- Email: [email protected]
Toluene and Temporal Discrimination in Rats
Neurotoxicology and Teratology, Vol. 21, No. 6, pp. 709–718, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0892-0362/99 $–see front matter
PII S0892-0362(99)00033-1
Toluene and Temporal Discrimination in Rats: Effects on Accuracy, Discriminability, and Time Estimation HIROMI WADA Hokkaido University, College of Medical Technology, Sapporo, Japan Received 6 April 1997; Accepted 19 April 1999 WADA, H. Toluene and temporal discrimination in rats: Effects on accuracy, discriminability, and time estimation. NEUROTOXICOL TERATOL 21(6) 709–718, 1999.—Thirty-five rats acquired a temporal discrimination of seven signal durations in an operant chamber with two levers. If a 2-s or an 8-s tone signal was presented, rats were required to press one lever (”short” response) or the other lever (”long” response) to be reinforced, respectively. Neither response was reinforced when five intermediate signals were presented. Percentages of a long response in each of the seven signals were calculated and the psychophysical function between signal durations and long response percentages was obtained. Intraperitoneal injections of 50 mg/kg and 100 mg/kg toluene steepened a gradient of the psychophysical function and elevated correct responses in 2-s and 8-s signals. The function showed lower difference limen and Weber fraction. Accuracy and discriminability of temporal discrimination were enhanced. However, 400 mg/kg and 600 mg/kg toluene resulted in a shallower gradient and reduced correct responses. Higher difference limen and Weber fraction were also obtained. Accuracy and discriminability deteriorated and, in particular, behavioral depression was observed for 600 mg/kg toluene. The point of indifference was changed and dose-related overestimation or underestimation of time were suggested to occur. Blood toluene levels were reported to be 11–18 mg/ml for 50–100 mg/kg toluene and 47–73 mg/ml for 400–600 mg/kg toluene. It is speculated that temporal discrimination was sharpened by 10–20 mg/ml toluene in blood and disrupted by 50–70 mg/ml toluene in blood. © 1999 Elsevier Science Inc. All rights reserved. Toluene
Temporal discrimination
Time estimation
Rat
animals if they did not respond within an interval of time. Thus, toluene induced deterioration of temporal differentiation and it was hypothesized that toluene disturbed the timing of responding (21,27,30,31). The other type of reinforcement procedure concerning temporal discrimination requires animals to estimate temporal durations of signals (time estimation methods). Animals can be trained to press one lever if a signal duration is estimated as “short” and to press the other lever if it is estimated as “long” (7,18,19). In a preliminary report (29), the author applied this temporal discrimination to investigate acute effects of 100, 200, and 400 mg/kg toluene and revealed that toluene affected accuracy and discriminability. Toluene interferes with temporal differentiation and discrimination (13,21,22,27,29–31) and it causes hazardous effects in our daily life and workplaces (e.g., playing games with
TEMPORAL differentiation and discrimination are important functions of humans and other animals that affect their cognitive processes, and toluene has been reported to disturb temporal behavior in various reinforcement schedules (6,11– 13,21,22,27,29–31). For example, a fixed interval (FI) schedule is characterized by an initial pause and a following response acceleration (FI scallop), but toluene disrupts this response pattern (13,22). A differential reinforcement of low rate (DRL) schedule reinforces specified interresponse times (IRTs) and, accordingly, animals learn to withhold a response and produce particular IRTs. However, toluene-exposed mice responded with shorter IRTs and, occasionally, bursts of high-frequency responding were observed (21). The shortening of IRTs and response latencies (RLs) was found in shock avoidance schedules of the Sidman type (27) and the shuttle type (30,31) in which an electrical shock was presented to the
Requests for reprints should be addressed to H. Wada, Hokkaido University, College of Medical Technology, Kita 12, Nishi 5, Kita-Ku, Sapporo, 060-0812 Japan. Tel: 181-11-706-3321; Fax: 181-11-706-4916; E-mail: [email protected]
709
710
WADA
a moving ball, driving a car, and operating machines on an assembly line) because toluene-exposed workers responded slowly and showed poorer performance of perceptual speed and reaction time (9,15). The present research focused on accuracy, discriminability, and time estimation of a temporal discrimination, clarified dose-dependent effects of toluene, and determined blood and brain toluene levels that affected temporal discrimination. METHODS
Subjects Thirty-five male rats of the Wistar strain, about 100 days old, were used for this experiment. They were individually housed in plastic cages under a 12-h light-dark cycle (light 1900–700, dark 700–1900). All experiments were carried out during the dark period. Their body weights were maintained at 250 6 10 g to control motivation levels, with additional food provided after the daily experiment to do so. Tap water was always available in the home cage. The room temperature was maintained at 22 6 28C and relative humidity was 50 6 10%. Environmental conditions were in accordance with the Guide for the Care and Use of Laboratory Animals (Hokkaido University School of Medicine, 1992). Apparatus Five standard operant chambers (27 cm in height, 25 cm in width, and 30 cm in length) with two levers were used. They consisted of acrylic plastic panels and the front panels were of clear plastic so the subjects could be observed. The flooring was made of stainless steel grids 5 mm diameter spaced 2 cm apart. A lamp, a food cup, and two levers were located on the side panel. A lamp was mounted on the center 11 cm above the floor and provided dim light to observe a subject. The food cup was 10 cm below the lamp; a food pellet (45 mg) could be delivered into the cup from a pellet dispenser. Two levers (3 cm in width and 3 cm in length) protruded from the panel, one 8 cm to the right and the other 8 cm to the left of the food cup and 3 cm above the floor. A house light and a tone signal were presented from the ceiling of the chamber. A speaker with a diameter of 17 cm was installed outside of the chamber and white noise (70 dB) was provided for masking extraneous sounds. The operant chamber was set into an insulated box to attenuate external light and sound. A personal computer (PC9801-RX, NEC, Japan) controlled the reinforcement schedule and data recording. The software was written in MS-C. Procedure Temporal discrimination of two signals. After training to press each lever, rats were trained to press one lever after 2-s tone signals (short response) and the other lever after 8-s tone signals (long response). The tone signal was a complex sound composed of 3 kHz and 6 kHz in frequency and 80 dB in loudness. Assignment of signal durations (2 s or 8 s) and levers (right or left) was counterbalanced among rats. A trial started with the house light on and then one of two signals was presented after 1 s. Each signal was presented 25 times with a probability of 0.5. If the rat pressed the correct lever, the response was reinforced by a food pellet, but if it pressed the incorrect lever, no reinforcer was delivered. Then the house light was turned off and the next trial began following an intertrial interval (ITI) of 10 s. When the rat pressed a lever during a signal, the trial was terminated and the same
trial started again after a 10-s ITI. A limited hold (LH) of 30 s was employed: If no response occurred within 30 s of the end of the signal a 10-s ITI was then initiated and followed by the next trial. All responses during ITIs and intervals between the house light and the signal did not affect the reinforcement schedule. A session comprised 50 trials and one session was run every other day for 30 sessions. The percentage of a correct response in 2-s and 8-s signals reached 85 6 2.2% and 80 6 2.8%, respectively (mean 6 SEM). Temporal discrimination of seven signals. Five intermediate signals of 2.5-, 3.2-, 4.0-, 5.0-, and 6.3-s duration spaced at equal logarithmic intervals between 2-s and 8-s signals were introduced. The rat was trained to establish a temporal discrimination of the seven signals. Signals of 2-s and 8-s durations were presented 25 times each with a probability of 0.25 and five signals of intermediate durations were presented ten times each with a probability of 0.1. A trial started with the house light on and then one of seven signals was pseudorandomly presented after 1 s. When a 2-s or an 8-s signal was presented, the correct response was reinforced and the incorrect one was not reinforced. When one of five intermediate signals was presented, no response was reinforced. Then the house light was turned off and the next trial began following a 10-s ITI. When the rat responded during a signal, the trial was terminated and the same trial started again after a 10-s ITI. An LH of 30 s terminated the trial with no response. A session comprised 100 trials and one session was run every other day for seven sessions. The percentage of a correct response in 2-s and 8-s signals reached 86 6 1.6% and 88 6 2.0%, respectively (mean 6 SEM). These data served as precontrols (noninjection). Toluene injections. All rats next received seven-signal temporal discrimination training every other day for six sessions. They were injected with 0.1 ml olive oil intraperitoneally 1 h before the training. Finally, the percentage of a correct response in 2-s and 8-s signals reached 92 6 1.1% and 91 6 1.4%, respectively (mean 6 SEM) and the behavioral data of the last session served as vehicle controls (olive oil injection). The next day, rats were divided into five groups of seven per group and received intraperitoneal injections of toluene at dosages of 50, 100, 200, 400, or 600 mg/kg. Toluene was dissolved in an equal volume of olive oil to prevent inflammation of internal organs and the injection was carried out 1 h before the experiment. Retraining of temporal discrimination. Following toluene injections, all rats were kept in home cages for a month and then received retraining of seven-signal temporal discrimination for six sessions. A session comprised 100 trials and one session was run every other day. The data of the last session served as postcontrols (noninjection). Statistical Analysis Behavioral data were analyzed by a two-factor analysis of variance (groups and sessions) with repeated measures of sessions (32). If a significant effect was found for groups, the Spjøtvoll–Stoline test, which is a generalization of Tukey’s HSD test appropriate for unequal sample sizes, was employed for multiple comparisons (16). RESULTS
General Observations All rats established a stable and robust baseline of temporal discrimination but two rats of the 400-mg/kg–injected
TOLUENE AND TEMPORAL DISCRIMINATION group and three rats of the 600-mg/kg–injected group did not press the lever. Although no response during the five intermediate signals provided a reinforcer, the rats’ performance was not affected and there was no evidence of extinction found in this experiment. Percentage of Trials With a Response The percentage of trials with a response was calculated as (the number of trials with a response/the number of total trials) 3 100. Figure 1 shows the effects of toluene on the percentage of trials with a response. Precontrols versus vehicle controls. The percentage was nearly 100% for all groups in precontrols and vehicle controls. No significant difference was obtained for groups, sessions (precontrols vs. vehicle controls), and interactions. The groups were equivalent in the response percentage and effects of olive oil were not found. Vehicle controls versus toluene injections. There was a steep decrease in the percentage for the 400-mg/kg– and the 600mg/kg–injected groups. It was below 10% for two rats after 400 mg/kg toluene and for four rats after 600 mg/kg toluene. The 50-, 100-, and 200-mg/kg–injected groups did not alter the response percentage. They responded at the same level as vehicle controls. Significant effects were found for groups (dosages) [F(4, 30) 5 11.88, p , 0.01]; sessions (vehicle controls vs. toluene injections) [F(1, 30) 5 26.45, p , 0.01]; and interactions [F(4, 30) 5 11.94, p , 0.01]. The 600-mg/kg–injected group showed a significant decrease of the percentage compared with the 50-, 100-, and 200-mg/kg–injected groups (p , 0.01) and the 400-mg/kg–injected group (p , 0.05) by a multiple comparison test. Toluene reduced the response percentage in a dose-dependent manner and, in particular, 600 mg/kg toluene caused a severe decrease. Vehicle controls versus postcontrols. The 400-mg/kg– and the 600-mg/kg–injected groups recovered and showed the same percentage as vehicle controls. Significant differences were not observed for groups, sessions (vehicle controls vs. postcontrols), and interactions, and effects of high-volume toluene disappeared. The response percentage in each of the seven signals was
711 calculated. Each rat responded equally to all signals whether it could receive a reinforcer or not. Psychophysical Function Each rat was trained to press one of two levers after an 8-s signal and it was called a long response. The percentage of trials with a long response in each of the seven signals was calculated as (the number of trials with a long response/the number of trials with a response) 3 100. The psychophysical function between signal durations and long response percentages was obtained for 35 rats by linear regression using the method of least squares. The gradient, point of indifference (PI), difference limen (DL), and Weber fraction (WF), which were important measures for discrimination behavior (10), were estimated from the individual psychophysical functions of 35 rats. The regression coefficient of the psychophysical function served as the gradient. The PI was the signal duration associated with a 50% long response, which was a 50% choice point for a short or a long response. We can consider overestimation or underestimation of signal durations by the shift of the PI. Signal durations associated with a 25% long response and a 75% long response were calculated, and one-half of the range between these two durations was defined as the DL. The DL is a duration from the PI, which is necessary to estimate a signal as short or long with a 75% probability. The WF was defined as DL/PI, which was a constant ratio between an arbitrary duration and its DL (Weber’s law). Small DLs and WFs indicate higher discriminability and large ones indicate lower discriminability. The effects of toluene on these measures are presented in Table 1, and the psychophysical function for groups is indicated in Fig. 2. Precontrols versus vehicle controls. The gradient of vehicle controls was larger than that of precontrols for all groups and the psychophysical function became steeper. Groups and interactions were not significant, and the gradient was equivalent between groups. Sessions (precontrols vs. vehicle controls) were significant [F(1, 30) 5 14.65, p , 0.01]. There was no significant difference of groups, sessions, and interactions on the PI. All groups were equivalent in the PI and olive oil injections did not affect it. The DL and the WF were de-
FIG. 1. Effects of toluene on the percentage of trials with a response. Data are mean and SEM.
712
WADA TABLE 1 EFFECTS OF TOLUENE ON THE PSYCHOPHYSICAL FUNCTION Dosage (mg/kg)
Gradient Pre Vehicle Toluene Post Point of indifference Pre Vehicle Toluene Post Difference limen Pre Vehicle Toluene Post Weber fraction Pre Vehicle Toluene Post
50
100
200
11.87 6 0.86 12.19 6 0.69 14.12 6 0.70 11.97 6 0.67
13.21 6 0.84 14.59 6 0.34 15.62 6 0.74 12.53 6 0.84
13.75 6 0.85 15.82 6 0.69 14.55 6 1.39 14.46 6 0.82
5.05 6 0.43 5.46 6 0.27 5.00 6 0.17 5.68 6 0.37
4.93 6 0.16 4.72 6 0.21 4.81 6 0.19 4.81 6 0.19
2.19 6 0.17 2.10 6 0.12 1.80 6 0.09 2.14 6 0.14 0.47 6 0.08 0.39 6 0.02 0.36 6 0.03 0.38 6 0.02
400
600
12.47 6 1.18 16.04 6 0.77 8.59 6 2.30 13.40 6 1.23
13.20 6 0.44 15.21 6 1.20 3.19 6 4.21 14.52 6 1.45
5.37 6 0.28 4.96 6 0.19 5.39 6 0.34 5.41 6 0.27
5.23 6 0.47 5.18 6 0.25 39.51 6 30.01 4.93 6 0.36
5.06 6 0.16 4.93 6 0.13 68.51 6 49.20 4.94 6 0.11
1.95 6 0.13 1.72 6 0.04 1.63 6 0.08 2.06 6 0.15
1.88 6 0.14 1.60 6 0.07 1.89 6 0.26 1.77 6 0.11
2.18 6 0.28 1.58 6 0.07 19.87 6 15.50 1.99 6 0.20
1.91 6 0.06 1.74 6 0.19 37.12 6 27.33 1.88 6 0.23
0.40 6 0.04 0.37 6 0.02 0.34 6 0.03 0.43 6 0.03
0.35 6 0.02 0.32 6 0.01 0.34 6 0.03 0.33 6 0.02
0.41 6 0.02 0.31 6 0.01 0.46 6 0.05 0.43 6 0.07
0.38 6 0.02 0.35 6 0.04 0.53 6 0.09 0.37 6 0.04
Data are calculated from individual psychophysical functions of 35 rats. Values are mean 6 SEM.
creased for vehicle controls compared with precontrols. Sessions were significant for the DL [F(1, 30) 5 8.99, p , 0.01] and the WF [F(1, 30) 5 6.27, p , 0.05]. However, there was no difference for groups and interactions. Vehicle controls versus toluene injections. The 50-mg/kg– and the 100-mg/kg–injected groups showed a higher gradient compared with vehicle controls. In the 200-mg/kg–injected group, the gradient was lowered. That of the 400-mg/kg– and the 600-mg/kg–injected groups decreased. Significant effects were found for groups (dosages) [F(4, 25) 5 3.67, p , 0.05] and sessions (vehicle controls vs. toluene injections) [F(1, 25) 5 18.89, p , 0.01]. Interactions were also significant [F(4, 25) 5 10.47, p , 0.01]. The gradient reflects discriminability of stimulus durations. A steep gradient indicates high discriminability and a shallow one indicates low discriminability. The 50mg/kg– and the 100-mg/kg–injected groups were sensitive to discriminate temporal durations but the 400-mg/kg– and the 600-mg/kg–injected groups became blunt in discriminability. Most severe degradation was revealed for the 600-mg/kg–injected group compared with the 100-mg/kg–injected group (p , 0.01), the 50-mg/kg–, and the 200-mg/kg–injected groups (p , 0.05) by a multiple comparison test. The PI was reduced for the 50mg/kg–injected group but the other groups enhanced it. There were significant effects for sessions [F(1, 25) 5 4.48, p , 0.05]. The change of a PI means the temporal shift of a psychophysical function. The decrease of a PI shifts the function leftward and its increase shifts the function rightward. The injection with 50 mg/kg toluene reduced the PI and shifted the function leftward. The percentage of trials with a long response was increased in longer durations and overestimation of temporal durations could occur (Fig. 2a). By contrast, toluene injections of 200, 400, and 600 mg/kg increased PIs. The 200-mg/ kg–injected group shifted the functions rightward, the long response percentage was decreased, and signal durations could be underestimated (Fig. 2c). The most dramatic effect was ob-
served for the 400-mg/kg– and the 600-mg/kg–injected group, which shorter durations were overestimated and longer ones were underestimated (Figs. 2d–2e). Thus, toluene seemed to affect time estimation and cause dose-dependent overestimation or underestimation of temporal durations. The DL and the WF were reduced for the 50-mg/kg– and the 100-mg/kg–injected groups but increased for the 200-, 400-, and 600-mg/kg–injected groups. Sessions were significant for the DL [F(1, 25) 5 4.60, p , 0.05] and the WF [F(1, 25) 5 12.27, p , 0.01]. Interactions were also significant for the WF [F(4, 25) 5 6.38, p , 0.01]. Small DLs and WFs show higher discriminability of temporal durations and large ones show lower discriminability (18,19). Toluene affected discriminability, and low volumes of toluene elevated it and high volumes of toluene degraded it. Vehicle controls versus postcontrols. The gradient was decreased for all groups, and sessions (vehicle controls vs. postcontrols) were significant [F(1, 30) 5 8.75, p , 0.01]. The DL and the WF were increased and significant differences were found for sessions [F(1, 30) 5 8.19, p , 0.01] and [F(1, 30) 5 4.42, p , 0.01]. However, groups and interactions were not significant. No significant difference was obtained for the PI. Thus, groups were equivalent in these measures and the highvolume–injected groups recaptured temporal discrimination at the same level as the low-volume–injected groups. Accuracy in 2-s and 8-s Signals When the signal duration was 2 s or 8 s, rats could receive a reinforcer if they pressed the correct lever. Two-second and 8-s durations were standards to classify a signal duration as short or long. The percentage of trials with a correct response in 2-s signals (short response percentage) and 8 s signals (long response percentage) is indicated in Fig. 3. Precontrols versus vehicle controls. The correct response percentages in 2-s signals were enhanced for all groups. Significant
TOLUENE AND TEMPORAL DISCRIMINATION
713
FIG. 2. Effects of toluene on the percentage of trials with a long response. The point in the diagram indicates long response percentages in signal durations of 2, PI 2 DL, PI, PI 1 DL, and 8 s. The PI 2 DL, PI, and PI 1 DL are signal durations matching to 25, 50, and 75% long responses, respectively. The psychophysical function for groups was calculated between these signal durations and long response percentages. Data are mean.
differences were obtained for sessions (precontrols vs. vehicle controls) [F(1, 30) 5 14.36, p , 0.01], but groups and interactions were not significant. The correct response percentages in 8-s signals did not show any difference of groups, sessions, and interactions. The groups were equivalent in accuracy. Vehicle controls versus toluene injections. The 50-mg/kg– and the 100-mg/kg–injected groups showed a higher correct percentage in 2-s signals. A lower percentage was found for the 400-mg/kg– and the 600-mg/kg–injected groups. The same result was observed in 8-s signals with a dose-dependent manner. Significant effects were found for sessions (vehicle controls vs. toluene injections) in 2-s signals [F(1, 25) 5 4.74, p , 0.05]. Groups (dosages), sessions, and interactions were significant in 8-s signals [F(4, 25) 5 5.48, p , 0.01; F(1, 25) 5 14.58, p , 0.01; and F(4, 25) 5 10.94, p , 0.01, respectively]. The 600-mg/kg–injected group showed significant differences of the correct response percentage in 8-s signals compared with the 50-, 100-, and 200-mg/kg–injected groups (p , 0.01) and the 400-mg/kg–injected group (p , 0.05) by a multiple comparison test. The percentage was increased by 50 mg/kg toluene and low volumes of toluene enhanced the accuracy of
temporal discrimination. However, the percentage was decreased by 400 mg/kg and 600 mg/kg toluene and high volumes caused accuracy to deteriorate. Vehicle controls versus postcontrols. No significant differences were observed for groups, sessions (vehicle controls vs. postcontrols), and interactions. The 400-mg/kg– and the 600mg/kg–injected groups showed the same correct response percentage as vehicle controls. They could recover accuracy in temporal discrimination.
ITI Response Rate A response in a 10-s ITI did not have any influence on the reinforcement schedule. An ITI response rate was calculated as (the total number of responses in ITIs/the number of ITIs) and is presented in Table 2. The 600-mg/kg–injected group decreased the ITI response rate markedly but recovered in postcontrols. There was no significant effect for groups (dosages), sessions (vehicle controls vs. toluene injections), and interactions. No differences
714
WADA
FIG. 3. Effects of toluene on the percentage of trials with a correct response in 2-s and 8-s signals. Data are mean and SEM.
were obtained in precontrols versus vehicle controls and vehicle controls versus postcontrols. Response Latency A response latency (RL) was the time interval from the termination of a signal to the initiation of a response. Table 3
indicates the changes in RLs. Because the rat could receive a reinforcer in 2-s and 8-s signals, RLs in these signals were separately obtained. Precontrols versus vehicle controls. The RL in total responses was shortened except for the 50-mg/kg–injected group and significant differences were found [F(4, 30) 5 2.87, p , 0.05], which indicated groups were not equivalent. Then,
TABLE 2 EFFECTS OF TOLUENE ON THE ITI RESPONSE RATE Dosage (mg/kg)
Pre Vehicle Toluene Post
50
100
200
400
600
0.26 6 0.05 0.28 6 0.06 0.23 6 0.05 0.27 6 0.06
0.45 6 0.08 0.37 6 0.08 0.41 6 0.10 0.46 6 0.07
0.23 6 0.04 0.28 6 0.04 0.26 6 0.03 0.29 6 0.06
0.25 6 0.04 0.32 6 0.05 0.25 6 0.10 0.30 6 0.07
0.30 6 0.07 0.27 6 0.06 0.04 6 0.02 0.41 6 0.10
Values are mean 6 SEM.
TOLUENE AND TEMPORAL DISCRIMINATION
715 TABLE 3
EFFECTS OF TOLUENE ON THE RESPONSE LATENCY Dosage (mg/kg)
Total Pre Vehicle Toluene Post 2s Pre Vehicle Toluene Post 8s Pre Vehicle Toluene Post
50
100
200
400
600
2.44 6 0.24 2.51 6 0.28 2.16 6 0.22 2.49 6 0.27
3.33 6 0.45 2.81 6 0.34 2.26 6 0.24 3.52 6 0.38
2.30 6 0.27 2.18 6 0.41 2.48 6 0.49 2.94 6 0.56
2.75 6 0.36 2.22 6 0.17 3.75 6 0.70 2.58 6 0.23
3.51 6 0.18 2.96 6 0.29 7.44 6 2.37 3.40 6 0.20
3.05 6 0.21 2.69 6 0.40 2.04 6 0.14 2.54 6 0.16
4.38 6 0.63 3.06 6 0.50 2.55 6 0.31 4.12 6 0.56
2.68 6 0.53 2.65 6 0.56 2.57 6 0.68 3.48 6 0.73
3.19 6 0.57 1.93 6 0.24 3.37 6 0.82 2.60 6 0.39
3.77 6 0.38 3.03 6 0.30 6.05 6 2.68 3.65 6 0.43
1.79 6 0.35 1.15 6 0.12 1.37 6 0.28 1.49 6 0.21
2.32 6 0.40 1.65 6 0.21 1.33 6 0.11 2.19 6 0.39
1.50 6 0.25 1.16 6 0.17 1.38 6 0.30 1.83 6 0.33
1.98 6 0.51 1.48 6 0.28 4.54 6 11.10 1.53 6 0.18
2.47 6 0.56 2.34 6 0.33 6.38 6 3.20 2.23 6 0.40
Values are mean 6 SEM.
the RL in toluene injections and postcontrols was recalculated as the percentage of that in vehicle controls for following analyses. RLs in 2-s and 8-s signals were also shortened. Neither groups nor interactions showed any difference but sessions (precontrols vs. vehicle controls) were significant in 2-s signals [F(1, 30) 5 6.90, p , 0.05] and 8-s signals [F(1, 30) 5 4.85, p , 0.05]. Vehicle controls versus toluene injections. The 50-mg/kg– and the 100-mg/kg–injected groups decreased RLs in total responses and responded quickly. The 200-, 400-, and 600-mg/ kg–injected groups increased RLs and responded slowly. Significant effects were obtained for groups (dosages) [F(4, 29) 5 2.85, p , 0.05], sessions (vehicle controls vs. toluene injections) [F(1, 29) 5 5.53, p , 0.05], and interactions [F(4, 29) 5 2.85, p , 0.05]. The RL in 8-s signals was lengthened and in particular, the 400-mg/kg– and the 600-mg/kg–injected groups responded with a longer RL. It was significant for groups [F(4, 28) 5 3.56, p , 0.05] and sessions [F(1, 28) 5 6.51, p , 0.05]. The RL in 2-s signals was shortened for the 50-mg/kg– and the 100-mg/kg–injected groups and lengthened for the 400-mg/ kg– and the 600-mg/kg–injected groups. However, there was no significant effect for groups, sessions, and interactions. Vehicle controls versus postcontrols. The 400-mg/kg– and the 600-mg/kg–injected groups were reinstated in RLs and indicated the same ones as the other groups. Neither groups nor interactions were significant for RLs in total responses, RLs in 2-s signals and those in 8-s signals, but sessions (vehicle controls vs. postcontrols) were significant [F(1, 30) 5 13.89, p , 0.01; F(1, 30) 5 11.55, p , 0.01; and F(1, 30) 5 4.98, p , 0.05, respectively].
p , 0.01]. For the compensation of the group inequality, the number of restarted trials in toluene injections and postcontrols was recalculated as the percentage of that in vehicle controls. Vehicle controls versus toluene injections. There was no significant effect of groups, sessions (vehicle controls vs. toluene injections), and interactions, but the 600-mg/kg–injected group eliminated restarted trials almost completely. The other groups increased them and in particular, the 400-mg/kg–injected group clearly elevated restarted trials. It was evidenced that 400 mg/kg toluene did not cause ataxia or paralysis. Vehicle controls versus postcontrols. The 600-mg/kg–injected group indicated the same level of restarted trials as vehicle controls and recovered them. No significant differences were observed for groups, sessions (vehicle controls vs. postcontrols), and interactions. The effects of toluene disappeared in postcontrols. ITI Response Rate After Reset An ITI response rate after a reset was calculated as (the total number of responses in ITIs after a reset/the number of resets) and it is presented in Figure 4 (bottom). The 600-mg/kg–injected group showed a marked decrease in ITI response rate after a reset and significant effects were obtained for groups (dosages) [F(4, 23) 5 3.96, p , 0.05]. However, the 600-mg/kg–injected group recovered in postcontrols. Neither precontrols versus vehicle controls nor vehicle controls versus postcontrols had any significant difference. DISCUSSION
Restarted Trial When rats responded during signal presentations, the trial was reset and started again following a 10-s ITI. The rat was required to withhold a response during signal durations. Figure 4 (top) indicates the number of restarted trials. Precontrols versus vehicle controls. The number of restarted trials was higher for the 200-mg/kg–injected group and significant differences were found for groups [F(4, 30) 5 5.29,
It has been revealed that toluene has dose-dependent biphasic effects on animal behavior, that is, excitatory effects at low dosages and depressive effects at high dosages (3,14, 20,31,33). The same effect was found for temporal differentiation behavior. IRTs in a DRL schedule were shortened at 1600-ppm and 3200-ppm toluene exposures and their distributions showed leftward shifts, but 6400-ppm toluene made distributions flat (21). Decreased IRTs were also observed in
716
WADA
FIG. 4. Effects of toluene on the number of restarted trials (top) and the ITI response rate after a reset (bottom). Data are mean and SEM.
Sidman avoidance learning at concentrations of 1000-ppm and 3000-ppm toluene vapor (27). In particular, 3000-ppm toluene exposure caused a marked shortening of IRTs. Sequential changes of RLs following toluene exposures were analyzed in shock avoidance learning (30,31). Immediately after exposure, RL histograms were shifted leftward at 2000-ppm toluene vapor and RLs were shortened. Avoidance responses were decreased in 6000-ppm and 8000-ppm toluene-exposed rats and RL histograms became flat; nevertheless they recovered avoidance performance after 3 h when a shortening of RLs was observed. These results indicate that lower levels of toluene (1000–3000 ppm) produce shortening of IRTs or RLs and quicken responses, that is, excitatory effects on temporal differentiation, and higher levels of toluene (6000–8000 ppm) produce flattening of their histograms, that is, depressive effects on temporal differentiation. Biphasic effects of toluene on temporal discrimination were observed in the present experiment using the time estimation method. Analyzing the psychophysical function between signal durations and long responses, the 50-mg/kg– and the 100-mg/kg–injected groups had elevated the gradient of the function and decreased the DL and the WF. These mea-
sures are indices of discriminability and showed that the rats’ discriminability was elevated (18,19,29). Moreover, these injections increased correct responses in the standard 2-s and 8-s training signals with shorter RLs. The rats responded more quickly and accurately. Lower volumes of toluene enhanced accuracy and discriminability, and improved temporal discrimination due to excitatory effects. By contrast, the 400mg/kg– and the 600-mg/kg–injected groups decreased the gradient and the psychophysical function became moderate and shallow. The DL and the WF were increased, which showed that discriminability had deteriorated at these doses. Correct response percentages were lowered in the standard two training signals and the rats responded slowly. Higher volumes of toluene degraded accuracy and discriminability, and disrupted temporal discrimination due to depressive effects. A toluene dose of 50 mg/kg increased the gradient, decreased the PI, and produced a leftward shift in the temporal gradient of the psychophysical function. Toluene doses of 200, 400, and 600 mg/kg decreased the gradient and increased the PI dose dependently. The 50-mg/kg–injected group estimated signal durations as being longer (overestimation) and the 200-, 400-, and 600-mg/kg–injected groups estimated durations as
TOLUENE AND TEMPORAL DISCRIMINATION being shorter (underestimation). Further research is necessary to clarify acute effects of toluene on time estimation. High volumes of toluene affect motor activity and cause ataxia or paralysis (3,14,31,33). For the 50-, 100-, and 200-mg/ kg–injected groups, the percentages of trials with a response were at the same level as vehicle controls and depressive effects on behavior were not found. The 400-mg/kg–injected group decreased response percentages and depressive effects were observed. However, the number of restarted trials was definitely elevated and the rats frequently responded during signal presentations. They could not withhold a response and thus, behavioral depression did occur but ataxia or paralysis did not occur. The 400-mg/kg–injected group produced longer RLs and responded more slowly to the signal. It has been hypothesized that longer latencies cause attentional lapses, which are responsible for poor performance in temporal discriminations (18,19,29). The 600-mg/kg–injected group degraded all of the behavioral measures. Behavioral depression was marked and its nonspecific effects seemed to impair temporal discrimination. It was most likely that a larger volume of toluene and olive oil caused behavioral depression. The 200-mg/kg–injected group decreased the gradient of the psychophysical function. The DL and the WF were increased, which indicated that discriminability had been lowered. The injection of 200 mg/kg toluene was suggested to be the lowest level that produced depressive effects on temporal discrimination behavior. Toluene elevated auditory thresholds (25,26,28) and hearing loss occurred (4,8,25,28). Damage to cochlea hair cells has been detected (5,28). High volumes of toluene decreased accuracy in standard signals and induced severe impairments of temporal discrimination. However, there was no difference of groups in postcontrols and even the 400-mg/kg– and the 600mg/kg–injected groups could recapture baseline performance. We therefore conclude that acute effects of toluene are transient and reversible. In precontrols versus vehicle controls or vehicle controls versus postcontrols, several measures indicated significant differences of sessions. It was possible that olive oil injections affected performance, but we should notice that the rats received additional training between precontrols and vehicle controls, and they were kept in home cages for a month be-
717 fore retraining (postcontrols). Additional learning or forgetfulness could explain these differences. The type of experimental animals including strain, age, and gender is an important factor that can influence the pharmacokinetics of toluene. Although we must be cautious, efforts to determine the critical level of toluene associated with behavioral changes are of value in comparing a number of research findings, which employed different behavioral tests and solvent administrations (e.g., injection or inhalation). The toluene level in blood has been reported to be 19.6, 30.2, and 50.0 mg/ml after inhalation of 750-, 1500-, and 2700-ppm toluene vapor, respectively (1,2). Because toluene vapor that induced shorter IRTs or RLs is 1000–3000 ppm (21,27,30,31), it can be estimated that IRTs or RLs were shortened at blood toluene levels of 23–55 mg/ml. The response rates in a shock avoidance task were increased and acceleration of reaction times was observed at toluene levels of 27 mg/ml in blood and 32 mg/g in brain, and anesthetic effects occurred at 120 mg/ml in blood and 160 mg/g in brain (17). In the present research, toluene was dissolved with an equal volume of olive oil and injected intraperitoneally. For the same method of administration toluene levels in blood have been determined in rats (23,24). Toluene in blood at 30–60 min following injections reached maximum levels of 10.5, 17.9, 24.7, 47.2, and 73.2 mg/ml in blood with injections of 50, 100, 200, 400, and 600 mg/kg toluene, respectively. Thus, we can estimate that toluene induces excitatory effects on temporal discrimination at levels of 11–18 mg/ml in blood and depressive effects at 47–73 mg/ml in blood. These toluene levels are much lower than those that affected temporal differentiation, and temporal discrimination using the time estimation was more sensitive to acute toxicity of toluene. Therefore, the author concludes that the time estimation methods can be applied to analyze the toxicity of other chemicals as well.
ACKNOWLEDGEMENTS
This research was supported by a Grant-in-Aid for the Encouragement of Young Scientists no. 02851011 from the Ministry of Education, Science, Sports and Culture, Japan.
REFERENCES 1. Aikawa, H.; Momotani, H.; Shigeta, S.: Changes in blood toluene concentrations in rats under various kinds of exposure conditions and physical loads. (Japanese text with English abstract) Jpn. J. Ind. Health 30:379–384; 1988. 2. Arito, H.; Tsuruta, H.; Oguri, M.: Changes in sleep and wakefulness following single and repeated exposures to toluene vapor in rats. Arch. Toxicol. 62:76–80; 1988. 3. Bushnell, P. J.; Evans, H. L.; Palmes, E. D.: Effects of toluene inhalation on carbon dioxide production and locomotor activity in mice. Fundam. Appl. Toxicol. 5:971–977; 1985. 4. Bushnell, P. J.; Kelly, K. L.; Crofton, K. M.: Effects of toluene inhalation on detection of auditory signals in rats. Neurotoxicol. Teratol. 16:149–160; 1994. 5. Campo, P.; Lataye, R.; Cossec, B.; Placidi, V.: Toluene-induced hearing loss: A mid-frequency location of the cochlear lesions. Neurotoxicol. Teratol. 19:129–140; 1997. 6. Colotla, V. A.; Bautista, S.: Effects of solvents on schedule-controlled behavior. Neurobehav. Toxicol. 1(Suppl. 1):113–118; 1979. 7. Daniel, S. A.; Thompson, T.: Methadone-induced attenuation of
8. 9.
10. 11. 12. 13.
the effects of D9-tetrahydrocannabinol on temporal discrimination in pigeons. J. Pharmacol. Exp. Ther. 213:247–253; 1980. Ehyai, A.; Freemon, F. R.: Progressive optic neuropathy and sensorineural hearing loss due to chronic glue sniffing. J. Neurol. Neurosurg. Psychiatry 46:349–351; 1983. Elofsson, S. A.; Gamberale, F.; Hindmarsh, T.; Iregren, A.; Isaksson, A.; Johnsson, I.; Knave, B.; Lydahl, E.; Mindus, P.; Persson, H. E.; Philipson, B.; Steby, M.; Struwe, G.; Söderman, E.; Wennberg, A.; Widén, L.: Exposure to organic solvents: A cross-sectional epidemiologic investigation on occupationally exposed car and industrial spray painters with special reference to the nervous system. Scand. J. Work Environ. Health 6:239–273; 1980. Evans, H. L.; Daniel, S. A.: Discriminative behavior as an index of toxicity. Advances in Behavioral Pharmacology 4:257–283; 1984. Geller, I.; Hartmann, R. J.; Randle, S. R.; Gause, E. M.: Effects of acetone and toluene vapors on multiple schedule performance of rats. Pharmacol. Biochem. Behav. 11:395–399; 1979. Glowa, J. R.: Comparisons of some behavioral effects of d-amphetamine and toluene. Neurotoxicology 8:237–248; 1987. Glowa, J. R.; DeWeese, J.; Natale, M. E.; Holland, J. J.; Dews, P. B.:
718
14. 15.
16. 17. 18. 19. 20.
21. 22.
WADA Behavioral toxicology of volatile organic solvents I. Methods: Acute effects of toluene. J. Environ. Pathol. Toxicol. Oncol. 6:153–168; 1986. Hinman, D. J.: Biphasic dose-response relationship for effects of toluene inhalation on locomotor activity. Pharmacol. Biochem. Behav. 26:65–69; 1987. Iregren, A.: Effects on psychological test performance of workers exposed to a single solvent (toluene)—A comparison with effects of exposure to a mixture of organic solvents. Neurobehav. Toxicol. Teratol. 4:695–701; 1982. Kirk, R. E.: Experimental design: Procedures for the behavioral sciences, 2nd ed. Monterey, CA: Brooks/Cole Publishing Company; 1982. Kishi, R.; Harabuchi, I.; Ikeda, T.; Yokota, H.; Miyake, H.: Neurobehavioral effects and pharmacokinetics of toluene in rats and their relevance to man. Br. J. Ind. Med. 45:396–408; 1988. Maricq, A. V.; Church, R. M.: The differential effects of haloperidol and methamphetamine on time estimation in the rat. Psychopharmacology 79:10–15; 1983. Maricq, A. V.; Roberts, S.; Church, R. M.: Methamphetamine and time estimation. J. Exp. Psychol. (Anim. Behav. Process.) 7:18–30; 1981. Molnár, J.; Paksy, K. Á.; Náray, M.: Changes in the rat’s motor behaviour during 4-hr inhalation exposure to prenarcotic concentrations of benzene and its derivatives. Acta Physiol. Hung. 67:349–354; 1986. Moser, V. C.; Balster, R. L.: The effects of acute and repeated toluene exposure on operant behavior in mice. Neurobehav. Toxicol. Teratol. 3:471–475; 1981. Moser, V. C.; Balster, R. L.: The effects of inhaled toluene, halothane, 1,1,1-trichloroethane, and ethanol on fixed-interval responding. Neurobehav. Toxicol. Teratol. 8:525–531; 1986.
23. Nakagaki, K.; Arito, H.; Tsuruta, H.: Changes in sleep-waking rhythms of rats following a single injection of toluene. Ind. Health 21:165–174; 1983. 24. Nakagaki, K.; Tsuruta, H.; Arito, H.: Determination of toluene concentrations in blood intermittently sampled from jugular veincatheterized rats. Ind. Health 20:147–150; 1982. 25. Pryor, G. T.: A toluene-induced motor syndrome in rats resembling that seen in some human solvent abusers. Neurotoxicol. Teratol. 13:387–400; 1991. 26. Rebert, C. S.; Sorenson, S. S.; Howd, R. A.; Pryor, G. T.: Toluene-induced hearing loss in rats evidenced by the brainstem auditory-evoked response. Neurobehav. Toxicol. Teratol. 5:59–62; 1983. 27. Shigeta, S.; Aikawa, H.; Misawa, T.; Kondo, A.: Effect of single exposure to toluene on Sidman avoidance response in rats. J. Toxicol. Sci. 3:305–312; 1978. 28. Sullivan, M. J.; Rarey, K. E.; Conolly, R. B.: Ototoxicity of toluene in rats. Neurotoxicol. Teratol. 10:525–530; 1989. 29. Wada, H.: Toluene and temporal discrimination behavior in the rat. Neurotoxicol. Teratol. 19:399–403; 1997. 30. Wada, H.; Hosokawa, T.; Saito, K.: Effects of single exposure to toluene on shock avoidance and time estimation in rats. Neurobehav. Toxicol. Teratol. 8:727–730; 1986. 31. Wada, H.; Hosokawa, T.; Saito, K.: Single toluene exposure and changes of response latency in shock avoidance performance. Neurotoxicol. Teratol. 11:265–272; 1989. 32. Winer, B. J.: Statistical principles in experimental design, 2nd ed. New York: McGraw-Hill; 1971. 33. Wood, R. W.; Colotla, V. A.: Biphasic changes in mouse motor activity during exposure to toluene. Fundam. Appl. Toxicol. 14:6–14; 1990.
PII S0892-0362(99)00033-1
Toluene and Temporal Discrimination in Rats: Effects on Accuracy, Discriminability, and Time Estimation HIROMI WADA Hokkaido University, College of Medical Technology, Sapporo, Japan Received 6 April 1997; Accepted 19 April 1999 WADA, H. Toluene and temporal discrimination in rats: Effects on accuracy, discriminability, and time estimation. NEUROTOXICOL TERATOL 21(6) 709–718, 1999.—Thirty-five rats acquired a temporal discrimination of seven signal durations in an operant chamber with two levers. If a 2-s or an 8-s tone signal was presented, rats were required to press one lever (”short” response) or the other lever (”long” response) to be reinforced, respectively. Neither response was reinforced when five intermediate signals were presented. Percentages of a long response in each of the seven signals were calculated and the psychophysical function between signal durations and long response percentages was obtained. Intraperitoneal injections of 50 mg/kg and 100 mg/kg toluene steepened a gradient of the psychophysical function and elevated correct responses in 2-s and 8-s signals. The function showed lower difference limen and Weber fraction. Accuracy and discriminability of temporal discrimination were enhanced. However, 400 mg/kg and 600 mg/kg toluene resulted in a shallower gradient and reduced correct responses. Higher difference limen and Weber fraction were also obtained. Accuracy and discriminability deteriorated and, in particular, behavioral depression was observed for 600 mg/kg toluene. The point of indifference was changed and dose-related overestimation or underestimation of time were suggested to occur. Blood toluene levels were reported to be 11–18 mg/ml for 50–100 mg/kg toluene and 47–73 mg/ml for 400–600 mg/kg toluene. It is speculated that temporal discrimination was sharpened by 10–20 mg/ml toluene in blood and disrupted by 50–70 mg/ml toluene in blood. © 1999 Elsevier Science Inc. All rights reserved. Toluene
Temporal discrimination
Time estimation
Rat
animals if they did not respond within an interval of time. Thus, toluene induced deterioration of temporal differentiation and it was hypothesized that toluene disturbed the timing of responding (21,27,30,31). The other type of reinforcement procedure concerning temporal discrimination requires animals to estimate temporal durations of signals (time estimation methods). Animals can be trained to press one lever if a signal duration is estimated as “short” and to press the other lever if it is estimated as “long” (7,18,19). In a preliminary report (29), the author applied this temporal discrimination to investigate acute effects of 100, 200, and 400 mg/kg toluene and revealed that toluene affected accuracy and discriminability. Toluene interferes with temporal differentiation and discrimination (13,21,22,27,29–31) and it causes hazardous effects in our daily life and workplaces (e.g., playing games with
TEMPORAL differentiation and discrimination are important functions of humans and other animals that affect their cognitive processes, and toluene has been reported to disturb temporal behavior in various reinforcement schedules (6,11– 13,21,22,27,29–31). For example, a fixed interval (FI) schedule is characterized by an initial pause and a following response acceleration (FI scallop), but toluene disrupts this response pattern (13,22). A differential reinforcement of low rate (DRL) schedule reinforces specified interresponse times (IRTs) and, accordingly, animals learn to withhold a response and produce particular IRTs. However, toluene-exposed mice responded with shorter IRTs and, occasionally, bursts of high-frequency responding were observed (21). The shortening of IRTs and response latencies (RLs) was found in shock avoidance schedules of the Sidman type (27) and the shuttle type (30,31) in which an electrical shock was presented to the
Requests for reprints should be addressed to H. Wada, Hokkaido University, College of Medical Technology, Kita 12, Nishi 5, Kita-Ku, Sapporo, 060-0812 Japan. Tel: 181-11-706-3321; Fax: 181-11-706-4916; E-mail: [email protected]
709
710
WADA
a moving ball, driving a car, and operating machines on an assembly line) because toluene-exposed workers responded slowly and showed poorer performance of perceptual speed and reaction time (9,15). The present research focused on accuracy, discriminability, and time estimation of a temporal discrimination, clarified dose-dependent effects of toluene, and determined blood and brain toluene levels that affected temporal discrimination. METHODS
Subjects Thirty-five male rats of the Wistar strain, about 100 days old, were used for this experiment. They were individually housed in plastic cages under a 12-h light-dark cycle (light 1900–700, dark 700–1900). All experiments were carried out during the dark period. Their body weights were maintained at 250 6 10 g to control motivation levels, with additional food provided after the daily experiment to do so. Tap water was always available in the home cage. The room temperature was maintained at 22 6 28C and relative humidity was 50 6 10%. Environmental conditions were in accordance with the Guide for the Care and Use of Laboratory Animals (Hokkaido University School of Medicine, 1992). Apparatus Five standard operant chambers (27 cm in height, 25 cm in width, and 30 cm in length) with two levers were used. They consisted of acrylic plastic panels and the front panels were of clear plastic so the subjects could be observed. The flooring was made of stainless steel grids 5 mm diameter spaced 2 cm apart. A lamp, a food cup, and two levers were located on the side panel. A lamp was mounted on the center 11 cm above the floor and provided dim light to observe a subject. The food cup was 10 cm below the lamp; a food pellet (45 mg) could be delivered into the cup from a pellet dispenser. Two levers (3 cm in width and 3 cm in length) protruded from the panel, one 8 cm to the right and the other 8 cm to the left of the food cup and 3 cm above the floor. A house light and a tone signal were presented from the ceiling of the chamber. A speaker with a diameter of 17 cm was installed outside of the chamber and white noise (70 dB) was provided for masking extraneous sounds. The operant chamber was set into an insulated box to attenuate external light and sound. A personal computer (PC9801-RX, NEC, Japan) controlled the reinforcement schedule and data recording. The software was written in MS-C. Procedure Temporal discrimination of two signals. After training to press each lever, rats were trained to press one lever after 2-s tone signals (short response) and the other lever after 8-s tone signals (long response). The tone signal was a complex sound composed of 3 kHz and 6 kHz in frequency and 80 dB in loudness. Assignment of signal durations (2 s or 8 s) and levers (right or left) was counterbalanced among rats. A trial started with the house light on and then one of two signals was presented after 1 s. Each signal was presented 25 times with a probability of 0.5. If the rat pressed the correct lever, the response was reinforced by a food pellet, but if it pressed the incorrect lever, no reinforcer was delivered. Then the house light was turned off and the next trial began following an intertrial interval (ITI) of 10 s. When the rat pressed a lever during a signal, the trial was terminated and the same
trial started again after a 10-s ITI. A limited hold (LH) of 30 s was employed: If no response occurred within 30 s of the end of the signal a 10-s ITI was then initiated and followed by the next trial. All responses during ITIs and intervals between the house light and the signal did not affect the reinforcement schedule. A session comprised 50 trials and one session was run every other day for 30 sessions. The percentage of a correct response in 2-s and 8-s signals reached 85 6 2.2% and 80 6 2.8%, respectively (mean 6 SEM). Temporal discrimination of seven signals. Five intermediate signals of 2.5-, 3.2-, 4.0-, 5.0-, and 6.3-s duration spaced at equal logarithmic intervals between 2-s and 8-s signals were introduced. The rat was trained to establish a temporal discrimination of the seven signals. Signals of 2-s and 8-s durations were presented 25 times each with a probability of 0.25 and five signals of intermediate durations were presented ten times each with a probability of 0.1. A trial started with the house light on and then one of seven signals was pseudorandomly presented after 1 s. When a 2-s or an 8-s signal was presented, the correct response was reinforced and the incorrect one was not reinforced. When one of five intermediate signals was presented, no response was reinforced. Then the house light was turned off and the next trial began following a 10-s ITI. When the rat responded during a signal, the trial was terminated and the same trial started again after a 10-s ITI. An LH of 30 s terminated the trial with no response. A session comprised 100 trials and one session was run every other day for seven sessions. The percentage of a correct response in 2-s and 8-s signals reached 86 6 1.6% and 88 6 2.0%, respectively (mean 6 SEM). These data served as precontrols (noninjection). Toluene injections. All rats next received seven-signal temporal discrimination training every other day for six sessions. They were injected with 0.1 ml olive oil intraperitoneally 1 h before the training. Finally, the percentage of a correct response in 2-s and 8-s signals reached 92 6 1.1% and 91 6 1.4%, respectively (mean 6 SEM) and the behavioral data of the last session served as vehicle controls (olive oil injection). The next day, rats were divided into five groups of seven per group and received intraperitoneal injections of toluene at dosages of 50, 100, 200, 400, or 600 mg/kg. Toluene was dissolved in an equal volume of olive oil to prevent inflammation of internal organs and the injection was carried out 1 h before the experiment. Retraining of temporal discrimination. Following toluene injections, all rats were kept in home cages for a month and then received retraining of seven-signal temporal discrimination for six sessions. A session comprised 100 trials and one session was run every other day. The data of the last session served as postcontrols (noninjection). Statistical Analysis Behavioral data were analyzed by a two-factor analysis of variance (groups and sessions) with repeated measures of sessions (32). If a significant effect was found for groups, the Spjøtvoll–Stoline test, which is a generalization of Tukey’s HSD test appropriate for unequal sample sizes, was employed for multiple comparisons (16). RESULTS
General Observations All rats established a stable and robust baseline of temporal discrimination but two rats of the 400-mg/kg–injected
TOLUENE AND TEMPORAL DISCRIMINATION group and three rats of the 600-mg/kg–injected group did not press the lever. Although no response during the five intermediate signals provided a reinforcer, the rats’ performance was not affected and there was no evidence of extinction found in this experiment. Percentage of Trials With a Response The percentage of trials with a response was calculated as (the number of trials with a response/the number of total trials) 3 100. Figure 1 shows the effects of toluene on the percentage of trials with a response. Precontrols versus vehicle controls. The percentage was nearly 100% for all groups in precontrols and vehicle controls. No significant difference was obtained for groups, sessions (precontrols vs. vehicle controls), and interactions. The groups were equivalent in the response percentage and effects of olive oil were not found. Vehicle controls versus toluene injections. There was a steep decrease in the percentage for the 400-mg/kg– and the 600mg/kg–injected groups. It was below 10% for two rats after 400 mg/kg toluene and for four rats after 600 mg/kg toluene. The 50-, 100-, and 200-mg/kg–injected groups did not alter the response percentage. They responded at the same level as vehicle controls. Significant effects were found for groups (dosages) [F(4, 30) 5 11.88, p , 0.01]; sessions (vehicle controls vs. toluene injections) [F(1, 30) 5 26.45, p , 0.01]; and interactions [F(4, 30) 5 11.94, p , 0.01]. The 600-mg/kg–injected group showed a significant decrease of the percentage compared with the 50-, 100-, and 200-mg/kg–injected groups (p , 0.01) and the 400-mg/kg–injected group (p , 0.05) by a multiple comparison test. Toluene reduced the response percentage in a dose-dependent manner and, in particular, 600 mg/kg toluene caused a severe decrease. Vehicle controls versus postcontrols. The 400-mg/kg– and the 600-mg/kg–injected groups recovered and showed the same percentage as vehicle controls. Significant differences were not observed for groups, sessions (vehicle controls vs. postcontrols), and interactions, and effects of high-volume toluene disappeared. The response percentage in each of the seven signals was
711 calculated. Each rat responded equally to all signals whether it could receive a reinforcer or not. Psychophysical Function Each rat was trained to press one of two levers after an 8-s signal and it was called a long response. The percentage of trials with a long response in each of the seven signals was calculated as (the number of trials with a long response/the number of trials with a response) 3 100. The psychophysical function between signal durations and long response percentages was obtained for 35 rats by linear regression using the method of least squares. The gradient, point of indifference (PI), difference limen (DL), and Weber fraction (WF), which were important measures for discrimination behavior (10), were estimated from the individual psychophysical functions of 35 rats. The regression coefficient of the psychophysical function served as the gradient. The PI was the signal duration associated with a 50% long response, which was a 50% choice point for a short or a long response. We can consider overestimation or underestimation of signal durations by the shift of the PI. Signal durations associated with a 25% long response and a 75% long response were calculated, and one-half of the range between these two durations was defined as the DL. The DL is a duration from the PI, which is necessary to estimate a signal as short or long with a 75% probability. The WF was defined as DL/PI, which was a constant ratio between an arbitrary duration and its DL (Weber’s law). Small DLs and WFs indicate higher discriminability and large ones indicate lower discriminability. The effects of toluene on these measures are presented in Table 1, and the psychophysical function for groups is indicated in Fig. 2. Precontrols versus vehicle controls. The gradient of vehicle controls was larger than that of precontrols for all groups and the psychophysical function became steeper. Groups and interactions were not significant, and the gradient was equivalent between groups. Sessions (precontrols vs. vehicle controls) were significant [F(1, 30) 5 14.65, p , 0.01]. There was no significant difference of groups, sessions, and interactions on the PI. All groups were equivalent in the PI and olive oil injections did not affect it. The DL and the WF were de-
FIG. 1. Effects of toluene on the percentage of trials with a response. Data are mean and SEM.
712
WADA TABLE 1 EFFECTS OF TOLUENE ON THE PSYCHOPHYSICAL FUNCTION Dosage (mg/kg)
Gradient Pre Vehicle Toluene Post Point of indifference Pre Vehicle Toluene Post Difference limen Pre Vehicle Toluene Post Weber fraction Pre Vehicle Toluene Post
50
100
200
11.87 6 0.86 12.19 6 0.69 14.12 6 0.70 11.97 6 0.67
13.21 6 0.84 14.59 6 0.34 15.62 6 0.74 12.53 6 0.84
13.75 6 0.85 15.82 6 0.69 14.55 6 1.39 14.46 6 0.82
5.05 6 0.43 5.46 6 0.27 5.00 6 0.17 5.68 6 0.37
4.93 6 0.16 4.72 6 0.21 4.81 6 0.19 4.81 6 0.19
2.19 6 0.17 2.10 6 0.12 1.80 6 0.09 2.14 6 0.14 0.47 6 0.08 0.39 6 0.02 0.36 6 0.03 0.38 6 0.02
400
600
12.47 6 1.18 16.04 6 0.77 8.59 6 2.30 13.40 6 1.23
13.20 6 0.44 15.21 6 1.20 3.19 6 4.21 14.52 6 1.45
5.37 6 0.28 4.96 6 0.19 5.39 6 0.34 5.41 6 0.27
5.23 6 0.47 5.18 6 0.25 39.51 6 30.01 4.93 6 0.36
5.06 6 0.16 4.93 6 0.13 68.51 6 49.20 4.94 6 0.11
1.95 6 0.13 1.72 6 0.04 1.63 6 0.08 2.06 6 0.15
1.88 6 0.14 1.60 6 0.07 1.89 6 0.26 1.77 6 0.11
2.18 6 0.28 1.58 6 0.07 19.87 6 15.50 1.99 6 0.20
1.91 6 0.06 1.74 6 0.19 37.12 6 27.33 1.88 6 0.23
0.40 6 0.04 0.37 6 0.02 0.34 6 0.03 0.43 6 0.03
0.35 6 0.02 0.32 6 0.01 0.34 6 0.03 0.33 6 0.02
0.41 6 0.02 0.31 6 0.01 0.46 6 0.05 0.43 6 0.07
0.38 6 0.02 0.35 6 0.04 0.53 6 0.09 0.37 6 0.04
Data are calculated from individual psychophysical functions of 35 rats. Values are mean 6 SEM.
creased for vehicle controls compared with precontrols. Sessions were significant for the DL [F(1, 30) 5 8.99, p , 0.01] and the WF [F(1, 30) 5 6.27, p , 0.05]. However, there was no difference for groups and interactions. Vehicle controls versus toluene injections. The 50-mg/kg– and the 100-mg/kg–injected groups showed a higher gradient compared with vehicle controls. In the 200-mg/kg–injected group, the gradient was lowered. That of the 400-mg/kg– and the 600-mg/kg–injected groups decreased. Significant effects were found for groups (dosages) [F(4, 25) 5 3.67, p , 0.05] and sessions (vehicle controls vs. toluene injections) [F(1, 25) 5 18.89, p , 0.01]. Interactions were also significant [F(4, 25) 5 10.47, p , 0.01]. The gradient reflects discriminability of stimulus durations. A steep gradient indicates high discriminability and a shallow one indicates low discriminability. The 50mg/kg– and the 100-mg/kg–injected groups were sensitive to discriminate temporal durations but the 400-mg/kg– and the 600-mg/kg–injected groups became blunt in discriminability. Most severe degradation was revealed for the 600-mg/kg–injected group compared with the 100-mg/kg–injected group (p , 0.01), the 50-mg/kg–, and the 200-mg/kg–injected groups (p , 0.05) by a multiple comparison test. The PI was reduced for the 50mg/kg–injected group but the other groups enhanced it. There were significant effects for sessions [F(1, 25) 5 4.48, p , 0.05]. The change of a PI means the temporal shift of a psychophysical function. The decrease of a PI shifts the function leftward and its increase shifts the function rightward. The injection with 50 mg/kg toluene reduced the PI and shifted the function leftward. The percentage of trials with a long response was increased in longer durations and overestimation of temporal durations could occur (Fig. 2a). By contrast, toluene injections of 200, 400, and 600 mg/kg increased PIs. The 200-mg/ kg–injected group shifted the functions rightward, the long response percentage was decreased, and signal durations could be underestimated (Fig. 2c). The most dramatic effect was ob-
served for the 400-mg/kg– and the 600-mg/kg–injected group, which shorter durations were overestimated and longer ones were underestimated (Figs. 2d–2e). Thus, toluene seemed to affect time estimation and cause dose-dependent overestimation or underestimation of temporal durations. The DL and the WF were reduced for the 50-mg/kg– and the 100-mg/kg–injected groups but increased for the 200-, 400-, and 600-mg/kg–injected groups. Sessions were significant for the DL [F(1, 25) 5 4.60, p , 0.05] and the WF [F(1, 25) 5 12.27, p , 0.01]. Interactions were also significant for the WF [F(4, 25) 5 6.38, p , 0.01]. Small DLs and WFs show higher discriminability of temporal durations and large ones show lower discriminability (18,19). Toluene affected discriminability, and low volumes of toluene elevated it and high volumes of toluene degraded it. Vehicle controls versus postcontrols. The gradient was decreased for all groups, and sessions (vehicle controls vs. postcontrols) were significant [F(1, 30) 5 8.75, p , 0.01]. The DL and the WF were increased and significant differences were found for sessions [F(1, 30) 5 8.19, p , 0.01] and [F(1, 30) 5 4.42, p , 0.01]. However, groups and interactions were not significant. No significant difference was obtained for the PI. Thus, groups were equivalent in these measures and the highvolume–injected groups recaptured temporal discrimination at the same level as the low-volume–injected groups. Accuracy in 2-s and 8-s Signals When the signal duration was 2 s or 8 s, rats could receive a reinforcer if they pressed the correct lever. Two-second and 8-s durations were standards to classify a signal duration as short or long. The percentage of trials with a correct response in 2-s signals (short response percentage) and 8 s signals (long response percentage) is indicated in Fig. 3. Precontrols versus vehicle controls. The correct response percentages in 2-s signals were enhanced for all groups. Significant
TOLUENE AND TEMPORAL DISCRIMINATION
713
FIG. 2. Effects of toluene on the percentage of trials with a long response. The point in the diagram indicates long response percentages in signal durations of 2, PI 2 DL, PI, PI 1 DL, and 8 s. The PI 2 DL, PI, and PI 1 DL are signal durations matching to 25, 50, and 75% long responses, respectively. The psychophysical function for groups was calculated between these signal durations and long response percentages. Data are mean.
differences were obtained for sessions (precontrols vs. vehicle controls) [F(1, 30) 5 14.36, p , 0.01], but groups and interactions were not significant. The correct response percentages in 8-s signals did not show any difference of groups, sessions, and interactions. The groups were equivalent in accuracy. Vehicle controls versus toluene injections. The 50-mg/kg– and the 100-mg/kg–injected groups showed a higher correct percentage in 2-s signals. A lower percentage was found for the 400-mg/kg– and the 600-mg/kg–injected groups. The same result was observed in 8-s signals with a dose-dependent manner. Significant effects were found for sessions (vehicle controls vs. toluene injections) in 2-s signals [F(1, 25) 5 4.74, p , 0.05]. Groups (dosages), sessions, and interactions were significant in 8-s signals [F(4, 25) 5 5.48, p , 0.01; F(1, 25) 5 14.58, p , 0.01; and F(4, 25) 5 10.94, p , 0.01, respectively]. The 600-mg/kg–injected group showed significant differences of the correct response percentage in 8-s signals compared with the 50-, 100-, and 200-mg/kg–injected groups (p , 0.01) and the 400-mg/kg–injected group (p , 0.05) by a multiple comparison test. The percentage was increased by 50 mg/kg toluene and low volumes of toluene enhanced the accuracy of
temporal discrimination. However, the percentage was decreased by 400 mg/kg and 600 mg/kg toluene and high volumes caused accuracy to deteriorate. Vehicle controls versus postcontrols. No significant differences were observed for groups, sessions (vehicle controls vs. postcontrols), and interactions. The 400-mg/kg– and the 600mg/kg–injected groups showed the same correct response percentage as vehicle controls. They could recover accuracy in temporal discrimination.
ITI Response Rate A response in a 10-s ITI did not have any influence on the reinforcement schedule. An ITI response rate was calculated as (the total number of responses in ITIs/the number of ITIs) and is presented in Table 2. The 600-mg/kg–injected group decreased the ITI response rate markedly but recovered in postcontrols. There was no significant effect for groups (dosages), sessions (vehicle controls vs. toluene injections), and interactions. No differences
714
WADA
FIG. 3. Effects of toluene on the percentage of trials with a correct response in 2-s and 8-s signals. Data are mean and SEM.
were obtained in precontrols versus vehicle controls and vehicle controls versus postcontrols. Response Latency A response latency (RL) was the time interval from the termination of a signal to the initiation of a response. Table 3
indicates the changes in RLs. Because the rat could receive a reinforcer in 2-s and 8-s signals, RLs in these signals were separately obtained. Precontrols versus vehicle controls. The RL in total responses was shortened except for the 50-mg/kg–injected group and significant differences were found [F(4, 30) 5 2.87, p , 0.05], which indicated groups were not equivalent. Then,
TABLE 2 EFFECTS OF TOLUENE ON THE ITI RESPONSE RATE Dosage (mg/kg)
Pre Vehicle Toluene Post
50
100
200
400
600
0.26 6 0.05 0.28 6 0.06 0.23 6 0.05 0.27 6 0.06
0.45 6 0.08 0.37 6 0.08 0.41 6 0.10 0.46 6 0.07
0.23 6 0.04 0.28 6 0.04 0.26 6 0.03 0.29 6 0.06
0.25 6 0.04 0.32 6 0.05 0.25 6 0.10 0.30 6 0.07
0.30 6 0.07 0.27 6 0.06 0.04 6 0.02 0.41 6 0.10
Values are mean 6 SEM.
TOLUENE AND TEMPORAL DISCRIMINATION
715 TABLE 3
EFFECTS OF TOLUENE ON THE RESPONSE LATENCY Dosage (mg/kg)
Total Pre Vehicle Toluene Post 2s Pre Vehicle Toluene Post 8s Pre Vehicle Toluene Post
50
100
200
400
600
2.44 6 0.24 2.51 6 0.28 2.16 6 0.22 2.49 6 0.27
3.33 6 0.45 2.81 6 0.34 2.26 6 0.24 3.52 6 0.38
2.30 6 0.27 2.18 6 0.41 2.48 6 0.49 2.94 6 0.56
2.75 6 0.36 2.22 6 0.17 3.75 6 0.70 2.58 6 0.23
3.51 6 0.18 2.96 6 0.29 7.44 6 2.37 3.40 6 0.20
3.05 6 0.21 2.69 6 0.40 2.04 6 0.14 2.54 6 0.16
4.38 6 0.63 3.06 6 0.50 2.55 6 0.31 4.12 6 0.56
2.68 6 0.53 2.65 6 0.56 2.57 6 0.68 3.48 6 0.73
3.19 6 0.57 1.93 6 0.24 3.37 6 0.82 2.60 6 0.39
3.77 6 0.38 3.03 6 0.30 6.05 6 2.68 3.65 6 0.43
1.79 6 0.35 1.15 6 0.12 1.37 6 0.28 1.49 6 0.21
2.32 6 0.40 1.65 6 0.21 1.33 6 0.11 2.19 6 0.39
1.50 6 0.25 1.16 6 0.17 1.38 6 0.30 1.83 6 0.33
1.98 6 0.51 1.48 6 0.28 4.54 6 11.10 1.53 6 0.18
2.47 6 0.56 2.34 6 0.33 6.38 6 3.20 2.23 6 0.40
Values are mean 6 SEM.
the RL in toluene injections and postcontrols was recalculated as the percentage of that in vehicle controls for following analyses. RLs in 2-s and 8-s signals were also shortened. Neither groups nor interactions showed any difference but sessions (precontrols vs. vehicle controls) were significant in 2-s signals [F(1, 30) 5 6.90, p , 0.05] and 8-s signals [F(1, 30) 5 4.85, p , 0.05]. Vehicle controls versus toluene injections. The 50-mg/kg– and the 100-mg/kg–injected groups decreased RLs in total responses and responded quickly. The 200-, 400-, and 600-mg/ kg–injected groups increased RLs and responded slowly. Significant effects were obtained for groups (dosages) [F(4, 29) 5 2.85, p , 0.05], sessions (vehicle controls vs. toluene injections) [F(1, 29) 5 5.53, p , 0.05], and interactions [F(4, 29) 5 2.85, p , 0.05]. The RL in 8-s signals was lengthened and in particular, the 400-mg/kg– and the 600-mg/kg–injected groups responded with a longer RL. It was significant for groups [F(4, 28) 5 3.56, p , 0.05] and sessions [F(1, 28) 5 6.51, p , 0.05]. The RL in 2-s signals was shortened for the 50-mg/kg– and the 100-mg/kg–injected groups and lengthened for the 400-mg/ kg– and the 600-mg/kg–injected groups. However, there was no significant effect for groups, sessions, and interactions. Vehicle controls versus postcontrols. The 400-mg/kg– and the 600-mg/kg–injected groups were reinstated in RLs and indicated the same ones as the other groups. Neither groups nor interactions were significant for RLs in total responses, RLs in 2-s signals and those in 8-s signals, but sessions (vehicle controls vs. postcontrols) were significant [F(1, 30) 5 13.89, p , 0.01; F(1, 30) 5 11.55, p , 0.01; and F(1, 30) 5 4.98, p , 0.05, respectively].
p , 0.01]. For the compensation of the group inequality, the number of restarted trials in toluene injections and postcontrols was recalculated as the percentage of that in vehicle controls. Vehicle controls versus toluene injections. There was no significant effect of groups, sessions (vehicle controls vs. toluene injections), and interactions, but the 600-mg/kg–injected group eliminated restarted trials almost completely. The other groups increased them and in particular, the 400-mg/kg–injected group clearly elevated restarted trials. It was evidenced that 400 mg/kg toluene did not cause ataxia or paralysis. Vehicle controls versus postcontrols. The 600-mg/kg–injected group indicated the same level of restarted trials as vehicle controls and recovered them. No significant differences were observed for groups, sessions (vehicle controls vs. postcontrols), and interactions. The effects of toluene disappeared in postcontrols. ITI Response Rate After Reset An ITI response rate after a reset was calculated as (the total number of responses in ITIs after a reset/the number of resets) and it is presented in Figure 4 (bottom). The 600-mg/kg–injected group showed a marked decrease in ITI response rate after a reset and significant effects were obtained for groups (dosages) [F(4, 23) 5 3.96, p , 0.05]. However, the 600-mg/kg–injected group recovered in postcontrols. Neither precontrols versus vehicle controls nor vehicle controls versus postcontrols had any significant difference. DISCUSSION
Restarted Trial When rats responded during signal presentations, the trial was reset and started again following a 10-s ITI. The rat was required to withhold a response during signal durations. Figure 4 (top) indicates the number of restarted trials. Precontrols versus vehicle controls. The number of restarted trials was higher for the 200-mg/kg–injected group and significant differences were found for groups [F(4, 30) 5 5.29,
It has been revealed that toluene has dose-dependent biphasic effects on animal behavior, that is, excitatory effects at low dosages and depressive effects at high dosages (3,14, 20,31,33). The same effect was found for temporal differentiation behavior. IRTs in a DRL schedule were shortened at 1600-ppm and 3200-ppm toluene exposures and their distributions showed leftward shifts, but 6400-ppm toluene made distributions flat (21). Decreased IRTs were also observed in
716
WADA
FIG. 4. Effects of toluene on the number of restarted trials (top) and the ITI response rate after a reset (bottom). Data are mean and SEM.
Sidman avoidance learning at concentrations of 1000-ppm and 3000-ppm toluene vapor (27). In particular, 3000-ppm toluene exposure caused a marked shortening of IRTs. Sequential changes of RLs following toluene exposures were analyzed in shock avoidance learning (30,31). Immediately after exposure, RL histograms were shifted leftward at 2000-ppm toluene vapor and RLs were shortened. Avoidance responses were decreased in 6000-ppm and 8000-ppm toluene-exposed rats and RL histograms became flat; nevertheless they recovered avoidance performance after 3 h when a shortening of RLs was observed. These results indicate that lower levels of toluene (1000–3000 ppm) produce shortening of IRTs or RLs and quicken responses, that is, excitatory effects on temporal differentiation, and higher levels of toluene (6000–8000 ppm) produce flattening of their histograms, that is, depressive effects on temporal differentiation. Biphasic effects of toluene on temporal discrimination were observed in the present experiment using the time estimation method. Analyzing the psychophysical function between signal durations and long responses, the 50-mg/kg– and the 100-mg/kg–injected groups had elevated the gradient of the function and decreased the DL and the WF. These mea-
sures are indices of discriminability and showed that the rats’ discriminability was elevated (18,19,29). Moreover, these injections increased correct responses in the standard 2-s and 8-s training signals with shorter RLs. The rats responded more quickly and accurately. Lower volumes of toluene enhanced accuracy and discriminability, and improved temporal discrimination due to excitatory effects. By contrast, the 400mg/kg– and the 600-mg/kg–injected groups decreased the gradient and the psychophysical function became moderate and shallow. The DL and the WF were increased, which showed that discriminability had deteriorated at these doses. Correct response percentages were lowered in the standard two training signals and the rats responded slowly. Higher volumes of toluene degraded accuracy and discriminability, and disrupted temporal discrimination due to depressive effects. A toluene dose of 50 mg/kg increased the gradient, decreased the PI, and produced a leftward shift in the temporal gradient of the psychophysical function. Toluene doses of 200, 400, and 600 mg/kg decreased the gradient and increased the PI dose dependently. The 50-mg/kg–injected group estimated signal durations as being longer (overestimation) and the 200-, 400-, and 600-mg/kg–injected groups estimated durations as
TOLUENE AND TEMPORAL DISCRIMINATION being shorter (underestimation). Further research is necessary to clarify acute effects of toluene on time estimation. High volumes of toluene affect motor activity and cause ataxia or paralysis (3,14,31,33). For the 50-, 100-, and 200-mg/ kg–injected groups, the percentages of trials with a response were at the same level as vehicle controls and depressive effects on behavior were not found. The 400-mg/kg–injected group decreased response percentages and depressive effects were observed. However, the number of restarted trials was definitely elevated and the rats frequently responded during signal presentations. They could not withhold a response and thus, behavioral depression did occur but ataxia or paralysis did not occur. The 400-mg/kg–injected group produced longer RLs and responded more slowly to the signal. It has been hypothesized that longer latencies cause attentional lapses, which are responsible for poor performance in temporal discriminations (18,19,29). The 600-mg/kg–injected group degraded all of the behavioral measures. Behavioral depression was marked and its nonspecific effects seemed to impair temporal discrimination. It was most likely that a larger volume of toluene and olive oil caused behavioral depression. The 200-mg/kg–injected group decreased the gradient of the psychophysical function. The DL and the WF were increased, which indicated that discriminability had been lowered. The injection of 200 mg/kg toluene was suggested to be the lowest level that produced depressive effects on temporal discrimination behavior. Toluene elevated auditory thresholds (25,26,28) and hearing loss occurred (4,8,25,28). Damage to cochlea hair cells has been detected (5,28). High volumes of toluene decreased accuracy in standard signals and induced severe impairments of temporal discrimination. However, there was no difference of groups in postcontrols and even the 400-mg/kg– and the 600mg/kg–injected groups could recapture baseline performance. We therefore conclude that acute effects of toluene are transient and reversible. In precontrols versus vehicle controls or vehicle controls versus postcontrols, several measures indicated significant differences of sessions. It was possible that olive oil injections affected performance, but we should notice that the rats received additional training between precontrols and vehicle controls, and they were kept in home cages for a month be-
717 fore retraining (postcontrols). Additional learning or forgetfulness could explain these differences. The type of experimental animals including strain, age, and gender is an important factor that can influence the pharmacokinetics of toluene. Although we must be cautious, efforts to determine the critical level of toluene associated with behavioral changes are of value in comparing a number of research findings, which employed different behavioral tests and solvent administrations (e.g., injection or inhalation). The toluene level in blood has been reported to be 19.6, 30.2, and 50.0 mg/ml after inhalation of 750-, 1500-, and 2700-ppm toluene vapor, respectively (1,2). Because toluene vapor that induced shorter IRTs or RLs is 1000–3000 ppm (21,27,30,31), it can be estimated that IRTs or RLs were shortened at blood toluene levels of 23–55 mg/ml. The response rates in a shock avoidance task were increased and acceleration of reaction times was observed at toluene levels of 27 mg/ml in blood and 32 mg/g in brain, and anesthetic effects occurred at 120 mg/ml in blood and 160 mg/g in brain (17). In the present research, toluene was dissolved with an equal volume of olive oil and injected intraperitoneally. For the same method of administration toluene levels in blood have been determined in rats (23,24). Toluene in blood at 30–60 min following injections reached maximum levels of 10.5, 17.9, 24.7, 47.2, and 73.2 mg/ml in blood with injections of 50, 100, 200, 400, and 600 mg/kg toluene, respectively. Thus, we can estimate that toluene induces excitatory effects on temporal discrimination at levels of 11–18 mg/ml in blood and depressive effects at 47–73 mg/ml in blood. These toluene levels are much lower than those that affected temporal differentiation, and temporal discrimination using the time estimation was more sensitive to acute toxicity of toluene. Therefore, the author concludes that the time estimation methods can be applied to analyze the toxicity of other chemicals as well.
ACKNOWLEDGEMENTS
This research was supported by a Grant-in-Aid for the Encouragement of Young Scientists no. 02851011 from the Ministry of Education, Science, Sports and Culture, Japan.
REFERENCES 1. Aikawa, H.; Momotani, H.; Shigeta, S.: Changes in blood toluene concentrations in rats under various kinds of exposure conditions and physical loads. (Japanese text with English abstract) Jpn. J. Ind. Health 30:379–384; 1988. 2. Arito, H.; Tsuruta, H.; Oguri, M.: Changes in sleep and wakefulness following single and repeated exposures to toluene vapor in rats. Arch. Toxicol. 62:76–80; 1988. 3. Bushnell, P. J.; Evans, H. L.; Palmes, E. D.: Effects of toluene inhalation on carbon dioxide production and locomotor activity in mice. Fundam. Appl. Toxicol. 5:971–977; 1985. 4. Bushnell, P. J.; Kelly, K. L.; Crofton, K. M.: Effects of toluene inhalation on detection of auditory signals in rats. Neurotoxicol. Teratol. 16:149–160; 1994. 5. Campo, P.; Lataye, R.; Cossec, B.; Placidi, V.: Toluene-induced hearing loss: A mid-frequency location of the cochlear lesions. Neurotoxicol. Teratol. 19:129–140; 1997. 6. Colotla, V. A.; Bautista, S.: Effects of solvents on schedule-controlled behavior. Neurobehav. Toxicol. 1(Suppl. 1):113–118; 1979. 7. Daniel, S. A.; Thompson, T.: Methadone-induced attenuation of
8. 9.
10. 11. 12. 13.
the effects of D9-tetrahydrocannabinol on temporal discrimination in pigeons. J. Pharmacol. Exp. Ther. 213:247–253; 1980. Ehyai, A.; Freemon, F. R.: Progressive optic neuropathy and sensorineural hearing loss due to chronic glue sniffing. J. Neurol. Neurosurg. Psychiatry 46:349–351; 1983. Elofsson, S. A.; Gamberale, F.; Hindmarsh, T.; Iregren, A.; Isaksson, A.; Johnsson, I.; Knave, B.; Lydahl, E.; Mindus, P.; Persson, H. E.; Philipson, B.; Steby, M.; Struwe, G.; Söderman, E.; Wennberg, A.; Widén, L.: Exposure to organic solvents: A cross-sectional epidemiologic investigation on occupationally exposed car and industrial spray painters with special reference to the nervous system. Scand. J. Work Environ. Health 6:239–273; 1980. Evans, H. L.; Daniel, S. A.: Discriminative behavior as an index of toxicity. Advances in Behavioral Pharmacology 4:257–283; 1984. Geller, I.; Hartmann, R. J.; Randle, S. R.; Gause, E. M.: Effects of acetone and toluene vapors on multiple schedule performance of rats. Pharmacol. Biochem. Behav. 11:395–399; 1979. Glowa, J. R.: Comparisons of some behavioral effects of d-amphetamine and toluene. Neurotoxicology 8:237–248; 1987. Glowa, J. R.; DeWeese, J.; Natale, M. E.; Holland, J. J.; Dews, P. B.:
718
14. 15.
16. 17. 18. 19. 20.
21. 22.
WADA Behavioral toxicology of volatile organic solvents I. Methods: Acute effects of toluene. J. Environ. Pathol. Toxicol. Oncol. 6:153–168; 1986. Hinman, D. J.: Biphasic dose-response relationship for effects of toluene inhalation on locomotor activity. Pharmacol. Biochem. Behav. 26:65–69; 1987. Iregren, A.: Effects on psychological test performance of workers exposed to a single solvent (toluene)—A comparison with effects of exposure to a mixture of organic solvents. Neurobehav. Toxicol. Teratol. 4:695–701; 1982. Kirk, R. E.: Experimental design: Procedures for the behavioral sciences, 2nd ed. Monterey, CA: Brooks/Cole Publishing Company; 1982. Kishi, R.; Harabuchi, I.; Ikeda, T.; Yokota, H.; Miyake, H.: Neurobehavioral effects and pharmacokinetics of toluene in rats and their relevance to man. Br. J. Ind. Med. 45:396–408; 1988. Maricq, A. V.; Church, R. M.: The differential effects of haloperidol and methamphetamine on time estimation in the rat. Psychopharmacology 79:10–15; 1983. Maricq, A. V.; Roberts, S.; Church, R. M.: Methamphetamine and time estimation. J. Exp. Psychol. (Anim. Behav. Process.) 7:18–30; 1981. Molnár, J.; Paksy, K. Á.; Náray, M.: Changes in the rat’s motor behaviour during 4-hr inhalation exposure to prenarcotic concentrations of benzene and its derivatives. Acta Physiol. Hung. 67:349–354; 1986. Moser, V. C.; Balster, R. L.: The effects of acute and repeated toluene exposure on operant behavior in mice. Neurobehav. Toxicol. Teratol. 3:471–475; 1981. Moser, V. C.; Balster, R. L.: The effects of inhaled toluene, halothane, 1,1,1-trichloroethane, and ethanol on fixed-interval responding. Neurobehav. Toxicol. Teratol. 8:525–531; 1986.
23. Nakagaki, K.; Arito, H.; Tsuruta, H.: Changes in sleep-waking rhythms of rats following a single injection of toluene. Ind. Health 21:165–174; 1983. 24. Nakagaki, K.; Tsuruta, H.; Arito, H.: Determination of toluene concentrations in blood intermittently sampled from jugular veincatheterized rats. Ind. Health 20:147–150; 1982. 25. Pryor, G. T.: A toluene-induced motor syndrome in rats resembling that seen in some human solvent abusers. Neurotoxicol. Teratol. 13:387–400; 1991. 26. Rebert, C. S.; Sorenson, S. S.; Howd, R. A.; Pryor, G. T.: Toluene-induced hearing loss in rats evidenced by the brainstem auditory-evoked response. Neurobehav. Toxicol. Teratol. 5:59–62; 1983. 27. Shigeta, S.; Aikawa, H.; Misawa, T.; Kondo, A.: Effect of single exposure to toluene on Sidman avoidance response in rats. J. Toxicol. Sci. 3:305–312; 1978. 28. Sullivan, M. J.; Rarey, K. E.; Conolly, R. B.: Ototoxicity of toluene in rats. Neurotoxicol. Teratol. 10:525–530; 1989. 29. Wada, H.: Toluene and temporal discrimination behavior in the rat. Neurotoxicol. Teratol. 19:399–403; 1997. 30. Wada, H.; Hosokawa, T.; Saito, K.: Effects of single exposure to toluene on shock avoidance and time estimation in rats. Neurobehav. Toxicol. Teratol. 8:727–730; 1986. 31. Wada, H.; Hosokawa, T.; Saito, K.: Single toluene exposure and changes of response latency in shock avoidance performance. Neurotoxicol. Teratol. 11:265–272; 1989. 32. Winer, B. J.: Statistical principles in experimental design, 2nd ed. New York: McGraw-Hill; 1971. 33. Wood, R. W.; Colotla, V. A.: Biphasic changes in mouse motor activity during exposure to toluene. Fundam. Appl. Toxicol. 14:6–14; 1990.