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Effects of ethanol and toluene on fixed-ratio performance in short sleep and long sleep mice
Drug and Alcohol Dependence, 31 (1992) 65 - 75
Elsevier Scientific Publishers
Effects
of ethanol and toluene on fixed-ratio performance short sleep and long sleep mice*
Robert L. Balster, Department
65
Ireland Ltd.
of Pharmacology
in
Jenny L. Wiley, Mary E. Tokarz and Janet S. Knisely*
and Toxicology,
Virginia Commonwealth University, Richmond, Virginia
23298-0613
(USA)
(Accepted April 20th, 1992) This study examined the effects of ethanol (EtOH) and toluene on fixed-ratio (FR) responding in mice selectively bred for sensitivity to the effects of EtOH on sleep time. Although the more sensitive long sleep (LS) mice showed greater EtOH-induced impairment in a motor performance task than did the less sensitive short sleep (SS) mice, changes in FR performance in the two lines did not differ in response to EtOH, regardless of route (oral or intraperitoneal) or time (40 vs. 60 min pre-session) of administration. These results emphasize the importance of considering task variables in determination of the behavior of different genotypes. In contrast to results with EtOH, the volatile inhalant toluene produced different effects on FR responding in the selected lines, with SS mice being more sensitive than LS mice. Key words: ethanol; toluene; long-sleep
mice; short-sleep
mice; behavior; selected lines
Introduction Research on genetic factors related to alcoholism (e.g., Crabbe et al., 1985; Goodwin, 1980; Schukit, 1986) and to its biological determinants is hampered by the difficulty of directly manipulating the relevant variables in humans; hence, an important research strategy has been to use selective breeding in animals. One approach is to use animals selectively bred for differential responses to ethanol (EtOH). An advantage of this strategy is that the relationship between the selected response and other behaviors or biochemical measures can be determined. Behavioral differences between the groups may be influenced by the selected genes; more importantly, a lack of differences can help rule out genotype as a causative factor in producing the behavior. Correspondence to: Robert L. Balster, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298-0613, USA. *Present address: Department of Psychiatry, Medical College of Virginia, Richmond, VA 23298-0109, USA.
0376~8716/92/$05.00 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
Several selected lines of rodents have been developed for sensitivity to acute effects of ethanol, including high alcohol sensitive (HAS) and low alcohol sensitive (LAS) rats (Draski et al., 1992), alcohol tolerant (AT) and alcohol nontolerant (ANT) rats (Eriksson, 1988) and longsleep (LS) and short-sleep (SS) mice (M&learn and Kakihana, 1973). The latter group has been bred for differences in hypnotic response as measured by length of time the righting reflex is lost after initial exposure to ethanol. Duration of the loss of the righting reflex is several-fold longer in LS mice than in SS mice (M&learn and Kakihana, 1981). Sleep time is an indicator of the sensitivity of the brain to ethanol (Heston et al., 1974; Kakihana et al., 1966); thus, LS mice are more sensitive to the effects of EtOH than are SS mice. Studies have revealed other differences between these two lines of mice in EtOH-induced physiological and behavioral measures (for reviews see Collins, 1981 and Phillips et al., 1989). LS mice are more sensitive to a number of EtOH effects, including its hypnotic (Erwin et al., 1976), hypothermic (Howerton et al., 1983a) and coordination-impairing Ireland Ltd.
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(Dudek et al., 1984) effects. SS mice are more sensitive to other EtOH effects; e.g., locomotor stimulation (Church et al., 1977; Sanders, 1976) and severity of EtOH withdrawal seizures (Goldstein and Kakihana, 1974). Of particular importance to the investigation of genetic influences in alcohol abuse are the results of studies that examine the relationship between genetic differences in sensitivity to the effects of EtOH and its reinforcing efficacy. In the case of LS and SS mice the data are inconclusive. The results of preference procedures reveal that, while both lines showed low preference for unsweetened EtOH (Chan, 1976), SS mice consumed more glucose-sweetened EtOH than did LS mice (Church et al., 1979; Fuller, 1980). In contrast, the results of oral self-administration studies reveal that EtOH served as a reinforcer only in LS mice (Elmer et al., 1990; George, 1988). Whereas LS mice showed concentrationdependent increases in responding for EtOH, SS mice responded similarly for water and for all concentrations of EtOH (Elmer et al., 1990). Thus, the relationship between neurosensitivity to EtOH and its reinforcing efficacy in SS and LS mice seems to depend upon the type of procedure. Studies comparing LS and SS mice on their sensitivity to the effect of EtOH on learned schedule-controlled behavior have not been conducted. This comparison seems important to examine since impairment of schedule-controlled behavior could result in reinforcement loss, a factor which can markedly alter behavioral sensitivity to drug treatments (e.g., Carlton and Wolgin, 1971; Schuster et al., 1966). Experiment 1 of the present study was conducted to investigate the effect of EtOH on fixed-ratio (FR) performance in LS and SS mice with the use of a traditional reinforcer (i.e., sweetened condensed milk). The effects of route of administration (oral vs. intraperitoneal) and length of presession injection interval (40 vs. 60 min) were also investigated. A similar study with the alcohol accepting (AA) and alcohol nonaccepting (ANA) selected rat lines found few between-group differences (George et al., 1990). Experiment 2 was conducted to verify that the two lines of mice differed in their sensitivity to
the effects of EtOH on a non-operant measure of motor behavior. A standard measure of motor coordination, the inverted screen test (Coughenour et al., 1977), was used to determine the duration of EtOH intoxication in each line. Experiment 3 examined the effects of the volatile inhalant toluene on FR performance in LS and SS mice. Many of the acute behavioral effects of toluene resemble those of EtOH and other CNS depressants (for a review, see Evans and Balster, 1991). Both solvents produce concentration-dependent decreases in FR responding (Moser and Balster, 1985a), similar motor performance in an inverted screen test (Moser and Balster, 1985b) and antipunishment effects in conflict procedures (Cook and Davidson, 1973; Glowa and Barrett, 1976; Wood et al., 1984). Like EtOH, toluene can serve as a reinforcer in self-administration studies with animals (Wood, 1982) and may have abuse potential in humans (Balster, 1987). Toluene also has been shown to substitute for EtOH in a drug discrimination procedure with EtOHtrained mice (Rees et al., 1987). These similarities in the behavioral pharmacological profiles of EtOH and toluene suggest that they may share a common mechanism of action. If toluene and EtOH have identical neural effects, then mice selectively bred for EtOH sensitivity should also be differentially sensitive to toluene. Methods Subjects
Experimentally naive LS (28 -36 g) and SS (23 - 30 g) male mice, selectively bred for differing sleep times in response to ethanol, were obtained from the Institute for Behavioral Genetics (University of Colorado, Boulder, CO). All mice were individually housed in clear plastic cages with wood-chip bedding in a temperature controlled (22 - 24°C) animal colony room (12-h light/dark cycle) and were transported to the laboratory on weekdays for testing. Mice were maintained at 85% of their free-feeding body weights by restriction of daily food ration. Water was available ad libitum in the home cages. All SS mice (n = 10) were tested in each
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mortality within the procedure; however, original LS group of mice (n = 12) resulted in a decrease in the number of these mice tested as the study progressed. Apparatus
Standard mouse operant chambers housed in sound-attenuated cubicles (described previously in Balster and Baird, 1979) were used for operant tests. A lever was mounted on the right side of one wall of each chamber. A liquid dipper system (Model E14-05, Coulbourn Instruments, Lehigh Valley, PA), located to the left of the lever in the center of the wall, allowed access to 0.02 ml of sweetened condensed milk (1 part sugar, 1 part condensed milk, 2 parts water by volume), contingent upon the required number of responses. A house light located above the lever indicated that the session was in progress. Experimental contingencies were controlled by a microcomputer (Carroll et al., 1981), which also recorded data for each session. The inverted screen apparatus consisted of six square screen sections (12 x 12 cm), each mounted horizontally on a metal rod. The metal rod was attached to a handle and could be rotated 180”. Chambers used for static inhalation exposure to toluene have been previously described (Woolverton and Balster, 1981). Briefly, mice were exposed to toluene in 29-l gas chromatography jars for 20 min prior to operant sessions. Toluene was administered by injection onto filter paper suspended above the chamber. A fan dispersed toluene vapors within the chamber. Infrared spectrophotometry (Miran lA, Foxboro Analytical, South Norwalk, CT) was used to monitor chamber concentrations of toluene. Chemicals EtOH (U.S.P.) was diluted with sterile distilled water and administered orally (p.0.) or intraperitoneally (i.p.) in a volume of 10 ml/kg. Toluene (T-324, Fisher Scientific Co.) was administered via 20-min exposure in static inhalation chambers. Concentrations of toluene are expressed as ppm. Procedure
Mice were trained to lever press under a FRl
reinforcement schedule for 0.02 ml of sweetened condensed milk during daily 15-min sessions. Gradually, the ratio requirement was increased to FR20. When stable rates of responding had been established under the FR20 schedule, testing with EtOH began. Tests were usually conducted on Tuesdays and Fridays. In order to be tested, responding during the session on the preceding day was required to be within 20% of the mouse’s average response rate during the previous three training sessions. Experiment 1 consisted of three EtOH doseresponse determinations in which route of administration and length of pre-session injection interval were varied. During the first doseresponse determination, E tOH (1.5 - 4 g/kg) was administered orally 40 min before the start of the operant session. During subsequent doseresponse determinations, EtOH (0.5 - 2 g/kg) was injected intraperitoneally (i.p.) at 40 or at 60 min pre-session. For each dose-effect curve, EtOH doses were administered in random order. The saline vehicle was tested before and after each dose-effect curve determination. Following completion of tests for EtOH effects on operant behavior, mice were trained on the inverted screen apparatus and then tested with E tOH in this procedure. During each test, six mice were injected i.p. with 2 glkg of EtOH and placed individually on top of the six screens at the following post-injection intervals: 10, 25, 40, 55, 60, 75 and 90 min. LS mice were also tested at 100, 110 and 120 min postinjection. At each of these intervals, the rod was rotated 180 degrees so that the mice were at the bottom of the screens. The number of mice that remained on the screens for 60 s was recorded. Between tests, mice were returned to their home cages. Experiment 3 consisted of testing the mice in the operant procedure described above for Experiment 1 following 20-min inhalation exposure to various concentrations of toluene (1000 - 8000 ppm). Toluene concentrations were administered in random order. Statistical
analysis
Response rates (responses/s) were calculated for the entire session during the three EtOH
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dose-response determinations and for the first 5 min of the session during the toluene concentration-effect determination. For the purposes of data analysis, response rate was converted to a percentage of the mean response rate during the pre- and post-saline tests or pre- and post-air tests in Experiments 1 and 3, respectively. In Experiments 1 and 3, separate repeated measures analyses of variance (ANOVA) and one between/within (split-plot) ANOVA were conducted for each route and pre-session time of administration of EtOH or toluene. The repeated measures ANOVA’s, responding during the pre- and post-control tests, were compared for LS and SS mice, respectively. The split-plot ANOVA compared pre-control responding to each dose of EtOH or toluene (within group) in each line of mice (between group). Due to evidence of toxicity, three LS mice were not tested at the highest dose of EtOH. In each case, the group mean for the remaining LS mice was substituted for the missing data. Tukey post hoc tests were used to specify differences revealed by significant ANOVA’s. When the dose-effect curves of SS and LS mice differed, EDso’s (with 95% confidence intervals) were calculated with the least squares method of linear regression on the linear part of each curves (Goldstein, 1964). In order to reveal differences in baseline response rates between SS and LS mice, a t-test for independent samples was performed on response rates averaged across all pre- and post-control tests for each line. Results Experiment
I
Both SS and LS mice acquired lever-pressing Table I.
SS mice LS mice
Mean responses/second
behavior under the fixed-ratio 20 schedule with similar training durations. Table I presents the response rates after saline control injections tested before each EtOH dose-effect curve determination. During Experiment 1, response rates after saline injections averaged less than 1.0/s, with higher rates consistently seen in the SS compared to the LS mice. In addition, there was a tendency for rates of responding in SS mice to increase over the course of the study, from an average after saline injection of 0.61 response/s to 1.88 responses/s following chamber air exposure during experiment 3. When averaged across all control tests, response rates for SS mice were significantly higher than those for LS mice. Because of these differences in baseline rates of responding in LS and SS mice, the effects of EtOH are expressed as percentage of the mean control level for each dose effect determination. No difference was found in the effects of EtOH on operant behavior in LS and SS mice. The ANOVA’s revealed that there were no main effect group differences after EtOH administration in LS and SS mice by either p.o. (Fig. 1) or i.p. (Fig. 2) routes of administration (P > 0.05). When given 40-min pre-session, EtOH produced significant decreases in rates of responding after both p.o. (P < 0.04) and i.p. (P < 0.001) administration. Individual comparisons at each dose showed that 4 g/kg p.o. significantly decreased rates compared to the 2 g/kg dose (P < 0.05), but not compared to saline control (Fig. 1). With i.p. injection at 40-min pre-session (Fig. 2, panel A), the highest dose of 2 g/kg produced a significant difference from the presaline control values (P < 0.05) but the difference between LS and SS mice at this dose
(+ S.E.M.) during control sessions for SS and LS mice.
Saline oral-40 min
Saline i.p.-40 min
Saline i.p.-60 min
Chamber Air exposure
0.61 (0.09) n = 10 0.45 (0.06) 72= 12
0.86 (0.12) 12= 10 0.45 (0.04) n=9
0.91 (0.20) n = 10 0.42 (0.06) n=7
1.88 (0.20) ?z = 10 1.04 (0.21) n=6
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was not significant (P > 0.05). When given i.p. 1 h before testing (Fig. 2, panel B), none of the doses of EtOH produced a significant change in response rates (P > 0.05).
160
1 140 1 120 -
d loo_ E
B
3 = z
w_
6
a 6
Experiment
A
60-
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Experiment
20
i 0
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F&-&T
2
Between-group differences in motor performance and duration of EtOH intoxication were apparent during testing in the inverted screen procedure. With the exception of the test 10 min after EtOH injection, a greater percentage of SS mice remained on the screens for the entire 60 s at all pre-treatment intervals (Fig. 3).
I
,
1
I
I
SALINE
ETOH
.
3
2 DOSE
,
4
(g/kg)
Fig. 1. Effects of oral EtOH administered 40 min presession on FR20 responding in SS and LS mice. Mean percentage of mean control response rates (* S.E.M.) is shown.
3
Unlike EtOH, toluene produced different effects in LS and SS mice, with SS mice (ED,, = 3317 ppm, 95% CI = 2871-3832 ppm) being more sensitive than LS mice (EDso = 6940 ppm, 95% CI = 4482- 10 745 ppm) to the response-rate decreasing effects (Fig. 4). A split-plot ANOVA revealed significant main
160_ 140-
120-
‘,O: T loo- 6
6 F 5
loo-
0 = % 0”
eo-
w-
60_
w-
4ll-
40-
20 -
20 -
O-
1
1
PRE
POST
SALINE
I
0
I
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2
ETOH DOSE (g/kg)
&
f
OJ
I P&-&T SALINE
Fig. 2. Effects of i.p. EtOH administered 40 min (panel A) or 60 min (panel B) pre-session LS mice. Mean percentage of mean control response rates (+ S.E.M.) is shown.
0
I 1
! 2
ETOH DOSE (g/kg)
on FR20 responding
in SS and
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effects of line (P c 0.001) and dose (P < 0.001) and a significant interaction (P < 0.01). Responding in both lines was similar during pre-air control tests and following inhalation of 1000 ppm toluene. At higher concentrations, response rates after toluene exposure differed in LS and SS mice. The 4000 ppm, 6000 ppm and 8000 ppm concentrations of toluene decreased responding in SS mice (P < 0.05). In contrast, only the highest concentration of toluene (8000 ppm) produced a significant decrease in responding in LS mice (P < 0.05).
so-
B Y
s: E
40-
s
10
25
40
60
PRE-TREATMENT
75 INTERVAL
I
I
I
I
90
100
110
120
(MN)
Fig. 3. Effects of 2 g/kg of i.p. EtOH on motor performance in an inverted screen task over time in SS (n = 10) and LS (n = 7) mice. Percentage of mice that remained on the screens for the 60-s test is shown.
I
PRE AIR
POST
1000
I
2000 TOLUENE
4000
1
moo
(PPM)
Fig. 4. Effects of 20-min inhalation exposure to toluene on FR20 responding in SS and LS mice. Mean percentage of mean control response rates (+ S.E.M.) is shown. Asterisks indicate significant differences from responding during the pre-air session.
Discussion LS and SS mice did not differ in their sensitivity to the effects of EtOH on schedule controlled operant behavior. Dose effect curves for EtOH after either oral or i.p. administration in mice trained to lever press under a fixed-ratio schedule of food reinforcement did not significantly differ in the two lines, despite substantial differences in their sensitivity to recovery of the righting reflex after high dose ethanol treatment (McClearn and Kakihana, 1981). These results are rather surprising, but are consistent with those of the previous operant study with AA and ANA selected rat lines (George et al., 1990). In that study ethanol produced similar dose-dependent decreases in rates of responding under a fixed-ratio 32 schedule of water reinforcement in AA and ANA rats. George et al. (1990) also reported unpublished observations that they had not found a difference in the sensitivity of LS and SS mice to EtOH effects on FR responding. These results also offer support for the idea that self-administration of EtOH by LS mice, but not by SS mice (Elmer et al., 1990; George, 1988), is due to differences in the reinforcing efficacy of EtOH in these lines rather than to EtOH-induced differences in rates of schedule controlled responding. Further, the fact that responding in both lines is similarly affected by subhypnotic doses of EtOH suggests that lack of behavioral activity at these lower doses cannot account for the failure of SS mice to selfadminister EtOH in operant procedures (e.g.,
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George, 1988). Since we did not test higher doses of EtOH, it is unknown whether doses nearer to the dose used for selection (i.e., 4.2 g/kg) would have produced differential effects in SS and LS mice. Although the effects of EtOH were similar in LS and SS mice, actual response rates of SS mice were consistently higher than those of LS mice during all but one EtOH test. These differences in response rates reflect differences in baseline rates of responding during pre-saline control tests rather than a differential effect of EtOH on behavior (see Table I). This finding emphasizes the importance of controlling for baseline response rate in operant studies with selected lines. LS and SS mice often exhibit clear differences in their behavioral responses to EtOH in nonoperant procedures. The results of Experiment 2 show that the two lines differ in duration of EtOH-induced motor impairment in the inverted screen test. Following injection with 2 g/kg of EtOH, more than half of the LS mice were impaired for 75 min. SS mice recovered more quickly from EtOH intoxication; half of the SS mice had recovered by 40 min after injection with this same dose of EtOH. EtOHinduced differences between LS and SS mice are also evident on other behavioral measures such as locomotor activity (Church et al., 1977; Sanders, 1976), coordination (Dudek et al., 1984), and grooming behavior (Allan and Isaacson, 1985); hence, the nature of the differences between these selected lines is task-specific. One difference between these types of tasks and schedule controlled operant behavior is that, whereas decreases in performance on nonoperant tasks are without consequences, decreases in response rate under fixed-ratio schedules results in loss of reinforcement. Reinforcement loss is one factor that influences the development of tolerance to the rate-decreasing effects of drugs in operant procedures (Carlton and Wolgin, 1971; Schuster et al., 1966; Smith, 1990). Similarly, it is possible that the environmental contingencies of operant procedures (e.g., reinforcement loss) may mitigate against the behavioral expression of differences
in genetic sensitivity to EtOH. Alternatively, between line differences in sensitivity to the acute effects of EtOH may be limited to responses mediated through receptors or neuronal pathways which are inherently different between these lines of animals (e.g., see Allan and Harris, 1991). It is possible that the neuronal pathways involved in control of operant behavior may differ from those mediating nonoperant behaviors. In Experiment 3 we examined the effects of toluene on FR responding in these mice. Drug discrimination studies have demonstrated that toluene shares discriminative stimulus effects with EtOH (Rees et al., 1987). Toluene also has many other EtOH-like behavioral and pharmacological effects (Evans and Balster, 1991). We hypothesized that, if toluene and EtOH had a common neural basis for these shared effects, then they would produce similar patterns of effects in mice selectively bred for neural sensitivity to EtOH. This hypothesis was not supported. While toluene, like EtOH, dosedependently decreased FR responding in both lines of mice, the concentrations of toluene at which responding was decreased differed between the lines. Responding was similar in the two lines following inhalation of the lowest concentration of toluene (1000 ppm). As concentration of toluene increased, response rates of SS mice showed a linear decrease. In contrast, the relationship between toluene concentration and responding was curvilinear for LS mice, response rates decreasing only at the highest concentration (8000 ppm). Thus, SS mice showed considerably greater behavioral sensitivity to the effects of toluene than did LS mice. Previous research provides evidence that the behavioral response used for selective breeding, duration of the loss of righting reflex, differs in LS and SS mice not only in response to EtOH (Erwin et al., 1976; M&learn and Kakihana, 1981), but also in response to other alcohols (Dudek et al., 1984) and to barbiturates (Alpern and McIntyre, 1985; Howerton et al., 1984; McIntyre and Alpern, 1986; O’Connor et al., 1982), although the direction of the barbiturateinduced difference is disputed (for a review, see
McIntyre and Alpern, 1985). Several previous studies have reported that pentobarbital produced longer sleep times in SS mice than in LS mice (Howerton et al., 1984; O’Connor et al., 1982; Siemens and Chan, 1976), suggesting that the effects of pentobarbital on other types of behavior in these mice may differ from those of EtOH. In the present study, toluene, like pentobarbital, was more potent in SS than LS mice. Several authors have suggested that the lipid solubility of alcohols and barbiturates may be related to the effects on duration of loss of righting reflex in LS and SS mice (Howerton et al., 1983a, 198313;Marley et al., 1986). Similarly, lipid solubility may be important in the behavioral actions of toluene. Moser and Balster (1985b) have shown that the relative lipid solubility of several solvents, including toluene and EtOH, is positively correlated with the behavioral activity of these substances in an inverted screen test. It is possible that the greater lipid solubility of toluene than of EtOH may be related to differences in the behavioral effects of toluene and EtOH in the present study. It may also be possible that differences in the elimination of toluene in LS and SS mice may account for the differences in sensitivity to toluene observed in these lines. At the time of awakening, LS mice exhibit lower blood EtOH levels than do SS mice, suggesting that LS mice are more sensitive to EtOH’s effects on the brain (Smolen and Smolen, 1987; Smolen et al., 1987). Although EtOH elimination rates are slightly higher in SS mice (Gilliam and Collins, 1983; Gilliam et al., 1983), this factor cannot account for the drastic differences in durations of loss of righting reflex (Phillips et al., 1989). In contrast, blood levels of pentobarbital at time of awakening are similar in each selected line; thus, pentobarbital elimination rates are slower in SS mice (O’Connor et al., 1982; Siemens and Chan, 1976). This similarity in awakening pentobarbital blood levels suggests that the lines do not differ in neural sensitivity to pentobarbital, but rather in pentobarbital elimination rates (O’Connor et al., 1982). Since toluene elimination rates have not been measured in these mice, it is impossible to determine whether the greater
behavioral sensitivity to toluene exhibited by SS mice in the present study is caused by greater neural sensitivity or by slower elimination rates. There is considerable data showing a substantial overlap in the behavioral and pharmacological effects of toluene, EtOH and the abused CNS depressant class of drugs (Evans and Balster, 1991). Despite these similarities, differences among them are also evident (Barry and Krimmer, 1978; DeFry and Slangen, 1986; Rees and Balster, 1988). These differences may be exploited in discovering the commonalities and differences in their neural bases of action. The fact that LS and SS lines of mice are differentially sensitive to ethanol and toluene on a measure of operant behavior provides a rationale for comparing cellular sites of action of these two agents. For example, cerebellar tissues from LS and SS mice have been shown to differ in EtOH-induced chloride flux (Allan and Harris, 1986). Since anesthetics have been EtOH-like effects on shown to produce GABAergic systems (Cheng and Brunner, 1981; Huidobro-Toro et al., 1987; Keane and Biziere, 1987; Nakahiro et al., 1989), it would be of interest to compare the in vitro actions of toluene and EtOH in these systems using tissues from LS and SS mice. Based on the results of this study, differences might be expected between these lines in response to EtOH and toluene which may contribute to our understanding of the neural basis of toluene’s behavioral effects and its abuse. In summary, the demonstration that LS and SS mice bred for different EtOH-induced hypnotic responses do not show differential behavioral sensitivity to EtOH in an operant paradigm emphasizes the need for caution in assuming that differences in specific phenotypic characteristics reflect differences in general sensitivity to behavioral manipulations. The behavioral responses required in operant procedures may involve neuronal pathways different from those involved in the hypnotic response. Alternatively, the nature of learned operant behavior with its consequences of reduced food reinforcement when the behavior is impaired may mitigate differences in sensitivity
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that are apparent in other behavioral procedures. Finally, the substantially greater sensitivity of SS than LS mice to the effects of toluene provides evidence for differences in neural substrate for EtOH and this solvent and provides a rationale for using this sensitivity difto explore possible ference to toluene mechanisms for its behavioral actions. Acknowledgment This research was supported by NIDA grant DA-03112. J.L. Wiley is a postdoctoral fellow supported by training grant DA-07027. References Allan, A.M. and Harris, R.A. (1986) Gamma-aminobutyric acid and alcohol actions: Neurochemical studies of long sleep and short sleep mice. Life Sci. 39, 2005 2015. Allan, A.M. and Harris, R.A. (1991) Neurochemieal studies of genetic differences in alcohol action. In: The Genetic Basis of Alcohol and Drug Actions (Crabbe, J.C., Jr. and Harris, R.A., eds.), pp. 105- 152. Plenum Press, New York. Allan, A.M. and Isaacson, R.L. (1985) Ethanol-induced grooming in mice selectively bred for differential sensitivity to ethanol. Behav. Neural Biol. 44, 386-392. Alpern, H.P. and McIntyre, T.D. (1985) Evidence that the selectively bred long- and short-sleep mouse lines display common narcotic reactions to many depressants. Psychopharmacology 85, 456 - 459. Balster, R.L. (1987) Abuse potential evaluation of inhalants. Drug Alcohol Depend. 49, 7 - 15. Balster, R.L. and Baird, J.B. (1979) The effects of phencyclidine, d-amphetamine and pentobarbital on spaced responding in mice. Pharmacol. Bioehem. Behav. 11, 617 - 623. Barry III, H. and Krimmer, E.C. (1978) Similarities and differences in discriminative stimulus effects of chlordiazepoxide, pentobarbital, ethanol and other sedatives. In: Stimulus Properties of Drugs. Ten Years of Progress (Colpaert, F.C. and Rosecrans, J.A., eds.), pp. 31-51. Elsevier, Amsterdam. Carlton, P.L. and Wolgin, D.L. (1971) Contingent tolerance to the anorexigenic effects of amphetamine. Physiol. Behav. 7, 221- 223. Carroll, M.E., Santi, P.A. and Kliner, D.J. (1981) A microcomputer system for the control of behavioral experiments. Pharmacol. Biochem. Behav. 4, 415-417. Chan, A.W.K. (1976) Gamma aminobutyric acid in different strains of mice. Effect of ethanol. Life Sci. 19, 597 - 604. Cheng, S. and Brunner, E.A. (1981) Effects of anesthetic agents on synaptosomal GABA disposal. Anesthesiology 55, 34-40.
Church, AC., Fuller, J.L. and Dann, L. (1979) Alcohol intake in selected lines of mice: Importance of sex and genotype. J. Comp. Physiol. Psychol. 93, 242-246. Church, A.C., Fuller, J.L. and Dudek, B.C. (1977) Behavioral effects of salsolinol and ethanol on mice selected for sensitivity to alcohol-induced sleep time. Drug Alcohol Depend. 2, 443 - 452. Collins, A.C. (1981) A review of research using Short-sleep and Long-sleep mice. In: Development of Animal Models as Pharmacogenetic Tools (McClearn, G.E., Deitrich, R.A. and Erwin, V.G., eds.), pp. 161- 170. National Institute on Alcohol Abuse and Alcoholism, Research Monograph 6, DHEW Publication No. (ADM) 81- 1133, Washington, D.C. Cook, L. and Davidson, A.B. (1973) Effects of behaviorally active drugs in a conflict-punishment procedure. In: The Benzodiazepines (Garattini, S., Mussini, E. and Randall, L.O., eds.), pp. 327-345. Raven Press, New York. Coughenour, L.I., McClearn, J.R. and Parker, R.B. (1977) A new device for rapid measurement of impaired motor function in mice. Pharmacol. Biochem. Behav. 6, 351- 353. Crabbe, J.C., McSwigan, J.D. and Belknap, J.K. (1985) The role of genetics in substance abuse. In: Individual Differences in Substance Abuse (Maisto, S. and Galizio, M., eds.), pp. 13-64. Plenum Press, New York. De Vry, J. and Slangen, J.L. (1986) Effects of training dose on discrimination and cross-generalization of chlordiazepoxide, pentobarbital and ethanol in the rat. Psychopharmacology 88, 341- 345. Draski, L.J., Spuhler, K.P., Erwin, V.G., Baker, R.C. and Deitrich, R.A. (1992) Selective breeding of rats differing in sensitivity to the effects of acute ethanol administration. Alcoholism: Clin. Exp. Res. 16, 48-54. Dudek, B.C., Abbott, M.E. and Phillips, T.J. (1984) Stimulant and depressant properties of sedativehypnotics in mice selectively bred for differential sensitivity to ethanol. Psychopharmacology 82, 46- 51. Elmer, G.I., Meisch, R.A., Goldberg, S.R. and George, F.R. (1990) Ethanol self-administration in long sleep and short sleep mice indicates reinforcement is not inversely related to neurosensitivity. J. Pharmacol. Exp. Ther. 254, 1054 - 1062. Eriksson, C.J.P. (1988) Finnish selective breeding studies for initial sensitivity to ethanol: Update 1988 on the AT and ANT rat lines. In: Initial Sensitivity to Alcohol (Deitrich, R.A. and Pawlowski, A.A., eds.), pp. 61-86. National Institute on Alcohol Abuse and Alcoholism, Research Monograph 6, Washington, D.C. Erwin, V.G., Heston, W.D.W., M&learn, G.E. and Deitrich, R.A. (1976) Effect of hypnotics on mice genetically selected for sensitivity to ethanol. Pharmacol. Biochem. Behav. 4, 679-683. Evans, E.B. and Balster, R.L. (1991) CNS depressant effects of volatile organic solvents. Neurosci. Biobehav. Rev. 15, 233-241. Fuller, J.L. (1980) Regulation of ethanol intake in long- and short-sleep mice. In: Animal Models in Alcohol Research
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(Eriksson, K., Sinclair, J.D. and Kiianmaa, K., eds.), pp. 57-62. Academic Press, New York. George, F.R. (1988) Genetic tools in the study of drug selfadministration. Alcoholism: Clin. Exp. Res. 12, 586 - 590. George, F.R., Ritz, MC. and Meisch, R.A. (1990) Ratedepressant effects of ethanol on fixed-ratio responding in ALKO AA and ANA rats. Adv. Alcohol Subst. Abuse 9, 31-42. Gilliam, D.M. and Collins, A.C. (1983) Concentrationdependent effects of ethanol in long-sleep and short-sleep mice. Alcoholism: Clin. Exp. Res. 7, 337-342. Gilliam, D.M., Bloedow, D.C. and Collins, A.C. (1983) Nonlinear pharmacokinetics of ethanol in long sleep and short sleep mice. Alcoholism: Clin. Exp. Res. 7, 95-99. Glowa, J.R. and Barrett, J.E. (1976) Effects of alcohol on punished and unpunished responding of squirrel monkeys. Pharmacol. Biochem. Behav. 4, 169- 173. Goldstein, A. (1964) Biostatistics: An Introductory Text, MacMillan Company, New York. Goldstein, D.B. and Kakihana, R. (1974) Alcohol withdrawal reactions and reserpine effects in inbred strains of mice. Life Sci. 15, 415-425. Goodwin, D.W. (1980) The genetics of alcoholism. Subst. Alcohol Action/Misuse 1, 101 - 117. Heston, W.D.W., Erwin, V.G., Anderson, S.M. and Robbins, H. (1974) A comparison of the effects of alcohol on mice selectively bred for differences in ethanol sleeptime. Life Sci. 14, 365-370. Howerton, T.C., O’Connor, M.F. and Collins, A.C. (1983a) Differential effects of long-chain alcohols in long- and short-sleep mice. Psychopharmacology 79, 313-317. Howerton, T.C., O’Connor, M.F. and Collins, A.C. (198313) Lipid solubility is correlated with hypnotic and hypothermic responses of long-sleep (LS) and short-sleep (SS) mice to various depressant drugs. J. Pharmacol. Exp. Ther. 227, 389-393. Howerton, T.C., Burch, J.B., O’Connor, M.F., Miner, L.L. and Collins, A.C. (1984) A genetic analysis of ethanol, pentobarbital and methyprylon sleep-time response. Alcoholism: Clin. Exp. Res. 8, 546-550. Huidobro-Toro, J.P., Bleck, V., Allan, A.M. and Harris, R.A. (1987) Neurochemical actions of anesthetic drugs on the gamma-aminobutyric acid receptor-chloride channel complex. J. Pharmacol. Exp. Ther. 242, 963-969. Kakihana, R., Brown, D.R., McClearn, G.E. and Tabershaw, I.R. (1966) Brain sensitivity to alcohol in inbred mouse strains. Science 154, 1574- 1575. Keane, P.E. and Biziere, K. (1987) The effects of general anaesthetics on GABAergic synaptic transmission. Life Sci. 41, 1437 - 1448. Marley, R.J., Miner, L.L., Wehner, J.M. and Collins, A.C. (1986) Differential effects of central nervous system depressants in long-sleep and short-sleep mice. J. Pharmacol. Exp. Ther. 238, 1028-1033. McClearn, G.E. and Kakihana, R. (1973) Selective breeding for ethanol sensitivity in mice. Behav. Genetics 3, 409-410.
McClearn, G.E. and Kakihana, R. (1981) Selective breeding for ethanol sensitivity: short sleep and long sleep mice. In: Development of Animal Models as Pharmacogenetic Tools (McClearn, G.E., Deitrich, R.A. and Erwin, V.G., eds.), pp. 147 - 160. National Institute on Alcohol Abuse and Alcoholism, Research Monograph 6, DHEW Publication No. (AOM) 81- 1133 Washington, D.C. McIntyre, T.D. and Alpern, H.P. (1985) Reinterpretation of the literature indicates differential sensitivities of longsleep and short-sleep mice are not specific to alcohol. Psychopharmacology 87, 379 - 389. McIntyre, T.D. and Alpern, H.P. (1986) Thiopental, phenobarbital and chlordiazepoxide induce the same differences in narcotic reaction as ethanol in long-sleep and short-sleep selectively-bred mice. Pharmacol. Biochem. Behav. 24, 895-898. Moser, V.C. and Balster, R.L. (1985a) Effects of toluene, halothane and ethanol vapor on fixed-ratio performance in mice. Pharmacol. Biochem. Behav. 22, 797-802. Moser, V.C. and Balster, R.L. (1985b) Acute motor and lethal effects of inhaled toluene, l,l,l-trichloroethane, halothane, and ethanol in mice: Effects of exposure duration. Toxicol. Appl. Pharmacol. 77, 285-291. Nakahiro, M., Yeh, J.Z., Brunner, E. and Narahashi, T. (1989) General anesthetics modulate GABA receptor channel complex in rat dorsal root ganglion neurons. FASEB J. 3, 1850 - 1854. O’Connor, M.F., Howerton, T.C. and Collins, AC. (1982) Effects of pentobarbital in mice selected for differential sensitivity to ethanol. Pharmacol. Biochem. Behav. 17, 245 - 248. Phillips, T.J., Feller, D.J. and Crabbe, J.C. (1989) Selected mouse lines, alcohol and behavior. Experientia 45, 805-827. Rees, D.C. and Balster, R.L. (1988) Attenuation of the discriminative stimulus properties of ethanol and oxazepam, but not of pentobarbital, by Ro 15-4513 in mice. J. Pharmacol. Exp. Ther. 244, 592-598. Rees, DC., Knisely, J.S., Breen, T.J. and Balster, R.L. (1987) Toluene, halothane, l,l,l-trichloroethane and oxazepam produce ethanol-like discriminative stimulus effects in mice. J. Pharmacol. Exp. Ther. 243, 931- 937. Sanders, B. (1976) Sensitivity to low doses of ethanol and pentobarbital in mice selected for sensitivity to hypnotic doses of ethanol. J. Comp. Physiol. Psychol. 90, 394 - 398. Schukit, M.A. (1986) Genetic aspects of alcoholism. Ann. Emer. Med. 15, 991-996. Schuster, C.R., Dockens, W.S. and Woods, J.H. (1966) Behavioral variables affecting the development of amphetamine tolerance. Psychopharmacologia 9, 170- 182. Siemens, A.J. and Chan, A.W.K. (1976) Differential effects of pentobarbital and ethanol in mice. Life Sci. 19, 581- 590. Smith, J.B. (1990) Effects of fixed-ratio requirement on observed tolerance to decreased responding by clonidine. Pharmacol. Biochem. Behav. 36, 993 - 995. Smolen, A. and Smolen, T.N. (1987) Demonstration of a
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threshold for ethanol at the time of regaining the righting response in long-sleep and short-sleep mice. Alcohol Drug Res. 7, 279-283. Smolen, A., Smolen, T.N. and van de Kamp, J.L. (1987) Sensitivity of inbred and selectively bred mice to ethanol. Alcohol 4, 57 - 62. Wood, R.W. (1982) Stimulus properties of inhaled substances: An update. In: Nervous System Toxicology (Mitchell, CL., ed.), pp. 199-213. Raven Press, New York.
Wood, R.W., Coleman, J.B., Schuler, R. and Cox, C. (1984) Anticonvulsant and antipunishment effects of toluene. J. Pharmacol. Exp. Ther. 230, 407-412. Woolverton, W.L. and Balster, R.L. (1981) Behavioral and lethal effects of combinations of oral ethanol and inhaled l,l,l-trichlorethane in mice. Toxicol. Appl. Pharmacol. 59, l-7.
Elsevier Scientific Publishers
Effects
of ethanol and toluene on fixed-ratio performance short sleep and long sleep mice*
Robert L. Balster, Department
65
Ireland Ltd.
of Pharmacology
in
Jenny L. Wiley, Mary E. Tokarz and Janet S. Knisely*
and Toxicology,
Virginia Commonwealth University, Richmond, Virginia
23298-0613
(USA)
(Accepted April 20th, 1992) This study examined the effects of ethanol (EtOH) and toluene on fixed-ratio (FR) responding in mice selectively bred for sensitivity to the effects of EtOH on sleep time. Although the more sensitive long sleep (LS) mice showed greater EtOH-induced impairment in a motor performance task than did the less sensitive short sleep (SS) mice, changes in FR performance in the two lines did not differ in response to EtOH, regardless of route (oral or intraperitoneal) or time (40 vs. 60 min pre-session) of administration. These results emphasize the importance of considering task variables in determination of the behavior of different genotypes. In contrast to results with EtOH, the volatile inhalant toluene produced different effects on FR responding in the selected lines, with SS mice being more sensitive than LS mice. Key words: ethanol; toluene; long-sleep
mice; short-sleep
mice; behavior; selected lines
Introduction Research on genetic factors related to alcoholism (e.g., Crabbe et al., 1985; Goodwin, 1980; Schukit, 1986) and to its biological determinants is hampered by the difficulty of directly manipulating the relevant variables in humans; hence, an important research strategy has been to use selective breeding in animals. One approach is to use animals selectively bred for differential responses to ethanol (EtOH). An advantage of this strategy is that the relationship between the selected response and other behaviors or biochemical measures can be determined. Behavioral differences between the groups may be influenced by the selected genes; more importantly, a lack of differences can help rule out genotype as a causative factor in producing the behavior. Correspondence to: Robert L. Balster, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298-0613, USA. *Present address: Department of Psychiatry, Medical College of Virginia, Richmond, VA 23298-0109, USA.
0376~8716/92/$05.00 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
Several selected lines of rodents have been developed for sensitivity to acute effects of ethanol, including high alcohol sensitive (HAS) and low alcohol sensitive (LAS) rats (Draski et al., 1992), alcohol tolerant (AT) and alcohol nontolerant (ANT) rats (Eriksson, 1988) and longsleep (LS) and short-sleep (SS) mice (M&learn and Kakihana, 1973). The latter group has been bred for differences in hypnotic response as measured by length of time the righting reflex is lost after initial exposure to ethanol. Duration of the loss of the righting reflex is several-fold longer in LS mice than in SS mice (M&learn and Kakihana, 1981). Sleep time is an indicator of the sensitivity of the brain to ethanol (Heston et al., 1974; Kakihana et al., 1966); thus, LS mice are more sensitive to the effects of EtOH than are SS mice. Studies have revealed other differences between these two lines of mice in EtOH-induced physiological and behavioral measures (for reviews see Collins, 1981 and Phillips et al., 1989). LS mice are more sensitive to a number of EtOH effects, including its hypnotic (Erwin et al., 1976), hypothermic (Howerton et al., 1983a) and coordination-impairing Ireland Ltd.
66
(Dudek et al., 1984) effects. SS mice are more sensitive to other EtOH effects; e.g., locomotor stimulation (Church et al., 1977; Sanders, 1976) and severity of EtOH withdrawal seizures (Goldstein and Kakihana, 1974). Of particular importance to the investigation of genetic influences in alcohol abuse are the results of studies that examine the relationship between genetic differences in sensitivity to the effects of EtOH and its reinforcing efficacy. In the case of LS and SS mice the data are inconclusive. The results of preference procedures reveal that, while both lines showed low preference for unsweetened EtOH (Chan, 1976), SS mice consumed more glucose-sweetened EtOH than did LS mice (Church et al., 1979; Fuller, 1980). In contrast, the results of oral self-administration studies reveal that EtOH served as a reinforcer only in LS mice (Elmer et al., 1990; George, 1988). Whereas LS mice showed concentrationdependent increases in responding for EtOH, SS mice responded similarly for water and for all concentrations of EtOH (Elmer et al., 1990). Thus, the relationship between neurosensitivity to EtOH and its reinforcing efficacy in SS and LS mice seems to depend upon the type of procedure. Studies comparing LS and SS mice on their sensitivity to the effect of EtOH on learned schedule-controlled behavior have not been conducted. This comparison seems important to examine since impairment of schedule-controlled behavior could result in reinforcement loss, a factor which can markedly alter behavioral sensitivity to drug treatments (e.g., Carlton and Wolgin, 1971; Schuster et al., 1966). Experiment 1 of the present study was conducted to investigate the effect of EtOH on fixed-ratio (FR) performance in LS and SS mice with the use of a traditional reinforcer (i.e., sweetened condensed milk). The effects of route of administration (oral vs. intraperitoneal) and length of presession injection interval (40 vs. 60 min) were also investigated. A similar study with the alcohol accepting (AA) and alcohol nonaccepting (ANA) selected rat lines found few between-group differences (George et al., 1990). Experiment 2 was conducted to verify that the two lines of mice differed in their sensitivity to
the effects of EtOH on a non-operant measure of motor behavior. A standard measure of motor coordination, the inverted screen test (Coughenour et al., 1977), was used to determine the duration of EtOH intoxication in each line. Experiment 3 examined the effects of the volatile inhalant toluene on FR performance in LS and SS mice. Many of the acute behavioral effects of toluene resemble those of EtOH and other CNS depressants (for a review, see Evans and Balster, 1991). Both solvents produce concentration-dependent decreases in FR responding (Moser and Balster, 1985a), similar motor performance in an inverted screen test (Moser and Balster, 1985b) and antipunishment effects in conflict procedures (Cook and Davidson, 1973; Glowa and Barrett, 1976; Wood et al., 1984). Like EtOH, toluene can serve as a reinforcer in self-administration studies with animals (Wood, 1982) and may have abuse potential in humans (Balster, 1987). Toluene also has been shown to substitute for EtOH in a drug discrimination procedure with EtOHtrained mice (Rees et al., 1987). These similarities in the behavioral pharmacological profiles of EtOH and toluene suggest that they may share a common mechanism of action. If toluene and EtOH have identical neural effects, then mice selectively bred for EtOH sensitivity should also be differentially sensitive to toluene. Methods Subjects
Experimentally naive LS (28 -36 g) and SS (23 - 30 g) male mice, selectively bred for differing sleep times in response to ethanol, were obtained from the Institute for Behavioral Genetics (University of Colorado, Boulder, CO). All mice were individually housed in clear plastic cages with wood-chip bedding in a temperature controlled (22 - 24°C) animal colony room (12-h light/dark cycle) and were transported to the laboratory on weekdays for testing. Mice were maintained at 85% of their free-feeding body weights by restriction of daily food ration. Water was available ad libitum in the home cages. All SS mice (n = 10) were tested in each
67
mortality within the procedure; however, original LS group of mice (n = 12) resulted in a decrease in the number of these mice tested as the study progressed. Apparatus
Standard mouse operant chambers housed in sound-attenuated cubicles (described previously in Balster and Baird, 1979) were used for operant tests. A lever was mounted on the right side of one wall of each chamber. A liquid dipper system (Model E14-05, Coulbourn Instruments, Lehigh Valley, PA), located to the left of the lever in the center of the wall, allowed access to 0.02 ml of sweetened condensed milk (1 part sugar, 1 part condensed milk, 2 parts water by volume), contingent upon the required number of responses. A house light located above the lever indicated that the session was in progress. Experimental contingencies were controlled by a microcomputer (Carroll et al., 1981), which also recorded data for each session. The inverted screen apparatus consisted of six square screen sections (12 x 12 cm), each mounted horizontally on a metal rod. The metal rod was attached to a handle and could be rotated 180”. Chambers used for static inhalation exposure to toluene have been previously described (Woolverton and Balster, 1981). Briefly, mice were exposed to toluene in 29-l gas chromatography jars for 20 min prior to operant sessions. Toluene was administered by injection onto filter paper suspended above the chamber. A fan dispersed toluene vapors within the chamber. Infrared spectrophotometry (Miran lA, Foxboro Analytical, South Norwalk, CT) was used to monitor chamber concentrations of toluene. Chemicals EtOH (U.S.P.) was diluted with sterile distilled water and administered orally (p.0.) or intraperitoneally (i.p.) in a volume of 10 ml/kg. Toluene (T-324, Fisher Scientific Co.) was administered via 20-min exposure in static inhalation chambers. Concentrations of toluene are expressed as ppm. Procedure
Mice were trained to lever press under a FRl
reinforcement schedule for 0.02 ml of sweetened condensed milk during daily 15-min sessions. Gradually, the ratio requirement was increased to FR20. When stable rates of responding had been established under the FR20 schedule, testing with EtOH began. Tests were usually conducted on Tuesdays and Fridays. In order to be tested, responding during the session on the preceding day was required to be within 20% of the mouse’s average response rate during the previous three training sessions. Experiment 1 consisted of three EtOH doseresponse determinations in which route of administration and length of pre-session injection interval were varied. During the first doseresponse determination, E tOH (1.5 - 4 g/kg) was administered orally 40 min before the start of the operant session. During subsequent doseresponse determinations, EtOH (0.5 - 2 g/kg) was injected intraperitoneally (i.p.) at 40 or at 60 min pre-session. For each dose-effect curve, EtOH doses were administered in random order. The saline vehicle was tested before and after each dose-effect curve determination. Following completion of tests for EtOH effects on operant behavior, mice were trained on the inverted screen apparatus and then tested with E tOH in this procedure. During each test, six mice were injected i.p. with 2 glkg of EtOH and placed individually on top of the six screens at the following post-injection intervals: 10, 25, 40, 55, 60, 75 and 90 min. LS mice were also tested at 100, 110 and 120 min postinjection. At each of these intervals, the rod was rotated 180 degrees so that the mice were at the bottom of the screens. The number of mice that remained on the screens for 60 s was recorded. Between tests, mice were returned to their home cages. Experiment 3 consisted of testing the mice in the operant procedure described above for Experiment 1 following 20-min inhalation exposure to various concentrations of toluene (1000 - 8000 ppm). Toluene concentrations were administered in random order. Statistical
analysis
Response rates (responses/s) were calculated for the entire session during the three EtOH
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dose-response determinations and for the first 5 min of the session during the toluene concentration-effect determination. For the purposes of data analysis, response rate was converted to a percentage of the mean response rate during the pre- and post-saline tests or pre- and post-air tests in Experiments 1 and 3, respectively. In Experiments 1 and 3, separate repeated measures analyses of variance (ANOVA) and one between/within (split-plot) ANOVA were conducted for each route and pre-session time of administration of EtOH or toluene. The repeated measures ANOVA’s, responding during the pre- and post-control tests, were compared for LS and SS mice, respectively. The split-plot ANOVA compared pre-control responding to each dose of EtOH or toluene (within group) in each line of mice (between group). Due to evidence of toxicity, three LS mice were not tested at the highest dose of EtOH. In each case, the group mean for the remaining LS mice was substituted for the missing data. Tukey post hoc tests were used to specify differences revealed by significant ANOVA’s. When the dose-effect curves of SS and LS mice differed, EDso’s (with 95% confidence intervals) were calculated with the least squares method of linear regression on the linear part of each curves (Goldstein, 1964). In order to reveal differences in baseline response rates between SS and LS mice, a t-test for independent samples was performed on response rates averaged across all pre- and post-control tests for each line. Results Experiment
I
Both SS and LS mice acquired lever-pressing Table I.
SS mice LS mice
Mean responses/second
behavior under the fixed-ratio 20 schedule with similar training durations. Table I presents the response rates after saline control injections tested before each EtOH dose-effect curve determination. During Experiment 1, response rates after saline injections averaged less than 1.0/s, with higher rates consistently seen in the SS compared to the LS mice. In addition, there was a tendency for rates of responding in SS mice to increase over the course of the study, from an average after saline injection of 0.61 response/s to 1.88 responses/s following chamber air exposure during experiment 3. When averaged across all control tests, response rates for SS mice were significantly higher than those for LS mice. Because of these differences in baseline rates of responding in LS and SS mice, the effects of EtOH are expressed as percentage of the mean control level for each dose effect determination. No difference was found in the effects of EtOH on operant behavior in LS and SS mice. The ANOVA’s revealed that there were no main effect group differences after EtOH administration in LS and SS mice by either p.o. (Fig. 1) or i.p. (Fig. 2) routes of administration (P > 0.05). When given 40-min pre-session, EtOH produced significant decreases in rates of responding after both p.o. (P < 0.04) and i.p. (P < 0.001) administration. Individual comparisons at each dose showed that 4 g/kg p.o. significantly decreased rates compared to the 2 g/kg dose (P < 0.05), but not compared to saline control (Fig. 1). With i.p. injection at 40-min pre-session (Fig. 2, panel A), the highest dose of 2 g/kg produced a significant difference from the presaline control values (P < 0.05) but the difference between LS and SS mice at this dose
(+ S.E.M.) during control sessions for SS and LS mice.
Saline oral-40 min
Saline i.p.-40 min
Saline i.p.-60 min
Chamber Air exposure
0.61 (0.09) n = 10 0.45 (0.06) 72= 12
0.86 (0.12) 12= 10 0.45 (0.04) n=9
0.91 (0.20) n = 10 0.42 (0.06) n=7
1.88 (0.20) ?z = 10 1.04 (0.21) n=6
69
was not significant (P > 0.05). When given i.p. 1 h before testing (Fig. 2, panel B), none of the doses of EtOH produced a significant change in response rates (P > 0.05).
160
1 140 1 120 -
d loo_ E
B
3 = z
w_
6
a 6
Experiment
A
60-
4oEzl
Experiment
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i 0
J
F&-&T
2
Between-group differences in motor performance and duration of EtOH intoxication were apparent during testing in the inverted screen procedure. With the exception of the test 10 min after EtOH injection, a greater percentage of SS mice remained on the screens for the entire 60 s at all pre-treatment intervals (Fig. 3).
I
,
1
I
I
SALINE
ETOH
.
3
2 DOSE
,
4
(g/kg)
Fig. 1. Effects of oral EtOH administered 40 min presession on FR20 responding in SS and LS mice. Mean percentage of mean control response rates (* S.E.M.) is shown.
3
Unlike EtOH, toluene produced different effects in LS and SS mice, with SS mice (ED,, = 3317 ppm, 95% CI = 2871-3832 ppm) being more sensitive than LS mice (EDso = 6940 ppm, 95% CI = 4482- 10 745 ppm) to the response-rate decreasing effects (Fig. 4). A split-plot ANOVA revealed significant main
160_ 140-
120-
‘,O: T loo- 6
6 F 5
loo-
0 = % 0”
eo-
w-
60_
w-
4ll-
40-
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O-
1
1
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POST
SALINE
I
0
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ETOH DOSE (g/kg)
&
f
OJ
I P&-&T SALINE
Fig. 2. Effects of i.p. EtOH administered 40 min (panel A) or 60 min (panel B) pre-session LS mice. Mean percentage of mean control response rates (+ S.E.M.) is shown.
0
I 1
! 2
ETOH DOSE (g/kg)
on FR20 responding
in SS and
70
effects of line (P c 0.001) and dose (P < 0.001) and a significant interaction (P < 0.01). Responding in both lines was similar during pre-air control tests and following inhalation of 1000 ppm toluene. At higher concentrations, response rates after toluene exposure differed in LS and SS mice. The 4000 ppm, 6000 ppm and 8000 ppm concentrations of toluene decreased responding in SS mice (P < 0.05). In contrast, only the highest concentration of toluene (8000 ppm) produced a significant decrease in responding in LS mice (P < 0.05).
so-
B Y
s: E
40-
s
10
25
40
60
PRE-TREATMENT
75 INTERVAL
I
I
I
I
90
100
110
120
(MN)
Fig. 3. Effects of 2 g/kg of i.p. EtOH on motor performance in an inverted screen task over time in SS (n = 10) and LS (n = 7) mice. Percentage of mice that remained on the screens for the 60-s test is shown.
I
PRE AIR
POST
1000
I
2000 TOLUENE
4000
1
moo
(PPM)
Fig. 4. Effects of 20-min inhalation exposure to toluene on FR20 responding in SS and LS mice. Mean percentage of mean control response rates (+ S.E.M.) is shown. Asterisks indicate significant differences from responding during the pre-air session.
Discussion LS and SS mice did not differ in their sensitivity to the effects of EtOH on schedule controlled operant behavior. Dose effect curves for EtOH after either oral or i.p. administration in mice trained to lever press under a fixed-ratio schedule of food reinforcement did not significantly differ in the two lines, despite substantial differences in their sensitivity to recovery of the righting reflex after high dose ethanol treatment (McClearn and Kakihana, 1981). These results are rather surprising, but are consistent with those of the previous operant study with AA and ANA selected rat lines (George et al., 1990). In that study ethanol produced similar dose-dependent decreases in rates of responding under a fixed-ratio 32 schedule of water reinforcement in AA and ANA rats. George et al. (1990) also reported unpublished observations that they had not found a difference in the sensitivity of LS and SS mice to EtOH effects on FR responding. These results also offer support for the idea that self-administration of EtOH by LS mice, but not by SS mice (Elmer et al., 1990; George, 1988), is due to differences in the reinforcing efficacy of EtOH in these lines rather than to EtOH-induced differences in rates of schedule controlled responding. Further, the fact that responding in both lines is similarly affected by subhypnotic doses of EtOH suggests that lack of behavioral activity at these lower doses cannot account for the failure of SS mice to selfadminister EtOH in operant procedures (e.g.,
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George, 1988). Since we did not test higher doses of EtOH, it is unknown whether doses nearer to the dose used for selection (i.e., 4.2 g/kg) would have produced differential effects in SS and LS mice. Although the effects of EtOH were similar in LS and SS mice, actual response rates of SS mice were consistently higher than those of LS mice during all but one EtOH test. These differences in response rates reflect differences in baseline rates of responding during pre-saline control tests rather than a differential effect of EtOH on behavior (see Table I). This finding emphasizes the importance of controlling for baseline response rate in operant studies with selected lines. LS and SS mice often exhibit clear differences in their behavioral responses to EtOH in nonoperant procedures. The results of Experiment 2 show that the two lines differ in duration of EtOH-induced motor impairment in the inverted screen test. Following injection with 2 g/kg of EtOH, more than half of the LS mice were impaired for 75 min. SS mice recovered more quickly from EtOH intoxication; half of the SS mice had recovered by 40 min after injection with this same dose of EtOH. EtOHinduced differences between LS and SS mice are also evident on other behavioral measures such as locomotor activity (Church et al., 1977; Sanders, 1976), coordination (Dudek et al., 1984), and grooming behavior (Allan and Isaacson, 1985); hence, the nature of the differences between these selected lines is task-specific. One difference between these types of tasks and schedule controlled operant behavior is that, whereas decreases in performance on nonoperant tasks are without consequences, decreases in response rate under fixed-ratio schedules results in loss of reinforcement. Reinforcement loss is one factor that influences the development of tolerance to the rate-decreasing effects of drugs in operant procedures (Carlton and Wolgin, 1971; Schuster et al., 1966; Smith, 1990). Similarly, it is possible that the environmental contingencies of operant procedures (e.g., reinforcement loss) may mitigate against the behavioral expression of differences
in genetic sensitivity to EtOH. Alternatively, between line differences in sensitivity to the acute effects of EtOH may be limited to responses mediated through receptors or neuronal pathways which are inherently different between these lines of animals (e.g., see Allan and Harris, 1991). It is possible that the neuronal pathways involved in control of operant behavior may differ from those mediating nonoperant behaviors. In Experiment 3 we examined the effects of toluene on FR responding in these mice. Drug discrimination studies have demonstrated that toluene shares discriminative stimulus effects with EtOH (Rees et al., 1987). Toluene also has many other EtOH-like behavioral and pharmacological effects (Evans and Balster, 1991). We hypothesized that, if toluene and EtOH had a common neural basis for these shared effects, then they would produce similar patterns of effects in mice selectively bred for neural sensitivity to EtOH. This hypothesis was not supported. While toluene, like EtOH, dosedependently decreased FR responding in both lines of mice, the concentrations of toluene at which responding was decreased differed between the lines. Responding was similar in the two lines following inhalation of the lowest concentration of toluene (1000 ppm). As concentration of toluene increased, response rates of SS mice showed a linear decrease. In contrast, the relationship between toluene concentration and responding was curvilinear for LS mice, response rates decreasing only at the highest concentration (8000 ppm). Thus, SS mice showed considerably greater behavioral sensitivity to the effects of toluene than did LS mice. Previous research provides evidence that the behavioral response used for selective breeding, duration of the loss of righting reflex, differs in LS and SS mice not only in response to EtOH (Erwin et al., 1976; M&learn and Kakihana, 1981), but also in response to other alcohols (Dudek et al., 1984) and to barbiturates (Alpern and McIntyre, 1985; Howerton et al., 1984; McIntyre and Alpern, 1986; O’Connor et al., 1982), although the direction of the barbiturateinduced difference is disputed (for a review, see
McIntyre and Alpern, 1985). Several previous studies have reported that pentobarbital produced longer sleep times in SS mice than in LS mice (Howerton et al., 1984; O’Connor et al., 1982; Siemens and Chan, 1976), suggesting that the effects of pentobarbital on other types of behavior in these mice may differ from those of EtOH. In the present study, toluene, like pentobarbital, was more potent in SS than LS mice. Several authors have suggested that the lipid solubility of alcohols and barbiturates may be related to the effects on duration of loss of righting reflex in LS and SS mice (Howerton et al., 1983a, 198313;Marley et al., 1986). Similarly, lipid solubility may be important in the behavioral actions of toluene. Moser and Balster (1985b) have shown that the relative lipid solubility of several solvents, including toluene and EtOH, is positively correlated with the behavioral activity of these substances in an inverted screen test. It is possible that the greater lipid solubility of toluene than of EtOH may be related to differences in the behavioral effects of toluene and EtOH in the present study. It may also be possible that differences in the elimination of toluene in LS and SS mice may account for the differences in sensitivity to toluene observed in these lines. At the time of awakening, LS mice exhibit lower blood EtOH levels than do SS mice, suggesting that LS mice are more sensitive to EtOH’s effects on the brain (Smolen and Smolen, 1987; Smolen et al., 1987). Although EtOH elimination rates are slightly higher in SS mice (Gilliam and Collins, 1983; Gilliam et al., 1983), this factor cannot account for the drastic differences in durations of loss of righting reflex (Phillips et al., 1989). In contrast, blood levels of pentobarbital at time of awakening are similar in each selected line; thus, pentobarbital elimination rates are slower in SS mice (O’Connor et al., 1982; Siemens and Chan, 1976). This similarity in awakening pentobarbital blood levels suggests that the lines do not differ in neural sensitivity to pentobarbital, but rather in pentobarbital elimination rates (O’Connor et al., 1982). Since toluene elimination rates have not been measured in these mice, it is impossible to determine whether the greater
behavioral sensitivity to toluene exhibited by SS mice in the present study is caused by greater neural sensitivity or by slower elimination rates. There is considerable data showing a substantial overlap in the behavioral and pharmacological effects of toluene, EtOH and the abused CNS depressant class of drugs (Evans and Balster, 1991). Despite these similarities, differences among them are also evident (Barry and Krimmer, 1978; DeFry and Slangen, 1986; Rees and Balster, 1988). These differences may be exploited in discovering the commonalities and differences in their neural bases of action. The fact that LS and SS lines of mice are differentially sensitive to ethanol and toluene on a measure of operant behavior provides a rationale for comparing cellular sites of action of these two agents. For example, cerebellar tissues from LS and SS mice have been shown to differ in EtOH-induced chloride flux (Allan and Harris, 1986). Since anesthetics have been EtOH-like effects on shown to produce GABAergic systems (Cheng and Brunner, 1981; Huidobro-Toro et al., 1987; Keane and Biziere, 1987; Nakahiro et al., 1989), it would be of interest to compare the in vitro actions of toluene and EtOH in these systems using tissues from LS and SS mice. Based on the results of this study, differences might be expected between these lines in response to EtOH and toluene which may contribute to our understanding of the neural basis of toluene’s behavioral effects and its abuse. In summary, the demonstration that LS and SS mice bred for different EtOH-induced hypnotic responses do not show differential behavioral sensitivity to EtOH in an operant paradigm emphasizes the need for caution in assuming that differences in specific phenotypic characteristics reflect differences in general sensitivity to behavioral manipulations. The behavioral responses required in operant procedures may involve neuronal pathways different from those involved in the hypnotic response. Alternatively, the nature of learned operant behavior with its consequences of reduced food reinforcement when the behavior is impaired may mitigate differences in sensitivity
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that are apparent in other behavioral procedures. Finally, the substantially greater sensitivity of SS than LS mice to the effects of toluene provides evidence for differences in neural substrate for EtOH and this solvent and provides a rationale for using this sensitivity difto explore possible ference to toluene mechanisms for its behavioral actions. Acknowledgment This research was supported by NIDA grant DA-03112. J.L. Wiley is a postdoctoral fellow supported by training grant DA-07027. References Allan, A.M. and Harris, R.A. (1986) Gamma-aminobutyric acid and alcohol actions: Neurochemical studies of long sleep and short sleep mice. Life Sci. 39, 2005 2015. Allan, A.M. and Harris, R.A. (1991) Neurochemieal studies of genetic differences in alcohol action. In: The Genetic Basis of Alcohol and Drug Actions (Crabbe, J.C., Jr. and Harris, R.A., eds.), pp. 105- 152. Plenum Press, New York. Allan, A.M. and Isaacson, R.L. (1985) Ethanol-induced grooming in mice selectively bred for differential sensitivity to ethanol. Behav. Neural Biol. 44, 386-392. Alpern, H.P. and McIntyre, T.D. (1985) Evidence that the selectively bred long- and short-sleep mouse lines display common narcotic reactions to many depressants. Psychopharmacology 85, 456 - 459. Balster, R.L. (1987) Abuse potential evaluation of inhalants. Drug Alcohol Depend. 49, 7 - 15. Balster, R.L. and Baird, J.B. (1979) The effects of phencyclidine, d-amphetamine and pentobarbital on spaced responding in mice. Pharmacol. Bioehem. Behav. 11, 617 - 623. Barry III, H. and Krimmer, E.C. (1978) Similarities and differences in discriminative stimulus effects of chlordiazepoxide, pentobarbital, ethanol and other sedatives. In: Stimulus Properties of Drugs. Ten Years of Progress (Colpaert, F.C. and Rosecrans, J.A., eds.), pp. 31-51. Elsevier, Amsterdam. Carlton, P.L. and Wolgin, D.L. (1971) Contingent tolerance to the anorexigenic effects of amphetamine. Physiol. Behav. 7, 221- 223. Carroll, M.E., Santi, P.A. and Kliner, D.J. (1981) A microcomputer system for the control of behavioral experiments. Pharmacol. Biochem. Behav. 4, 415-417. Chan, A.W.K. (1976) Gamma aminobutyric acid in different strains of mice. Effect of ethanol. Life Sci. 19, 597 - 604. Cheng, S. and Brunner, E.A. (1981) Effects of anesthetic agents on synaptosomal GABA disposal. Anesthesiology 55, 34-40.
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