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Concentration-dependent conditioned place preference to inhaled toluene vapors in rats
Drug and Alcohol Dependence 85 (2006) 87–90
Short communication
Concentration-dependent conditioned place preference to inhaled toluene vapors in rats Dianne E. Lee b , Madina R. Gerasimov a , Wynne K Schiffer b , Andrew N. Gifford a,∗ a b
Medical Department, Brookhaven National Laboratory, Upton, NY 11973, United States Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973, United States
Received 29 August 2005; received in revised form 13 March 2006; accepted 17 March 2006
Abstract Objectives: Toluene is present in many commercial products and is subject to abuse by inhalation. The goal of this study was to extend previous reports indicating that rats will exhibit a positive conditioned place preference to inhaled toluene vapors and to determine the dose–response relationship for inhaled toluene in terms of exposure concentration and number of exposures. For the conditioned place preference experiments rats were exposed to toluene vapors at concentrations of 800, 2000, 3000 or 5000 ppm in one compartment of a three-compartment box. Results: Following six conditioning sessions with toluene, a significant place preference was obtained at 2000 and 3000 ppm, but not at 800 or 5000 ppm. Extending the number of toluene pairings at the 2000 and 3000 ppm concentration to 12 significantly enhanced the place preference compared to that at six pairings. Conclusions: These experiments extend our previous finding that rats will show a conditioned place preference to inhaled toluene, and indicate that a reinforcing “dose” of toluene depends on both the concentration and number of pairings. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Toluene; Inhalant abuse; Conditioned place preference; Rats; Drugs-of-abuse
1. Introduction Voluntary inhalation of volatile solvents found in inexpensive commercial products such as glues, paint products, cleaning fluids and lighter fluids is prevalent in industrialized cities throughout the world, especially amongst juveniles and adolescent children (Andersen and Loomis, 2003; Basu et al., 2004). However, despite the widespread nature of solvent-abuse, also termed “glue-sniffing” there have been relatively few studies aimed at developing animal models for the abuse of these substances, especially when compared with the extensive literature on animals models for the reinforcing properties of cocaine, opiates and alcohol (Tzschentke, 1998; Bardo and Bevins, 2000). Part of the explanation for this may be related to the fact that these solvents are abused by inhalation, making them hard to administer to animals in controlled amounts. Additionally, the CNS target site for abused-solvents has been less well
∗
Corresponding author. Tel.: +1 631 344 7069; fax: +1 631 344 5311. E-mail address: [email protected] (A.N. Gifford).
0376-8716/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.drugalcdep.2006.03.013
defined than that for other drugs-of-abuse and this has deterred studies on the neurobiological basis for their reinforcing effects. Toluene is a component of many of the commercial products used for solvent-abuse and has been the focus of much of the existing animal literature relating to this topic. The acute behavioral effects of toluene-intoxication in animals have been well documented and include ataxic gait, biphasic effects on motor activity and anxiolytic actions (Garriott et al., 1981; King, 1982; Wood et al., 1984; Balster, 1998; Bowen et al., 1999). In drugdiscrimination studies toluene shows cross-generalization with ethanol and barbiturates (Rees et al., 1985, 1987; Knisely et al., 1990). Animal models of the reinforcing effects of toluene have been studied in several diverse studies using both inhaled and intravenous routes of administration (Weiss et al., 1979; Riegel and French, 2002; Bespalov et al., 2003; Blokhina et al., 2004). We chose to model the inhaled route of administration, as this is directly relevant to the route of administration characteristic of recreational use. In our own studies (Gerasimov et al., 2003; Lee et al., 2004) and those by Yavich et al. (1994) and
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D.E. Lee et al. / Drug and Alcohol Dependence 85 (2006) 87–90
Funada et al. (2002), inhaled toluene produces a positive place preference in the conditioned place preference (CPP) model for drug-seeking behavior. The purpose of the present investigation was to optimize the paradigm for producing CPP to toluene vapors in rats by investigating the effect of extra conditioning days and a broader range of toluene concentrations on the degree of place preference.
For the place preference test, animals were placed in the middle compartment for 5 min with the guillotine doors closed for an initial acclimation. Subsequently, the animals were allowed free access to all three compartments for 15 min (900 s) and the time spent in each compartment of the box was digitally recorded.
Animal procedures were in accordance with the NIH guide for the care and use of all laboratory animals. Male Sprague–Dawley rats (150–250 g; Taconic Farms, Germantown, NY) were housed in pairs on a 12 h light/dark cycle.
2.2.3. Statistical analysis. Place preference on the test days were expressed using a preference score, representing the mean time spent in toluene-paired chamber minus the mean time spent in air-paired chamber (in seconds). For the determination of a significant place preference the preference scores were compared to the null hypothesis of a zero preference score using a pairedsample t-test. For determination of the effect of the number of conditioning trials on the place preference the 2000 and 3000 ppm preference scores were analyzed with a two-factor analysis of variance (ANOVA) with one factor being the number of conditioning trials and the second factor the concentration of toluene.
2.2. Conditioned place preference (CPP)
3. Results
2. Materials and methods 2.1. Subjects
2.2.1. Apparatus. The place preference box (ENV-013, MED Associates, Inc. St. Albans, VT) consisted of three distinct compartments separated by two guillotine doors. The walls of the middle chamber were gray (21 cm × 21 cm × 11 cm) with a smooth PVC floor while the outer conditioning compartments (21 cm × 21 cm × 27.5 cm, internal volume 12 L) had black walls with a smooth floor and white walls with a textured floor, respectively. Toluene vapors were generated by bubbling air through a flask filled with toluene. The vapors were delivered through an opening on the top of the side of both black and white compartments under positive pressure, and the atmosphere was exhausted through an opening at the bottom of both chambers. The toluene-saturated air was then mixed with filtered laboratory air in defined ratios set by computercontrolled flow regulators (Dyna-Blender, Matheson, PA), with a total flow rate set at 2 L/min. Before experiments with toluene were carried out toluene vapor was introduced at 2 L/min for at least 1 h so that the chamber volume (∼12 L) was exchanged a minimum of 10 times. The toluene concentration in the chambers for specific toluene:air ratios was determined in our previous study (Gerasimov et al., 2003) by taking air samples from the chambers and assaying toluene levels using a capillary gas chromatograph and flame ionization detector. All experiments were performed in a temperature-controlled room and CPP boxes were located in fume hoods. Time spent by the rat in each side of the box was automatically recorded by infrared photocells interfaced with MED-PC for Windows Version IV and Delphi TM 4 (SOF-735; MedAssociates). 2.2.2. Procedure. These studies consisted of a pre-conditioning phase, a conditioning phase and a testing phase. During the first three pre-conditioning days the animals were handled and acclimated to the test room. On the fourth pre-conditioning day the animals were allowed to freely explore all three (white/gray/black) compartments of the CPP box for 15 min and the time spent in each compartment was recorded. Using these chambers we have previously found that rats exposed to air on both sides (no toluene) do not exhibit an overall preference for either black or white sides on the test day (Gerasimov et al., 2003). However, they do spend between 30 and 50% of their time in the central corridor. This may be related to the fact that this chamber is about a quarter of the size of the two larger, more open, conditioning chambers. For the conditioning phase rats were assigned to receive toluene vapors (800, 2000, 3000 or 5000 ppm) in their least preferred compartment and air in the opposite compartment. The flow of vapors was initiated at least an hour prior to the beginning of the exposure to allow for chamber concentrations to equilibrate. Cage mates were exposed to toluene and air in pairs, so that on any given conditioning day both sides of the apparatus were filled with either air or toluene vapors. Conditioning sessions were performed with animal confined to either side for 30 min with both doors closed. Tests of CPP were conducted 24 h after the completion of each conditioning phase, in the absence of any toluene in the exposure chamber. Conditioning phases consisted of six pairing sessions (six air and six toluene exposures), conducted over 2 weeks. At 2000 and 3000 ppm conditioning was continued for an additional 6 sessions, giving 12 conditioning sessions over 4 weeks.
In the pre-conditioning tests animals showed no bias to either conditioning chamber with approximately equal time spent in the black and white sides of the box (black: 271 ± 10 s; white: 306 ± 4 s, p not significant (t-test)). As observed in our previous study (Gerasimov et al., 2003), the animals showed most preference for the central choice chamber (mean of 323 s). This may be related to the relatively enclosed space in this compartment compared to the larger, more open, pairing chambers. Animals conditioned with six pairings of 800 ppm toluene vapors exhibited no overall place preference with equal time spent in toluene- and air-paired chamber (Fig. 1). However, animals conditioned with 2000 and 3000 ppm toluene spent significantly (p < 0.05) more time on the toluene-paired side than on the air-paired side. No overall place preference was observed at the highest toluene concentration of 5000 ppm. Extending the number of toluene pairings at the 2000 and 3000 ppm level of toluene to 12 significantly enhanced the time spent in the toluene-paired chamber over the air-paired chamber compared to that obtained following six pairings ((F(1,60) = 11.2, p < 0.01 for effect of pairings on place preference using a two-factor ANOVA, with one factor representing the number of conditioning pairings and one factor the toluene concentration).
Fig. 1. Place preference following 6 or 12 conditioning pairings of inhaled toluene at the concentrations indicated. Data are mean differences (±S.E.M.) between the times spent in the toluene- and air-paired sides of the CPP apparatus from 8 to 24 rats; * p < 0.05, ** p < 0.001 vs. null hypothesis of zero preference (paired t-test).
D.E. Lee et al. / Drug and Alcohol Dependence 85 (2006) 87–90
4. Discussion Rats will show a CPP for a variety of drugs that are abused by humans (Tzschentke, 1998; Bardo and Bevins, 2000). Cocaine, amphetamines, and opiates produce especially robust place preferences (Carr and White, 1986; Nomikos and Spyraki, 1988). Alcohol and nicotine will also produce conditioned place preferences (Shoaib et al., 1994; Risinger and Oakes, 1996). The data in the present study confirms previous reports (Yavich et al., 1994; Funada et al., 2002; Gerasimov et al., 2003; Lee et al., 2004), that animals will show CPP to toluene vapors, a commonly abused organic solvent present in household products. In the current study, we examined several concentrations of toluene and a varying number of conditioning trials on the degree of CPP. Overall, 12 pairings produced the strongest CPP and the optimal toluene concentration was 2000–3000 ppm. With higher levels of toluene the CPP was not present, perhaps because the animal was too sedated to be aware of its surroundings. However, it should be noted that in our previous study (Gerasimov et al., 2003) a significant CPP was apparent at a 5000 ppm concentration of toluene. In this study, two out of eight animals expressed a CPP at this concentration, representing a smaller proportion than our previous study, where four out of eight animals preferred the toluene-paired chamber (Gerasimov et al., 2003). Taken together it appears that 5000 ppm may be toward the high end of the well-established inverted u-shaped concentration-response for toluene-induced changes in behavior (Moser and Balster, 1981; Himnan, 1984; Funada et al., 2002). The lowest concentration of toluene tested, 800 ppm, did not produce a CPP in the current study, and a similar lack of effect of this concentration of toluene in producing CPP was observed in our previous study (Gerasimov et al., 2003). The fact that a CPP was not obtained at this lower concentration is of significance since even this low concentration of toluene has a strong odor. Therefore, the observed place preference to higher levels of toluene is not simply due to the animals’ response to the toluene odor. The toluene concentrations that produced a CPP in the present study are similar to those documented to produce behavioral and neurochemical effects in rodents. Examples include enhancement of electrical brain self stimulation behavior (Bespalov et al., 2003) changes in motor activity (Moser and Balster, 1985; Bowen and Balster, 1998a,b), anxiolytic and antipunishment effects (Wood et al., 1984), and increases in dopamine release in several brain regions (Stengard et al., 1994; Gerasimov et al., 2002). Higher toluene concentrations, in the range of 8000 ppm and above, produce anesthesia-like effects and strong sedation (Bowen and Balster, 1998a,b; Beyer et al., 2001; D. Lee and A.N. Gifford, unpublished observations). The inverted-u-shaped response to the reinforcing salience of inhaled toluene observed in the current study is also consistent with toluene-induced changes in other behaviors, where low doses are stimulatory and higher doses depress locomotion (Hinman, 1987; Riegel and French, 1999) and operant behaviors (Evans and Balster, 1991; Bowen and Balster, 1998a,b).
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PET studies in our own lab (Gerasimov et al., 2002) using [11 C]toluene showed a rapid uptake of [11 C]toluene into the brain after intravenous administration, with maximum uptake into brain regions in both primates and rodents at 1–3 min. Such a rapid rise in brain concentrations following systemic administration appears to be a common feature of many of drugs-of-abuse (Volkow et al., 1996; Gatley et al., 1997; Quinn et al., 1997) and thus may be related to the abuse liability of toluene. Although in the present study brain levels of toluene during the toluene exposure were not measured, toluene levels in the blood and brain of rats after respiratory exposure have been documented, albeit at different concentrations, by Benignus et al. (1984) and blood to breath ratios documented by Garriott et al. (1981). In conclusion our data confirms and extends previous findings that rats show a positive CPP response to toluene, when given at appropriate concentrations. Acknowledgements Supported by National Institute of Health grant DA017349 and performed under Brookhaven Science Associates contract No. DE-AC02-98CH10886 with the U.S. Department of Energy. References Andersen, C.E., Loomis, G.A., 2003. Recognition and prevention of inhalant abuse. Am. Fam. Physician 68, 869–874. Balster, R.L., 1998. Neural basis of inhalant abuse. Drug Alcohol Depend. 51, 207–214. Bardo, M.T., Bevins, R.A., 2000. Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology (Berl.) 153, 31–43. Basu, D., Jhirwal, O.P., Singh, J., Kumar, S., Mattoo, S.K., 2004. Inhalant abuse by adolescents: a new challenge for Indian physicians. Ind. J. Med. Sci. 58, 245–249. Benignus, V.A., Muller, K.E., Graham, J.A., Barton, C.N., 1984. Toluene levels in blood and brain of rats as a function of toluene level in inspired air. Environ. Res. 33, 39–46. Bespalov, A., Sukhotina, I., Medvedev, I., Malyshkin, A., Belozertseva, I., Balster, R., Zvartau, E., 2003. Facilitation of electrical brain self-stimulation behavior by abused solvents. Pharmacol. Biochem. Behav. 75, 199–208. Beyer, C.E., Stafford, D., LeSage, M.G., Glowa, J.R., Steketee, J.D., 2001. Repeated exposure to inhaled toluene induces behavioral and neurochemical cross-sensitization to cocaine in rats. Psychopharmacology (Berl.) 154, 198–204. Blokhina, E.A., Dravolina, O.A., Bespalov, A.Y., Balster, R.L., Zvartau, E.E., 2004. Intravenous self-administration of abused solvents and anesthetics in mice. Eur. J. Pharmacol. 485, 211–218. Bowen, S.E., Balster, R.L., 1998a. A direct comparison of inhalant effects on locomotor activity and schedule-controlled behavior in mice. Exp. Clin. Psychopharmacol. 6, 235–247. Bowen, S.E., Balster, R.L., 1998b. The effects of inhaled isoparaffins on locomotor activity and operant performance in mice. Pharmacol. Biochem. Behav. 61, 271–280. Bowen, S.E., Wiley, J.L., Jones, H.E., Balster, R.L., 1999. Phencyclidine- and diazepam-like discriminative stimulus effects of inhalants in mice. Exp. Clin. Psychopharmacol. 7, 28–37. Carr, G.D., White, N.M., 1986. Anatomical disassociation of amphetamine’s rewarding and aversive effects: an intracranial microinjection study. Psychopharmacology (Berl.) 89, 340–346. Evans, E.B., Balster, R.L., 1991. CNS depressant effects of volatile organic solvents. Neurosci. Biobehav. Rev. 15, 233–241.
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Funada, M., Sato, M., Makino, Y., Wada, K., 2002. Evaluation of rewarding effect of toluene by the conditioned place preference procedure in mice. Brain Res. Protoc. 10, 47–54. Gatley, S.J., Volkow, N.D., Gifford, A.N., Ding, Y.S., Logan, J., Wang, G.J., 1997. Model for estimating dopamine transporter occupancy and subsequent increases in synaptic dopamine using positron emission tomography and carbon-11-labeled cocaine. Biochem. Pharmacol. 53, 43–52. Garriott, J.C., Foerster, E., Juarez, L., de la Garza, F., Mendiola, I., Curoe, J., 1981. Measurement of toluene in blood and breath in cases of solvent abuse. Clin. Toxicol. 18, 471–479. Gerasimov, M.R., Collier, L., Ferrieri, A., Alexoff, D., Lee, D., Gifford, A.N., Balster, R.L., 2003. Toluene inhalation produces a conditioned place preference in rats. Eur. J. Pharmacol. 477, 45–52. Gerasimov, M.R., Schiffer, W.K., Marstellar, D., Ferrieri, R., Alexoff, D., Dewey, S.L., 2002. Toluene inhalation produces regionally specific changes in extracellular dopamine. Drug Alcohol Depend. 65, 243– 251. Himnan, D., 1984. Tolerance and reverse tolerance to toluene inhalation: effects on open-field behavior. Pharmacol. Biochem. Behav. 21, 625– 631. Hinman, D.J., 1987. Biphasic dose–response relationship for effects of toluene inhalation on locomotor activity. Pharmacol. Biochem. Behav. 26, 65–69. King, M.D., 1982. Neurological sequelae of toluene abuse. Hum. Toxicol. 1, 281–287. Knisely, J.S., Rees, D.C., Balster, R.L., 1990. Discriminative stimulus properties of toluene in the rat. Neurotoxicol. Teratol. 12, 129–133. Lee, D.E., Schiffer, W.K., Dewey, S.L., 2004. Gamma-vinyl GABA (vigabatrin) blocks the expression of toluene-induced conditioned place preference (CPP). Synapse 54, 183–185. Moser, V.C., Balster, R.L., 1981. The effects of acute and repeated toluene exposure on operant behavior in mice. Neurobehav. Toxicol. Teratol. 3, 471–475. Moser, V.C., Balster, R.L., 1985. Acute motor and lethal effects of inhaled toluene, 1,1,1-trichloroethane, halothane, and ethanol in mice: effects of exposure duration. Toxicol. Appl. Pharmacol. 77, 285– 291. Nomikos, G.G., Spyraki, C., 1988. Cocaine-induced place conditioning: importance of route of administration and other procedural variables. Psychopharmacology (Berl.) 94, 119–125.
Quinn, D.I., Wodak, A., Day, R.O., 1997. Pharmacokinetic and pharmacodynamic principles of illicit drug use and treatment of illicit drug users. Clin. Pharmacokinet. 33, 344–400. Rees, D.C., Coggeshall, E., Balster, R.L., 1985. Inhaled toluene produces pentobarbital-like discriminative stimulus effects in mice. Life Sci. 37, 1319–1325. Rees, D.C., Knisely, J.S., Breen, T.J., Balster, R.L., 1987. Toluene, halothane, 1,1,1-trichloroethane and oxazepam produce ethanol-like discriminative stimulus effects in mice. J. Pharmacol. Exp. Ther. 243, 931–937. Riegel, A.C., French, E.D., 1999. Acute toluene induces biphasic changes in rat spontaneous locomotor activity which are blocked by remoxipride. Pharmacol. Biochem. Behav. 62, 399–402. Riegel, A.C., French, E.D., 2002. Abused inhalants and central reward pathways: electrophysiological and behavioral studies in the rat. Ann. N. Y. Acad. Sci. 965, 281–291. Risinger, F.O., Oakes, R.A., 1996. Dose- and conditioning trial-dependent ethanol-induced conditioned place preference in Swiss–Webster mice. Pharmacol. Biochem. Behav. 55, 117–123. Shoaib, M., Stolerman, I.P., Kumar, R.C., 1994. Nicotine-induced place preferences following prior nicotine exposure in rats. Psychopharmacology (Berl.) 113, 445–452. 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., 1998. Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues. Prog. Neurobiol. 56, 613–672. Volkow, N.D., Wang, G.J., Gatley, S.J., Fowler, J.S., Ding, Y.S., Logan, J., Hitzemann, R., Angrist, B., Lieberman, J., 1996. Temporal relationships between the pharmacokinetics of methylphenidate in the human brain and its behavioral and cardiovascular effects. Psychopharmacology (Berl.) 123, 26–33. Weiss, B., Wood, R.W., Macys, D.A., 1979. Behavioral toxicology of carbon disulfide and toluene. Environ. Health Perspect. 30, 39–45. 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.
Short communication
Concentration-dependent conditioned place preference to inhaled toluene vapors in rats Dianne E. Lee b , Madina R. Gerasimov a , Wynne K Schiffer b , Andrew N. Gifford a,∗ a b
Medical Department, Brookhaven National Laboratory, Upton, NY 11973, United States Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973, United States
Received 29 August 2005; received in revised form 13 March 2006; accepted 17 March 2006
Abstract Objectives: Toluene is present in many commercial products and is subject to abuse by inhalation. The goal of this study was to extend previous reports indicating that rats will exhibit a positive conditioned place preference to inhaled toluene vapors and to determine the dose–response relationship for inhaled toluene in terms of exposure concentration and number of exposures. For the conditioned place preference experiments rats were exposed to toluene vapors at concentrations of 800, 2000, 3000 or 5000 ppm in one compartment of a three-compartment box. Results: Following six conditioning sessions with toluene, a significant place preference was obtained at 2000 and 3000 ppm, but not at 800 or 5000 ppm. Extending the number of toluene pairings at the 2000 and 3000 ppm concentration to 12 significantly enhanced the place preference compared to that at six pairings. Conclusions: These experiments extend our previous finding that rats will show a conditioned place preference to inhaled toluene, and indicate that a reinforcing “dose” of toluene depends on both the concentration and number of pairings. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Toluene; Inhalant abuse; Conditioned place preference; Rats; Drugs-of-abuse
1. Introduction Voluntary inhalation of volatile solvents found in inexpensive commercial products such as glues, paint products, cleaning fluids and lighter fluids is prevalent in industrialized cities throughout the world, especially amongst juveniles and adolescent children (Andersen and Loomis, 2003; Basu et al., 2004). However, despite the widespread nature of solvent-abuse, also termed “glue-sniffing” there have been relatively few studies aimed at developing animal models for the abuse of these substances, especially when compared with the extensive literature on animals models for the reinforcing properties of cocaine, opiates and alcohol (Tzschentke, 1998; Bardo and Bevins, 2000). Part of the explanation for this may be related to the fact that these solvents are abused by inhalation, making them hard to administer to animals in controlled amounts. Additionally, the CNS target site for abused-solvents has been less well
∗
Corresponding author. Tel.: +1 631 344 7069; fax: +1 631 344 5311. E-mail address: [email protected] (A.N. Gifford).
0376-8716/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.drugalcdep.2006.03.013
defined than that for other drugs-of-abuse and this has deterred studies on the neurobiological basis for their reinforcing effects. Toluene is a component of many of the commercial products used for solvent-abuse and has been the focus of much of the existing animal literature relating to this topic. The acute behavioral effects of toluene-intoxication in animals have been well documented and include ataxic gait, biphasic effects on motor activity and anxiolytic actions (Garriott et al., 1981; King, 1982; Wood et al., 1984; Balster, 1998; Bowen et al., 1999). In drugdiscrimination studies toluene shows cross-generalization with ethanol and barbiturates (Rees et al., 1985, 1987; Knisely et al., 1990). Animal models of the reinforcing effects of toluene have been studied in several diverse studies using both inhaled and intravenous routes of administration (Weiss et al., 1979; Riegel and French, 2002; Bespalov et al., 2003; Blokhina et al., 2004). We chose to model the inhaled route of administration, as this is directly relevant to the route of administration characteristic of recreational use. In our own studies (Gerasimov et al., 2003; Lee et al., 2004) and those by Yavich et al. (1994) and
88
D.E. Lee et al. / Drug and Alcohol Dependence 85 (2006) 87–90
Funada et al. (2002), inhaled toluene produces a positive place preference in the conditioned place preference (CPP) model for drug-seeking behavior. The purpose of the present investigation was to optimize the paradigm for producing CPP to toluene vapors in rats by investigating the effect of extra conditioning days and a broader range of toluene concentrations on the degree of place preference.
For the place preference test, animals were placed in the middle compartment for 5 min with the guillotine doors closed for an initial acclimation. Subsequently, the animals were allowed free access to all three compartments for 15 min (900 s) and the time spent in each compartment of the box was digitally recorded.
Animal procedures were in accordance with the NIH guide for the care and use of all laboratory animals. Male Sprague–Dawley rats (150–250 g; Taconic Farms, Germantown, NY) were housed in pairs on a 12 h light/dark cycle.
2.2.3. Statistical analysis. Place preference on the test days were expressed using a preference score, representing the mean time spent in toluene-paired chamber minus the mean time spent in air-paired chamber (in seconds). For the determination of a significant place preference the preference scores were compared to the null hypothesis of a zero preference score using a pairedsample t-test. For determination of the effect of the number of conditioning trials on the place preference the 2000 and 3000 ppm preference scores were analyzed with a two-factor analysis of variance (ANOVA) with one factor being the number of conditioning trials and the second factor the concentration of toluene.
2.2. Conditioned place preference (CPP)
3. Results
2. Materials and methods 2.1. Subjects
2.2.1. Apparatus. The place preference box (ENV-013, MED Associates, Inc. St. Albans, VT) consisted of three distinct compartments separated by two guillotine doors. The walls of the middle chamber were gray (21 cm × 21 cm × 11 cm) with a smooth PVC floor while the outer conditioning compartments (21 cm × 21 cm × 27.5 cm, internal volume 12 L) had black walls with a smooth floor and white walls with a textured floor, respectively. Toluene vapors were generated by bubbling air through a flask filled with toluene. The vapors were delivered through an opening on the top of the side of both black and white compartments under positive pressure, and the atmosphere was exhausted through an opening at the bottom of both chambers. The toluene-saturated air was then mixed with filtered laboratory air in defined ratios set by computercontrolled flow regulators (Dyna-Blender, Matheson, PA), with a total flow rate set at 2 L/min. Before experiments with toluene were carried out toluene vapor was introduced at 2 L/min for at least 1 h so that the chamber volume (∼12 L) was exchanged a minimum of 10 times. The toluene concentration in the chambers for specific toluene:air ratios was determined in our previous study (Gerasimov et al., 2003) by taking air samples from the chambers and assaying toluene levels using a capillary gas chromatograph and flame ionization detector. All experiments were performed in a temperature-controlled room and CPP boxes were located in fume hoods. Time spent by the rat in each side of the box was automatically recorded by infrared photocells interfaced with MED-PC for Windows Version IV and Delphi TM 4 (SOF-735; MedAssociates). 2.2.2. Procedure. These studies consisted of a pre-conditioning phase, a conditioning phase and a testing phase. During the first three pre-conditioning days the animals were handled and acclimated to the test room. On the fourth pre-conditioning day the animals were allowed to freely explore all three (white/gray/black) compartments of the CPP box for 15 min and the time spent in each compartment was recorded. Using these chambers we have previously found that rats exposed to air on both sides (no toluene) do not exhibit an overall preference for either black or white sides on the test day (Gerasimov et al., 2003). However, they do spend between 30 and 50% of their time in the central corridor. This may be related to the fact that this chamber is about a quarter of the size of the two larger, more open, conditioning chambers. For the conditioning phase rats were assigned to receive toluene vapors (800, 2000, 3000 or 5000 ppm) in their least preferred compartment and air in the opposite compartment. The flow of vapors was initiated at least an hour prior to the beginning of the exposure to allow for chamber concentrations to equilibrate. Cage mates were exposed to toluene and air in pairs, so that on any given conditioning day both sides of the apparatus were filled with either air or toluene vapors. Conditioning sessions were performed with animal confined to either side for 30 min with both doors closed. Tests of CPP were conducted 24 h after the completion of each conditioning phase, in the absence of any toluene in the exposure chamber. Conditioning phases consisted of six pairing sessions (six air and six toluene exposures), conducted over 2 weeks. At 2000 and 3000 ppm conditioning was continued for an additional 6 sessions, giving 12 conditioning sessions over 4 weeks.
In the pre-conditioning tests animals showed no bias to either conditioning chamber with approximately equal time spent in the black and white sides of the box (black: 271 ± 10 s; white: 306 ± 4 s, p not significant (t-test)). As observed in our previous study (Gerasimov et al., 2003), the animals showed most preference for the central choice chamber (mean of 323 s). This may be related to the relatively enclosed space in this compartment compared to the larger, more open, pairing chambers. Animals conditioned with six pairings of 800 ppm toluene vapors exhibited no overall place preference with equal time spent in toluene- and air-paired chamber (Fig. 1). However, animals conditioned with 2000 and 3000 ppm toluene spent significantly (p < 0.05) more time on the toluene-paired side than on the air-paired side. No overall place preference was observed at the highest toluene concentration of 5000 ppm. Extending the number of toluene pairings at the 2000 and 3000 ppm level of toluene to 12 significantly enhanced the time spent in the toluene-paired chamber over the air-paired chamber compared to that obtained following six pairings ((F(1,60) = 11.2, p < 0.01 for effect of pairings on place preference using a two-factor ANOVA, with one factor representing the number of conditioning pairings and one factor the toluene concentration).
Fig. 1. Place preference following 6 or 12 conditioning pairings of inhaled toluene at the concentrations indicated. Data are mean differences (±S.E.M.) between the times spent in the toluene- and air-paired sides of the CPP apparatus from 8 to 24 rats; * p < 0.05, ** p < 0.001 vs. null hypothesis of zero preference (paired t-test).
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4. Discussion Rats will show a CPP for a variety of drugs that are abused by humans (Tzschentke, 1998; Bardo and Bevins, 2000). Cocaine, amphetamines, and opiates produce especially robust place preferences (Carr and White, 1986; Nomikos and Spyraki, 1988). Alcohol and nicotine will also produce conditioned place preferences (Shoaib et al., 1994; Risinger and Oakes, 1996). The data in the present study confirms previous reports (Yavich et al., 1994; Funada et al., 2002; Gerasimov et al., 2003; Lee et al., 2004), that animals will show CPP to toluene vapors, a commonly abused organic solvent present in household products. In the current study, we examined several concentrations of toluene and a varying number of conditioning trials on the degree of CPP. Overall, 12 pairings produced the strongest CPP and the optimal toluene concentration was 2000–3000 ppm. With higher levels of toluene the CPP was not present, perhaps because the animal was too sedated to be aware of its surroundings. However, it should be noted that in our previous study (Gerasimov et al., 2003) a significant CPP was apparent at a 5000 ppm concentration of toluene. In this study, two out of eight animals expressed a CPP at this concentration, representing a smaller proportion than our previous study, where four out of eight animals preferred the toluene-paired chamber (Gerasimov et al., 2003). Taken together it appears that 5000 ppm may be toward the high end of the well-established inverted u-shaped concentration-response for toluene-induced changes in behavior (Moser and Balster, 1981; Himnan, 1984; Funada et al., 2002). The lowest concentration of toluene tested, 800 ppm, did not produce a CPP in the current study, and a similar lack of effect of this concentration of toluene in producing CPP was observed in our previous study (Gerasimov et al., 2003). The fact that a CPP was not obtained at this lower concentration is of significance since even this low concentration of toluene has a strong odor. Therefore, the observed place preference to higher levels of toluene is not simply due to the animals’ response to the toluene odor. The toluene concentrations that produced a CPP in the present study are similar to those documented to produce behavioral and neurochemical effects in rodents. Examples include enhancement of electrical brain self stimulation behavior (Bespalov et al., 2003) changes in motor activity (Moser and Balster, 1985; Bowen and Balster, 1998a,b), anxiolytic and antipunishment effects (Wood et al., 1984), and increases in dopamine release in several brain regions (Stengard et al., 1994; Gerasimov et al., 2002). Higher toluene concentrations, in the range of 8000 ppm and above, produce anesthesia-like effects and strong sedation (Bowen and Balster, 1998a,b; Beyer et al., 2001; D. Lee and A.N. Gifford, unpublished observations). The inverted-u-shaped response to the reinforcing salience of inhaled toluene observed in the current study is also consistent with toluene-induced changes in other behaviors, where low doses are stimulatory and higher doses depress locomotion (Hinman, 1987; Riegel and French, 1999) and operant behaviors (Evans and Balster, 1991; Bowen and Balster, 1998a,b).
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PET studies in our own lab (Gerasimov et al., 2002) using [11 C]toluene showed a rapid uptake of [11 C]toluene into the brain after intravenous administration, with maximum uptake into brain regions in both primates and rodents at 1–3 min. Such a rapid rise in brain concentrations following systemic administration appears to be a common feature of many of drugs-of-abuse (Volkow et al., 1996; Gatley et al., 1997; Quinn et al., 1997) and thus may be related to the abuse liability of toluene. Although in the present study brain levels of toluene during the toluene exposure were not measured, toluene levels in the blood and brain of rats after respiratory exposure have been documented, albeit at different concentrations, by Benignus et al. (1984) and blood to breath ratios documented by Garriott et al. (1981). In conclusion our data confirms and extends previous findings that rats show a positive CPP response to toluene, when given at appropriate concentrations. Acknowledgements Supported by National Institute of Health grant DA017349 and performed under Brookhaven Science Associates contract No. DE-AC02-98CH10886 with the U.S. Department of Energy. References Andersen, C.E., Loomis, G.A., 2003. Recognition and prevention of inhalant abuse. Am. Fam. Physician 68, 869–874. Balster, R.L., 1998. Neural basis of inhalant abuse. Drug Alcohol Depend. 51, 207–214. Bardo, M.T., Bevins, R.A., 2000. Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology (Berl.) 153, 31–43. Basu, D., Jhirwal, O.P., Singh, J., Kumar, S., Mattoo, S.K., 2004. Inhalant abuse by adolescents: a new challenge for Indian physicians. Ind. J. Med. Sci. 58, 245–249. Benignus, V.A., Muller, K.E., Graham, J.A., Barton, C.N., 1984. Toluene levels in blood and brain of rats as a function of toluene level in inspired air. Environ. Res. 33, 39–46. Bespalov, A., Sukhotina, I., Medvedev, I., Malyshkin, A., Belozertseva, I., Balster, R., Zvartau, E., 2003. Facilitation of electrical brain self-stimulation behavior by abused solvents. Pharmacol. Biochem. Behav. 75, 199–208. Beyer, C.E., Stafford, D., LeSage, M.G., Glowa, J.R., Steketee, J.D., 2001. Repeated exposure to inhaled toluene induces behavioral and neurochemical cross-sensitization to cocaine in rats. Psychopharmacology (Berl.) 154, 198–204. Blokhina, E.A., Dravolina, O.A., Bespalov, A.Y., Balster, R.L., Zvartau, E.E., 2004. Intravenous self-administration of abused solvents and anesthetics in mice. Eur. J. Pharmacol. 485, 211–218. Bowen, S.E., Balster, R.L., 1998a. A direct comparison of inhalant effects on locomotor activity and schedule-controlled behavior in mice. Exp. Clin. Psychopharmacol. 6, 235–247. Bowen, S.E., Balster, R.L., 1998b. The effects of inhaled isoparaffins on locomotor activity and operant performance in mice. Pharmacol. Biochem. Behav. 61, 271–280. Bowen, S.E., Wiley, J.L., Jones, H.E., Balster, R.L., 1999. Phencyclidine- and diazepam-like discriminative stimulus effects of inhalants in mice. Exp. Clin. Psychopharmacol. 7, 28–37. Carr, G.D., White, N.M., 1986. Anatomical disassociation of amphetamine’s rewarding and aversive effects: an intracranial microinjection study. Psychopharmacology (Berl.) 89, 340–346. Evans, E.B., Balster, R.L., 1991. CNS depressant effects of volatile organic solvents. Neurosci. Biobehav. Rev. 15, 233–241.
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