exposure produced persistent impairment in initiating novel behaviors or integrating sensory information. Structural changes observed in the cerebral cortex served as the foundation for their proposal that a dysfunction of glutamatergic corticostriatal pyramidal cells may be responsible for the observed changes in spatial memory. Additionally, excessive PFC DA activity has been linked to decreased spatial working memory in rats and monkeys (Murphy et al., 1996). Therefore, it is conceivable that memory and cognition impairment reported in humans following toluene exposure could be related, in part, to the increased cortical DA activity reported here. In addition, changes in DA levels reported in the present study may be of clinical relevance to toluene’s abuse liability, as brief exposure to high levels
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( 10 000 ppm) produces psychotropic effects including euphoria, intoxication, slurred speech, ataxia, hallucinations, and dizziness (Flanagan and Ives, 1994). Although direct comparisons across species must be interpreted with caution, work by Kishi (Kishi et al., 1988) indicates that plasma toluene concentrations are very close in rats and humans following exposure to similar toluene concentrations. Furthermore, Garriott et al., 1981 described symptoms of mild intoxication and euphoria at plasma levels of approximately 30 mg/ml in human toluene abusers. In this study, cocaine was much less effective at increasing extracellular DA levels in the PFC than in the NACC. This finding is consistent with a reportedly low number of DA transporters (DAT) in the PFC over striatal regions (Sesack et al., 1998) and with earlier finding from Moghaddam and Bunney, 1989. The fact that cocaine-induced DAT blockade had no effect on PFC extracellular DA levels following toluene inhalation could be explained by differential feedback regulation of mesolimbic and mesocortical DA neurons (Carr and Sesack, 2000). It is possible, based on a greater sensitivity of mesocortical neurons over mesolimbic neurons to glutamate antagonists (Westerink et al., 1996), that the balance between toluene-mediated NMDA antagonism and GABAA receptor activation in PFC DA is less sensitive to changes arising from cocaine-induced blockade of a restricted number of PFC DAT. Accordingly, not only is the number of DAT higher in the NACC, but also regional variations exist in DA reuptake mechanisms that could presumably render cocaine a less effective reuptake blocker in the PFC (Hadfield and Nugent, 1983). In fact, it appears that toluene exposure had an inhibitory effect on cocaine-induced increases in PFC DA, since the peak increase following the combined challenge was lower than the increase produced by cocaine alone. In marked contrast to the results observed in the PFC, simultaneous administration of cocaine and toluene produced synergistic elevations in extracellular NACC DA, the nature of which is unclear. One could hypothesize that cocaine-induced blockade of the DAT contributed in a supradditive manner to changes in extracellular NACC DA activity secondary to toluenemediated increases in VTA A10 DA neuron cell firing and bursting activity (Riegel and French, 1999a). Alternatively, toluene-induced blockade of NMDA receptors on VTA GABA interneurons and the subsequent removal of this inhibitory output might be expected to produce enhanced DA cell firing through a disinhibitory mechanism (Wang and French, 1995). This notion is further supported by findings that toluene exposure decreases the firing rate of presumed VTA GABAergic interneurons proportional to exposure duration (Riegel and French, 1999b). Thus, toluene may transiently increase DAergic activity in the NACC by
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either directly stimulating A10 DA cell bodies (which project to the NACC) or indirectly by inhibition of VTA GABA interneurons. This interpretation should be viewed with caution, as recordings of DA cell firing in the VTA were performed in ketamine-anaesthetized animals exposed to toluene at concentrations several times higher (10 000 ppm) than those used here (3000 ppm). The synergistic DAergic response observed following toluene inhalation and cocaine administration in the NACC is similar to what we reported earlier following a cocaine and heroin or a cocaine and nicotine challenge (Gerasimov et al., 2000). Combined, these data may suggest a potential neurochemical basis for the significant correlation reported between the abuse of inhalants and other addictive drugs (Schu¨ tz et al., 1994) Finally, in an effort to investigate the pharmacokinetics and central distribution of toluene in vivo, we synthesized 11C-toluene and performed imaging studies in primates using positron emission tomography (Gerasimov et al., 2001). The rapid uptake and clearance observed in these studies is in agreement with the long-standing hypothesis of a link between the rate of drug delivery, its effects in the brain, and its reinforcing properties (Balster and Schuster, 1973). In the present study we observed regionally specific increases in DA levels following toluene inhalation as well as marked differential effects of cocaine on these increases. Taken together, these findings suggest that specific regional protein binding affinities following rapid brain delivery may underlie the addictive liability of toluene. Acknowledgements Authors are thankful to Dr J. Fowler for helpful discussions. This research was carried out under contract with the US Department of Energy Office of Biological and Environmental Research (USDOE/ OBER DE-AC02-98CH10886). References Ameno, K., Kiriu, T., Fuke, C., Ameno, S., Shinohara, T., Ijiri, I., 1992. Regional brain distribution of toluene in rats and in a human autopsy. Arch. Toxicol. 66, 153156. Balster, R.L., 1998. Neural basis of inhalant abuse. Drug Alcohol Depend. 51, 207 – 214. Balster, R.L., Schuster, C.R., 1973. Fixed interval schedule of cocaine reinforcement: effects of dose and infusion duration. J. Exp. Anal. Behav. 20, 119 – 129. Beckstead, M.J., Weiner, J.L., Eger, E.I.N., Gong, D.H., Mihic, S.J., 2000. Glycine and gamma-aminobutyric acid (A) receptor function is enhanced by inhaled drugs of abuse. Mol. Pharmacol. 57, 1199 – 1205. Bowen, S.E., Wiley, J.L., Balster, R.L., 1996. The effects of abused inhalants on mouse behavior in an elevated plus-maze. Eur. J. Pharmacol. 312, 131 – 136.
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King, M.D., 1982. Neurological sequelae of toluene abuse. Hum. Toxicol. 1, 281 – 287. Kiriu, T., Ameno, K., Fuke, C., Ameno, S., Ijiri, I., 1990. The distribution of toluene in the brain and its effects on the brain catecholamines in acute toluene poisoning. Nippon. Hoigaku. Zasshi. 44, 25 – 33. Kishi, R., Harabuchi, I., Ikeda, T., Yokota, H., Miyake, H., 1988. Neurobehavioural effects and pharmacokinetics of toluene in rats and their relevance to man. Br. J. Ind. Med. 45, 396 – 408. Kondo, H., Huang, J., Ichihara, G., Kamijima, M., Saito, I., Shibata, E., Ono, Y., Hisanaga, N., Takeuchi, Y., Nakahara, D., 1995. Toluene induces behavioral activation without affecting striated dopamine metabolism in the rat: behavioral and microdialysis studies. Pharmacol. Biochem. Behav. 51, 97 – 101. Kozel, N. Epidemiology of inhalant abuse. An international perspective. (NIDA Res Monograph 148, NIH public c95-3831) 1995. Department of Health and Human Services, Rockville, MD, US. Lopez-Rubaclava, C., Hen, R., Cruz, S.L., 2000. Anxiolytic-like actions of toluene in the burying behavior and plus-maze test: differences in sensitivity between 5-HT1B knockout and wild-type mice. Behav. Brain. Res. 115, 85 – 94. Moghaddam, B., Bunney, B.S., 1989. Differential effect of cocaine on extracellular dopamine levels in rat medial prefrontal cortex and nucleus accumbens: comparison to amphetamine. Synapse 4, 156 – 161. Murphy, B.L., Arnsten, A.F., Goldman-Rakic, P.S., Roth, R.H., 1996. Increased dopamine turnover in the prefrontal cortex impairs spatial working memory performance in rats and monkeys. Proc. Natl. Acad. Sci. USA 93, 1325 – 1329. Nishijima, K., Kashiwa, A., Nishikawa, T., 1994. Preferential stimulation of extracellular release of dopamine in rat frontal cortex to striatum following competitive inhibition of the N-methyl-D-aspartate receptor. J. Neurochem. 63, 375 – 378. Rees, D.C., Knisely, J.S., Breen, T.J., Balster, R.L., 1987a. Toluene, halothane, 1, 1, 1-trichloroethane and oxazepam produce ethanollike discriminative stimulus effects in mice. J. Pharmacol. Exp. Ther. 243, 931 – 937. Rees, D.C., Knisely, J.S., Jordan, S., Balster, R.L., 1987b. Discriminative stimulus properties of toluene in the mouse. Toxicol. Appl. Pharmacol. 88, 97 – 104. Riegel, A.C., French, E.D., 1999a. An electrophysiological analysis of rat ventral tegmental dopamine neuronal activity during acute toluene exposure. Pharmacol. Toxicol. 85, 37 – 43. Riegel, A.C., French, E.D., 1999b. The susceptibility of rat non-dopamine ventral segmental neurons to inhibition during toluene exposure. Pharmacol. Toxicol. 85, 44 – 46. Schutz, C.G., Chilcoat, H.D., Anthony, J.C., 1994. The association between sniffing inhalants and injecting drugs. Compr. Psychiatry 35, 99 – 105. Sesack, S.R., Hawrylak, V., Matus, C., Guido, M.A., Levey, A.L., 1998. Dopamine axon varicosities in the prelimbic division of the rat prefrontal cortex exhibit sparse immunoreactivity for the dopamine transporter. J. Neurosci. 18, 2697 – 2708. Stengard, K., Hoglund, G., Ungerstedt, U., 1994. Extracellular dopamine levels within the striatum increase during inhalation exposure to toluene: a microdialysis study in awake, freely moving rats. Toxicol. Lett. 71, 245 – 255. Tzschentke, T.M., 2000. The medial prefrontal cortex as a part of the brain reward system. Amino Acids 19, 211 – 219. von Euler, G., 1994. Toluene and dopamenergic transmission. In: Isaacson, R.L., Jensen, K.F. (Eds.), The Vulnerable Brain and Environmental risks, vol. 3. Plenum Press, New York. von Euler, G., Fuxe, K., Hansson, T., Eneroth, P., Agnati, L.F., Harfstrand, A., Gustafsson, J., 1988. Effects of haloperidol treatment on regional catecholamine levels and utilization in rats exposed to toluene. Acta Physiol. Scand. 132, 199 – 208.
M.R. Gerasimo6 et al. / Drug and Alcohol Dependence 65 (2002) 243–251 von Euler, G., Ogren, S.O., Li, X.M., Fuxe, K., Gustafsson, J.A., 1993. Persistent effects of subtonic toluene exposure on spatial learning and memory, dopamine-mediated locomotion activity and dopamine D2 agents binding in the rat. Toxicology 77, 223 – 232. von Euler, M., Pham, T.M., Hillefors, M., Bjelke, B., Henriksson, B., von Euler, G., 2000. Inhalation of low concentrations of toluene induces persistent effects on a learning retention task, beam-walk performance, and cerebrocortical size in the rat. Exp. Neurol. 163, 1–8. Wang, T., French, E.D., 1995. NMDA, quaint, and AMPA depolarize nondopamine neurons in the rat ventral tegmentum. Brain Res. Bull. 36, 39 – 43.
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Weiss, B., Wood, R.W., Macys, D.A., 1979. Behavioral toxicology of carbon disulfide and toluene. Environ. Health Perspect. 30, 39 – 45. Westerink, B.H., Kwint, H.F., deVries, J.B., 1996. The pharmacology of mesolimbic dopamine neurons: a dual-probe microdialysis study in the ventral segmental area and nucleus accumbens of the rat brain. J. Neurosci. 16, 2605 – 2611. Wood, R.W., Coleman, J.B., Schuler, R., Cox, C., 1984. Anticonvulsant and antipunishment effects of toluene. J. Pharmacol. Exp. Ther. 230, 407 – 412. Yavich, L., Patkina, N., Zvartau, E., 1994. Experimental estimation of addictive potential of a mixture of organic solvents. Eur. Neuropsychopharmacol. 4, 111 – 118.
Toluene inhalation produces regionally specific changes in extracellular dopamine Madina R. Gerasimov *, Wynne K. Schiffer, Douglas Marstellar, Richard Ferrieri, David Alexoff, Stephen L. Dewey Chemistry Department, Brookha6en National Laboratory, Upton, NY 11973, USA Received 20 March 2001; received in revised form 9 May 2001; accepted 11 May 2001
Abstract The aim of the present study was to investigate the effect of toluene inhalation on dopaminergic transmission in two distinct brain areas presumably involved in mediating the reward processes important for toluene abuse. Extracellular dopamine (DA) levels were measured in prefrontal cortex (PFC) and nucleus accumbens (NACC) of freely moving rats using in vivo microdialysis. Inhalation of a behaviorally relevant concentration of toluene (3000 ppm) produced a significant increase in the PFC but not in the NACC. However, the odorant isoamyl acetate, increased PFC DA levels by only 37%, significantly less than the 96% increase observed following toluene exposure. When toluene inhalation was combined with cocaine administration (20 mg/kg i.p.), the response to the combined challenge was not different from the response to toluene alone in the PFC. However, the combination of these two drugs produced a supradditive response of 802% in the NACC, compared with the 450% increase observed following cocaine alone. Recent reports indicate that toluene influences the function of several ionotropic receptors in a subunit specific manner. As further evidence of specific effects, our results indicate regionally specific changes in dopaminergic transmission following toluene exposure. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Toluene; Dopamine; Microdialysis; Nucleus accumbens; Prefrontal cortex
1. Introduction Solvent abuse, mainly among adolescents, has been reported to occur worldwide (Flanagan and Ives 1994; Kozel, 1995). In the US, for example, inhaled agents rank fourth behind alcohol, marijuana and tobacco in their incidence of abuse, while in Japan, organic solvents and amphetamine are two of the most commonly abused substances. Toluene is the principal intoxicant in many of the products involved in ‘sniffing’. Although inhalant abuse is now recognized as a worldwide problem, organic solvents are currently the least studied drugs of abuse. For example, relatively little is known about the underlying cellular mechanisms of action through which these substances produce their effects in the central nervous system (CNS). * Corresponding author. Tel.: + 1-631-3444395; fax: +1-6313447902. E-mail address: [email protected] (M.R. Gerasimov).
A review of the current literature clearly indicates the paucity of in vivo research designed to specifically address the acute affects of toluene on neurotransmitter concentration, release, or the response to pharmacological challenge (Balster, 1998). Exposure to toluene may produce behaviors consistent with many other drugs of abuse. Toluene, like benzodiazepines, alcohol and phencyclidine (PCP), has a demonstrated abuse liability in humans (Howard et al., 2001). Furthermore, its rewarding/reinforcing effects have been demonstrated in animal studies where it increased the response rate of intracranial self-stimulation (ICSS), induced conditioned place preference (CPP) (Yavich et al., 1994), and maintained self-administration in squirrel monkeys (Weiss et al., 1979). In view of the well established role of dopamine (DA) in underlying the reinforcing effects of many abused drugs, earlier ex vivo studies assessed changes in brain DA content and turnover following acute and chronic toluene exposure (von Euler, 1994). To our knowledge,
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however, only two studies assessed brain DA levels in freely moving animals during toluene inhalation. These studies measured DA levels in the corpus striatum and provided contradictory results. Stengard et al., 1994 found that exposure to toluene increased striatal DA 47% above baseline while Kondo et al., 1995 failed to observe any significant change. Due to its high lipid solubility, toluene has long been considered to act in a ‘non-specific’ manner, through the disordering of cell membrane lipids. In fact, behavioral evidence indicates that toluene shares many properties with other small lipophilic agents like inhaled anesthetics and ethanol (Rees et al., 1987a; Bowen et al., 1996). In addition, there exists behavioral similarities between toluene and classical non-volatile CNS depressants like benzodiazepines and barbiturates (Evans and Balster, 1991), compounds that produce their effects directly through the gamma-aminobutyric acid (GABA) receptor complex. For example, toluene, ethanol, midazolam and pentobarbital effectively decreased withdrawal symptoms produced by trichloroethane inhalation (Evans and Balster, 1993). Furthermore, mice trained to discriminate toluene from vehicle generalized to pentobarbital, but not morphine (Rees et al., 1987b). Toluene resembles benzodiazepines in its ability to produce anticonvulsant, antipunishment and antianxiety effects (Bowen et al., 1996; Wood et al., 1984). Finally, recent work by Bowen et al., 1999 demonstrated that toluene produced concentration-related partial substitution for phencyclidine (PCP) in a drug discrimination assay. Taken together, these findings suggest some specificity in the relevance of both NMDA receptor antagonism and GABA receptor stimulation in the mechanism of action of toluene. It appears that several neurotransmitter systems may mediate the complex symptomatology associated with toluene abuse. While nucleus accumbens (NACC) DA activity is considered to be the principal element in generating reward-related behaviors (Di Chiara, 1995; Gardner, 1997), recent studies utilizing ICSS, CPP and self-administration paradigms suggest that the prefrontal cortex (PFC) is also involved in the generation of reward (Tzschentke, 2000). Therefore, the present study was designed to test whether acute toluene inhalation altered extracellular DA levels in the NACC and PFC in freely moving animals. Furthermore, since earlier work (Gerasimov and Dewey, 1999; Gerasimov et al., 2000; Hemby et al., 1999) has demonstrated that addictive drugs often produce synergistic effects when co-administered, we also investigated possible interactions between cocaine and toluene. These data may provide insight into the apparent association between drug inhalation and other drugs of abuse where inhalant users were five times more likely than non-users to abuse illicit drugs (Schu¨ tz et al., 1994). Finally, in order to rule out possible effects of stress on extracellular DA
levels caused by a non-specific environmental stimuli (e.g. a pronounced odor) or a possible change in air composition, we exposed animals to the odorant isoamyl acetate under identical conditions to tolueneexposed animals.
2. Materials and methods
2.1. Animals and surgery All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local animal care committee. Experimentally naı¨ve, male Sprague– Dawley rats were used in all experiments (200–300 g, Taconic Farms, Germantown, NY) and were given food and water ad libitum. Each animal was housed individually on a 12-h light:12-h dark cycle. Animals were anesthetized with ketamine/xylazine mixture and placed in a stereotaxic frame. Siliconized guide cannulae (BAS, West Lafayette, INF) were implanted in the brain above the PFC (anterior-posterior to bregma 3.7, medial-lateral to bregma 1.0, dorsal-ventral to bregma 1.0) or NACC (anterior-posterior 1.5, medial-lateral 1.0, dorsal-ventral 5.6) 2 days before the microdialysis experiment. These coordinates were calculated according to the Paxinos and Watson atlas for the rat brain (Second edition, 1986). Dental acrylic and machine screws held the cannulae in place throughout the study. On the day of the experiment, animals were removed from their home cage and placed in the dialysis chamber. Concentric flow dialysis probes (4 mm for PFC, 2 mm for NACC) were lowered through the guide cannulae in awake animals, terminating in the structures of interest. The dialysis probes were perfused with Ringer’s solution (McGaw, Inc. Irvine, CA) at a flow rate of 2.0 ml/min. Dialysate samples were collected for 20 min and analyzed on-line. After all experiments, animals were euthanized with an overdose of chloral hydrate and brains dissected to verify probe placement.
2.2. Analytical procedure Dialysate samples were assayed for DA content by high-pressure liquid chromatography (HPLC) coupled with electrochemical detection. This HPLC system consists of a BAS reverse-phase microbore column (150×1 mm, 5 mm microbore column with C18 packing; BAS) directly attached to a conventional flow electrochemical detector equipped with a glassy carbon working electrode maintained at + 650 mV relative to a Ag/AgCl reference electrode (LC-4C, BAS). The microbore column was kept at a constant temperature of 27.6 °C. The range was set to 0.2 nA, and the electrical noise and output filter at 0.02 Hz. The mobile phase con-
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sisted of 8.0% acetonitrile, 1.0% tetrahydrofuran, 14.5 mM sodium phosphate monobasic, 30 mM sodium citrate, 27 mM disodium-EDTA, 10 mM diethylamine HCl, 1.95 mM decanesulfonic acid, sodium salt, and pH 4.0. An online degasser was used to ensure that the mobile phase was free of air. The net flow rate calibrated from the microbore column was 0.10 ml min − 1, obtained by using a flow splitting technique. Data was collected on-line using chronograph® software (BAS) as well as with a dual-pen strip chart recorder that records chemical activity, detection voltage on the chart recorder was set at 1 V.
2.3. Exposure to toluene and isoamyl acetate. Animals were exposed to toluene and isoamyl acetate for 40 min in a microdialysis chamber modified for this particular experiment (Fig. 1). The use of a rotating bowl with an optical monitoring device (Raturn®, BAS) eliminates the need for a liquid swivel, thus dramatically improving the recovery of DA in the dialysate. Rotation is prompted by rat’s movement, thus preventing the connecting lines from damage due to entanglement. An opening in the lid of the chamber allowed for free movement of the arm with the tether hooked to the animal collar, together with the tubing directly connecting the microdialysis probe with the on-line injector.
Fig. 1. Diagram demonstrating an animal undergoing simultaneous dialysis and toluene exposure. (A). Syringe pumping CSF at 2.0 ml/min, (B). On-line injector and LC analysis system, (C). Glass vestibule with sand and heated toluene, (D). Ceramic plate with holes allowed the heated vapor to rise into the microdialysis chamber
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After baseline measurements of dopamine were obtained (defined by at least four consecutive DA samples differing by less than 10%) the ceramic plate was lifted without disturbing the animal and a glass sand bath filled with toluene or isoamyl acetate (100 ml, Sigma– Aldrich, Milwaukee, WI) heated to boiling point was placed underneath the platform. The heating of the toluene-filled sand bath was performed in a ventilated hood using Thermolyne® heating plate. A grated floor allowed the heated vapor to rise into the microdialysis chamber. Air samples drawn every 10 min from the bowl at the level of the animal head indicated that initial levels of the vapors (immediately following introduction of the source into the bowl) were significantly lower (300–350 ppm) than stable concentrations of toluene and isoamyl acetate levels that were reached at 10 min. These vapor levels maintained at 3000950 ppm and 2500950 ppm, respectively, throughout the exposure period, and declined thereafter. Thus, the reported numbers actually represent the highest vapor levels that were achieved by this method. Air sampling for measurement of airborne toluene levels in the exposure chamber was conducted using a 10 ml glass, gas-tight syringe (Hamilton Valve Co.) equipped with a shut-off valve. The syringe was coupled to a 25× 0.16 cm Teflon tube. These syringes have Teflon barrels and stainless heads with a locking Teflon insert. Any other plastic material in the syringe could cause problems through solvent adsorption which will confuse a trace level analysis. The air sample withdrawn from the bowl was immediately locked inside the syringe to allow time to remove the Teflon sampling tube without complications of loss of sample from the syringe. The sample was then slowly dispelled through 0.5 ml of acetonitrile that was in a small Teflon sealed vial (crimp seal type). Head space volume was minimized by using these vials. The vial had to be vented while the 10 cm3 of air sample was dispelled through the solvent. We ran the outlet gas flow through a second volume of solvent to ensure that no toluene was passing through the first solvent trap and out. We used acetonitrile as a solvent because it does not carry a trace load of toluene. Toluene losses from the solvent were not a problem once it was entrained in the solvent. This was verified by standardizing the flame ionization detector response to amounts of toluene, by measuring standard stability over time (a few h). There was no noticeable toluene loss from the standards as long as the vials remained sealed. Actual samples were analyzed within 15 min of drawing the sample. Gas chromatographic analysis was carried out using a Hewlett–Packard 5890A capillary gas chromatograph equipped with a flame ionization detector whose output was interfaced with a Vision 4 chromatography acquisition system (Scientific Systems, State College, PA). One microliter
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samples was injected and split 1:100 onto a 60 m× 0.025 mm SE-30 column (J and W Capillary) initially maintained at 50 °C, and then temperature programmed to 100 °C at a 2 °C/min rate. The injector temperature was maintained at 225 °C and the flame ionization detector maintained at 350 °C throughout the analysis. Standard samples of toluene were prepared in two ways to effect an accurate range of amounts of substance from below 5 to 50 000 ppm. Standards were prepared for the detector response measurements from the below 5 to 5000 ppm range by pipetting 1– 10 ml volumes of toluene using calibrated analytical pipettes into 1–10 ml volumetric flasks containing HPLC grade acetonitrile solvent. Standards were made up in triplicate, as well as detector response measurements carried out in triplicate. Standards for higher concentrations of toluene ( \5000 ppm) were made up in HPLC grade hexane solvent using the same procedures described. In these instances, the slight impurity level of toluene in the hexane solvent was insignificant relative to the sample injected. Standards were typically kept refrigerated and maintained their integrity for several days without a loss of sample. A rigorous calibration was performed on detector response at the onset of the study and then spot checked daily within the critical range to ensure that response was within 2% of the expected valve. In the control (air exposure) studies, animals remained in the modified chambers with the lid in place. In the studies designed to examine a potential synergistic effect between toluene and cocaine, cocaine (20 mg/kg i.p.) was administered immediately prior to toluene exposure.
2.4. Statistics Baseline OA level was calculated for each animal separately as the average of three pre-drug samples not differing from each other by more than 10%. This average number was subsequently used for each animal to calculate individual drug response. Peak increases in extracellular DA, expressed as a percent change from these baseline values, were compared for every group with a one-way analysis of variance (ANOVA) followed by a post hoc Student’s Newman – Keuls test. Significance levels were set at P B0.05.
3. Results
3.1. Effect of 6apor exposure alone and in combination with cocaine on extracellular DA le6els in the PFC. Extracellular DA levels increased immediately following the onset of toluene inhalation and reached a
Fig. 2. Effect of toluene (3000 ppm) and isoamyl acetate (2500 ppm) vapors on extracellular DA levels in the prefrontal cortex of rats. Values are mean 9S.E.M. and expressed as percent of basal levels (n = 5 – 9 per group). Exposure started at time 0 and ended at time 40 min. *, PB 0.05 different from the peak effect in animals exposed to isoamyl acetate (ANOVA and post-hoc Student’s Newman–Keuls test).
maximum of 96% above baseline by the end of the 40 min exposure (n=9) (Fig. 2). DA levels returned to baseline 100 min after the source of toluene vapor was removed. This peak increase was significantly different (P B0.05) from the increase following isoamyl acetate exposure (37% above baseline, n= 6). The peak increase of 71% following combined cocaine and toluene administration (n= 9) was not significantly different from elevations observed in animals exposed to toluene alone (96% above baseline). In addition, the temporal parameters of this peak increase and subsequent return to baseline were not different between these two groups either. This increase, however, was significantly different from the cocaine-induced increase of 165% above baseline (n= 6) (P=0.01) (Fig. 3).
3.2. Effect of 6apor exposure alone and in combination with cocaine on extracellular DA le6els in the NACC. There was no significant difference between the DAergic response to air (n= 4), isoamyl acetate (n=4) or toluene (n=9) alone (data not shown). Conversely, cocaine administration produced an increase of 450% above baseline (n= 9). When combined with toluene, however, extracellular DA increased by 802% (n =9). This increase was significantly different from the response to cocaine or toluene alone (PB0.001). In both groups DA peaked 40 min post exposure and returned to baseline in 90 min (Fig. 4).
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4. Discussion Toluene increased extracellular PFC DA to a greater extent than did isoamyl acetate, while neither had any effect on extracellular NACC DA. In addition, inhalation of toluene immediately following cocaine administration, had no effect on increases in PFC DA but significantly potentiated the response observed in the NACC. The majority of studies reported in the literature were designed to address the effects of toluene on DA neurons by utilizing biochemical assays mainly reflective of intracellular concentrations (Fuxe et al., 1982; von Euler et al., 1988; von Euler, 1994). While these levels may be altered by both synthesis and exocytotic release of DA, they may not indicate whether DAergic transmission is increased or decreased. Additionally, these studies focused primarily on persistent and shortterm effects of chronic toluene exposure and are thus of limited relevance to understanding the potential acute effects. Mesocorticolimbic DA activity is regulated by both GABAergic and glutamatergic input. Beckstead et al., 2000 demonstrated that toluene significantly enhanced GABAA receptor function in a dose-dependent and reversible manner. Of particular significance, however, is their observation that the application of the highest concentration of toluene did not influence GABAA receptor activity in the absence of GABA. Toluene might also disrupt excitatory amino acid input by in-
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hibiting NMDA-mediated currents in vitro (Cruz et al., 1998) and in vivo (Cruz et al., 1999). Taken together, these data suggest that neurochemically specific as well as non-specific mechanisms of action need to be considered. The moderately high concentrations of toluene used in this study (3000 ppm) are self-administered (Weiss et al., 1979), produce discriminative stimulus effects (Rees et al., 1987a) and have a distinct antipunishment effect (Wood et al., 1984). In addition, this concentration exerts anxyolitic-like action on burying behavior and in elevated plus-maze test performance (Lopez-Rubaclava et al., 2000). In the present study, toluene exposure increased extracellular DA in the PFC (Fig. 1) while having no effect in the NACC (Fig. 3). To our knowledge, this is the first direct in vivo evidence that toluene inhalation alters extracellular DA in this cortical region. Furthermore, the unique finding of specific regional effects supports the hypothesis that, notwithstanding toluene’s reported distribution according to lipid content (Gospe and Calaban, 1988), some of its effects may be due to selective interactions with neurotransmitter receptor proteins. That is, even though the lowest brain/blood toluene concentration ratio was found in cerebral cortex (Ameno et al., 1992; Kiriu et al., 1990), we observed significant changes in extracellular DA levels following toluene inhalation in this brain region. DA cell bodies in the VTA receive reciprocal excitatory amino acid input from PFC, and in turn project
Fig. 3. Effect of combined toluene and cocaine administration on extracellular DA levels in the prefrontal cortex of rats. Values are means9S.E.M. expressed as percent of basal levels (n= 6–9/group). Exposure started at time 0 and ended at time 40 min. **, PB 0.01 different from the peak effect in animals exposed to either toluene alone or to combination
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Fig. 4. Effect of combined toluene and cocaine administration on extracellular DA levels in the nucleus accumbens of rats. Values are means of9 S.E.M. expressed as percent of basal levels (n= 9 per group). Exposure started at time 0 and ended at time 40 min. ***, P= 0.001 different from the peak effect in animals exposed to either toluene or cocaine alone.
back to the NACC. Recent evidence indicates that PFC afferents synapse on either mesocortical DA neurons or on mesolimbic GABA neurons (Carr and Sesack, 2000). Therefore, it is conceivable that the regionally specific changes observed in the present study represent indirect effects of toluene on these subpopulations of VTA cell bodies. In fact, PFC DA increased immediately following toluene inhalation, suggesting that transient activation of GABAA receptors and/or non-competitive inhibition of NMDA receptors might have indirectly contributed to this marked increase. It should be noted, however, that isoamyl acetate increased PFC DA levels as well, but to a much lesser degree. This difference addresses the argument that increases observed following toluene exposure are directly related to a non-specific olfactory mechanism or to a change in the molecular composition of breathed air. Beckstead et al., 2000 demonstrated that toluene, at concentrations of 0.2– 0.9 mM, or 20 – 90 mg/ml (which approximately corresponds to 30 mg/g in rats exposed to 1000–2000 ppm of toluene (Kishi et al., 1988), significantly increased ligand-gated currents in GABAA and glycine receptors. Accordingly, human plasma toluene concentrations in abusers have been reported to be in the 0.01–0.1 mM range (King, 1982). Interestingly, toluene IC50 values for inhibiting NMDA-induced currents is in the same range of 0.2–2 mM (Cruz et al., 1998). This is also close to the effective concentrations
of other non-competitive NMDA receptor antagonists that preferentially stimulate PFC DA (Hata et al., 1990; Nishijima et al., 1994) via GABA-mediated disinhibition. The notion that toluene produces neurobehavioral effects similar to those of non-competitive NMDA antagonists is further supported by evidence from Bowen et al., 1999 who demonstrated that toluene, at a concentration of 1000–4000 ppm, partially substituted for a pharmacologically relevant dose of PCP (2 mg/ kg). The clinical significance of the present findings is further emphasized by the work of (von Euler et al., 1993, 2000) who proposed that subchronic toluene exposure produced persistent impairment in initiating novel behaviors or integrating sensory information. Structural changes observed in the cerebral cortex served as the foundation for their proposal that a dysfunction of glutamatergic corticostriatal pyramidal cells may be responsible for the observed changes in spatial memory. Additionally, excessive PFC DA activity has been linked to decreased spatial working memory in rats and monkeys (Murphy et al., 1996). Therefore, it is conceivable that memory and cognition impairment reported in humans following toluene exposure could be related, in part, to the increased cortical DA activity reported here. In addition, changes in DA levels reported in the present study may be of clinical relevance to toluene’s abuse liability, as brief exposure to high levels
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( 10 000 ppm) produces psychotropic effects including euphoria, intoxication, slurred speech, ataxia, hallucinations, and dizziness (Flanagan and Ives, 1994). Although direct comparisons across species must be interpreted with caution, work by Kishi (Kishi et al., 1988) indicates that plasma toluene concentrations are very close in rats and humans following exposure to similar toluene concentrations. Furthermore, Garriott et al., 1981 described symptoms of mild intoxication and euphoria at plasma levels of approximately 30 mg/ml in human toluene abusers. In this study, cocaine was much less effective at increasing extracellular DA levels in the PFC than in the NACC. This finding is consistent with a reportedly low number of DA transporters (DAT) in the PFC over striatal regions (Sesack et al., 1998) and with earlier finding from Moghaddam and Bunney, 1989. The fact that cocaine-induced DAT blockade had no effect on PFC extracellular DA levels following toluene inhalation could be explained by differential feedback regulation of mesolimbic and mesocortical DA neurons (Carr and Sesack, 2000). It is possible, based on a greater sensitivity of mesocortical neurons over mesolimbic neurons to glutamate antagonists (Westerink et al., 1996), that the balance between toluene-mediated NMDA antagonism and GABAA receptor activation in PFC DA is less sensitive to changes arising from cocaine-induced blockade of a restricted number of PFC DAT. Accordingly, not only is the number of DAT higher in the NACC, but also regional variations exist in DA reuptake mechanisms that could presumably render cocaine a less effective reuptake blocker in the PFC (Hadfield and Nugent, 1983). In fact, it appears that toluene exposure had an inhibitory effect on cocaine-induced increases in PFC DA, since the peak increase following the combined challenge was lower than the increase produced by cocaine alone. In marked contrast to the results observed in the PFC, simultaneous administration of cocaine and toluene produced synergistic elevations in extracellular NACC DA, the nature of which is unclear. One could hypothesize that cocaine-induced blockade of the DAT contributed in a supradditive manner to changes in extracellular NACC DA activity secondary to toluenemediated increases in VTA A10 DA neuron cell firing and bursting activity (Riegel and French, 1999a). Alternatively, toluene-induced blockade of NMDA receptors on VTA GABA interneurons and the subsequent removal of this inhibitory output might be expected to produce enhanced DA cell firing through a disinhibitory mechanism (Wang and French, 1995). This notion is further supported by findings that toluene exposure decreases the firing rate of presumed VTA GABAergic interneurons proportional to exposure duration (Riegel and French, 1999b). Thus, toluene may transiently increase DAergic activity in the NACC by
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either directly stimulating A10 DA cell bodies (which project to the NACC) or indirectly by inhibition of VTA GABA interneurons. This interpretation should be viewed with caution, as recordings of DA cell firing in the VTA were performed in ketamine-anaesthetized animals exposed to toluene at concentrations several times higher (10 000 ppm) than those used here (3000 ppm). The synergistic DAergic response observed following toluene inhalation and cocaine administration in the NACC is similar to what we reported earlier following a cocaine and heroin or a cocaine and nicotine challenge (Gerasimov et al., 2000). Combined, these data may suggest a potential neurochemical basis for the significant correlation reported between the abuse of inhalants and other addictive drugs (Schu¨ tz et al., 1994) Finally, in an effort to investigate the pharmacokinetics and central distribution of toluene in vivo, we synthesized 11C-toluene and performed imaging studies in primates using positron emission tomography (Gerasimov et al., 2001). The rapid uptake and clearance observed in these studies is in agreement with the long-standing hypothesis of a link between the rate of drug delivery, its effects in the brain, and its reinforcing properties (Balster and Schuster, 1973). In the present study we observed regionally specific increases in DA levels following toluene inhalation as well as marked differential effects of cocaine on these increases. Taken together, these findings suggest that specific regional protein binding affinities following rapid brain delivery may underlie the addictive liability of toluene. Acknowledgements Authors are thankful to Dr J. Fowler for helpful discussions. This research was carried out under contract with the US Department of Energy Office of Biological and Environmental Research (USDOE/ OBER DE-AC02-98CH10886). References Ameno, K., Kiriu, T., Fuke, C., Ameno, S., Shinohara, T., Ijiri, I., 1992. Regional brain distribution of toluene in rats and in a human autopsy. Arch. Toxicol. 66, 153156. Balster, R.L., 1998. Neural basis of inhalant abuse. Drug Alcohol Depend. 51, 207 – 214. Balster, R.L., Schuster, C.R., 1973. Fixed interval schedule of cocaine reinforcement: effects of dose and infusion duration. J. Exp. Anal. Behav. 20, 119 – 129. Beckstead, M.J., Weiner, J.L., Eger, E.I.N., Gong, D.H., Mihic, S.J., 2000. Glycine and gamma-aminobutyric acid (A) receptor function is enhanced by inhaled drugs of abuse. Mol. Pharmacol. 57, 1199 – 1205. Bowen, S.E., Wiley, J.L., Balster, R.L., 1996. The effects of abused inhalants on mouse behavior in an elevated plus-maze. Eur. J. Pharmacol. 312, 131 – 136.
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