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The effects of ethanol and Ro 15–4513 on elevated plus-maze and rotarod performance in long-sleep and short-sleep mice
Alcohol, Vol. 6, pp. 369-376. ©Pergamon Press plc, 1989. Printed in the U.S.A.
0741-8329/89 $3.00 + .00
The Effects of Ethanol and Ro 15-4513 on Elevated Plus-Maze and Rotarod Performance in Long-Sleep and Short-Sleep Mice AUDRA STINCHCOMB,*t
B A R B A R A J. B O W E R S * $ A N D JEANNE M. W E H N E R * t 1
*Institute for Behavioral Genetics, ~'School of Pharmacy and e,:Department of Psychology, University of Colorado, Boulder, CO 80309 R e c e i v e d 17 M a r c h 1988; A c c e p t e d 29 M a y 1989
STINCHCOMB, A., B. J. BOWERS AND J. M. WEHNER. The effectsof ethanol and Ro 15-4513 on elevatedplus-maze and rotarod performance in long-sleep and short-sleep mice. ALCOHOL 6(5) 369-376, 1989.--The effects of ethanol and diazepam were examined in long-sleep (LS) and short-sleep (SS) mice using the elevated plus-maze. Ethanol had more pronounced effects in SS mice than in LS mice. In contrast, LS mice were more sensitive to the effects of diazepam on the elevated plus-maze. The ataxic effects of ethanol were measured by rotarod performance. SS mice were more resistent to the ataxic effects of a 2.0 g/kg dose of ethanol than LS mice. Ro 15-4513 reversed ethanol's ataxic effects when administered after ethanol in both LS mice and SS mice. Pentobarbital-induced ataxia was unaffected by treatment with Ro 15-4513. Studies of competition of Ro 15-4513 on 3H-flunitrazepam binding indicated that LS and SS mice did not differ in this measure in cortex, cerebellum or hippocampus. LS/SS mice
Ethanol
Ro 15-4513
Elevated plus-maze
PREVIOUS behavioral and biochemical studies have implicated the GABA/benzodiazepine/barbiturate receptor complex in the mediation of some of ethanl's effects, Both benzodiazepines and ethanol have anxiolytic effects (7,30). Furthermore, GABA mimetics prolong the duration of ethanol-induced anesthesia (14), increase motor incoordination produced by ethanol (8), and there is some degree of cross tolerance between ethanol and benzodiazepines (13). At the receptor level, biochemical studies have indicated that at physiologically relevant concentrations, ethanol potentiates muscimol-stimulated C1 flux in isolated rat cortical vesicles (33) and primary cultures of spinal cord neurons (35). This potentiation can be blocked, to some degree, by the imidazobenzodiazepine, Ro 15-4513, thus leading some investigators to suggest that Ro 15-4513 may be a specific ethanol antagonist (32). The examinations of the behavioral specificity of Ro 15-4513 on ethanol's actions have been more controversial (4,20). Although there have been reports indicating that it is specific in comparison to another inverse agonist, FG 7142 (32), others have indicated that FG 7142 can block ethanol's effects provided higher doses are administered to compensate for its reduced receptor affinity (16,18). Ro 15-4513 lowers seizure thresholds in mice (29) and increases seizure susceptibility during ethanol withdrawal thereby limiting its clinical value (19). Nevertheless, Ro 15-4513
Ataxia
and other benzodiazepines do provide a tool to determine whether a behavioral action of ethanol is mediated via an interaction with the GABA/benzodiazepine receptor complex. We and others (1, 24-28) have used a pharmacogenetic approach to analyze the mechanisms of ethanol's actions on the GABAergic system by using long-sleep (LS) and short-sleep (SS) mice which were selectively bred for a differential sleep-time response to ethanol (22). It has been reported previously that LS and SS mice differ not only in their response to ethanol, but also in behavioral responses related to the GABAergic system including incoordination activity (28), seizure sensitivity (26), benzodiazepine-induced loss of righting response (24), and the anticonvulsant effects of benzodiazepines on GABAergic seizures (24). Interestingly, the differences between these two lines of mice depend on the dose and specific behavior tested so that a simple relationship does not exist. Characterization of the GABA/benzodiazepine/barbiturate receptor complex in these mice has indicated that LS mice are more sensitive to ethanol potentiation of muscimol-stimulated C1- flux (1). Studies have shown that the high affinity form of the GABA receptor is not different between the LS and SS mice (26). The number or K d of benzodiazepine receptors in cortex, cerebellum or hippocampus also are not different (27). Differences between LS
~Requests for reprints should be addressed to Jeanne M. Wehner, University of Colorado, Institute for Behavioral Genetics, Box 447, Boulder, CO 80309.
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STINCHCOMB, BOWERS AND WEHNER
and SS mice in the degree of coupling between the GABA receptor and the benzodiazepine receptor in cortical and cerebellar tissue are evident from the fact that GABA differentially enhances benzodiazepine binding in these regions (25). We have focused on the differences at the receptor level because other aspects of the GABA system do not appear to be different between these two lines of mice. For example, brain GABA levels in LS and SS mice are not different (5), nor are these levels differentially affected by ethanol. There also does not appear to be a differential effect of ethanol on reuptake of GABA (12). There was no difference in effect of ethanol on spontaneous release of 3H-GABA from cerebellar slices from LS and SS mice, but LS cortical slices were more sensitive to ethanol inhibition of K~-stimulated 3H-GABA release than were SS cortical slices, albeit at high ethanol concentrations (170-500 mM) (11). Because these LS/SS differences in GABA release are not evident at lower more physiologically relevant doses of ethanol, it does not appear that components of the GABAergic system regulating uptake and release may be as important as receptor sensitivity in determining the differential sensitivity of LS and SS mice to the behavioral actions of ethanol. In order to understand further the differential responses of LS and SS mice to benzodiazepines and ethanol, and possible receptor differences that may mediate these responses, we have examined the anxiolytic effects of benzodiazepines and ethanol in these mice using the elevated plus-maze. In this maze mice are scored on the number of entrances into and time spent in two open versus two closed arms of a four-armed maze (17,30). Previous studies have indicated that this test provides a reliable method for assessing benzodiazepines' actions (17,30). It is thought that a less anxious rodent will enter the open arms and spend more time in the open space than in the closed area of the maze. Total entries into all arms of the maze can provide an assessment of activity so that a drug-induced change in locomotor activity of the animal is not confused with an anxiolytic effect. As a second measure, the ataxic effects of ethanol were examined using rotarod performance, The ability of Ro 15-4513 to block these behavioral effects of ethanol and competition of benzodiazepine binding by Ro 15-4513 were also measured in brain tissue from LS and SS mice. METHOD
Animals Male and female 60-90 day old LS and SS mice from the Institute for Behavioral Genetics were housed in groups of 2-5 with like-sex litter mates. Animals were maintained on a 12 hr light/dark cycle (lights on at 0700 to 1900) and were permitted free access to food (Wayne Lab Blox) and water.
Apparatus The apparatus was patterned after that described by Pellow et at. (17). The elevated plus-maze consisted of two open arms (30 × 5 cm) opposite to each other and two enclosed arms (30 × 5 cm) opposite to each other with an open roof. The two enclosed arms were surrounded by clear acrylic plastic enclosures that were 14 cm high. The maze was elevated to a height of 35 cm. The behavior was videotaped for 5 rain by an observer who remained in the room during testing. The maze was cleaned with 10% ethanol between testing of animals. The rotarod (Ugo Basile Co., Milan, Italy) consisted of a rotating rod with a rotation speed of 8 rpm. All mice were trained to walk on the rotating rod until they were able to complete three 100 sec periods. Each mouse was allowed a 100 sec walk before
each experiment to check training memory. At test times, the mice were placed on the rotating rod for 100 sec or until they felt off the apparatus.
Chemicals Flurazepam, flunitrazepam, diazepam, and Ro 15-1788 were generous gifts from Dr. P. Sorter of Hoffmann-La Roche (Nutley, N J). Ro 15-4513 was a generous gift from Dr. W. Haefely and Dr. R. Eigenmann of Hoffmann-La Roche (Basel, Switzerland). [3H] Flunitrazepam (FNZ) was obtained from NEN Products (Boston, MA), specific activity=65 Ci/mmol. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO),
Elevated Plus-Maze: Diazepam and Ethanol Dose Re.wonse Six groups of SS mice and five groups of LS mice were tested in the elevated plus-maze experiments after ethanol treatment (n = 10 at each dose). Mice were injected 20 rain before testing with either a range of ethanol (20% w/v) (1.0, 2.0, 2.5, 3.0, and 3.5 g/kg for SS mice; 0.5, 0.75, 1.0, and 2.0 g/kg for LS mice) or saline (0.01 ml/g body weight). The effects of an acute dose of diazepam were evaluated in six groups of SS mice and five groups of LS mice (n = 10 at each dose). Thirty minutes before testing, mice were injected intraperitoneally tIP) with a range of diazepam doses prepared in distilled water, acidified with hydrochloric acid to p H = 2 (0.02 ml/g body weight). Control groups received a vehicle injection. SS mice received 0.5, 1.0, 2.0, 3.0, and 4.0 mg/kg diazepam; LS mice received 0.5, 1.0, 2.0, and 3.0 mg/kg diazepam. Thirty seconds before testing, the mouse was placed in an opaque plastic tube in the center of the maze to prevent a predetermined choice of arms by the mouse. After removal of the tube, animals remained in the maze for five minutes. Videotapes were scored for number of entries into open and closed arms, as well as time spent on the arms. A score qualified as an arm entry when all four feet were in the arm. Entry percentages were defined as total entries into the open arm divided by total entries into both arm types. Time percentages were also determined by total time spent on the open a n n divided by total time spent on both arm types. Total entries were recorded as an index of overall activity.
Ethanol/Ro 15-4513 Dose Response Four groups of SS mice (n = 12-34) were preinjected tIP) with a range of Ro 15-4513 doses (0, 0.5, 1.0, 3.0 mg/kg) suspended in 0.5% gelatin 5 rain before receiving an ethanol (2.5 g/kg) or saline injection. Twenty minutes after the second injection, the mice were tested and scored in the plus-maze as described in the ethanol dose-response procedure. Data were analyzed using Analysis of Variance (ANOVA) techniques followed by post hoc analysis using Tukey's B. In order to measure possible intrinsic effects of Ro 15-4513, two groups of SS mice (n = 10) were injected with vehicle ( 0 . 5 ~ gelatin) or 3.0 mg/kg Ro 15-4513. To increase overall baseline activity in the plus-maze, these groups of animals were exposed to a novel environment; i.e., an open-field arena, 5 min prior to testing in the maze. This procedure has been shown to raise baseline activity in the open arms so that potential anxiogenic effects of a drug can be measured (17). Mice received vehicle or Ro-compound injections 20 min before a 5 min exposure to the open field; that is, 25 rain before testing in the plus-maze. Data were analyzed using one-way ANOVA.
Rotarod Performance: Ethanol and Pentobarbital Effects Potential ataxic effects of ethanol as measured on a rotarod
EFFECTS OF ETHANOL AND Ro 15-4513
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were examined in five groups of LS and SS mice (n = 5-27 at each dose). Each group received an IP injection of saline or ethanol (2.0 g/kg, LS; 2.0 or 2.5 g/kg, SS) followed 5 min later by a second injection, consisting of either 0.5% gelatin (control), Ro 15-4513 (20 mg/kg), Ro 15-1788 (20 mg/kg), or a combination of Ro 15-4513 (20 mg/kg) and Ro 15-1788 (20 mg/kg). Mice were tested 20 min later on the rotarod as described. Both Ro-compounds were suspended in 0.5% gelatin. LS and SS mice were also tested on the rotarod after pentobarbital administration. Each group received an injection of saline or 20, 25, 27.5 or 30 mg/kg pentobarbital 5 min before an IP injection of either 20 mg/kg Ro 15-4513 suspended in 0.5% gelatin or 0.5% gelatin (control). Mice were tested twenty minutes following the second injection. Data were analyzed using either Student's t-test, Mann-Whitney U-tests, or by ANOVA techniques followed by post hoc analysis using Tukey's B.
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3H-Flunitrazepam Binding Assays The washed pellet was resuspended in 50 mM Na/K phosphate buffer for use in the assays. The assays were conducted in the manner outlined previously (9, 24, 25). Nonspecific binding was determined in the presence of 10 -5 M flurazepam. NaC1 (0.2 M) was present in all assays. 3H-FNZ was used at concentrations of 0.5-1.0 nM. Competition assays were performed by the addition of varying concentrations of Ro 15-4513. Ro 15-4513 was dissolved in a l: 1 mixture of DMSO and 50 mM Na/K phosphate buffer. Concentration points for Ro 15-4513 ranged from 1 x 10-1o M to 5 × 10 - 7 M. Vehicle was added to all assay tubes. Samples were incubated on ice at 4 degrees for 90 min. The reaction was stopped with the addition of 3 ml of ice-cold buffer and filtration through glass fiber filters, which were washed 3 times with buffer. Filters were counted in toluene scintillation fluid using a Beckman 7000 liquid scintillation counter. Protein concentrations were determined by the methods of Lowry et al. (19). Competition curves were analyzed using the EBDA program of McPherson (23) for the IBM P.C. RESULTS
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plus-maze in LS and SS mice are shown in Fig. 2. The total number of entrances into the open and closed arms of the maze was significantly increased in SS mice after IP administration of ethanol, F(5,59) = 6.51, p<0.001. Post hoc analysis indicated that this effect may be due to the activation produced by the 2.0 g/kg dose (p<0.01); total number of entrances began to decrease at ethanol doses greater than 2.0 g/kg. LS mice exhibited a decrease in total number of entrances after a 2.0 g/kg ethanol dose, F(4,49) = 4.53, p<0.01. The effects of a range of doses of Ro 15-4513 (0.5-3.0 mg/kg) on the ethanol response in the elevated plus-maze were evaluated
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Membrane Preparationfor 3H-Flunitrazepam Binding Naive mice were sacrificed by cervical dislocation and the brains removed. Brains were dissected on ice into three regions; cortex, cerebellum, and hippocampus and were homogenized in 100 volumes of ice-cold 50 mM Na/K phosphate buffer, pH = 7.4. The homogenate was centrifuged at 1,000 x g for 10 min and the resulting supernatant centrifuged at 40,000 x g for 10 min. This pellet was washed five times by a repeated procedure of resuspension in 100 volumes of buffer followed by centrifugation at 40,000 × g for 10 min. Membrane protein concentrations ranged from 250-500 Ixg protein/50 ILl membrane,
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VEH 0.5 1.0 in SS mice. This line of mice was chosen to investigate the potential reversal of ethanol's effects by Ro 15-4513 because of the magnitude of their mean response in the maze at 2.5 g/kg ethanol. The 51% and 64% increase for entrances into and time spent in the open arms at this dose was the largest response exhibited by either line of mice at any dose (see Fig. 1). A replication of the original ethanol response was observed in this set of experiments; a 2.5 g/kg injection of ethanol significantly increased entrances (not shown) and time spent in the open arms over saline-treated animals (Fig. 3), t(46) = 3.88, p<0.001; t(45) = 4.73, p<0.001; entrances and time, respectively. Treatment with three doses of Ro 15-4513 attenuated but did not significantly reverse the ethanol-induced increase in time spent in the open arm, F(3,90), p = 0.846, n.s. Of the 34 S S mice treated with 3.0 mg/kg of Ro 15-4513, 44% were totally reversed and returned to baseline scores of 0-20% for time spent in the open arm. Thus an extremely variable response was observed with Ro 15-4513. The effects of a single Ro 15-4513 injection are shown in the inset of Fig. 3. An anxiogenic-like response was observed in SS mice that had been exposed to the open field and had received a 3.0 mg/kg dose of the Ro-compound prior to testing in the plus-maze. The number of entrances into the open arms was significantly decreased compared to vehicle-injected animals, F(I, 17) = 7.1, p < 0 . 0 2 , and a reduction of time spent in the open arms approached significance, F(I, 17) = 4.1, p = 0.06. This attentuation of activity in the open arms was not explained by a decrease in locomotor activity; no differences in the number of total entrances into all arms occurred between the two treatment groups, F(1,17), p = 0 . 6 6 8 , n.s. For comparison with ethanol, the effects of diazepam were also examined. Dose-response curves of the effects of diazepam on the number of entrances into and amount of time spent in the open arms of the elevated plus-maze in LS and SS mice are shown in Fig. 4. The percentage of time spent in the open arms of the maze was significantly increased in LS mice after IP administration of diazepam, F(4,49)=2.54, p
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any significant change in behavior due to acute injections of diazepam. The total number of entries into all arms of the maze was used as a measure of activation. Dose-response curves of the effects of diazepam on the total number of entrances into open and closed arms of the elevated plus-maze in LS and SS mice are shown in Fig. 5. Total entries were significantly decreased in SS mice as a function of increased diazepam doses, 0.5-4.0 mg/kg; F(5,58)= 2.85, p < 0 . 0 5 . LS mice exhibited an increase in the number of
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FIG. 6. Effects of ethanol (g/kg) on rotarod performance with blockade by Ro 15-4513 and reversal by Ro 15-1788. Values represent means _+S.E.M. of 5 to 14 mice. S=saline treated, V=vehicle treated, 2E=2.0 g/kg ethanol treated, RO = 20 mg/kg Ro 15-4513, C = combination treatment of 20 mg/kg Ro 15-4513 and 20 mg/kg Ro 15-1788, 2.5E=2.5 g/kg ethanol.
entrances at the 1.0 mg/kg dose of diazepam, F(4,49)= 2.82, p<0.05.
Rotarod Performance Because LS mice were not affected by ethanol in the elevated plus-maze, a second measure of ethanol sensitivity was used to analyze: 1) the effects of ethanol on rotarod performance; 2) potential blockade of ethanol-induced ataxia by Ro 15-4513; and 3) inhibitory effects of the benzodiazepine antagonist, Ro 151788, on the reversal of the ethanol response by Ro 15-4513. Figure 6 presents the results of this series of experiments. A one-way ANOVA of the effect of a 2.0 g/kg dose of ethanol on rotarod performance in LS and SS mice indicated a significant difference between line of mouse, F(1,24) = 11.49, p<0.01. Also, Mann-Whitney U-tests showed significant impairment due to ethanol in both lines of mice (p<0.01). One-way ANOVAS indicated that LS mice were impaired at the 2.0 g/kg dose of ethanol, F(1,16)= 19.94, p
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Neither Ro-compound produced intrinsic effects on its own. Separate acute IP injections of Ro 15-4513 and Ro 15-1788 following saline injections had no effects on locomotor activity on the rotarod (not shown); scores were 100 sec as in saline/vehicle controls. To evaluate better the antagonist effects of Ro 15-4513 and Ro 15-1788, postinjection dose-response curves were generated for both compounds (Fig. 7). Mice received an injection of ethanol followed 5 min later by a range of doses of either Ro-compound (2.5-20.0 mg/kg) or 0.5% gelatin (vehicle control). In this set of experiments LS mice were administered 2.25 g/kg ethanol and SS, 2.5 g/kg. These doses produced an equivalent degree of ethanolinduced impairment on the rotarod (10 __ 2.5 sec; 8 - 1.6 sec; LS and SS, respectively). In both lines of mice, injections of Ro 15-4513 improved rotarod scores over vehicle postinjected controis, F(4,47) = 5.3, p<0.01; F(4,43) = 5.4, p
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and 22% for SS mice).
Competition of Flunitrazepam Binding We have previously shown that LS and SS mice do not differ in benzodiazepine receptor number or affinity in whole brain (24) cortex and cerebellum (27). To determine whether there are differential effects of Ro 15-4513 in LS and SS mice that could be observed at the level of the benzodiazepine receptor, competition of 3H-FNZ binding by Ro 15-4513 was evaluated in specific brain regions in both lines of mice. Membrane preparations from cortex, cerebellum and hippocampus were incubated with increasing concentrations of Ro 15-4513 (10 - m - 5 × - 1 0 7 M). Table t presents mean K~ and IC5o values from 3-6 assays. Ro 15-4513 competitively inhibited 3H-FNZ binding in both LS and SS mice; however, the degree of competition did not differ between the lines in any brain region. DISCUSSION As has been observed with other behavioral measures of GABA/benzodiazepine receptor function in LS and SS mice (24,28), these mice exhibit differential sensitivity to the effects of ethanol and diazepam in the plus-maze. The dose-response curves
TABLE 1 COMPETITION OF RO 15-4513 ON 3H-FLUNITRAZEPAMBINDING Line Region SS cortex LS cortex SS cerebellum LS cerebellum SS hippocampus LS hippocampus
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*K~ Values are expressed as nanomolar units. tlCso Values are expressed as nanomolar units. Results are the mean values + S.E.M. from three to six experiments done in duplicate.
for ethanol's effects do not overlap between LS and SS mice: ethanol was more effective in SS mice at doses that produce sedation in LS mice. Some of ethanol's effects in SS mice appear to be due to an increase in overall locomotor activity at low doses: however, at higher doses the observed decrease in locomotion is not associated with a concomitant decrease in activity in the open arms of the maze. In contrast to SS mice, low doses of ethanol that do not cause sedation in LS mice also do not produce a significant response in the elevated plus-maze. In order to validate whether the effects of ethanol in SS mice were true anxiolytic responses in the plus-maze, diazepam, a prototypic anxiolytic agent was also evaluated in LS and SS mice. Unlike the response to ethanol, SS mice are relatively insensitive to the anxiolytic effects of diazepam, whereas LS mice exhibit a response. These results suggest that different genes regulate the sensitivity to the effects of ethanol and diazepam. However, it appears that the plus-maze may not measure a true anxiolytic response in these mice because of the lack of diazepam effect in the SS mice. As with low doses of ethanol, some of the effects of diazepam at low doses in LS mice may be due to an increase in overall locomotor activation. A recent study by Lister (17) that examined the effects of various drugs on behavior by mice in the elevated plus-maze and holeboard demonstrated an increase in entries into and time spent in the open arms of the maze after chlordiazepoxide or ethanol administration. Both drugs also produced an increase in total arm entries. Locomotor activity as measured in the holeboard did not increase as a result of these drug treatments, suggesting that in their study the plus-maze was measuring true anxiolytic responses. We did not observe the marked attenuation of ethanol's effects in the plus-maze reported by Lister (18). Although 44% of SS mice tested were sensitive to complete reversal by Ro 15-4513, a significant dose-response relationship was not observed. This variability may be due to the fact that SS mice are not inbred and therefore may express differential sensitivities to Ro 15-4513 across individuals or due to the extremely difficult nature of measuring anxiolytic responses in the elevated plus-maze. It has been suggested that Ro 15-4513 may have intrinsic properties that are opposite of those produced by ethanol such that the observed consequence of ethanol plus Ro 15-4513 treatment is a summation effect rather than a true blockade (4). The response demonstrated by the SS mice in the plus-maze after a 3.0 mg/kg IP injection of Ro 15-4513 supports the conclusion that SS mice do respond as would be predicted to the anxiogenic effects of Ro 15-4513. After raising overall baseline activity in the maze by initially exposing the mice to another novel environment, the effects of Ro 15-4513 were measured. This low dose did not affect locomotor activity as no reduction in total arm entries were observed. Therefore, any attenuation of ethanol's effects by this compound appears to be due to an interaction of its own effects with those of ethanol, rather than a true antagonism at the receptor level (2). The difference observed in this study between LS and SS mice in response to diazepam emphasizes the complexities of analyzing behavioral effects of benzodiazepines and deciphering genetic regulation of these drug responses. Previous work in our laboratory with flurazepam indicated that SS mice were more sensitive to the anticonvulsant effects of flurazepam than were LS mice in a dose range of 1-10 mg/kg (24). However, in the present study LS mice were more sensitive to the effects of diazepam in a similar dose range. The complex metabolism of flurazepam compared to diazepam and differing receptor affinities of the two benzodiazepines may contribute to these complexities. A more plausible explanation, however, is that specific brain regions may mediate the different behaviors so that generalizations concerning benzodiazepines' effects cannot he made. This lack of generalization concerning behavioral responses m
EFFECTS OF ETHANOL AND Ro 15-4513
375
benzodiazepines is also true for the behavioral actions of ethanol. LS mice are more sensitive to high-dose sedative effects of ethanol while SS mice are more sensitive to the low-dose activating properties (6,31). However, the ataxic properties of ethanol as measured by rotarod performance indicated that SS mice were less sensitive than LS mice. This may also indicate brain regional mediation of various behaviors or complex interaction of ethanol with other neurotransmitter systems. In SS mice, Ro 15-4513 reversed ethanol's ataxic effects. The actions of Ro 15-4513 appear to be mediated by its binding at the benzodiazepine receptor because treatment with the antagonist Ro 15-1788 prevented the reversal of ethanol's ataxic effects. However, posttreatment with Ro 15-1788 alone had no antagonistic effect on ethanol-induced ataxia. These results are similar to those reported by Liljequist and Engel (15), in which anxiolytic effects of ethanol in an anticonflict procedure were not blocked by Ro 15-1788. This suggests that although the reversal of Ro 15-4513's blockade of ethanol's effects may be mediated via the BZ receptor, the ataxic effects of ethanol cannot be antagonized directly at the receptor level. According to Lilequist and Engel, such direct blockade can be achieved through GABAergic mechanisms, not benzodiazepine-receptor mediated antagonism. In contrast to the behavior observed in the elevated plus-maze, no intrinsic effects of the Ro-compounds were observed on the rotarod i.e., neither compound appeared to alter rotarod performance. However, with this method of training mice to a criterion of 100 sec, it would not be possible to measure improvements in baseline behavior due to the drug. This method of rotarod training may also be the reason that we observed results in conflict with those previously reported by Hoffman et al. (10) using C57BL mice that were not pretrained on the rotarod. Alternately, because LS and SS mice are not fully inbred and we did observe reversal in a subset of these mice, some
of these animals may carry a set of genes that are sensitive to the effects of Ro 15-4513 while other individuals do not. The data presented here support other research concerning the ability of Ro 15-4513 to reverse some of ethanol's effects (3, 10, 29, 34). However, in another extensive study of Ro 15-4513's ability to antagonize ethanol's effects in LS and SS mice completed in this laboratory (unpublished data), successful antagonism of ethanol-induced changes in body temperature, heart rate, or Y-maze activity was not observed. Our data would support the conclusion made by others that Ro 15-4513 is effective in reversing only certain behavioral impairments produced by ethanol (34). The binding data presented here indicated that the inhibition of flunitrazepam binding with Ro 15-4513 does not differ in cortex, cerebellum, or hippocampus between LS and SS mice. Therefore, the behavioral differences observed here and those previously reported are consistent with the hypothesis that the recognition site for benzodiazepines is not different between LS and SS mice. However, other data support the hypothesis that coupling between the receptor in the G A B A receptor complex (25) and/or the C1ionophore may be different (1) in some brain regions of LS and SS mice. The role of such coupling phenomena in the mediation of the behavioral differences observed here remain to be determined. ACKNOWLEDGEMENTS We thank June Pounder and Thomas Bosy for excellent technical assistance. This work was supported by a grant #AA-03527 to J.M.W. and BRSG grants RR-07013-19 and RR-07012-20 awarded from the Biomedical Research Support Grant Program, Division of Research Resources, NIH, to the University of Colorado. A.S. is the recipient of an undergraduate research fellowship from American Association of Colleges of Pharmacy. B.J.B. is the recipient of a predoctoral traineeship (NIMH grant MH- 16880).
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12. Howerton, T. C.; Marks, M. J.; Collins, A. C. Norepinephrine, gamma-aminobutyric acid, and choline reuptake kinetics and the effects of ethanol in long-sleep mice. Subst. Alcohol Actions/Misuse 3:8%99; 1982. 13. Le, A. D.; Khanna, J. M.; Kalant, H.; Gross, F. Tolerance to and cross-tolerance among ethanol, pentobarbital, and chlordiazepoxide. Pharmacol. Biochem. Behav. 24:93-98; 1986. 14. Liljequist, S.; Engel, J. A. Effects of GABAergic agonists and antagonists on various ethanol-induced behavioral changes. Psychopharmacology (Berlin) 78:71-75; 1982. 15. Liljequist, S.; Engle, J. A. The effects of GABA and benzodiazepine receptor antagonists on the anti-conflict actions of diazepam and ethanol. Pharmacol. Biocbem. Behav. 21:521-525; 1984. 16. Lister, R. G. The benzodiazepine receptor inverse agonists FG 7142 and Ro 15-4513 both reverse some of the behavioral effects of ethanol in a holeboard test. Life Sci 41:1481-1489; 1987. 17. Lister, R. G. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology (Berlin) 92:180-185; 1987. 18. Lister, R. G. Interactions of three benzodiazepine receptor inverse agonists with ethanol in a plus-maze test of anxiety. Pharmacol. Biochem. Behav. 30:701-706; 1988. 19. Lister, R. G.; Karanian, J. W. Ro 15-4513 induces seizures in DBA/2 mice undergoing alcohol withdrawal. Alcohol 4:409-411; 1987. 20. Lister, R. G.; Nutt, D. J. Is Ro 15-4513 a specific alcohol antagonist? Trends Neurosci. 10:223-225; 1987. 21. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275; 1951. 22. McClearn, G. E.; Kakihana, R. Selective breeding for ethanol sensitivity: SS and LS mice. In: McClearn, G. E.; Deitrich, R. A.; Erwin, V. G., eds. Development of animal models as pharmacogenetic tools. Washington, DC: United States Government Printing Office: 1981:147-159.
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23. McPherson, G. A. A. A practical computer-based approach to the analysis of radioligand binding experiments. Comput. Programs Biomed. 17:107-114; 1983. 24. Marley, R. J.; Freund, R. K.; Wehner, J. M. Differential response to flurazepam in long-sleep and short-sleep mice. Pharmacol. Biochem. Behav. 31:453-458; 1988. 25. Marley, R. J.; Wehner, J. M. GABA enhancement of flunitrazepam binding in mice selectively bred for differential sensitivity to ethanol. Alcohol Drug Res. 7:25-32; 1986. 26. Marley, R. J.; Wehner, J. M. Correlation between the enhancement of flunitrazepam binding by GABA and seizure susceptibility in mice. Life Sci. 40:2215-2224; 1987. 27. Marley, R. l.; Stinchcomb, A.; Wehner, J. M. Further characterization of benzodiazepine receptor differences in long-sleep and shortsleep mice. Life Sci. 43:1223-1231; 1988. 28. Martz, A. R.; Deitrich, R. A.; Harris, R. A. Behavioral evidence for the involvement of gamma-aminobutyric acid in the actions of ethanol. Eur. J. Pharmacol. 89:53-62; 1983. 29, Nutt, D. J.; Lister, R. G. The effect of the imidazodiazepine Ro 15-4513 on the anticonvulsant effects of diazepam, sodium pentobar-
STINCHCOMB, BOWERS AND WEHNER
bital and ethanol. Brain Res.4t3:193-196; 1987. 30. Pellow, S,; Chopin, P.; File, S. E.; Briley, M. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods 14:149-167; 1985. 31. Sanders, B. 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; 1976. 32. Suzdak, P. D.; Glowa, J. R.; Crawley, J. N.; Schwartz, R. D.: Skolnick, P.; Paul, S. M. A selective imidazobenzodiazepine antagonist of ethanol in the rat. Science 234:1243-1247; 1986. 33. Suzdak, P. D.; Schwartz, R. D.; Skolnick, P.; Paul, S. M. Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes. Proc. Natl. Acad. Sci. USA 83:4071-4075; 1986. 34. Syapin, P. J.; Gee, K. W.; Alkana, R. L. Ro 15-4513 differentially affects ethanol-induced hypnosis and hypothermia. Brain Res. Bull. 19:603-605; 1987. 35. Ticku, M. K.; Lowrimore, P.; Lehoullier, P. Ethanol enhances GABA-Induced 36Cl-influx in primary spinal cord cultured neurons. Brain Res. Bull. 17:123-126; 1986.
0741-8329/89 $3.00 + .00
The Effects of Ethanol and Ro 15-4513 on Elevated Plus-Maze and Rotarod Performance in Long-Sleep and Short-Sleep Mice AUDRA STINCHCOMB,*t
B A R B A R A J. B O W E R S * $ A N D JEANNE M. W E H N E R * t 1
*Institute for Behavioral Genetics, ~'School of Pharmacy and e,:Department of Psychology, University of Colorado, Boulder, CO 80309 R e c e i v e d 17 M a r c h 1988; A c c e p t e d 29 M a y 1989
STINCHCOMB, A., B. J. BOWERS AND J. M. WEHNER. The effectsof ethanol and Ro 15-4513 on elevatedplus-maze and rotarod performance in long-sleep and short-sleep mice. ALCOHOL 6(5) 369-376, 1989.--The effects of ethanol and diazepam were examined in long-sleep (LS) and short-sleep (SS) mice using the elevated plus-maze. Ethanol had more pronounced effects in SS mice than in LS mice. In contrast, LS mice were more sensitive to the effects of diazepam on the elevated plus-maze. The ataxic effects of ethanol were measured by rotarod performance. SS mice were more resistent to the ataxic effects of a 2.0 g/kg dose of ethanol than LS mice. Ro 15-4513 reversed ethanol's ataxic effects when administered after ethanol in both LS mice and SS mice. Pentobarbital-induced ataxia was unaffected by treatment with Ro 15-4513. Studies of competition of Ro 15-4513 on 3H-flunitrazepam binding indicated that LS and SS mice did not differ in this measure in cortex, cerebellum or hippocampus. LS/SS mice
Ethanol
Ro 15-4513
Elevated plus-maze
PREVIOUS behavioral and biochemical studies have implicated the GABA/benzodiazepine/barbiturate receptor complex in the mediation of some of ethanl's effects, Both benzodiazepines and ethanol have anxiolytic effects (7,30). Furthermore, GABA mimetics prolong the duration of ethanol-induced anesthesia (14), increase motor incoordination produced by ethanol (8), and there is some degree of cross tolerance between ethanol and benzodiazepines (13). At the receptor level, biochemical studies have indicated that at physiologically relevant concentrations, ethanol potentiates muscimol-stimulated C1 flux in isolated rat cortical vesicles (33) and primary cultures of spinal cord neurons (35). This potentiation can be blocked, to some degree, by the imidazobenzodiazepine, Ro 15-4513, thus leading some investigators to suggest that Ro 15-4513 may be a specific ethanol antagonist (32). The examinations of the behavioral specificity of Ro 15-4513 on ethanol's actions have been more controversial (4,20). Although there have been reports indicating that it is specific in comparison to another inverse agonist, FG 7142 (32), others have indicated that FG 7142 can block ethanol's effects provided higher doses are administered to compensate for its reduced receptor affinity (16,18). Ro 15-4513 lowers seizure thresholds in mice (29) and increases seizure susceptibility during ethanol withdrawal thereby limiting its clinical value (19). Nevertheless, Ro 15-4513
Ataxia
and other benzodiazepines do provide a tool to determine whether a behavioral action of ethanol is mediated via an interaction with the GABA/benzodiazepine receptor complex. We and others (1, 24-28) have used a pharmacogenetic approach to analyze the mechanisms of ethanol's actions on the GABAergic system by using long-sleep (LS) and short-sleep (SS) mice which were selectively bred for a differential sleep-time response to ethanol (22). It has been reported previously that LS and SS mice differ not only in their response to ethanol, but also in behavioral responses related to the GABAergic system including incoordination activity (28), seizure sensitivity (26), benzodiazepine-induced loss of righting response (24), and the anticonvulsant effects of benzodiazepines on GABAergic seizures (24). Interestingly, the differences between these two lines of mice depend on the dose and specific behavior tested so that a simple relationship does not exist. Characterization of the GABA/benzodiazepine/barbiturate receptor complex in these mice has indicated that LS mice are more sensitive to ethanol potentiation of muscimol-stimulated C1- flux (1). Studies have shown that the high affinity form of the GABA receptor is not different between the LS and SS mice (26). The number or K d of benzodiazepine receptors in cortex, cerebellum or hippocampus also are not different (27). Differences between LS
~Requests for reprints should be addressed to Jeanne M. Wehner, University of Colorado, Institute for Behavioral Genetics, Box 447, Boulder, CO 80309.
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and SS mice in the degree of coupling between the GABA receptor and the benzodiazepine receptor in cortical and cerebellar tissue are evident from the fact that GABA differentially enhances benzodiazepine binding in these regions (25). We have focused on the differences at the receptor level because other aspects of the GABA system do not appear to be different between these two lines of mice. For example, brain GABA levels in LS and SS mice are not different (5), nor are these levels differentially affected by ethanol. There also does not appear to be a differential effect of ethanol on reuptake of GABA (12). There was no difference in effect of ethanol on spontaneous release of 3H-GABA from cerebellar slices from LS and SS mice, but LS cortical slices were more sensitive to ethanol inhibition of K~-stimulated 3H-GABA release than were SS cortical slices, albeit at high ethanol concentrations (170-500 mM) (11). Because these LS/SS differences in GABA release are not evident at lower more physiologically relevant doses of ethanol, it does not appear that components of the GABAergic system regulating uptake and release may be as important as receptor sensitivity in determining the differential sensitivity of LS and SS mice to the behavioral actions of ethanol. In order to understand further the differential responses of LS and SS mice to benzodiazepines and ethanol, and possible receptor differences that may mediate these responses, we have examined the anxiolytic effects of benzodiazepines and ethanol in these mice using the elevated plus-maze. In this maze mice are scored on the number of entrances into and time spent in two open versus two closed arms of a four-armed maze (17,30). Previous studies have indicated that this test provides a reliable method for assessing benzodiazepines' actions (17,30). It is thought that a less anxious rodent will enter the open arms and spend more time in the open space than in the closed area of the maze. Total entries into all arms of the maze can provide an assessment of activity so that a drug-induced change in locomotor activity of the animal is not confused with an anxiolytic effect. As a second measure, the ataxic effects of ethanol were examined using rotarod performance, The ability of Ro 15-4513 to block these behavioral effects of ethanol and competition of benzodiazepine binding by Ro 15-4513 were also measured in brain tissue from LS and SS mice. METHOD
Animals Male and female 60-90 day old LS and SS mice from the Institute for Behavioral Genetics were housed in groups of 2-5 with like-sex litter mates. Animals were maintained on a 12 hr light/dark cycle (lights on at 0700 to 1900) and were permitted free access to food (Wayne Lab Blox) and water.
Apparatus The apparatus was patterned after that described by Pellow et at. (17). The elevated plus-maze consisted of two open arms (30 × 5 cm) opposite to each other and two enclosed arms (30 × 5 cm) opposite to each other with an open roof. The two enclosed arms were surrounded by clear acrylic plastic enclosures that were 14 cm high. The maze was elevated to a height of 35 cm. The behavior was videotaped for 5 rain by an observer who remained in the room during testing. The maze was cleaned with 10% ethanol between testing of animals. The rotarod (Ugo Basile Co., Milan, Italy) consisted of a rotating rod with a rotation speed of 8 rpm. All mice were trained to walk on the rotating rod until they were able to complete three 100 sec periods. Each mouse was allowed a 100 sec walk before
each experiment to check training memory. At test times, the mice were placed on the rotating rod for 100 sec or until they felt off the apparatus.
Chemicals Flurazepam, flunitrazepam, diazepam, and Ro 15-1788 were generous gifts from Dr. P. Sorter of Hoffmann-La Roche (Nutley, N J). Ro 15-4513 was a generous gift from Dr. W. Haefely and Dr. R. Eigenmann of Hoffmann-La Roche (Basel, Switzerland). [3H] Flunitrazepam (FNZ) was obtained from NEN Products (Boston, MA), specific activity=65 Ci/mmol. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO),
Elevated Plus-Maze: Diazepam and Ethanol Dose Re.wonse Six groups of SS mice and five groups of LS mice were tested in the elevated plus-maze experiments after ethanol treatment (n = 10 at each dose). Mice were injected 20 rain before testing with either a range of ethanol (20% w/v) (1.0, 2.0, 2.5, 3.0, and 3.5 g/kg for SS mice; 0.5, 0.75, 1.0, and 2.0 g/kg for LS mice) or saline (0.01 ml/g body weight). The effects of an acute dose of diazepam were evaluated in six groups of SS mice and five groups of LS mice (n = 10 at each dose). Thirty minutes before testing, mice were injected intraperitoneally tIP) with a range of diazepam doses prepared in distilled water, acidified with hydrochloric acid to p H = 2 (0.02 ml/g body weight). Control groups received a vehicle injection. SS mice received 0.5, 1.0, 2.0, 3.0, and 4.0 mg/kg diazepam; LS mice received 0.5, 1.0, 2.0, and 3.0 mg/kg diazepam. Thirty seconds before testing, the mouse was placed in an opaque plastic tube in the center of the maze to prevent a predetermined choice of arms by the mouse. After removal of the tube, animals remained in the maze for five minutes. Videotapes were scored for number of entries into open and closed arms, as well as time spent on the arms. A score qualified as an arm entry when all four feet were in the arm. Entry percentages were defined as total entries into the open arm divided by total entries into both arm types. Time percentages were also determined by total time spent on the open a n n divided by total time spent on both arm types. Total entries were recorded as an index of overall activity.
Ethanol/Ro 15-4513 Dose Response Four groups of SS mice (n = 12-34) were preinjected tIP) with a range of Ro 15-4513 doses (0, 0.5, 1.0, 3.0 mg/kg) suspended in 0.5% gelatin 5 rain before receiving an ethanol (2.5 g/kg) or saline injection. Twenty minutes after the second injection, the mice were tested and scored in the plus-maze as described in the ethanol dose-response procedure. Data were analyzed using Analysis of Variance (ANOVA) techniques followed by post hoc analysis using Tukey's B. In order to measure possible intrinsic effects of Ro 15-4513, two groups of SS mice (n = 10) were injected with vehicle ( 0 . 5 ~ gelatin) or 3.0 mg/kg Ro 15-4513. To increase overall baseline activity in the plus-maze, these groups of animals were exposed to a novel environment; i.e., an open-field arena, 5 min prior to testing in the maze. This procedure has been shown to raise baseline activity in the open arms so that potential anxiogenic effects of a drug can be measured (17). Mice received vehicle or Ro-compound injections 20 min before a 5 min exposure to the open field; that is, 25 rain before testing in the plus-maze. Data were analyzed using one-way ANOVA.
Rotarod Performance: Ethanol and Pentobarbital Effects Potential ataxic effects of ethanol as measured on a rotarod
EFFECTS OF ETHANOL AND Ro 15-4513
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were examined in five groups of LS and SS mice (n = 5-27 at each dose). Each group received an IP injection of saline or ethanol (2.0 g/kg, LS; 2.0 or 2.5 g/kg, SS) followed 5 min later by a second injection, consisting of either 0.5% gelatin (control), Ro 15-4513 (20 mg/kg), Ro 15-1788 (20 mg/kg), or a combination of Ro 15-4513 (20 mg/kg) and Ro 15-1788 (20 mg/kg). Mice were tested 20 min later on the rotarod as described. Both Ro-compounds were suspended in 0.5% gelatin. LS and SS mice were also tested on the rotarod after pentobarbital administration. Each group received an injection of saline or 20, 25, 27.5 or 30 mg/kg pentobarbital 5 min before an IP injection of either 20 mg/kg Ro 15-4513 suspended in 0.5% gelatin or 0.5% gelatin (control). Mice were tested twenty minutes following the second injection. Data were analyzed using either Student's t-test, Mann-Whitney U-tests, or by ANOVA techniques followed by post hoc analysis using Tukey's B.
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3H-Flunitrazepam Binding Assays The washed pellet was resuspended in 50 mM Na/K phosphate buffer for use in the assays. The assays were conducted in the manner outlined previously (9, 24, 25). Nonspecific binding was determined in the presence of 10 -5 M flurazepam. NaC1 (0.2 M) was present in all assays. 3H-FNZ was used at concentrations of 0.5-1.0 nM. Competition assays were performed by the addition of varying concentrations of Ro 15-4513. Ro 15-4513 was dissolved in a l: 1 mixture of DMSO and 50 mM Na/K phosphate buffer. Concentration points for Ro 15-4513 ranged from 1 x 10-1o M to 5 × 10 - 7 M. Vehicle was added to all assay tubes. Samples were incubated on ice at 4 degrees for 90 min. The reaction was stopped with the addition of 3 ml of ice-cold buffer and filtration through glass fiber filters, which were washed 3 times with buffer. Filters were counted in toluene scintillation fluid using a Beckman 7000 liquid scintillation counter. Protein concentrations were determined by the methods of Lowry et al. (19). Competition curves were analyzed using the EBDA program of McPherson (23) for the IBM P.C. RESULTS
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plus-maze in LS and SS mice are shown in Fig. 2. The total number of entrances into the open and closed arms of the maze was significantly increased in SS mice after IP administration of ethanol, F(5,59) = 6.51, p<0.001. Post hoc analysis indicated that this effect may be due to the activation produced by the 2.0 g/kg dose (p<0.01); total number of entrances began to decrease at ethanol doses greater than 2.0 g/kg. LS mice exhibited a decrease in total number of entrances after a 2.0 g/kg ethanol dose, F(4,49) = 4.53, p<0.01. The effects of a range of doses of Ro 15-4513 (0.5-3.0 mg/kg) on the ethanol response in the elevated plus-maze were evaluated
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VEH 0.5 1.0 in SS mice. This line of mice was chosen to investigate the potential reversal of ethanol's effects by Ro 15-4513 because of the magnitude of their mean response in the maze at 2.5 g/kg ethanol. The 51% and 64% increase for entrances into and time spent in the open arms at this dose was the largest response exhibited by either line of mice at any dose (see Fig. 1). A replication of the original ethanol response was observed in this set of experiments; a 2.5 g/kg injection of ethanol significantly increased entrances (not shown) and time spent in the open arms over saline-treated animals (Fig. 3), t(46) = 3.88, p<0.001; t(45) = 4.73, p<0.001; entrances and time, respectively. Treatment with three doses of Ro 15-4513 attenuated but did not significantly reverse the ethanol-induced increase in time spent in the open arm, F(3,90), p = 0.846, n.s. Of the 34 S S mice treated with 3.0 mg/kg of Ro 15-4513, 44% were totally reversed and returned to baseline scores of 0-20% for time spent in the open arm. Thus an extremely variable response was observed with Ro 15-4513. The effects of a single Ro 15-4513 injection are shown in the inset of Fig. 3. An anxiogenic-like response was observed in SS mice that had been exposed to the open field and had received a 3.0 mg/kg dose of the Ro-compound prior to testing in the plus-maze. The number of entrances into the open arms was significantly decreased compared to vehicle-injected animals, F(I, 17) = 7.1, p < 0 . 0 2 , and a reduction of time spent in the open arms approached significance, F(I, 17) = 4.1, p = 0.06. This attentuation of activity in the open arms was not explained by a decrease in locomotor activity; no differences in the number of total entrances into all arms occurred between the two treatment groups, F(1,17), p = 0 . 6 6 8 , n.s. For comparison with ethanol, the effects of diazepam were also examined. Dose-response curves of the effects of diazepam on the number of entrances into and amount of time spent in the open arms of the elevated plus-maze in LS and SS mice are shown in Fig. 4. The percentage of time spent in the open arms of the maze was significantly increased in LS mice after IP administration of diazepam, F(4,49)=2.54, p
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any significant change in behavior due to acute injections of diazepam. The total number of entries into all arms of the maze was used as a measure of activation. Dose-response curves of the effects of diazepam on the total number of entrances into open and closed arms of the elevated plus-maze in LS and SS mice are shown in Fig. 5. Total entries were significantly decreased in SS mice as a function of increased diazepam doses, 0.5-4.0 mg/kg; F(5,58)= 2.85, p < 0 . 0 5 . LS mice exhibited an increase in the number of
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EFFECTS OF ETHANOL AND Ro 15-4513
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FIG. 6. Effects of ethanol (g/kg) on rotarod performance with blockade by Ro 15-4513 and reversal by Ro 15-1788. Values represent means _+S.E.M. of 5 to 14 mice. S=saline treated, V=vehicle treated, 2E=2.0 g/kg ethanol treated, RO = 20 mg/kg Ro 15-4513, C = combination treatment of 20 mg/kg Ro 15-4513 and 20 mg/kg Ro 15-1788, 2.5E=2.5 g/kg ethanol.
entrances at the 1.0 mg/kg dose of diazepam, F(4,49)= 2.82, p<0.05.
Rotarod Performance Because LS mice were not affected by ethanol in the elevated plus-maze, a second measure of ethanol sensitivity was used to analyze: 1) the effects of ethanol on rotarod performance; 2) potential blockade of ethanol-induced ataxia by Ro 15-4513; and 3) inhibitory effects of the benzodiazepine antagonist, Ro 151788, on the reversal of the ethanol response by Ro 15-4513. Figure 6 presents the results of this series of experiments. A one-way ANOVA of the effect of a 2.0 g/kg dose of ethanol on rotarod performance in LS and SS mice indicated a significant difference between line of mouse, F(1,24) = 11.49, p<0.01. Also, Mann-Whitney U-tests showed significant impairment due to ethanol in both lines of mice (p<0.01). One-way ANOVAS indicated that LS mice were impaired at the 2.0 g/kg dose of ethanol, F(1,16)= 19.94, p
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FIG. 7. Effects of a range of posttreatment doses (mg/kg) of Ro 15-4513 and Ro 15-1788 on ethanol-induced ataxia on the rotarod. Values represent means-S.E.M, of 5-13 mice per dose. The * represents the training criterion score of 100 sec.
Neither Ro-compound produced intrinsic effects on its own. Separate acute IP injections of Ro 15-4513 and Ro 15-1788 following saline injections had no effects on locomotor activity on the rotarod (not shown); scores were 100 sec as in saline/vehicle controls. To evaluate better the antagonist effects of Ro 15-4513 and Ro 15-1788, postinjection dose-response curves were generated for both compounds (Fig. 7). Mice received an injection of ethanol followed 5 min later by a range of doses of either Ro-compound (2.5-20.0 mg/kg) or 0.5% gelatin (vehicle control). In this set of experiments LS mice were administered 2.25 g/kg ethanol and SS, 2.5 g/kg. These doses produced an equivalent degree of ethanolinduced impairment on the rotarod (10 __ 2.5 sec; 8 - 1.6 sec; LS and SS, respectively). In both lines of mice, injections of Ro 15-4513 improved rotarod scores over vehicle postinjected controis, F(4,47) = 5.3, p<0.01; F(4,43) = 5.4, p
374
STINCHCOMB, BOWERS AND WEHNER
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FIG. 8. Effects of pentobarbital on rotarod performance with a demonstration of the specificity of Ro 15-4513 inhibitory effects for ethanol-induced ataxia. Values represent means -+ S.E.M. of 19 to 27 mice. S=saline treated, V = vehicle treated, P = 30 mg/kg pentobarbital, RO = 20 mg/kg Ro 15-4513.
and 22% for SS mice).
Competition of Flunitrazepam Binding We have previously shown that LS and SS mice do not differ in benzodiazepine receptor number or affinity in whole brain (24) cortex and cerebellum (27). To determine whether there are differential effects of Ro 15-4513 in LS and SS mice that could be observed at the level of the benzodiazepine receptor, competition of 3H-FNZ binding by Ro 15-4513 was evaluated in specific brain regions in both lines of mice. Membrane preparations from cortex, cerebellum and hippocampus were incubated with increasing concentrations of Ro 15-4513 (10 - m - 5 × - 1 0 7 M). Table t presents mean K~ and IC5o values from 3-6 assays. Ro 15-4513 competitively inhibited 3H-FNZ binding in both LS and SS mice; however, the degree of competition did not differ between the lines in any brain region. DISCUSSION As has been observed with other behavioral measures of GABA/benzodiazepine receptor function in LS and SS mice (24,28), these mice exhibit differential sensitivity to the effects of ethanol and diazepam in the plus-maze. The dose-response curves
TABLE 1 COMPETITION OF RO 15-4513 ON 3H-FLUNITRAZEPAMBINDING Line Region SS cortex LS cortex SS cerebellum LS cerebellum SS hippocampus LS hippocampus
Kr* 16 13 22 16 12 12
+ 5 - 2 ± 5 ± 3 -+ 2 +_ 3
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-- 5 ± 2 -+- 5 - 3 - 3 _+ 4
*K~ Values are expressed as nanomolar units. tlCso Values are expressed as nanomolar units. Results are the mean values + S.E.M. from three to six experiments done in duplicate.
for ethanol's effects do not overlap between LS and SS mice: ethanol was more effective in SS mice at doses that produce sedation in LS mice. Some of ethanol's effects in SS mice appear to be due to an increase in overall locomotor activity at low doses: however, at higher doses the observed decrease in locomotion is not associated with a concomitant decrease in activity in the open arms of the maze. In contrast to SS mice, low doses of ethanol that do not cause sedation in LS mice also do not produce a significant response in the elevated plus-maze. In order to validate whether the effects of ethanol in SS mice were true anxiolytic responses in the plus-maze, diazepam, a prototypic anxiolytic agent was also evaluated in LS and SS mice. Unlike the response to ethanol, SS mice are relatively insensitive to the anxiolytic effects of diazepam, whereas LS mice exhibit a response. These results suggest that different genes regulate the sensitivity to the effects of ethanol and diazepam. However, it appears that the plus-maze may not measure a true anxiolytic response in these mice because of the lack of diazepam effect in the SS mice. As with low doses of ethanol, some of the effects of diazepam at low doses in LS mice may be due to an increase in overall locomotor activation. A recent study by Lister (17) that examined the effects of various drugs on behavior by mice in the elevated plus-maze and holeboard demonstrated an increase in entries into and time spent in the open arms of the maze after chlordiazepoxide or ethanol administration. Both drugs also produced an increase in total arm entries. Locomotor activity as measured in the holeboard did not increase as a result of these drug treatments, suggesting that in their study the plus-maze was measuring true anxiolytic responses. We did not observe the marked attenuation of ethanol's effects in the plus-maze reported by Lister (18). Although 44% of SS mice tested were sensitive to complete reversal by Ro 15-4513, a significant dose-response relationship was not observed. This variability may be due to the fact that SS mice are not inbred and therefore may express differential sensitivities to Ro 15-4513 across individuals or due to the extremely difficult nature of measuring anxiolytic responses in the elevated plus-maze. It has been suggested that Ro 15-4513 may have intrinsic properties that are opposite of those produced by ethanol such that the observed consequence of ethanol plus Ro 15-4513 treatment is a summation effect rather than a true blockade (4). The response demonstrated by the SS mice in the plus-maze after a 3.0 mg/kg IP injection of Ro 15-4513 supports the conclusion that SS mice do respond as would be predicted to the anxiogenic effects of Ro 15-4513. After raising overall baseline activity in the maze by initially exposing the mice to another novel environment, the effects of Ro 15-4513 were measured. This low dose did not affect locomotor activity as no reduction in total arm entries were observed. Therefore, any attenuation of ethanol's effects by this compound appears to be due to an interaction of its own effects with those of ethanol, rather than a true antagonism at the receptor level (2). The difference observed in this study between LS and SS mice in response to diazepam emphasizes the complexities of analyzing behavioral effects of benzodiazepines and deciphering genetic regulation of these drug responses. Previous work in our laboratory with flurazepam indicated that SS mice were more sensitive to the anticonvulsant effects of flurazepam than were LS mice in a dose range of 1-10 mg/kg (24). However, in the present study LS mice were more sensitive to the effects of diazepam in a similar dose range. The complex metabolism of flurazepam compared to diazepam and differing receptor affinities of the two benzodiazepines may contribute to these complexities. A more plausible explanation, however, is that specific brain regions may mediate the different behaviors so that generalizations concerning benzodiazepines' effects cannot he made. This lack of generalization concerning behavioral responses m
EFFECTS OF ETHANOL AND Ro 15-4513
375
benzodiazepines is also true for the behavioral actions of ethanol. LS mice are more sensitive to high-dose sedative effects of ethanol while SS mice are more sensitive to the low-dose activating properties (6,31). However, the ataxic properties of ethanol as measured by rotarod performance indicated that SS mice were less sensitive than LS mice. This may also indicate brain regional mediation of various behaviors or complex interaction of ethanol with other neurotransmitter systems. In SS mice, Ro 15-4513 reversed ethanol's ataxic effects. The actions of Ro 15-4513 appear to be mediated by its binding at the benzodiazepine receptor because treatment with the antagonist Ro 15-1788 prevented the reversal of ethanol's ataxic effects. However, posttreatment with Ro 15-1788 alone had no antagonistic effect on ethanol-induced ataxia. These results are similar to those reported by Liljequist and Engel (15), in which anxiolytic effects of ethanol in an anticonflict procedure were not blocked by Ro 15-1788. This suggests that although the reversal of Ro 15-4513's blockade of ethanol's effects may be mediated via the BZ receptor, the ataxic effects of ethanol cannot be antagonized directly at the receptor level. According to Lilequist and Engel, such direct blockade can be achieved through GABAergic mechanisms, not benzodiazepine-receptor mediated antagonism. In contrast to the behavior observed in the elevated plus-maze, no intrinsic effects of the Ro-compounds were observed on the rotarod i.e., neither compound appeared to alter rotarod performance. However, with this method of training mice to a criterion of 100 sec, it would not be possible to measure improvements in baseline behavior due to the drug. This method of rotarod training may also be the reason that we observed results in conflict with those previously reported by Hoffman et al. (10) using C57BL mice that were not pretrained on the rotarod. Alternately, because LS and SS mice are not fully inbred and we did observe reversal in a subset of these mice, some
of these animals may carry a set of genes that are sensitive to the effects of Ro 15-4513 while other individuals do not. The data presented here support other research concerning the ability of Ro 15-4513 to reverse some of ethanol's effects (3, 10, 29, 34). However, in another extensive study of Ro 15-4513's ability to antagonize ethanol's effects in LS and SS mice completed in this laboratory (unpublished data), successful antagonism of ethanol-induced changes in body temperature, heart rate, or Y-maze activity was not observed. Our data would support the conclusion made by others that Ro 15-4513 is effective in reversing only certain behavioral impairments produced by ethanol (34). The binding data presented here indicated that the inhibition of flunitrazepam binding with Ro 15-4513 does not differ in cortex, cerebellum, or hippocampus between LS and SS mice. Therefore, the behavioral differences observed here and those previously reported are consistent with the hypothesis that the recognition site for benzodiazepines is not different between LS and SS mice. However, other data support the hypothesis that coupling between the receptor in the G A B A receptor complex (25) and/or the C1ionophore may be different (1) in some brain regions of LS and SS mice. The role of such coupling phenomena in the mediation of the behavioral differences observed here remain to be determined. ACKNOWLEDGEMENTS We thank June Pounder and Thomas Bosy for excellent technical assistance. This work was supported by a grant #AA-03527 to J.M.W. and BRSG grants RR-07013-19 and RR-07012-20 awarded from the Biomedical Research Support Grant Program, Division of Research Resources, NIH, to the University of Colorado. A.S. is the recipient of an undergraduate research fellowship from American Association of Colleges of Pharmacy. B.J.B. is the recipient of a predoctoral traineeship (NIMH grant MH- 16880).
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