Animals

Male (n = 96) and female (n = 99) adolescent Sprague Dawley (SD) rats at 21 days of age (P21) were purchased from BioLASCO (Taiwan). Rats of the same sex were pair-housed in polypropylene cages (425 × 266 × 185 mm) with corn cob beddings and given ad libitum access to water and feeds (Altromin 1324, Germany). The animal facility was a temperature- and humidity-controlled environment (20–22 °C, 40–70% humidity) with a 12-h reversed light–dark cycle (lights on: 1900–0700). Experimental procedures started in adolescent (P29) rats after 7 days of acclimatization and under the dark phase. All experiments were approved by the University of the Philippines Manila Institutional Animal Care and Use Committee (protocol no. 2021–008).

Conditioned place preference

The toluene conditioned place preference (CPP) protocol was adapted from previous studies in male SD rats (Gerasimov et al. 2003; Lee et al. 2004, 2006; Wayman and Woodward 2018) with some modifications.

The CPP apparatus (La Jolla Alcohol Research, Inc. (LJARI), USA) consisted of three distinct chambers: a smaller gray middle chamber with a smooth acrylic floor (21 × 13 × 21 cm), a white conditioning chamber with a smooth rubber floor (21 × 27 × 21 cm), and a black conditioning chamber with a textured rubber floor (21 × 27 × 21 cm). Manually operated guillotine doors separated the chambers. The black chamber where toluene was given had a gasket installed around the lid and an inlet for air or toluene vapor was installed at the top. The white chamber had holes at the lid to let air in. All three chambers had exhaust tubes installed. Liquid toluene (99.5% analytical grade, RCI Labscan, Thailand) was vaporized using custom-made bubblers attached to computer-controlled air flow regulators (LJARI, USA) at 2 or 3 lpm (for 1500 or 3000 ppm toluene vapor output, respectively). Toluene vapor concentrations within the black chamber were calibrated to averages of 1500 ppm (range: 1000 to 1900 ppm) or 3000 ppm (range: 2200 to 3600 ppm) using a portable toluene gas detector (Cosmos XP-3360II, DOD Technologies, USA) with measurements recorded every 15 s. Once rats were placed inside the chamber, there was an initial 5-min period to allow toluene vapor concentrations to reach target levels (1500 or 3000 ppm) which was then maintained for 30 min (Supplementary Fig. 1).

We chose 1500 and 3000 ppm to determine if there is a relationship between the toluene exposure concentration and behavioral effects. Toluene CPP has been previously demonstrated using a concentration of as low as 700 ppm in mice (Funada et al. 2002) and 1895 ppm in rats (Gerasimov et al. 2003), hence we explored whether 1500 ppm will produce CPP in SD rats. We chose 3000 ppm as the higher concentration based on previous studies demonstrating CPP at this level (Gerasimov et al. 2003; Lee et al. 2006; Wayman and Woodward 2018). We did not include 5000 ppm in our study based on prior reports that this concentration failed to produce CPP, and even triggered conditioned place aversion (Schiffer et al. 2006; Lee et al. 2006). In a pilot study in which we exposed rats to 5000 ppm toluene vapor, we observed lethargy and excessive salivation, which could interfere with learning drug-context associations during CPP conditioning.

The CPP protocol consisted of three phases: preconditioning, conditioning, and test phases. Preconditioning phase: Adolescent rats (P29 to P52) were placed in the center gray chamber with the guillotine doors closed. After 2 min, the doors were opened, and the rats were allowed to explore all three chambers for 15 min. Each rat’s initial preference score was calculated by the time spent in the black chamber (where toluene will be given) over the sum of the time spent in both black and white conditioning chambers. Rats were assigned to match the average group preference scores between air (control group), 1500 ppm toluene, or 3000 ppm toluene groups. Conditioning phase: Toluene groups were exposed to toluene vapor in the black chamber and air in the white chamber with the guillotine doors closed (Fig. 1A). The control group was exposed to air in both conditioning chambers. For 12 days, rats underwent one 30-min conditioning session per day. Rats received air on the first day, then air (control group) or toluene vapor (toluene groups) the next day, with alternating air and toluene conditioning sessions on subsequent days. The toluene groups received a total of six air and six toluene conditioning sessions, while the control group received 12 air conditioning sessions (Fig. 1B). Test phase: One day after the last toluene conditioning day (day 13, termed test day 1 (D1), rats were placed in the center gray chamber with the guillotine doors closed. After 2 min, the doors were opened, and the rats were allowed to explore all three chambers for 15 min. A CPP ratio score was calculated as the time spent in the black chamber (where toluene was given) divided by the sum of the time spent in both black and white chambers (Yates et al. 2013; Sun et al. 2017; Jia et al. 2023). A CPP difference score was calculated as the time spent in the black chamber minus the time spent in the white chamber during test. The CPP test was repeated on D8 and D22 of test phase.

Fig. 1

Experimental design. A Toluene CPP conditioning was performed using a three-chambered apparatus in which rats were exposed to air in a white chamber and to toluene vapor in a black chamber. Control rats were exposed to air in both chambers. During test, rats were tested for chamber preference. B The conditioning phase consisted of 12 days of alternating air and toluene exposures. One day after the last toluene exposure, rats initiated 22 days of drug-free abstinence and underwent a battery of behavioral tests. CPP Conditioned Place Preference, EPM Elevated Plus Maze, OFT Open Field Test, NOR Novel Object Recognition, SSN Sociability and Social Novelty Tests, Reversal Reversal Learning

Experimental design

To determine the behavioral effects of repeated toluene exposure during the test phase, a total of 87 male and 90 female adolescent SD rats were exposed to air, 1500 ppm toluene, or 3000 ppm toluene. These rats were then tested for various behaviors during the 22-d test phase (Fig. 1B). Due to a large number of behavioral tests, we did not run all tests in each rat. Prior to all behavioral tests, rats were allowed to habituate to the testing room for 1 h. Behavior was recorded using video cameras and analyzed using ANY-Maze version 7.33 (Stoelting Co., USA).

To determine the concentration of toluene in the blood following inhalation, 9 male and 9 female rats were exposed to air or 3000 ppm toluene vapor under the CPP conditioning schedule (Fig. 1B). Blood was drawn from the tail vein immediately after the first and last toluene vapor exposure and 24 h after the last exposure. Toluene levels from the blood samples were measured using a validated headspace gas chromatography method.

Elevated plus maze test

Rats underwent the elevated plus maze (EPM) test on D2, D6, and D20 of test phase. The EPM (Stoelting Co., USA) had two enclosed arms (50 × 10 cm) with 40 cm-high walls and two open arms without walls (50 × 10 cm). The maze was elevated 50 cm above the floor. Rats were placed in the center of the maze facing one of the open arms and allowed to explore freely for 5 min. The ratio of time spent on the open arms divided by the time spent in both open and closed arms was calculated. Two female rats were excluded from the D2 test because they fell from the apparatus.

Open field test

Rats underwent the open field test (OFT) on D3, D7, and D21 of test phase. The open field apparatus (Stoelting Co., USA) was a 100 × 100 cm box with 50 cm-high opaque walls and a smooth acrylic floor. Rats were placed in the center of the open field and allowed to explore freely for 10 min. Distance travelled and percentage of time spent in the center of the open field (50 × 50 cm, 25% of total area) were obtained.

Novel object recognition test

Rats underwent the novel object recognition (NOR) test on D5–D7 of test phase. The arena used for the NOR test was the open field apparatus (Stoelting, USA) divided into four quadrants by a wooden divider (height: 60 cm), which enabled rats to be tested four at a time. Each NOR arena measured 50 × 50 × 60 cm. The NOR test consisted of three phases: acclimatization, habituation, and testing phases. Acclimatization: On D5 of test phase, rats were placed in the empty arena for 5 min. Habituation: On D6 of test phase, the rats were presented with two identical objects in the arena for 10 min. We used circular glass jar lids (5.5 cm height × 11.5 cm diameter) in half of the rats and wooden spherical blocks (9 × 8 × 3.5 cm) in the other half. NOR Test: On D7 of test phase, rats were presented with one of the familiar objects from the previous day and a novel object (wooden block or glass lid) for 5 min. Exploration time for a given object was defined as the time spent sniffing or moving within 2 cm of the object. Time was measured manually with stopwatches, with observers blinded to the group and object novelty. The NOR score was calculated as the exploration time for the novel object divided by the sum of exploration times for both objects.

Sociability and social novelty test

Rats underwent the sociability and social novelty (SSN) test on D10–D12 of test phase. This task was based on a prior study that modeled social deficits in a mouse model of autism spectrum disorder (Moy et al. 2004). Several prior studies have adapted this task in rats to study social motivation and stress (Lukas et al. 2011; Netser et al. 2020; Potrebić et al. 2022; Shirenova et al. 2023), but not addiction. The social test apparatus (Ugo Basile, Italy) was a rectangular three-chamber testing apparatus with transparent acrylic walls, with each chamber measuring 40 × 40 × 40 cm. The middle chamber was separated from the left and right chambers by manually operated guillotine doors. Two identical cylindrical enclosures (25 cm height × 15 cm diameter) with metal railings and polyvinyl chloride roof and floor were placed in the middle of the left and right chambers. The SSN test consisted of three phases: habituation, sociability test, and social novelty test. Habituation: On D10 of test phase, test rats were initially placed in the closed center chamber for 2 min and were allowed to explore the three chambers and empty cylindrical enclosures for 10 min. Sociability test: On D11 of test phase, a demonstrator rat of the same sex and age as the test rat was placed in one of the cylindrical enclosures, while wooden blocks were placed in the other cylindrical enclosure. The chamber containing the demonstrator rat (left vs. right) was counterbalanced for all the groups. Each tested rat was given the opportunity to explore both side chambers for 10 min after being placed into the center chamber. Exploration time for a given enclosure was defined as the time spent sniffing or moving within 3 cm of the enclosure and excluded time when the center of the animal was on top of the enclosure. Exploration time was measured using ANY-Maze version 7.33 (Stoelting Co., USA). The sociability score was calculated as the exploration time for the cylindrical enclosure with the demonstrator rat divided by the sum of the exploration times for both cylindrical enclosures. Social novelty test: On D12 of test phase, demonstrator rats of the same sex and age as the test rat were placed in both cylindrical enclosures, one of which was the rat used during the sociability test (familiar rat) and the other rat was one that the test rat has not encountered prior to the test (novel rat). The position of the demonstrator rats was counterbalanced for all the groups. Each test rat was allowed to explore both enclosures for 10 min. The social novelty score was calculated as the exploration time for the cylindrical enclosure with the novel rat divided by the sum of the exploration times for both cylindrical enclosures.

Reversal learning

Appetitive reversal learning (adapted from Stephenson-Jones et al. 2016 with some modifications) was assessed in an operant conditioning apparatus with two bars (left and right) and a pellet feeder between the bars (Coulbourn Instruments, USA). The bars and feeder were controlled by ANY-Maze version 7.33 (Stoelting Co., USA). We performed reversal learning in a limited number of new animals that did not undergo CPP training and other behavioral tests. We chose to examine only the 3000 ppm concentration of toluene because the 1500 ppm concentration did not induce CPP. This subset of rats was food-restricted to maintain 90–95% of their free-feeding weight. The reversal learning experiment consisted of the following phases: initial training, toluene exposure, retraining, discrimination, reversal 1, reversal 2, and reversal 3, with each reversal of increasing difficulty. Initial training: Before toluene exposure, rats underwent bar press training for sucrose pellets for 10 days. Each press for either left or right bar was rewarded with one sucrose pellet (100% reinforcement). Toluene exposure: Rats were then exposed to 3000 ppm toluene vapor in the CPP apparatus for 30 min every other day across a 12-d period. During non-toluene exposure days, rats remained in their home cages. Retraining: On D1 of test phase, retraining for bar pressing was performed with 100% reinforcement. Discrimination: On D2–D3 of test phase, rats were trained to discriminate a bar that delivers pellets 3 out of 4 times (75% reinforcement) from a bar that never delivered pellets (0% reinforcement). Each session lasted 8 min. Reversal 1, 2, and 3: On D4 of test phase, for reversal 1, the positions of the 75%-bar and 0%-bar were reversed. On D5 of test phase, for reversal 2, the positions were reversed again, however, the reinforcement schedule of the 0%-bar was modified to 20% (2 out of every 5 presses). On D6 of test phase, for reversal 3, the positions were reversed again, however, the 20%-bar was modified to give 33% reinforcement (1 out of every 3 presses), and the 75%-bar was modified to give 67% reinforcement (2 out of every 3 presses). Each day started with the prior day’s reinforcement schedule for 2 min before switching to the new schedule for 8 min. For each minute, a bar preference score was calculated as the number of presses for the 75% bar during D3 divided by the total number of presses for both bars. The learning rate was calculated as the absolute value of the slope of the line of the bar preference score from minute 2 to minute 6 of the session.

Determination of blood toluene concentration

Rats were placed in toluene inhalation chambers measuring 21 × 27 × 21 cm (LJARI, USA) and exposed to 3000 ppm toluene vapor (toluene group). The toluene vapor concentration inside the chamber was calibrated with a portable toluene gas detector (Cosmos XP-3360II, DOD Technologies, USA). Toluene (99.5%, analytical grade, RCI Labscan, Thailand) was delivered to the chamber via custom-made bubblers attached to air flow regulators (LJARI, USA). The toluene group received six 30-min toluene exposure sessions on alternating days, similar to the conditioning schedule used for the behavioral studies (Fig. 1B). Control rats were only exposed to air. Blood samples (500 μl) were collected from the tail vein into heparin tubes 10 min after the first toluene exposure (D1), 10 min after the last toluene exposure (D6), and 24 h after the last toluene exposure (D7 or abstinence D1). From each tube, triplicates of 100-µL heparinized blood samples were immediately transferred to a headspace vial that was capped and sealed with parafilm before storage at 4 °C. Gas chromatographic analysis of the samples using a validated protocol was conducted the next day. The concentration of toluene in the blood samples was determined using static headspace gas chromatography with flame ionization detector (HS-GC-FID) using the GC-FID Nexus 2030 with HS-20 headspace autosampler and LabSolutions Post-Run Software (Shimadzu Corp., Japan). The GC column used was a silicone capillary column (SH-I-624Sil MS, 30 m × 0.25 mm × 1.40 µm, Shimadzu Asia Corp., Japan). Nitrogen carrier gas flow rate was set at 200 mL/min, headspace oven temperature was set at 80 °C, and column oven temperature program commenced at 50 °C for 2 min and ramped up to 175 °C at a rate of 25 °C/min, holding the final temperature for 5 min. FID was set at 250 °C.

A five-point calibration curve was used to estimate the levels of toluene in the blood. The calibration solutions in the concentration range of 0 to 25 µg/mL toluene were prepared using blood from naïve animals, spiked with appropriate amounts of stock standard toluene solutions (Chem-Supply Pty Ltd, Australia). Samples and calibration solutions in each headspace vial contained 5 µg/mL isobutanol (J.T Baker, USA) as internal standard (IS) and were dissolved in 2% ethanol. Deionized water was added to samples and calibration solutions to achieve a final volume of 500 µL. Calibration curves were constructed by plotting the peak area ratio of toluene to IS of each calibration solution against its toluene concentration.

Statistical analysis

Data were analyzed using Prism version 10.1.1 (GraphPad Software, USA). Two-way repeated measures ANOVA (for complete datasets) or mixed-effects analysis (for incomplete datasets) was used to analyze data for all behavioral tests and blood toluene levels, followed by post-hoc Dunnett’s test (for experiments with 3 groups: both toluene groups compared to air control group) or Šidák’s test (for experiments comparing 2 datasets: males compared to females; habituation compared to test scores in NOR, sociability, and social novelty; 3000 ppm toluene group compared to air control group in reversal learning). Differences were considered significant for p < 0.05. Data are presented as a mean and standard error of the mean (SEM). Linear regression analysis was performed on the GC calibration data using Microsoft Excel.