- Email: [email protected]
Time-dependent effect of ethanol upon discrimination behavior
Alcohol. Vol. 6. pp. 445-449~ © PergamonPress plc, 1989. Printed in the U.S.A.
0741-8329/89 $3.00 + .00
Time-Dependent Effect of Ethanol Upon Discrimination Behavior M A R T I N D. S C H E C H T E R
Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272 R e c e i v e d 17 M a r c h 1989; A c c e p t e d 29 J u n e 1989
SCHECHTER, M. D. Time-dependent effect of ethanol upon discrimination behavior. ALCOHOL 6(6) 445-449, 1989.--The discriminative stimulus properties produced by ethanol were employed to demonstrate differences in discriminative performance over time in rats trained at different postinjection times. Thus, one group of rats was trained to discriminate between intraperitoneally administered 600 mg/kg ethanol and its distilled water vehicle at 6-min postadministration, whereas a second group of rats was trained to make this discrimination when trained at 30-min postinjection. Subsequent to reaching criterion performance, dose-response relations to doses of ethanol from 150-900 mg/kg were determined to be similar in both groups. This indicated that the discriminative stimulus effects at the two postadministration times were equally effective for training of behavioral responding to ethanol. The rats trained at 30 min postadministration maintained criterion level discrimination performance when tested at 6-, 15- and 60-min postinjection. In contrast, the rats trained at 6-min postadministration discriminated ethanol at a reduced level (40%) when tested at 30-min postinjection. These results suggest that the nature of the discriminable stimuli produced by a low dose of ethanol are different at 6-min and at 30-min postadministration. Evidence is cited to further suggest that the earlier stimuli are excitatory whereas the later stimuli are sedative. Ethanol
Time course
Drug discrimination
Rats
Biphasic effects
THERE is a large body of evidence suggesting that the most prominent central effect of ethanol is depression, although behavioral excitation or stimulation can be observed at specific doses and times after its administration. The biphasic nature of ethanol has been documented to be dose-responsive in mice (13,21) and rats (4) which exhibit increased locomotor activity after low doses and decreased locomotion after higher doses. Although the majority of studies with ethanol concern dose-effect relationships at a fixed time postadministration, a few studies have examined the behavioral effects of ethanol as a function of the time after its administration (12). Thus,the observed biphasic action of ethanol may be considered to be both time dependent, as well as dose dependent, and ethanol can produce a transient state of excitation in man prior to the onset of its depressant effects with further ingestion (20,23). A behavioral paradigm which is particularly suited for assessing the subjective effects of drugs that act on the central nervous system is the drug discrimination procedure. Discriminative control of differential responding has been observed to be the property of virtually every psychoactive drug tested to date. Within the discriminative stimulus paradigm, a subject (generally a rat) comes under stimulus control of a drug whereby correct operant responding in a choice situation is contingent upon which drug was previously administered. Thus, in this paradigm, a food-deprived rat is trained to emit one response, i.e., to press one lever in a two-lever operant chmber, for a food reward following administration of a drug. The same subject must make the opposite response, i.e., press the other lever, following an injection of either a different training drug or a control solution (generally distilled water or 0.9% saline). The differential response is made
Excitation
Sedation
contingent upon the interoceptive stimuli that an animal perceives after a particular dose (the training dose) of a drug at a specific postadministration time. Many invetigators have varied the training dose of a particular drug and have, subsequently, tested the training dose at various novel times after training. However, the time course of training to a specific dose of a drug has rarely been used to investigate the time after administration effects on training [e.g., (15)]. In a series of reports (17-19), Altshuler and his colleagues trained rats to discriminate a fixed dose of ethanol (1000 mg/kg IP) at two different times postadministration, viz., at 6 min and at 30 min. These investigators reported that subsequent to ethanol training at the early phase, which they called "excitatory," the rats did not associate their drug state with ethanol when tested at 30-min postadministration (42.4% accuracy). Likewise, rats trained at 30-min postadministration (a group trained at the "sedative phase") could not accurately differentiate 1000 mg/kg ethanol when tested at 6-min postadministration (55% accuracy). These results suggest that the interoceptive stimulus that forms the basis for the ethanol discrimination cue at 6 min is qualitatively different than that utilized for differential learning at 30-min postadministration. Furthermore, these investigators reported (19) that naloxone pretreatment antagonized the ethanol cue in rats trained at 6-min postadministration but not those trained at 30 min, suggesting to the authors that the "excitatory" phase of ethanol discrimination may result from activation of endogenous opiate neurons. The purpose of the present experiment was to attempt to independently replicate the results of these published abstracts (17,18), as well as the full-length article published in this journal (19). An effort was made to expand the previous work by
445
446
SCHECHTER
employing an extended time course to test separate rats trained at 6- or 30-min postadministration. METHOD
Subjects Twelve experimentally-naive male Sprague-Dawley rats (Zivic Miller, Allison Park, PA) weighing approximately 260 g at the beginning of the experiment were individually housed and maintained on a 12-hour light(0600-1800)/12-hour dark cycle in a room kept at temperatures between 20-22°C. All rats received water ad lib and a daily rationing of a commercial rat chow so as to maintain their free-feeding weights at 80-85% of control values.
Apparatus Twelve standard rodent operant chambers (Lafayette Instruments Corp., Lafayette, IN) were used as the experimental space. Each chamber contained two levers situated 7 cm apart and 7 cm above a metal grid floor. A food receptacle was located equidistance between the levers and 2 cm above the floor. Each operant chamber was enclosed in a sound-attenuated cubicle with an exhaust fan and 9 W house light. Solid-state programming equipment (Med Associates, E. Fairfield, VT) was located in an adjacent room and was used to control and record discrimination sessions.
Group Selection and Lever Response Training Procedure Prior to discrimination training, the food-deprived rats were randomly divided into two equal groups ( n = 6 ) and trained (shaped) to press the levers in the operant chamber for food reinforcement (45 mg Noyes pellets). Training sessions were conducted once a day, 5 days a week. One lever in each chamber was designated as the "vehicle lever." That lever was located to the fight of the food receptacle for half of each of the groups and on the left of the food receptacle for the other half. For one group of six animals, training commenced 6 min after the intraperitoneal (IP) administration of 5 ml/kg of distilled water (vehicle) and they were rewarded solely for responses on a designated vehicle lever. For the other group, training commenced 30-rain postvehicle injection. Initially, all rats were trained to respond on the vehicle lever on a fixed ratio (FR) schedule of one, i.e., one response resulted in one reinforcement. During seven training sessions, the FR schedule was gradually increased to an FR10, i.e., 10 responses yielded one reinforcement, and animals were removed from the operant chamber and were returned to their home cages after receiving 40 reinforcements on the FRt0 schedule. For the other 6 rats, the identical training procedure was used at 30-rain postadministration of vehicle. Once an FR10 was established on the vehicle lever in both groups, training began on the opposite lever, the "ethanol lever.'" Following (600 mg/kg) ethanol administration to the 6-min group, all training began at 6-min postethanol administration, whereas in the 30-min group, training commenced 30-min postadministration. In all cases, rats were only rewarded for responses upon the ethanol lever and, as with previous vehicle training, the initial reinforcement schedule of FR1 was gradually increased to an FR10; this was done over a period of 4 days.
nation training began at either 6 min or 30 min after administration of vehicle on 600 mg/kg ethanol. Rats received either 5 ml/kg of (distilled water) vehicle (V) or vehicle containing 600 mg/mt ethanol (10% w/v; E) according to the following two-week repeating injection schedule: E,V,V,E,E; V,E,E,V,V. The first lever upon which 10 responses were made at the beginning of each session was considered the "selected" lever for that session. At the time of the 10th response, presses on both the selected and the unselected levers were recorded. The session was continued irregardless of the correctness of the selected lever until 400 responses were made on the correct lever for that daily session and, therefore, 40 reinforcements (on the FRI0 schedule) were received. Animals were required to choose the selected lever appropriate for the injection received in 8 out of 10 consecutive training sessions. The 80% performance level was required twice before dose-response testing commenced. The two-week pseudorandom injection schedule was repeated as often as necessary during this training period.
Dose-Response Testing Once the discriminative criterion was attained by all animals. the discrimination training regimen was limited to every other day to maintain discrimination. On intervening days, rats were tested at their particular postadministration time, i.e.. one group at 6-min and the second group at 30-min postinjection, after novel doses (150. 300, 450 and 900 mg/kg) of ethanol. Each dose was tested twice, once following a drug maintenance session and once following a vehicle maintenance session. This counter-balancing was used to control for any possible residual influence from the previous maintenance session. If at any time during testing a rat's maintenance discrimination fell below the 80% criterion, data on that animal would be dropped from the results. This. however, did not occur. During this series of experiments, one of the 6 animals in the 6-min training group died of causes unrelated to the procedure and this is reflected in the n = 5 that follows.
Phase Generalization Testing Discrimination of both ethanol and vehicle was tested at the novel time of 30 min in rats trained to discriminate ethanol at 6 min. Likewise. each treatment was tested at 6-min postinjection in those animals trained to discriminate between the ethanol and vehicle states at 30 rain. The novel time points were tested on two occasions; once following an ethanol and once following a vehicle maintenance session at the appropriate time interval used to train that particular group. This is a direct replication of the design reported by Shippenberg and Altshuler (19).
Time Course of Action of Ethanol Preliminary evidence (see Results, below) indicated that an expanded time course would be more informative. Thus, the 6-min group was further tested with the ~ n i n g dose of ethanol in two sessions each at 1-, 3-, 15-, 30. and 6 0 ~ postad~istration, whereas the group trained at 30,min postadministration were given the training dose of ethanol and tes~d at 15-, 60, and 90-min postadministration.
Measurements and Statistics Discrimination Training The rats were considered to be trained (shaped) to lever press once FR10 responding was established on both levers. Discrimi-
The data collected in the drug discrimination sessions are expressed as both quantal and quantitative measurements; Each of the individual measurements provides an indication of lever
TIME-DEPENDENT EFFECT OF ETHANOL
447
TABLE 1 DOSE RESPONSE AND PHASE GENERALIZATION IN RATS TRAINED TO DISCRIMINATE 600 mg/kg ETHANOL AT 6-MIN (n=5) AND 30-MIN ( n = 6 ) POSTADMINISTRATION
Dose Ethanol (mg/kg) 900 600 450 300 150 0 (veh) EDso (mg/kg) (95% conf. limit)
Trained at 6 min Quantal 100.0 91.4 70.0 60.0 20.0 8.0
Quantitative (SD) 87.1 75.2 55.3 48.2 22.5 18.5
265.3 (174.6--403.1)
Trained at 30 min
(5.4) (15.5) (8.6) (13.6) (0.2) (15.7)
324.6 (177.9-592.2)
Quantal 100.0 91.7 58.3 50.0 41.7 11.1
Quantitative (SD) 85.9 (15.4) 78.6 (7.4) 55.3 (14.3) 42.9 (7.9) 39.3 (6.2) 24.9 (11.4)
237.7 149.4-378.1)
275.9 (137.8-552.6)
Phase Generalization Tested at 30 min 600 0 (veh)
40.0 0.0
41.0 24.5
preference prior to any reinforcement. The quantal measurement is the percentage of rats that select the ethanol-appropriate lever as their selected lever, i.e., the lever to first accumulate 10 presses. The quantitative measurement is the number of responses on the ethanol lever divided by the total number of responses on both the ethanol and the vehicle lever at the time that 10 responses are accumulated on either single lever. This fraction is expressed as a percentage. Unlike the all-or-none quantal measurement, the quantitative measurement accounts for responses on both the selected and unselected levers and, thus, provides a relative measure of the magnitude, as well as the direction of lever preference. Additionally, statistics can be performed on the quantitative data. The advantages in using both types of measurements are more fully discussed by Stolerman and D'Mello (22). The Litchfield-Wilcoxon procedure (10), which employs probits vs. log-dose effects, was used to generate an EDso value from the ethanol dose-response data in each of the two postadministration trained groups. Quantitative dose-response measurements represent the average ( _ SD) of the individual sessions quantitative means for each treatment. RESULTS
The results of dose-response testing in the group of animals trained at 6 min and the other group trained at 30 min appear in Table 1. The " 6 - m i n " group, when tested at 6 min after receiving the training dose (600 mg/kg) of ethanol, selected the ethanolappropriate lever on 91.4% of all maintenance trials. Maintenance sessions with the (distilled water) vehicle resulted in the ethanolappropriate lever being chosen on 8% of all trials or, to look at it a different way, 92% of all first selections after vehicle were made on the vehicle-appropriate lever. Increasing the test dose of ethanol to 900 mg/kg or decreasing it to three lower doses (450, 300 and 150 mg/kg) produced a dose-response relationship and allowed a calculated quantal EDso value of 265.3 mg/kg. The quantitative measurement in the 6-min group also was shown to be dose-responsive and yielded a similar EDso value of 324.6 mg/kg. Likewise, the 6 rats trained at 30-min postadministration of 600 mg/kg ethanol maintained this discrimination on 91.7% of all trials
Tested at 6 min (4.6) (2.1)
91.7 0.0
80.6 (7.3) 14.4 (6.3)
and various test doses of ethanol tested at 30 min produced a dose-responsive relationship and allowed for a quantal EDso value of 237.7 mg/kg. Analysis of the slopes of the dose-response lines (10) indicates that they are parallel within 95% confidence limits with the critical t value (2.447) exceeding the calculated t for the quantal (0.5163) and quantitative (0.7798) lines. The results of phase-generalization testing in each group of animals (bottom portion, Table 1) indicate that the selected lever was always the vehicle-appropriate lever when vehicle was tested on two occasions at 30 min in the 6-min trained rats. In contrast, when 600 mg/kg was tested on two occasions in this group of animals at 30-min postadministration the ethanol-appropriate lever was the selected lever only 40% of the time. The quantitative measurement (41.0 - 4.6) was significantly less (t = 6.52;p<0.01) than the quantitative measurement at 6 rain (75.2+-15.5). In contrast, when 600 mg/kg ethanol was tested at 6-min postadministration in the group of animals trained to discriminate ethanol at 30-min postinjection there was no significant change in the ability to discriminate ethanol, i.e., they chose the ethanol-appropriate lever on the same percentage of trials as they had chosen it during maintenance sessions with 600 mg/kg ethanol. Results of the expanded time course studies with 600 mg/kg ethanol in each of the two postadministration-trained animals (Fig. 1) indicate that the group of animals trained at 6-min postadministration had their peak discriminative performance at their training time. In contrast, the group trained at 30-min postadministration showed a relatively constant, criterion level, discriminative performance between 6 and 30 min which continued to 60 min and then declined at 90 rain. DISCUSSION
It has been well established that in humans drinking alcohol a phase of excitation occurs prior to the depressant properties that ensue with further ingestion. These effects, although transient, can be measured in various tests of motor, cognitive and information processing function (12). This biphasic relationship after ethanol administration over time has also been reported to occur in rats (13,21) in which initial excitation was followed by later sedation.
~8
SCHECHTER
100.
ic~
90. 80. T O
~0
~ A '
0 - - 0 6 MIN ~--.A, 30 MIN
"A
70
L ~ ~ z O ~
6o 5o 4o 3o
©.____
zx
2o2 10-
o
I
0
I
I
I
I
I
I
:
~
~
~
1
',
1
1
1
:
1
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
TIME(MIN) FIG. 1. Time course of ethanol action in rats trained to discriminate 600 mg/kg ethanol from its vehicle at either 6-min (open circles) or 30-min (open triangles) postadministration. Ordinate: percentage of rats selecting the ethanol-appropriate lever; Abscissa: postadministration time in minutes. Each point is for two trials in each of either five (6-min group) or six (30-min group) rats.
Employing drugs as discriminative stimuli to allow rats to make differential responses has been shown to be a sensitive, stable and readily reproducible behavioral paradigm (16). In fact, ethanol was shown as early as 1951 by Conger to serve as a discrirninative stimulus and it, therefore, constitutes the first drug to be used in this behavioral technique (6). The present results indicate, once again, that rats can readily be trained to discriminate ethanol at the more usual postadministration time of 30 rain (2, 11, 14, 24). In addition, they can also be trained as early as 6-min postadministration. Indeed, others have trained rats to discriminate 1200 mg/kg ethanol IP (5,9) or 100 mg/kg intravenously (1) at 5-min postadministration. Administration of 150-900 mg/kg to rats trained at either 6- or 30-rain postadministration of 600 mg/kg ethanol produced a dose-responsive relationship with ED5o values of 265.3 and 237.7 mg/kg for the respective groups. This can be compared to the earlier study of Shippenberg and Altshuler (19) by employing the quantitative measurement as this is the quantification given in their work. Thus, in their study the EDso value in rats trained 6-rain postadministration was 520 mg/kg or 52% of the 1000 mg/kg training dose used. In the present study, the quantitative E D i t
value of 325 mg/kg is 54% of the 600 mg/kg training dose. Likewise, the EDso value in rats trained 30-rain postadministration of 1000 mg/kg ethanol was 580 mg/kg, whereas here it was 276 mg/kg; roughly equivalent percentages of the training doses employed. Thus, these E D i t values are similar and this would indicate that the discriminative stimulus effects of the two postadministration time phases were equally effective training conditions. In contrast to the Shippenberg and Altshuler study (t91 which showed symmetrical inability to generalize, the present research indicates that animals trained at 30-min postadministration can discriminate ethanol at 6-min postadministration but those trained at 6-min postadministration do not generalize this discrimination when tested at 30-min postadministration, The former observation had been made previously by Krimmer [(8). as cited in (31] who reportedly trained rats to 1000 mg/kg ethanol tP at 20 rain and showed a vigorous ethanol-like response at 2.5-, 5-. 40-. 60- and 120-min postadministration. The most parsimonious explanation for the present results is that during the drug onset period the animals trained at 30-min postadministration have had experience with the drug stimuli that are produced at both 6 and 30 min and, indeed, use them to allow for differential responding. In contrast, the animals who are trained at 6-min postadministration only have those stimuli that are produced from 0- to 6-min postadministration. If the stimulus that is produced 6 rain after administration of ethanol at this low dose is, as Shippenberg and Altshuler 119) suggest, excitatory in nature and the stimuli inherent in ethanol 30-min postadministration are sedative, then the animals trained at 6-rain postadministration use the excitation as their sole cue. whereas the animals who are trained 30-rain postadministration can identify the earlier stimulatory phase as well as the later sedative phase when they are tested at various postadministratio n times. This is substantiated by the results in Fig. I which further indicate that the excitation phase is present earlier than the 6-min postadministration "excitatory phase" and it lasts for 15 rain with a rapid decrease at 30 min. In contrast, the "' 30-rain" animals who were trained to the earlier stimulatory, as welt as with the later sedative phase, can discriminate ethanol equally well at 6-rain postadministration and that this discrimination continues to last until the ethanol is dissipated in the blood/brain. ACKNOWLEDGEMENTS The author would like to express his gratitude to Ms. Denise McBurney for her continued expertise in the laboratory and to Dr. Edward Krimmer for his advice on the manuscript.
REFERENCES 1. Ando, K. Discriminative control of operant behavior by intravenous administration of drugs in rats. Psychopharmacologia 45:47-50; 1975. 2. Barry, H., III. Classifications of drugs according to their discriminable effects in rats. Fed. Proc. 33:1814-1824; 1974. 3. Barry, H., HI; Krimmer, E. C. Discriminable stimuli produced by alcohol and other CNS depressants. In: Lal, H., ed. Advances in behavioral biology: Discriminative stimulus properties of drugs. New York: Plenum Press; 1977:73-92. 4. Buckalew, L. W.; Cartwright, G. W. General and differential behavioral effects of five ethanol doses in the albino rat. Physiol. Rep. 23:1151-1154; 1968. 5. Bueno, O. F. A.; Carlini, E. A.; Finkelfarb, E.; Suzuki, J. S. A 9-tetrahydrocannabinol, ethanol, and amphetamine as discriminative stimulus generalization tests with other drugs. Psychopharmacologia 46:235-243; 1976. 6. Conger, J. J. The effects of alcohol on conflict behavior in the albino rat. Q. J. Stud. Alcohol 12:1-29; 1951. 7. Glennon, R. A.; Rosecrans, J. A. Speculations on the mechanism of action of hallucinogenic indolealkylamines. Neurosci. Behav. Rev.
5:197-207; 1981. 8. Krimmer, E. D. Drugs as discriminative stimuli. Ph.D. Dissertation. University of Pittsburgh. Dissert. Abstr. Internat. 35:4572-B. 19741975. 9. Kubena, R. K.; Barry, H., III. Generalization by rats of alcohol and atropine stimulus characteristics to other drugs. Psychoph~acologia i5:196-206; 1969. 10. Litehfield. J. T.; Wilcoxon, F. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96:99--1 !3; !949. 11. Overton, D. A: State-dependent l e ~ g produced by depressant and atropine-like drugs. Psyehopharmacologia 10:6-31; 1966. 12. Pohorecky, L. A. Biphasic action of ethanol, Biobehav. Rev. 1: 231-240; 1977. 13. Read, G. W.; Cutting, W.; Furst, A. Comparison of excited phases after sedatives and tranquilizers. PsyChoph~cology 1:346-350; 1960. 14. Schechter, M. D. Extended schedule transfer of ethanol diScrimina: tion. Pharmacol. Bioehem. Behav. 14:23,25; 1981. 15. Scbechter, M. D. Serotonergic-dopaminergic mediation of 3,4 meth-
T I M E - D E P E N D E N T E F F E C T OF E T H A N O L
16. 17. 18.
19.
ylenedioxymethamphetamine (MDMA; "Ecstasy"). Pharmacol. Biochem. Behav. 31:817-824; 1988. Schechter, M. D.; Signs, S. A.; Boja, J. W. Stability of the stimulus properties of drugs over time. Pharmacol. Biochem. Behav. 32: 361-364; 1989. Shippenberg, T. S.; Knappenberger, E.; Altshuler, H. L. The discriminative stimulus effects of ethanol and morphine: Stimulant versus sedative effects. Alcohol.: Clin. Exp. Res. 7:121; 1983. (Abstract.) Shippenberg, T. S.; Altshuler, H. L. The biphasic effects of ethanol: Role of endogenous opiate pathways. Alcohol.: Clin. Exp. Res. 8:119; 1984. (Abstract.) Shippenberg, T. S.; Altshuler, H. L. A drug discrimination analysis of ethanol-induced behavioral excitation and sedation: The role of endogenous opiate pathways. Alcohol 2:197-201; 1985.
449
20. Sjoberg, L. V. Alcohol and gambling. Psychopharmacologia 14: 284-298; 1969. 21. Smoothy, R.; Berry, M. S. Time-course of the locomotor stimulant and depressant effects of a single low dose of ethanol in mice. Psychopharmacology (Berlin) 85:57-61; 1985. 22. Stolerman, I. P.; D'Mello, G. D. Role of training conditions in discrimination of central nervous system stimulants in rats. Psychopharmacology (Berlin) 73:295-303; 1981. 23. Williams, A. F. Social drinking, anxiety and depression. J. Pers. Soc. Psychol. 3:689-693; 1966. 24. York, J. L. Comparison of the discriminative effects of ethanol, barbital and phenobarbital in rats. Psychopharmacology (Berlin) 60:19-23; 1978.
0741-8329/89 $3.00 + .00
Time-Dependent Effect of Ethanol Upon Discrimination Behavior M A R T I N D. S C H E C H T E R
Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272 R e c e i v e d 17 M a r c h 1989; A c c e p t e d 29 J u n e 1989
SCHECHTER, M. D. Time-dependent effect of ethanol upon discrimination behavior. ALCOHOL 6(6) 445-449, 1989.--The discriminative stimulus properties produced by ethanol were employed to demonstrate differences in discriminative performance over time in rats trained at different postinjection times. Thus, one group of rats was trained to discriminate between intraperitoneally administered 600 mg/kg ethanol and its distilled water vehicle at 6-min postadministration, whereas a second group of rats was trained to make this discrimination when trained at 30-min postinjection. Subsequent to reaching criterion performance, dose-response relations to doses of ethanol from 150-900 mg/kg were determined to be similar in both groups. This indicated that the discriminative stimulus effects at the two postadministration times were equally effective for training of behavioral responding to ethanol. The rats trained at 30 min postadministration maintained criterion level discrimination performance when tested at 6-, 15- and 60-min postinjection. In contrast, the rats trained at 6-min postadministration discriminated ethanol at a reduced level (40%) when tested at 30-min postinjection. These results suggest that the nature of the discriminable stimuli produced by a low dose of ethanol are different at 6-min and at 30-min postadministration. Evidence is cited to further suggest that the earlier stimuli are excitatory whereas the later stimuli are sedative. Ethanol
Time course
Drug discrimination
Rats
Biphasic effects
THERE is a large body of evidence suggesting that the most prominent central effect of ethanol is depression, although behavioral excitation or stimulation can be observed at specific doses and times after its administration. The biphasic nature of ethanol has been documented to be dose-responsive in mice (13,21) and rats (4) which exhibit increased locomotor activity after low doses and decreased locomotion after higher doses. Although the majority of studies with ethanol concern dose-effect relationships at a fixed time postadministration, a few studies have examined the behavioral effects of ethanol as a function of the time after its administration (12). Thus,the observed biphasic action of ethanol may be considered to be both time dependent, as well as dose dependent, and ethanol can produce a transient state of excitation in man prior to the onset of its depressant effects with further ingestion (20,23). A behavioral paradigm which is particularly suited for assessing the subjective effects of drugs that act on the central nervous system is the drug discrimination procedure. Discriminative control of differential responding has been observed to be the property of virtually every psychoactive drug tested to date. Within the discriminative stimulus paradigm, a subject (generally a rat) comes under stimulus control of a drug whereby correct operant responding in a choice situation is contingent upon which drug was previously administered. Thus, in this paradigm, a food-deprived rat is trained to emit one response, i.e., to press one lever in a two-lever operant chmber, for a food reward following administration of a drug. The same subject must make the opposite response, i.e., press the other lever, following an injection of either a different training drug or a control solution (generally distilled water or 0.9% saline). The differential response is made
Excitation
Sedation
contingent upon the interoceptive stimuli that an animal perceives after a particular dose (the training dose) of a drug at a specific postadministration time. Many invetigators have varied the training dose of a particular drug and have, subsequently, tested the training dose at various novel times after training. However, the time course of training to a specific dose of a drug has rarely been used to investigate the time after administration effects on training [e.g., (15)]. In a series of reports (17-19), Altshuler and his colleagues trained rats to discriminate a fixed dose of ethanol (1000 mg/kg IP) at two different times postadministration, viz., at 6 min and at 30 min. These investigators reported that subsequent to ethanol training at the early phase, which they called "excitatory," the rats did not associate their drug state with ethanol when tested at 30-min postadministration (42.4% accuracy). Likewise, rats trained at 30-min postadministration (a group trained at the "sedative phase") could not accurately differentiate 1000 mg/kg ethanol when tested at 6-min postadministration (55% accuracy). These results suggest that the interoceptive stimulus that forms the basis for the ethanol discrimination cue at 6 min is qualitatively different than that utilized for differential learning at 30-min postadministration. Furthermore, these investigators reported (19) that naloxone pretreatment antagonized the ethanol cue in rats trained at 6-min postadministration but not those trained at 30 min, suggesting to the authors that the "excitatory" phase of ethanol discrimination may result from activation of endogenous opiate neurons. The purpose of the present experiment was to attempt to independently replicate the results of these published abstracts (17,18), as well as the full-length article published in this journal (19). An effort was made to expand the previous work by
445
446
SCHECHTER
employing an extended time course to test separate rats trained at 6- or 30-min postadministration. METHOD
Subjects Twelve experimentally-naive male Sprague-Dawley rats (Zivic Miller, Allison Park, PA) weighing approximately 260 g at the beginning of the experiment were individually housed and maintained on a 12-hour light(0600-1800)/12-hour dark cycle in a room kept at temperatures between 20-22°C. All rats received water ad lib and a daily rationing of a commercial rat chow so as to maintain their free-feeding weights at 80-85% of control values.
Apparatus Twelve standard rodent operant chambers (Lafayette Instruments Corp., Lafayette, IN) were used as the experimental space. Each chamber contained two levers situated 7 cm apart and 7 cm above a metal grid floor. A food receptacle was located equidistance between the levers and 2 cm above the floor. Each operant chamber was enclosed in a sound-attenuated cubicle with an exhaust fan and 9 W house light. Solid-state programming equipment (Med Associates, E. Fairfield, VT) was located in an adjacent room and was used to control and record discrimination sessions.
Group Selection and Lever Response Training Procedure Prior to discrimination training, the food-deprived rats were randomly divided into two equal groups ( n = 6 ) and trained (shaped) to press the levers in the operant chamber for food reinforcement (45 mg Noyes pellets). Training sessions were conducted once a day, 5 days a week. One lever in each chamber was designated as the "vehicle lever." That lever was located to the fight of the food receptacle for half of each of the groups and on the left of the food receptacle for the other half. For one group of six animals, training commenced 6 min after the intraperitoneal (IP) administration of 5 ml/kg of distilled water (vehicle) and they were rewarded solely for responses on a designated vehicle lever. For the other group, training commenced 30-rain postvehicle injection. Initially, all rats were trained to respond on the vehicle lever on a fixed ratio (FR) schedule of one, i.e., one response resulted in one reinforcement. During seven training sessions, the FR schedule was gradually increased to an FR10, i.e., 10 responses yielded one reinforcement, and animals were removed from the operant chamber and were returned to their home cages after receiving 40 reinforcements on the FRt0 schedule. For the other 6 rats, the identical training procedure was used at 30-rain postadministration of vehicle. Once an FR10 was established on the vehicle lever in both groups, training began on the opposite lever, the "ethanol lever.'" Following (600 mg/kg) ethanol administration to the 6-min group, all training began at 6-min postethanol administration, whereas in the 30-min group, training commenced 30-min postadministration. In all cases, rats were only rewarded for responses upon the ethanol lever and, as with previous vehicle training, the initial reinforcement schedule of FR1 was gradually increased to an FR10; this was done over a period of 4 days.
nation training began at either 6 min or 30 min after administration of vehicle on 600 mg/kg ethanol. Rats received either 5 ml/kg of (distilled water) vehicle (V) or vehicle containing 600 mg/mt ethanol (10% w/v; E) according to the following two-week repeating injection schedule: E,V,V,E,E; V,E,E,V,V. The first lever upon which 10 responses were made at the beginning of each session was considered the "selected" lever for that session. At the time of the 10th response, presses on both the selected and the unselected levers were recorded. The session was continued irregardless of the correctness of the selected lever until 400 responses were made on the correct lever for that daily session and, therefore, 40 reinforcements (on the FRI0 schedule) were received. Animals were required to choose the selected lever appropriate for the injection received in 8 out of 10 consecutive training sessions. The 80% performance level was required twice before dose-response testing commenced. The two-week pseudorandom injection schedule was repeated as often as necessary during this training period.
Dose-Response Testing Once the discriminative criterion was attained by all animals. the discrimination training regimen was limited to every other day to maintain discrimination. On intervening days, rats were tested at their particular postadministration time, i.e.. one group at 6-min and the second group at 30-min postinjection, after novel doses (150. 300, 450 and 900 mg/kg) of ethanol. Each dose was tested twice, once following a drug maintenance session and once following a vehicle maintenance session. This counter-balancing was used to control for any possible residual influence from the previous maintenance session. If at any time during testing a rat's maintenance discrimination fell below the 80% criterion, data on that animal would be dropped from the results. This. however, did not occur. During this series of experiments, one of the 6 animals in the 6-min training group died of causes unrelated to the procedure and this is reflected in the n = 5 that follows.
Phase Generalization Testing Discrimination of both ethanol and vehicle was tested at the novel time of 30 min in rats trained to discriminate ethanol at 6 min. Likewise. each treatment was tested at 6-min postinjection in those animals trained to discriminate between the ethanol and vehicle states at 30 rain. The novel time points were tested on two occasions; once following an ethanol and once following a vehicle maintenance session at the appropriate time interval used to train that particular group. This is a direct replication of the design reported by Shippenberg and Altshuler (19).
Time Course of Action of Ethanol Preliminary evidence (see Results, below) indicated that an expanded time course would be more informative. Thus, the 6-min group was further tested with the ~ n i n g dose of ethanol in two sessions each at 1-, 3-, 15-, 30. and 6 0 ~ postad~istration, whereas the group trained at 30,min postadministration were given the training dose of ethanol and tes~d at 15-, 60, and 90-min postadministration.
Measurements and Statistics Discrimination Training The rats were considered to be trained (shaped) to lever press once FR10 responding was established on both levers. Discrimi-
The data collected in the drug discrimination sessions are expressed as both quantal and quantitative measurements; Each of the individual measurements provides an indication of lever
TIME-DEPENDENT EFFECT OF ETHANOL
447
TABLE 1 DOSE RESPONSE AND PHASE GENERALIZATION IN RATS TRAINED TO DISCRIMINATE 600 mg/kg ETHANOL AT 6-MIN (n=5) AND 30-MIN ( n = 6 ) POSTADMINISTRATION
Dose Ethanol (mg/kg) 900 600 450 300 150 0 (veh) EDso (mg/kg) (95% conf. limit)
Trained at 6 min Quantal 100.0 91.4 70.0 60.0 20.0 8.0
Quantitative (SD) 87.1 75.2 55.3 48.2 22.5 18.5
265.3 (174.6--403.1)
Trained at 30 min
(5.4) (15.5) (8.6) (13.6) (0.2) (15.7)
324.6 (177.9-592.2)
Quantal 100.0 91.7 58.3 50.0 41.7 11.1
Quantitative (SD) 85.9 (15.4) 78.6 (7.4) 55.3 (14.3) 42.9 (7.9) 39.3 (6.2) 24.9 (11.4)
237.7 149.4-378.1)
275.9 (137.8-552.6)
Phase Generalization Tested at 30 min 600 0 (veh)
40.0 0.0
41.0 24.5
preference prior to any reinforcement. The quantal measurement is the percentage of rats that select the ethanol-appropriate lever as their selected lever, i.e., the lever to first accumulate 10 presses. The quantitative measurement is the number of responses on the ethanol lever divided by the total number of responses on both the ethanol and the vehicle lever at the time that 10 responses are accumulated on either single lever. This fraction is expressed as a percentage. Unlike the all-or-none quantal measurement, the quantitative measurement accounts for responses on both the selected and unselected levers and, thus, provides a relative measure of the magnitude, as well as the direction of lever preference. Additionally, statistics can be performed on the quantitative data. The advantages in using both types of measurements are more fully discussed by Stolerman and D'Mello (22). The Litchfield-Wilcoxon procedure (10), which employs probits vs. log-dose effects, was used to generate an EDso value from the ethanol dose-response data in each of the two postadministration trained groups. Quantitative dose-response measurements represent the average ( _ SD) of the individual sessions quantitative means for each treatment. RESULTS
The results of dose-response testing in the group of animals trained at 6 min and the other group trained at 30 min appear in Table 1. The " 6 - m i n " group, when tested at 6 min after receiving the training dose (600 mg/kg) of ethanol, selected the ethanolappropriate lever on 91.4% of all maintenance trials. Maintenance sessions with the (distilled water) vehicle resulted in the ethanolappropriate lever being chosen on 8% of all trials or, to look at it a different way, 92% of all first selections after vehicle were made on the vehicle-appropriate lever. Increasing the test dose of ethanol to 900 mg/kg or decreasing it to three lower doses (450, 300 and 150 mg/kg) produced a dose-response relationship and allowed a calculated quantal EDso value of 265.3 mg/kg. The quantitative measurement in the 6-min group also was shown to be dose-responsive and yielded a similar EDso value of 324.6 mg/kg. Likewise, the 6 rats trained at 30-min postadministration of 600 mg/kg ethanol maintained this discrimination on 91.7% of all trials
Tested at 6 min (4.6) (2.1)
91.7 0.0
80.6 (7.3) 14.4 (6.3)
and various test doses of ethanol tested at 30 min produced a dose-responsive relationship and allowed for a quantal EDso value of 237.7 mg/kg. Analysis of the slopes of the dose-response lines (10) indicates that they are parallel within 95% confidence limits with the critical t value (2.447) exceeding the calculated t for the quantal (0.5163) and quantitative (0.7798) lines. The results of phase-generalization testing in each group of animals (bottom portion, Table 1) indicate that the selected lever was always the vehicle-appropriate lever when vehicle was tested on two occasions at 30 min in the 6-min trained rats. In contrast, when 600 mg/kg was tested on two occasions in this group of animals at 30-min postadministration the ethanol-appropriate lever was the selected lever only 40% of the time. The quantitative measurement (41.0 - 4.6) was significantly less (t = 6.52;p<0.01) than the quantitative measurement at 6 rain (75.2+-15.5). In contrast, when 600 mg/kg ethanol was tested at 6-min postadministration in the group of animals trained to discriminate ethanol at 30-min postinjection there was no significant change in the ability to discriminate ethanol, i.e., they chose the ethanol-appropriate lever on the same percentage of trials as they had chosen it during maintenance sessions with 600 mg/kg ethanol. Results of the expanded time course studies with 600 mg/kg ethanol in each of the two postadministration-trained animals (Fig. 1) indicate that the group of animals trained at 6-min postadministration had their peak discriminative performance at their training time. In contrast, the group trained at 30-min postadministration showed a relatively constant, criterion level, discriminative performance between 6 and 30 min which continued to 60 min and then declined at 90 rain. DISCUSSION
It has been well established that in humans drinking alcohol a phase of excitation occurs prior to the depressant properties that ensue with further ingestion. These effects, although transient, can be measured in various tests of motor, cognitive and information processing function (12). This biphasic relationship after ethanol administration over time has also been reported to occur in rats (13,21) in which initial excitation was followed by later sedation.
~8
SCHECHTER
100.
ic~
90. 80. T O
~0
~ A '
0 - - 0 6 MIN ~--.A, 30 MIN
"A
70
L ~ ~ z O ~
6o 5o 4o 3o
©.____
zx
2o2 10-
o
I
0
I
I
I
I
I
I
:
~
~
~
1
',
1
1
1
:
1
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
TIME(MIN) FIG. 1. Time course of ethanol action in rats trained to discriminate 600 mg/kg ethanol from its vehicle at either 6-min (open circles) or 30-min (open triangles) postadministration. Ordinate: percentage of rats selecting the ethanol-appropriate lever; Abscissa: postadministration time in minutes. Each point is for two trials in each of either five (6-min group) or six (30-min group) rats.
Employing drugs as discriminative stimuli to allow rats to make differential responses has been shown to be a sensitive, stable and readily reproducible behavioral paradigm (16). In fact, ethanol was shown as early as 1951 by Conger to serve as a discrirninative stimulus and it, therefore, constitutes the first drug to be used in this behavioral technique (6). The present results indicate, once again, that rats can readily be trained to discriminate ethanol at the more usual postadministration time of 30 rain (2, 11, 14, 24). In addition, they can also be trained as early as 6-min postadministration. Indeed, others have trained rats to discriminate 1200 mg/kg ethanol IP (5,9) or 100 mg/kg intravenously (1) at 5-min postadministration. Administration of 150-900 mg/kg to rats trained at either 6- or 30-rain postadministration of 600 mg/kg ethanol produced a dose-responsive relationship with ED5o values of 265.3 and 237.7 mg/kg for the respective groups. This can be compared to the earlier study of Shippenberg and Altshuler (19) by employing the quantitative measurement as this is the quantification given in their work. Thus, in their study the EDso value in rats trained 6-rain postadministration was 520 mg/kg or 52% of the 1000 mg/kg training dose used. In the present study, the quantitative E D i t
value of 325 mg/kg is 54% of the 600 mg/kg training dose. Likewise, the EDso value in rats trained 30-rain postadministration of 1000 mg/kg ethanol was 580 mg/kg, whereas here it was 276 mg/kg; roughly equivalent percentages of the training doses employed. Thus, these E D i t values are similar and this would indicate that the discriminative stimulus effects of the two postadministration time phases were equally effective training conditions. In contrast to the Shippenberg and Altshuler study (t91 which showed symmetrical inability to generalize, the present research indicates that animals trained at 30-min postadministration can discriminate ethanol at 6-min postadministration but those trained at 6-min postadministration do not generalize this discrimination when tested at 30-min postadministration, The former observation had been made previously by Krimmer [(8). as cited in (31] who reportedly trained rats to 1000 mg/kg ethanol tP at 20 rain and showed a vigorous ethanol-like response at 2.5-, 5-. 40-. 60- and 120-min postadministration. The most parsimonious explanation for the present results is that during the drug onset period the animals trained at 30-min postadministration have had experience with the drug stimuli that are produced at both 6 and 30 min and, indeed, use them to allow for differential responding. In contrast, the animals who are trained at 6-min postadministration only have those stimuli that are produced from 0- to 6-min postadministration. If the stimulus that is produced 6 rain after administration of ethanol at this low dose is, as Shippenberg and Altshuler 119) suggest, excitatory in nature and the stimuli inherent in ethanol 30-min postadministration are sedative, then the animals trained at 6-rain postadministration use the excitation as their sole cue. whereas the animals who are trained 30-rain postadministration can identify the earlier stimulatory phase as well as the later sedative phase when they are tested at various postadministratio n times. This is substantiated by the results in Fig. I which further indicate that the excitation phase is present earlier than the 6-min postadministration "excitatory phase" and it lasts for 15 rain with a rapid decrease at 30 min. In contrast, the "' 30-rain" animals who were trained to the earlier stimulatory, as welt as with the later sedative phase, can discriminate ethanol equally well at 6-rain postadministration and that this discrimination continues to last until the ethanol is dissipated in the blood/brain. ACKNOWLEDGEMENTS The author would like to express his gratitude to Ms. Denise McBurney for her continued expertise in the laboratory and to Dr. Edward Krimmer for his advice on the manuscript.
REFERENCES 1. Ando, K. Discriminative control of operant behavior by intravenous administration of drugs in rats. Psychopharmacologia 45:47-50; 1975. 2. Barry, H., III. Classifications of drugs according to their discriminable effects in rats. Fed. Proc. 33:1814-1824; 1974. 3. Barry, H., HI; Krimmer, E. C. Discriminable stimuli produced by alcohol and other CNS depressants. In: Lal, H., ed. Advances in behavioral biology: Discriminative stimulus properties of drugs. New York: Plenum Press; 1977:73-92. 4. Buckalew, L. W.; Cartwright, G. W. General and differential behavioral effects of five ethanol doses in the albino rat. Physiol. Rep. 23:1151-1154; 1968. 5. Bueno, O. F. A.; Carlini, E. A.; Finkelfarb, E.; Suzuki, J. S. A 9-tetrahydrocannabinol, ethanol, and amphetamine as discriminative stimulus generalization tests with other drugs. Psychopharmacologia 46:235-243; 1976. 6. Conger, J. J. The effects of alcohol on conflict behavior in the albino rat. Q. J. Stud. Alcohol 12:1-29; 1951. 7. Glennon, R. A.; Rosecrans, J. A. Speculations on the mechanism of action of hallucinogenic indolealkylamines. Neurosci. Behav. Rev.
5:197-207; 1981. 8. Krimmer, E. D. Drugs as discriminative stimuli. Ph.D. Dissertation. University of Pittsburgh. Dissert. Abstr. Internat. 35:4572-B. 19741975. 9. Kubena, R. K.; Barry, H., III. Generalization by rats of alcohol and atropine stimulus characteristics to other drugs. Psychoph~acologia i5:196-206; 1969. 10. Litehfield. J. T.; Wilcoxon, F. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96:99--1 !3; !949. 11. Overton, D. A: State-dependent l e ~ g produced by depressant and atropine-like drugs. Psyehopharmacologia 10:6-31; 1966. 12. Pohorecky, L. A. Biphasic action of ethanol, Biobehav. Rev. 1: 231-240; 1977. 13. Read, G. W.; Cutting, W.; Furst, A. Comparison of excited phases after sedatives and tranquilizers. PsyChoph~cology 1:346-350; 1960. 14. Schechter, M. D. Extended schedule transfer of ethanol diScrimina: tion. Pharmacol. Bioehem. Behav. 14:23,25; 1981. 15. Scbechter, M. D. Serotonergic-dopaminergic mediation of 3,4 meth-
T I M E - D E P E N D E N T E F F E C T OF E T H A N O L
16. 17. 18.
19.
ylenedioxymethamphetamine (MDMA; "Ecstasy"). Pharmacol. Biochem. Behav. 31:817-824; 1988. Schechter, M. D.; Signs, S. A.; Boja, J. W. Stability of the stimulus properties of drugs over time. Pharmacol. Biochem. Behav. 32: 361-364; 1989. Shippenberg, T. S.; Knappenberger, E.; Altshuler, H. L. The discriminative stimulus effects of ethanol and morphine: Stimulant versus sedative effects. Alcohol.: Clin. Exp. Res. 7:121; 1983. (Abstract.) Shippenberg, T. S.; Altshuler, H. L. The biphasic effects of ethanol: Role of endogenous opiate pathways. Alcohol.: Clin. Exp. Res. 8:119; 1984. (Abstract.) Shippenberg, T. S.; Altshuler, H. L. A drug discrimination analysis of ethanol-induced behavioral excitation and sedation: The role of endogenous opiate pathways. Alcohol 2:197-201; 1985.
449
20. Sjoberg, L. V. Alcohol and gambling. Psychopharmacologia 14: 284-298; 1969. 21. Smoothy, R.; Berry, M. S. Time-course of the locomotor stimulant and depressant effects of a single low dose of ethanol in mice. Psychopharmacology (Berlin) 85:57-61; 1985. 22. Stolerman, I. P.; D'Mello, G. D. Role of training conditions in discrimination of central nervous system stimulants in rats. Psychopharmacology (Berlin) 73:295-303; 1981. 23. Williams, A. F. Social drinking, anxiety and depression. J. Pers. Soc. Psychol. 3:689-693; 1966. 24. York, J. L. Comparison of the discriminative effects of ethanol, barbital and phenobarbital in rats. Psychopharmacology (Berlin) 60:19-23; 1978.