- Email: [email protected]
Evaluation of rewarding effect of toluene by the conditioned place preference procedure in mice
Brain Research Protocols 10 (2002) 47–54 www.elsevier.com / locate / bres
Protocol
Evaluation of rewarding effect of toluene by the conditioned place preference procedure in mice Masahiko Funada a , *, Mio Sato a , Yukiko Makino b , Kiyoshi Wada a a
Section of Addictive Drugs Research, Division of Drug Dependence, National Institute of Mental Health, National Center of Neurology and Psychiatry, 1 -7 -3 Kohnodai, Ichikawa, Chiba 272 -0827, Japan b Narcotics Control Department, Kanto-Shin’ etsu Bureau of Health and Welfare, Ministry of Health, Labour and Welfare, 2 -4 -14 Nakameguro, Meguro-ku, Tokyo, 153 -0061, Japan Accepted 7 August 2002
Abstract Toluene and many toluene-containing products are abused via inhalation. Previous investigations have used the place preference paradigm to evaluate the rewarding effects of commonly abused drugs such as morphine, cocaine, and amphetamine. A conditioning paradigm of toluene inhalation was developed in order to estimate the rewarding effect in mice. Conditioning sessions (five for toluene, five for air) were conducted twice daily for 5 days using a newly developed airtight inhalation shuttlebox (15330315 cm: w3l3h), which was divided into two compartments of equal size. One compartment was white with a textured floor, and the other was black with a smooth floor. All conditioning sessions were 20 min in duration, with a minimum of 7 h between sessions. Test sessions were carried out 1 day after the final training session with mice in a drug-free state. The time spent in each compartment during a 20-min session was measured using a digital video camera. Exposure to toluene vapors (700–3200 ppm) produced a significant conditioned place preference in mice. These results suggest that the conditioned place preference procedure using the newly developed airtight inhalation shuttlebox constitutes an important tool for studying the rewarding effect of abused solvents. 2002 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Motivation and emotion Keywords: Abuse; Conditioned place preference; Inhalants; Reward; Toluene
1. Type of research • • • •
Drug effects on behavior Shuttlebox for exposure to toluene vapors Study of conditioned place preference Evaluation of toluene concentration in the brain
2. Time required The time required for the conditioned place preference
procedure using two sets of the inhalation shuttlebox and 54 mice is 21 days.
2.1. Conditioned place preference procedure: 6 days Duration of each step: • Conditioning procedure: twice a day (one for toluene, one for air) for 5 days • Each conditioning duration: 20 min • Test session: 20 min
2.2. Tissue preparation for measurement of toluene concentration: 40 min *Corresponding author. Tel.: 181-47-372-0141; fax: 181-47-3712900. E-mail address: [email protected] (M. Funada).
• Inhalation of toluene: 20 min • Brain removal and tissue homogenizing: 10 min • Extraction of toluene: 10 min
1385-299X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S1385-299X( 02 )00182-4
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
48
2.3. Evaluation of toluene concentration gas chromatography: 30 min • Column preparation: 20 min • Sample analysis: 10 min
3. Materials
3.1. Animals Male ICR mice (20–25 g) were obtained from Tokyo Animal Laboratories (Tokyo, Japan). The mice were maintained on a 12-h light, 12-h dark schedule, and laboratory mouse chow and water were provided ad libitum. All procedures were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, approved by the Japanese Pharmacological Society.
3.2. Special equipment The system for conditioned place preference by inhal-
ants consists of the following equipment (Neuroscience Co., Tokyo, Japan; Fig. 1A). • An airtight inhalation shuttlebox for conditioned place preference (15330315 cm: w3l3h, Neuroscience Co., Ltd., Tokyo, Japan) • Air pump (HPa10000 model; Nisso Co., Ltd., Tokyo, Japan) • Gas wash bottle (Muenke model, 500 ml, Sansyo Co., Ltd., Tokyo, Japan) • Gas flowmeter (RK-200V model; Sansyo Co., Ltd., Tokyo, Japan) • Thermostatic water bath (T2 model; Sansyo Co., Ltd., Tokyo, Japan) The concentrations of toluene in the shuttlebox and in the brain tissue were measured by a capillary gas chromatography (GC) system with a flame ionization detector (FID). • GC system with FID (HP6890; Hewlett Packard) • Capillary column (HP5, 30 m30.32 mm, i.d., 0.25mm film thickness)
Fig. 1. A newly developed airtight inhalation shuttlebox for establishing the conditioned place preference paradigm. (A) The apparatus for exposure to toluene vapors. In order to obtain a stable temperature of toluene, the regulated volume of toluene (250 ml) in the gas wash bottle is evaporated at 35 8C. Using an air pump, the vapor of toluene is delivered to the inside of the airtight shuttlebox at a uniform volume. (B) Photograph of the novel airtight inhalation shuttlebox with gas flowmeter for establishing the conditioned place preference paradigm during conditioning session. (C) Photograph of the inhalation shuttlebox with flowmeter for establishing the conditioned place preference paradigm during test session.
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
3.3. Chemicals and reagents Toluene and n-hexane were purchased from Wako (Tokyo, Japan).
4. Detailed procedure
4.1. Measurement of toluene concentration in the shuttlebox Standard line: toluene vapor concentrations were calculated using the ideal gas law: pV 5 0.082 n(t 1 273) where p, pressure; V, volume; n, mol; t, temperature. Gas chromatography (GC) analysis was carried out using a GC system (HP6890; Hewlett Packard) with a capillary column (HP5, 30 m30.32 mm, i.d., 0.25-mm film thickness). The column oven temperature was held at 25 8C for 10 min. The temperature of the injector port was set at 250 8C. A split injection mode was used (ratio5 3.3:1). The carrier gas (helium) flow rate was 1.8 ml / min. Using a gas-tight syringe (81317; Hamilton, NV, USA), 100 ml of the air sample containing standard toluene was taken initially from the exhaust valve in the lid of the shuttlebox for analysis. The detection limit of toluene concentration in the air sample was 3 ppm. The relationship of the standard line of the toluene concentration in standard samples (100–5600 ppm) to the peak high value from gas chromatography analysis was established (Fig. 2A). Based on this standard line, the relationship between toluene concentration and toluene outflow was analyzed.
4.1.1. Toluene vapor outflow–toluene concentration relationship Outflow of toluene vapors (0.1–0.8 l / min) was controlled by an air pump. After initiation of exposure of toluene vapors, for purposes of analysis, 100 ml of the air sample containing toluene was taken from the exhaust valve, which is attached to the lid of the shuttlebox, using a gas-tight syringe (81317; Hamilton). 4.2. Measurement of toluene concentration in brain tissue Mice were sacrificed by decapitation following a 20-min exposure to toluene vapors. The whole brain was removed, weighed, and immediately homogenized with a tissue grinder in 300 ml desalting water on ice. These homogenates were extracted using 300 ml n-hexane. Homogenates were centrifuged at 15 000 rev. / min for 5 min at 4 8C, and 200 ml of each supernatant was collected. GC analysis was performed using a GC system (HP6890; Hewlett Packard) with a capillary column (HP5, 30 m30.32 mm, i.d., 0.25-mm film thickness). The column oven temperature was held at 30 8C for 10 min. The temperatures of the injector port and the interface
49
were set at 250 and 280 8C, respectively. A splitless injection mode was used. The carrier gas (helium) flow rate was 4.7 ml / min. Using a glass syringe, 1 ml of the sample was injected. The relationship of the standard line of the toluene concentration in standard samples (1–85 ng) to peak height was established (Fig. 3A). Based on this standard line, toluene concentrations in the whole brain were calculated.
4.3. Conditioned place preference procedure Conditioning sessions (five for toluene, five for air) were conducted twice daily for 5 days using a novel airtight inhalation shuttlebox (15330315 cm: w3l3h), which was divided into two equal-sized compartments by a sliding wall (Fig. 1B). One compartment was white with a textured floor, and the other was black with a smooth floor. The front wall of each compartment is a transparent plastic wall. The treatment compartment of the shuttlebox and the order of exposure to toluene vapors and air were counterbalanced (Table 1). Following saturation of the compartments with different concentrations of toluene vapors for 30 min, mice were immediately placed in each compartment within 10 s after opening the lid. All conditioning sessions were 20 min in duration, and a minimum of 7 h separated exposure to toluene vapors and exposure to air. The central wall used for the conditioning session was replaced by a sliding wall in the shape of a T for each test session (Fig. 1C). Mice were allowed free access to both compartments (each was 636 cm: w3h), on either side of the sliding wall. Test sessions were carried out 1 day after the final training session with mice in a drug-free state. Mice were placed in the center of the shuttlebox, and allowed free access to both compartments. The total time spent in each compartment during a 20-min session was recorded by a digital video camera with an integrated stopwatch (DCR-PC100; Sony). Assignment to the white or black compartment was made on the basis of the position of the mouse’s head as determined by visual analysis of the recorded videotape. All sessions were conducted during the light phase of the light / dark cycle used, under conditions of dim illumination (18 lx).
4.4. Statistical analysis Statistical analyses were performed using commercially available software (StatView 5.0 for Macintosh; SAS Institute, Cary, NC, USA). Each toluene standard line was estimated using regression analysis. Conditioning scores represent the time spent in the toluene-paired compartment minus the time spent in the air-paired compartment, and are expressed as the mean6S.E.M. The concentration– response line was analyzed using a one-way random factorial analysis of variance (ANOVA). The Fisher LSD test was used to determine whether individual concen-
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M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
Fig. 2. Toluene concentrations in the shuttlebox. (A) Standard line of the toluene concentration–peak height value relationship. A linear relationship between the toluene concentration in the shuttlebox and the peak height value from gas chromatography analysis was established ( y50.8x1180.2; r50.992). (B) Standard line of the toluene concentration–toluene vapor outflow relationship. A linear relationship between the toluene concentration in the shuttlebox and the toluene outflow volumes was established ( y54085.1x2102.8, r50.999). (C) Time course changes in toluene concentration in the shuttlebox following exposure to toluene. The values in the small box (0.2, 0.4, 0.6 and 0.8) show the values of airflow (l / min) into the shuttlebox. Each point represents the mean6S.E.M. of six samples. (D) Time course changes in toluene concentration in the shuttlebox during conditioning session. After saturation with different concentrations of toluene vapors for 30 min, the lid of the shuttlebox was opened for 10 s to insert mice into the compartment. The values in the small box (0.2, 0.4, 0.6, and 0.8) show the values of airflow (l / min) into the shuttlebox. Each point represents the mean6S.E.M. of 16 samples.
trations produced a significant conditioning. The accepted level of significance for all tests was P,0.05.
5. Results
5.1. Toluene concentration in the shuttlebox As shown Fig. 2A, a linear relationship between peak height and toluene concentration in standard samples was
established ( y50.8x1180.2; r50.992). Using this standard line, the toluene concentration–toluene outflow relationship was analyzed. Fig. 2B shows the standard line of the toluene concentration–toluene vapor outflow relationship. A linear relationship between toluene concentration in the shuttlebox and toluene vapor outflow was established ( y54085.1x2102.8; r50.999). Fig. 2C shows the time course changes in toluene concentration in the shuttlebox following exposure to toluene vapors. After completion of the 30-min outflow of toluene vapors, the
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
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Fig. 3. Toluene concentrations in the whole brain. (A) Standard line of the toluene concentration–peak height relationship. The toluene concentration in the whole brain tissue and the peak height were significantly correlated ( y510.0x113.3, r50.999). (B) Effect of toluene inhalation on toluene levels in the whole brain tissue following 20-min inhalation at 700, 2500, and 3200 ppm, respectively. Each column represents the mean6S.E.M. of eight mice.
concentrations of toluene saturated the toluene vapor-outflow in a volume-dependent manner. Thirty minutes was therefore adopted as the duration of the saturation of toluene vapor period. Fig. 2D shows the time course changes in toluene concentration in the shuttlebox during a conditioning session. After saturation with different concentrations of toluene vapors for 30 min, the lid of the shuttlebox was opened for 10 s in order to insert mice into the compartments. These procedures did not significantly affect each saturated concentration of toluene.
brain tissue by detected peak height after a 20-min exposure to concentrations of 700, 2500, and 3200 ppm, respectively, following a 30-min pre-inhalation.
5.3. Conditioned place preference At the beginning of the experiments, there were no significant differences in mean body weight among the five groups of mice (air, 20.260.2 g; 350 ppm, 20.760.4 g; 700 ppm, 20.960.3 g; 2500 ppm, 20.160.2 g; 3200 ppm, ¨ mice exhibited no significant prefer21.060.7 g). Naıve ence for either compartment of the shuttlebox. The mean time spent in the white and black compartments was 642.7659.4 and 557.3660.8 s (n515), respectively. During the 20-min exposure to toluene vapors, mice displayed slight hyperactivity at concentrations of 2500 and 3200 ppm, though no convulsions or hypnotic effects were observed at any of the concentrations. Following exposure
5.2. Toluene concentration in the brain The standard line of the toluene concentration–peak height relationship is shown in Fig. 3A. A linear relationship between peak height and toluene concentration in standard samples was established ( y510.0x113.3; r5 0.999). Fig. 3B shows the amount of toluene found in the
Table 1 Schedule of conditioned place preference 1
2
3
4
5
6 (test)
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
White side
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
Black side
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
Drug-free
Conditioning sessions (five for toluene: TOL; five for air: AIR) were conducted twice daily for 5 days using a newly developed airtight inhalation shuttlebox. One compartment was white with a textured floor, and the other was black with a smooth floor. The treatment compartment of the shuttle box, and the order of exposure to toluene vapors and air, were counterbalanced. After saturation of the shuttlebox with different concentrations of toluene vapors for 30 min, mice were immediately placed in each compartment. The control group was exposed only to air. All conditioning sessions were 20 min in duration, and a minimum of 7 h separated the exposure to toluene vapors and that to air. Test sessions were carried out 1 day after the final training session, with mice in a drug-free state. The time spent in each compartment during a 20-min session was recorded by a digital video camera (DCR-PC100; Sony).
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M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
to toluene vapors or air, and at the test session, groups did not significantly differ in mean body weight (air, 26.660.3 g; 350 ppm, 27.560.2 g; 700 ppm, 26.560.2 g; 2500 ppm, 26.960.4 g; 3200 ppm, 27.060.5 g). The air-exposed mice exhibited no preference. The mean conditioning score was 254.8666.4 s (n58). The place conditioning effect produced by the exposure to toluene is shown in Fig. 4. Toluene caused a significant preference for the drug-filled compartment (F1,51 59.18; P50.004). Toluene (350 ppm) induced a slight place preference (mean conditioning score of 53.6657.3 s; n510; P50.161), but this effect was not statistically significant. Significant conditioning scores were observed at doses of 700 ppm (mean conditioning score of 181.9656.6 s; n512; P50.003), 2500 ppm (mean conditioning score of 137.8626.3 s; n512; P5 0.012) and 3200 ppm (mean conditioning score of 149.5643.6 s; n512; P50.008). At these doses, all of the animals exposed to toluene exhibited a preference for the toluene-associated compartment.
6. Discussion
6.1. Assessment of the protocol The conditioned place preference procedure has proven useful for demonstrating the rewarding properties of drugs [1,4,6]. In this paradigm, a drug-free animal that has previously received pairings of a drug with a particular environment is conditioned to favor this environment over
a neutral environment. Thus, the first advantage of this procedure is that the rewarding effects of a drug are measured in animals that are drug-free. Therefore, any changes in unconditioned motor activity do not directly influence measurement of the dependent variable (time spent in the drug-paired place). Second, the short-term nature of the experiments (lasting on average from 1 to 3 weeks) and the sensitivity of the paradigm for demonstrating place conditioning (preference and aversion) provide other obvious advantages. For example, large numbers of compounds can be quickly tested using this technique, making it an excellent method for the initial screening of drug reward. One of the most difficult problems in repeated administration of pharmacological agents to any animal is the administration of volatile agents by inhalation. In order to obtain stable concentrations of toluene in the airtight shuttlebox, the regulated volume of toluene was evaporated at 35 8C and mixed with a larger volume of clean air to achieve the target concentrations. Using an air pump, the vapor of toluene was delivered to the inside of the airtight shuttlebox at a uniform value. The concentrations of toluene in the airtight shuttlebox reached plateau levels within 30 min, and were reliable and stable over the 20-min exposure period. Exposure to concentrations of toluene from 500 to 3200 ppm for 20 min resulted in brain levels of 4.7–44.7 ng / mg of tissue, confirming results in a previous report [3]. Our present results of assessment of brain toluene concentration are consistent with those in that report. The target concentrations of toluene in the airtight shuttlebox could be regulated by a novel vaporexposing system that included an air pump. The present technique of exposing animals to inhalant vapor by confinement in an airtight shuttlebox may be useful for the evaluation of abused inhalant-induced pharmacological actions in vivo. Using a novel conditioned place preference evaluation system, we observed that exposure to toluene vapors caused a conditioned place preference in mice. These results are consistent with those of previous studies on abused drugs using the conditioned place preference procedure. Thus, morphine, cocaine, and amphetamine have been shown to produce a strongly conditioned place preference in rats and mice [2,4,6]. The present protocol of the conditioned place preference procedure makes it possible to assess the rewarding effect of toluene by inhalation.
6.2. Trouble shooting Fig. 4. Place conditioning produced by exposure to toluene vapors in mice. Ordinate: mean difference (s) between the times spent in the toluene- and air-paired sides of the test box. Each point represents the mean6S.E.M. of 8–12 mice. None of the mice that inhaled air during the conditioning sessions (Controls) exhibited a significant preference for either compartment of the test box; the mean conditioning score was 254.8666.4 s (n58). *P,0.05 versus the air-paired control group.
Handling-associated stress should be minimized and equated across all treatment conditions, since differences in handling can influence the place preferences seen after conditioning [1]. In addition, after the conditioning session has ended, mice should not be returned to their home cages immediately, since toluene vapors will have adhered to
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
their hair and skin. Rather, they should be left beside their home cages for at least 30 min at room temperature. Toluene, benzene, ether, and other solvents have different flow values. Thus, the vapor values of the flow meter should be adjusted according to the standard value for toluene. Furthermore, the selection of concentration of toluene is very important. The concentration should be determined by exposing mice to various concentrations of toluene and carefully observing their behavioral changes. Concentrations that induce a hypnotic effect should be avoided. Finally, because toluene vapors are toxic to humans, the exhaust pipe of the airtight inhalation shuttlebox should be equipped with an effective ventilation system.
6.3. Alternative and support protocols Previous studies have indicated that inhalation of toluene produced reinforcing and discriminative stimulus effects. First, self-inhalation of organic solvents such as toluene, chloroform, and ether has been investigated in monkeys [7,10]. And it has been well established that many self-administered, abused addictive drugs can also be used to establish a conditioned place preference [1,4]. Interestingly, inhalation of organic solvents for five conditioning trials produced a place preference in rats [8]. Second, in a previous study, the discriminative stimulus properties of toluene were examined in mice trained to discriminate toluene from vehicle administrations [9]. In that study, it should be noted that generalization occurred after 20 min of toluene (150–3600 ppm) inhalation. Similarly, it has been reported that the response under a differential-reinforcement-of-low-rates schedule in mice was also increased following exposure to toluene (1500– 1600 ppm) [5]. The present results indicate that a toluene concentration of 700–3200 ppm produced a significant conditioned place preference in mice. Together with the results of previous studies, these data suggest that this toluene concentration range may be appropriate for evaluating toluene-induced CNS effects, such as reinforcing effects, motivational effects, and discriminative stimulus effects. The conditioned place preference procedure using the newly developed airtight inhalation shuttlebox thus constitutes an important tool for studying the rewarding effect of abused solvents.
7. Quick procedure
53
outflow (0.1–0.8 l / min) is controlled by the air pump at 35 8C. After completion of the 30-min outflow of toluene vapors, the concentrations of toluene were saturated in toluene-outflow in a volume-dependent manner.
7.2. Place conditioning After the saturation with different concentrations of toluene vapors, mice were immediately placed in each compartment within 10 s after opening the lid. The treatment compartment of the shuttlebox and the order of exposure to toluene vapors and air were counterbalanced. All conditioning sessions were 20 min in duration, and a minimum of 7 h separated the exposure to toluene vapors and the exposure to air. Conditioning sessions (five for toluene, five for air) were conducted twice daily for 5 days.
7.3. Test session In a test session, the central wall used for the conditioning session was replaced by a sliding wall in the shape of a T (Fig. 1C). Mice were allowed free access to both compartments via the spaces (636 cm: w3h) created by the sliding wall. Test sessions were carried out 1 day after the final training session, with mice in a drug-free state. Mice were placed in the center of the shuttlebox, and allowed free access to both compartments. The time spent in each compartment during a 20-min session was recorded by a digital video camera with an integrated stopwatch (DCR-PC100; Sony). Assignment to the white or black compartment was made on the basis of the position of the mouse’s head as determined by visual analysis of the recorded videotape.
8. Essential literature references Refs. [1,8,9]
Acknowledgements We thank Mr. Makoto Ohue for technical assistance and Mr. Eumihiko Yokota for critical reading of the manuscript. This work was supported by a Research Grant (13A-3-19) for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare of Japan (to M.F.).
7.1. Preparation of an airtight inhalation shuttlebox In order to obtain a stable temperature and concentration of toluene, the regulated volume of toluene (250 ml) in the gas wash bottle is evaporated at 35 8C. Using an air pump, the vapor of toluene is delivered to the inside of the airtight shuttlebox at a uniform volume. Toluene vapor
References [1] M.A. Bozarth, An overview of assessing drug reinforcement, in: M.A. Bozarth (Ed.), Methods of Assessing the Reinforcing Properties of Abused Drugs, Springer, New York, 1987, pp. 635–658. [2] M. Funada, T. Suzuki, M. Narita, M. Misawa, H. Nagase, Blockade
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[3] [4]
[5]
[6]
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54 of morphine reward through the activation of k-opioid receptors in mice, Neuropharmacology 32 (1993) 1315–1323. J.R. Glowa, Comparisons of some behavioral effects of d-amphetamine and toluene, Neurotoxicology 8 (1987) 237–248. D.C. Hoffman, The use of place conditioning in studying the neuropharmacology of drug reinforcement, Brain Res. Bull. 23 (1989) 373–387. V.C. Moser, R.L. Balster, The effects of acute and repeated toluene exposure on operant behavior in mice, Neurobehav. Toxicol. Teratol. 3 (1981) 471–475. M. Narita, M. Funada, T. Suzuki, Regulations of opioid dependence by opioid receptor types, Pharmacol. Ther. 89 (2001) 1–15.
[7] T. Yanagita, S. Takahashi, K. Ishida, H. Funamoto, Voluntary inhalation of volatile anesthetics and organic solvents by monkeys, Jpn. J. Clin. Pharmacol. 1 (1970) 13–16. [8] L. Yavich, N. Patkina, E. Zvartau, Experimental estimation of addictive potential of a mixture of organic solvents, Eur. Neuropsychopharmacol. 4 (1994) 111–118. [9] D.C. Rees, J.S. Knisely, S. Jordan, R.L. Balster, Discriminative stimulus properties of toluene in the mouse, Toxicol. Appl. Pharmacol. 88 (1987) 97–104. [10] R.W. Wood, Stimulus properties of inhaled substances, Environ. Health Perspect. 26 (1978) 69–76.
Protocol
Evaluation of rewarding effect of toluene by the conditioned place preference procedure in mice Masahiko Funada a , *, Mio Sato a , Yukiko Makino b , Kiyoshi Wada a a
Section of Addictive Drugs Research, Division of Drug Dependence, National Institute of Mental Health, National Center of Neurology and Psychiatry, 1 -7 -3 Kohnodai, Ichikawa, Chiba 272 -0827, Japan b Narcotics Control Department, Kanto-Shin’ etsu Bureau of Health and Welfare, Ministry of Health, Labour and Welfare, 2 -4 -14 Nakameguro, Meguro-ku, Tokyo, 153 -0061, Japan Accepted 7 August 2002
Abstract Toluene and many toluene-containing products are abused via inhalation. Previous investigations have used the place preference paradigm to evaluate the rewarding effects of commonly abused drugs such as morphine, cocaine, and amphetamine. A conditioning paradigm of toluene inhalation was developed in order to estimate the rewarding effect in mice. Conditioning sessions (five for toluene, five for air) were conducted twice daily for 5 days using a newly developed airtight inhalation shuttlebox (15330315 cm: w3l3h), which was divided into two compartments of equal size. One compartment was white with a textured floor, and the other was black with a smooth floor. All conditioning sessions were 20 min in duration, with a minimum of 7 h between sessions. Test sessions were carried out 1 day after the final training session with mice in a drug-free state. The time spent in each compartment during a 20-min session was measured using a digital video camera. Exposure to toluene vapors (700–3200 ppm) produced a significant conditioned place preference in mice. These results suggest that the conditioned place preference procedure using the newly developed airtight inhalation shuttlebox constitutes an important tool for studying the rewarding effect of abused solvents. 2002 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Motivation and emotion Keywords: Abuse; Conditioned place preference; Inhalants; Reward; Toluene
1. Type of research • • • •
Drug effects on behavior Shuttlebox for exposure to toluene vapors Study of conditioned place preference Evaluation of toluene concentration in the brain
2. Time required The time required for the conditioned place preference
procedure using two sets of the inhalation shuttlebox and 54 mice is 21 days.
2.1. Conditioned place preference procedure: 6 days Duration of each step: • Conditioning procedure: twice a day (one for toluene, one for air) for 5 days • Each conditioning duration: 20 min • Test session: 20 min
2.2. Tissue preparation for measurement of toluene concentration: 40 min *Corresponding author. Tel.: 181-47-372-0141; fax: 181-47-3712900. E-mail address: [email protected] (M. Funada).
• Inhalation of toluene: 20 min • Brain removal and tissue homogenizing: 10 min • Extraction of toluene: 10 min
1385-299X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S1385-299X( 02 )00182-4
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
48
2.3. Evaluation of toluene concentration gas chromatography: 30 min • Column preparation: 20 min • Sample analysis: 10 min
3. Materials
3.1. Animals Male ICR mice (20–25 g) were obtained from Tokyo Animal Laboratories (Tokyo, Japan). The mice were maintained on a 12-h light, 12-h dark schedule, and laboratory mouse chow and water were provided ad libitum. All procedures were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, approved by the Japanese Pharmacological Society.
3.2. Special equipment The system for conditioned place preference by inhal-
ants consists of the following equipment (Neuroscience Co., Tokyo, Japan; Fig. 1A). • An airtight inhalation shuttlebox for conditioned place preference (15330315 cm: w3l3h, Neuroscience Co., Ltd., Tokyo, Japan) • Air pump (HPa10000 model; Nisso Co., Ltd., Tokyo, Japan) • Gas wash bottle (Muenke model, 500 ml, Sansyo Co., Ltd., Tokyo, Japan) • Gas flowmeter (RK-200V model; Sansyo Co., Ltd., Tokyo, Japan) • Thermostatic water bath (T2 model; Sansyo Co., Ltd., Tokyo, Japan) The concentrations of toluene in the shuttlebox and in the brain tissue were measured by a capillary gas chromatography (GC) system with a flame ionization detector (FID). • GC system with FID (HP6890; Hewlett Packard) • Capillary column (HP5, 30 m30.32 mm, i.d., 0.25mm film thickness)
Fig. 1. A newly developed airtight inhalation shuttlebox for establishing the conditioned place preference paradigm. (A) The apparatus for exposure to toluene vapors. In order to obtain a stable temperature of toluene, the regulated volume of toluene (250 ml) in the gas wash bottle is evaporated at 35 8C. Using an air pump, the vapor of toluene is delivered to the inside of the airtight shuttlebox at a uniform volume. (B) Photograph of the novel airtight inhalation shuttlebox with gas flowmeter for establishing the conditioned place preference paradigm during conditioning session. (C) Photograph of the inhalation shuttlebox with flowmeter for establishing the conditioned place preference paradigm during test session.
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
3.3. Chemicals and reagents Toluene and n-hexane were purchased from Wako (Tokyo, Japan).
4. Detailed procedure
4.1. Measurement of toluene concentration in the shuttlebox Standard line: toluene vapor concentrations were calculated using the ideal gas law: pV 5 0.082 n(t 1 273) where p, pressure; V, volume; n, mol; t, temperature. Gas chromatography (GC) analysis was carried out using a GC system (HP6890; Hewlett Packard) with a capillary column (HP5, 30 m30.32 mm, i.d., 0.25-mm film thickness). The column oven temperature was held at 25 8C for 10 min. The temperature of the injector port was set at 250 8C. A split injection mode was used (ratio5 3.3:1). The carrier gas (helium) flow rate was 1.8 ml / min. Using a gas-tight syringe (81317; Hamilton, NV, USA), 100 ml of the air sample containing standard toluene was taken initially from the exhaust valve in the lid of the shuttlebox for analysis. The detection limit of toluene concentration in the air sample was 3 ppm. The relationship of the standard line of the toluene concentration in standard samples (100–5600 ppm) to the peak high value from gas chromatography analysis was established (Fig. 2A). Based on this standard line, the relationship between toluene concentration and toluene outflow was analyzed.
4.1.1. Toluene vapor outflow–toluene concentration relationship Outflow of toluene vapors (0.1–0.8 l / min) was controlled by an air pump. After initiation of exposure of toluene vapors, for purposes of analysis, 100 ml of the air sample containing toluene was taken from the exhaust valve, which is attached to the lid of the shuttlebox, using a gas-tight syringe (81317; Hamilton). 4.2. Measurement of toluene concentration in brain tissue Mice were sacrificed by decapitation following a 20-min exposure to toluene vapors. The whole brain was removed, weighed, and immediately homogenized with a tissue grinder in 300 ml desalting water on ice. These homogenates were extracted using 300 ml n-hexane. Homogenates were centrifuged at 15 000 rev. / min for 5 min at 4 8C, and 200 ml of each supernatant was collected. GC analysis was performed using a GC system (HP6890; Hewlett Packard) with a capillary column (HP5, 30 m30.32 mm, i.d., 0.25-mm film thickness). The column oven temperature was held at 30 8C for 10 min. The temperatures of the injector port and the interface
49
were set at 250 and 280 8C, respectively. A splitless injection mode was used. The carrier gas (helium) flow rate was 4.7 ml / min. Using a glass syringe, 1 ml of the sample was injected. The relationship of the standard line of the toluene concentration in standard samples (1–85 ng) to peak height was established (Fig. 3A). Based on this standard line, toluene concentrations in the whole brain were calculated.
4.3. Conditioned place preference procedure Conditioning sessions (five for toluene, five for air) were conducted twice daily for 5 days using a novel airtight inhalation shuttlebox (15330315 cm: w3l3h), which was divided into two equal-sized compartments by a sliding wall (Fig. 1B). One compartment was white with a textured floor, and the other was black with a smooth floor. The front wall of each compartment is a transparent plastic wall. The treatment compartment of the shuttlebox and the order of exposure to toluene vapors and air were counterbalanced (Table 1). Following saturation of the compartments with different concentrations of toluene vapors for 30 min, mice were immediately placed in each compartment within 10 s after opening the lid. All conditioning sessions were 20 min in duration, and a minimum of 7 h separated exposure to toluene vapors and exposure to air. The central wall used for the conditioning session was replaced by a sliding wall in the shape of a T for each test session (Fig. 1C). Mice were allowed free access to both compartments (each was 636 cm: w3h), on either side of the sliding wall. Test sessions were carried out 1 day after the final training session with mice in a drug-free state. Mice were placed in the center of the shuttlebox, and allowed free access to both compartments. The total time spent in each compartment during a 20-min session was recorded by a digital video camera with an integrated stopwatch (DCR-PC100; Sony). Assignment to the white or black compartment was made on the basis of the position of the mouse’s head as determined by visual analysis of the recorded videotape. All sessions were conducted during the light phase of the light / dark cycle used, under conditions of dim illumination (18 lx).
4.4. Statistical analysis Statistical analyses were performed using commercially available software (StatView 5.0 for Macintosh; SAS Institute, Cary, NC, USA). Each toluene standard line was estimated using regression analysis. Conditioning scores represent the time spent in the toluene-paired compartment minus the time spent in the air-paired compartment, and are expressed as the mean6S.E.M. The concentration– response line was analyzed using a one-way random factorial analysis of variance (ANOVA). The Fisher LSD test was used to determine whether individual concen-
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M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
Fig. 2. Toluene concentrations in the shuttlebox. (A) Standard line of the toluene concentration–peak height value relationship. A linear relationship between the toluene concentration in the shuttlebox and the peak height value from gas chromatography analysis was established ( y50.8x1180.2; r50.992). (B) Standard line of the toluene concentration–toluene vapor outflow relationship. A linear relationship between the toluene concentration in the shuttlebox and the toluene outflow volumes was established ( y54085.1x2102.8, r50.999). (C) Time course changes in toluene concentration in the shuttlebox following exposure to toluene. The values in the small box (0.2, 0.4, 0.6 and 0.8) show the values of airflow (l / min) into the shuttlebox. Each point represents the mean6S.E.M. of six samples. (D) Time course changes in toluene concentration in the shuttlebox during conditioning session. After saturation with different concentrations of toluene vapors for 30 min, the lid of the shuttlebox was opened for 10 s to insert mice into the compartment. The values in the small box (0.2, 0.4, 0.6, and 0.8) show the values of airflow (l / min) into the shuttlebox. Each point represents the mean6S.E.M. of 16 samples.
trations produced a significant conditioning. The accepted level of significance for all tests was P,0.05.
5. Results
5.1. Toluene concentration in the shuttlebox As shown Fig. 2A, a linear relationship between peak height and toluene concentration in standard samples was
established ( y50.8x1180.2; r50.992). Using this standard line, the toluene concentration–toluene outflow relationship was analyzed. Fig. 2B shows the standard line of the toluene concentration–toluene vapor outflow relationship. A linear relationship between toluene concentration in the shuttlebox and toluene vapor outflow was established ( y54085.1x2102.8; r50.999). Fig. 2C shows the time course changes in toluene concentration in the shuttlebox following exposure to toluene vapors. After completion of the 30-min outflow of toluene vapors, the
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
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Fig. 3. Toluene concentrations in the whole brain. (A) Standard line of the toluene concentration–peak height relationship. The toluene concentration in the whole brain tissue and the peak height were significantly correlated ( y510.0x113.3, r50.999). (B) Effect of toluene inhalation on toluene levels in the whole brain tissue following 20-min inhalation at 700, 2500, and 3200 ppm, respectively. Each column represents the mean6S.E.M. of eight mice.
concentrations of toluene saturated the toluene vapor-outflow in a volume-dependent manner. Thirty minutes was therefore adopted as the duration of the saturation of toluene vapor period. Fig. 2D shows the time course changes in toluene concentration in the shuttlebox during a conditioning session. After saturation with different concentrations of toluene vapors for 30 min, the lid of the shuttlebox was opened for 10 s in order to insert mice into the compartments. These procedures did not significantly affect each saturated concentration of toluene.
brain tissue by detected peak height after a 20-min exposure to concentrations of 700, 2500, and 3200 ppm, respectively, following a 30-min pre-inhalation.
5.3. Conditioned place preference At the beginning of the experiments, there were no significant differences in mean body weight among the five groups of mice (air, 20.260.2 g; 350 ppm, 20.760.4 g; 700 ppm, 20.960.3 g; 2500 ppm, 20.160.2 g; 3200 ppm, ¨ mice exhibited no significant prefer21.060.7 g). Naıve ence for either compartment of the shuttlebox. The mean time spent in the white and black compartments was 642.7659.4 and 557.3660.8 s (n515), respectively. During the 20-min exposure to toluene vapors, mice displayed slight hyperactivity at concentrations of 2500 and 3200 ppm, though no convulsions or hypnotic effects were observed at any of the concentrations. Following exposure
5.2. Toluene concentration in the brain The standard line of the toluene concentration–peak height relationship is shown in Fig. 3A. A linear relationship between peak height and toluene concentration in standard samples was established ( y510.0x113.3; r5 0.999). Fig. 3B shows the amount of toluene found in the
Table 1 Schedule of conditioned place preference 1
2
3
4
5
6 (test)
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
White side
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
Black side
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
TOL AIR
AIR TOL
Drug-free
Conditioning sessions (five for toluene: TOL; five for air: AIR) were conducted twice daily for 5 days using a newly developed airtight inhalation shuttlebox. One compartment was white with a textured floor, and the other was black with a smooth floor. The treatment compartment of the shuttle box, and the order of exposure to toluene vapors and air, were counterbalanced. After saturation of the shuttlebox with different concentrations of toluene vapors for 30 min, mice were immediately placed in each compartment. The control group was exposed only to air. All conditioning sessions were 20 min in duration, and a minimum of 7 h separated the exposure to toluene vapors and that to air. Test sessions were carried out 1 day after the final training session, with mice in a drug-free state. The time spent in each compartment during a 20-min session was recorded by a digital video camera (DCR-PC100; Sony).
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M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
to toluene vapors or air, and at the test session, groups did not significantly differ in mean body weight (air, 26.660.3 g; 350 ppm, 27.560.2 g; 700 ppm, 26.560.2 g; 2500 ppm, 26.960.4 g; 3200 ppm, 27.060.5 g). The air-exposed mice exhibited no preference. The mean conditioning score was 254.8666.4 s (n58). The place conditioning effect produced by the exposure to toluene is shown in Fig. 4. Toluene caused a significant preference for the drug-filled compartment (F1,51 59.18; P50.004). Toluene (350 ppm) induced a slight place preference (mean conditioning score of 53.6657.3 s; n510; P50.161), but this effect was not statistically significant. Significant conditioning scores were observed at doses of 700 ppm (mean conditioning score of 181.9656.6 s; n512; P50.003), 2500 ppm (mean conditioning score of 137.8626.3 s; n512; P5 0.012) and 3200 ppm (mean conditioning score of 149.5643.6 s; n512; P50.008). At these doses, all of the animals exposed to toluene exhibited a preference for the toluene-associated compartment.
6. Discussion
6.1. Assessment of the protocol The conditioned place preference procedure has proven useful for demonstrating the rewarding properties of drugs [1,4,6]. In this paradigm, a drug-free animal that has previously received pairings of a drug with a particular environment is conditioned to favor this environment over
a neutral environment. Thus, the first advantage of this procedure is that the rewarding effects of a drug are measured in animals that are drug-free. Therefore, any changes in unconditioned motor activity do not directly influence measurement of the dependent variable (time spent in the drug-paired place). Second, the short-term nature of the experiments (lasting on average from 1 to 3 weeks) and the sensitivity of the paradigm for demonstrating place conditioning (preference and aversion) provide other obvious advantages. For example, large numbers of compounds can be quickly tested using this technique, making it an excellent method for the initial screening of drug reward. One of the most difficult problems in repeated administration of pharmacological agents to any animal is the administration of volatile agents by inhalation. In order to obtain stable concentrations of toluene in the airtight shuttlebox, the regulated volume of toluene was evaporated at 35 8C and mixed with a larger volume of clean air to achieve the target concentrations. Using an air pump, the vapor of toluene was delivered to the inside of the airtight shuttlebox at a uniform value. The concentrations of toluene in the airtight shuttlebox reached plateau levels within 30 min, and were reliable and stable over the 20-min exposure period. Exposure to concentrations of toluene from 500 to 3200 ppm for 20 min resulted in brain levels of 4.7–44.7 ng / mg of tissue, confirming results in a previous report [3]. Our present results of assessment of brain toluene concentration are consistent with those in that report. The target concentrations of toluene in the airtight shuttlebox could be regulated by a novel vaporexposing system that included an air pump. The present technique of exposing animals to inhalant vapor by confinement in an airtight shuttlebox may be useful for the evaluation of abused inhalant-induced pharmacological actions in vivo. Using a novel conditioned place preference evaluation system, we observed that exposure to toluene vapors caused a conditioned place preference in mice. These results are consistent with those of previous studies on abused drugs using the conditioned place preference procedure. Thus, morphine, cocaine, and amphetamine have been shown to produce a strongly conditioned place preference in rats and mice [2,4,6]. The present protocol of the conditioned place preference procedure makes it possible to assess the rewarding effect of toluene by inhalation.
6.2. Trouble shooting Fig. 4. Place conditioning produced by exposure to toluene vapors in mice. Ordinate: mean difference (s) between the times spent in the toluene- and air-paired sides of the test box. Each point represents the mean6S.E.M. of 8–12 mice. None of the mice that inhaled air during the conditioning sessions (Controls) exhibited a significant preference for either compartment of the test box; the mean conditioning score was 254.8666.4 s (n58). *P,0.05 versus the air-paired control group.
Handling-associated stress should be minimized and equated across all treatment conditions, since differences in handling can influence the place preferences seen after conditioning [1]. In addition, after the conditioning session has ended, mice should not be returned to their home cages immediately, since toluene vapors will have adhered to
M. Funada et al. / Brain Research Protocols 10 (2002) 47–54
their hair and skin. Rather, they should be left beside their home cages for at least 30 min at room temperature. Toluene, benzene, ether, and other solvents have different flow values. Thus, the vapor values of the flow meter should be adjusted according to the standard value for toluene. Furthermore, the selection of concentration of toluene is very important. The concentration should be determined by exposing mice to various concentrations of toluene and carefully observing their behavioral changes. Concentrations that induce a hypnotic effect should be avoided. Finally, because toluene vapors are toxic to humans, the exhaust pipe of the airtight inhalation shuttlebox should be equipped with an effective ventilation system.
6.3. Alternative and support protocols Previous studies have indicated that inhalation of toluene produced reinforcing and discriminative stimulus effects. First, self-inhalation of organic solvents such as toluene, chloroform, and ether has been investigated in monkeys [7,10]. And it has been well established that many self-administered, abused addictive drugs can also be used to establish a conditioned place preference [1,4]. Interestingly, inhalation of organic solvents for five conditioning trials produced a place preference in rats [8]. Second, in a previous study, the discriminative stimulus properties of toluene were examined in mice trained to discriminate toluene from vehicle administrations [9]. In that study, it should be noted that generalization occurred after 20 min of toluene (150–3600 ppm) inhalation. Similarly, it has been reported that the response under a differential-reinforcement-of-low-rates schedule in mice was also increased following exposure to toluene (1500– 1600 ppm) [5]. The present results indicate that a toluene concentration of 700–3200 ppm produced a significant conditioned place preference in mice. Together with the results of previous studies, these data suggest that this toluene concentration range may be appropriate for evaluating toluene-induced CNS effects, such as reinforcing effects, motivational effects, and discriminative stimulus effects. The conditioned place preference procedure using the newly developed airtight inhalation shuttlebox thus constitutes an important tool for studying the rewarding effect of abused solvents.
7. Quick procedure
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outflow (0.1–0.8 l / min) is controlled by the air pump at 35 8C. After completion of the 30-min outflow of toluene vapors, the concentrations of toluene were saturated in toluene-outflow in a volume-dependent manner.
7.2. Place conditioning After the saturation with different concentrations of toluene vapors, mice were immediately placed in each compartment within 10 s after opening the lid. The treatment compartment of the shuttlebox and the order of exposure to toluene vapors and air were counterbalanced. All conditioning sessions were 20 min in duration, and a minimum of 7 h separated the exposure to toluene vapors and the exposure to air. Conditioning sessions (five for toluene, five for air) were conducted twice daily for 5 days.
7.3. Test session In a test session, the central wall used for the conditioning session was replaced by a sliding wall in the shape of a T (Fig. 1C). Mice were allowed free access to both compartments via the spaces (636 cm: w3h) created by the sliding wall. Test sessions were carried out 1 day after the final training session, with mice in a drug-free state. Mice were placed in the center of the shuttlebox, and allowed free access to both compartments. The time spent in each compartment during a 20-min session was recorded by a digital video camera with an integrated stopwatch (DCR-PC100; Sony). Assignment to the white or black compartment was made on the basis of the position of the mouse’s head as determined by visual analysis of the recorded videotape.
8. Essential literature references Refs. [1,8,9]
Acknowledgements We thank Mr. Makoto Ohue for technical assistance and Mr. Eumihiko Yokota for critical reading of the manuscript. This work was supported by a Research Grant (13A-3-19) for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare of Japan (to M.F.).
7.1. Preparation of an airtight inhalation shuttlebox In order to obtain a stable temperature and concentration of toluene, the regulated volume of toluene (250 ml) in the gas wash bottle is evaporated at 35 8C. Using an air pump, the vapor of toluene is delivered to the inside of the airtight shuttlebox at a uniform volume. Toluene vapor
References [1] M.A. Bozarth, An overview of assessing drug reinforcement, in: M.A. Bozarth (Ed.), Methods of Assessing the Reinforcing Properties of Abused Drugs, Springer, New York, 1987, pp. 635–658. [2] M. Funada, T. Suzuki, M. Narita, M. Misawa, H. Nagase, Blockade
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[7] T. Yanagita, S. Takahashi, K. Ishida, H. Funamoto, Voluntary inhalation of volatile anesthetics and organic solvents by monkeys, Jpn. J. Clin. Pharmacol. 1 (1970) 13–16. [8] L. Yavich, N. Patkina, E. Zvartau, Experimental estimation of addictive potential of a mixture of organic solvents, Eur. Neuropsychopharmacol. 4 (1994) 111–118. [9] D.C. Rees, J.S. Knisely, S. Jordan, R.L. Balster, Discriminative stimulus properties of toluene in the mouse, Toxicol. Appl. Pharmacol. 88 (1987) 97–104. [10] R.W. Wood, Stimulus properties of inhaled substances, Environ. Health Perspect. 26 (1978) 69–76.