Alcohol, Vol. 9, pp. 117-122,1992
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Ethanol-Produced Interoceptive Stimuli Are Time Dependent in Selectively Bred HAS and LAS Rats M A R T I N D. S C H E C H T E R
Department o f Pharmacology, Northeastern Ohio Universities College o f Medicine, Rootstown, O H 44272 Received 16 M a y 1991; Accepted 30 A u g u s t 1991 SCHECHTER, M. D. Ethanol-produced interoceptive stimuli are time dependent in selectively bred HAS and LAS rats. ALCOHOL 9(2) 117-122, 1992.- Fourteenth generation high alcohol-sensitive (HAS) and low alcohol-sensitive (LAS) rats were trained to discriminate the effects of 600 mg/kg intraperitoneally administered ethanol from its vehicle at 6 and 30 min postadministration. Each of the earlier- and later-trained animals were given lower doses of ethanol and EDso values at their trained postadministration interval were found to be nonsignificantly different. Thus, there was no difference between HAS and LAS animals as to their sensitivity to the discriminative effects of ethanol. Phase-generalization studies, where rats trained at 6 min postadministration were tested with the drug at 30 rain postadministration were shown not to generalize, whereas the animals trained at 30 min postadministration and tested at 6 min postinjection were shown to readily discriminate the discriminative stimuli. This asymmetrical generalization lends evidence to the biphasic action of ethanol, and suggests that the earlier phase is quantitatively different than the latter phase. The similarity in sensitivity of the LAS and HAS animals, furthermore, suggests that the discrimination of ethanol is not based on its hypnotic effects. Ethanol
Drug discrimination
HAS
LAS
Biphasiceffects
A paradigm that is particularly well suited for assessing the subjective effects of drugs that act on the central nervous system is the drug discrimination procedure. Discriminative control of differential response has been observed to be the property of virtually every psychoactive drug tested to date, with ethanol being the first drug so employed (2). 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 on 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 chamber, 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°7o saline). The animal's differential response is made contingent on the interoceptive stimuli that it perceives after a particular dose (the training dose) of the drug at a specific postadministration time. The drug, at that time, functions as a discriminative stimulus or "cue" that signals the availability of reinforcement contingent on the occurrence of an appropriate choice response. The discriminability of a drug can be measured by
Rats
how many training sessions it takes before the animals learn to discriminate that particular drug, as well as the dose necessary to produce reliable discriminative performance. The drug-discrimination technique has been used to train rats to discriminate 1.0 g/kg intraperitoneally administered ethanol at two different postinjection intervals, i.e., at 6 and 30 min postinjection (16-18). These investigators reported that subsequent to ethanol training at the earlier time, which they call the excitatory phase, rats did not associate the drug state when tested at a 30-min postinjection interval (PI). Likewise, rats trained at the 30-rain PI (a group trained at what was termed the sedative phase) (17) could not accurately discriminate ethanol when tested at the 6-rain PI. These observations led to the suggestion that the interoceptive stimuli that formed the basis for the ethanol discriminative cue at 6-rain PI are qualitatively different from those that are utilized for differential responding at the 30-min PI. More recent work from this laboratory (14), using a lower training dose (600 mg/kg) of ethanol to train one group of animals at 6 min PI and a second group at 30 min PI, expanded on the Altshuler group's work in that each of the two groups received different doses of ethanol at the trained PI, and the EDso values between earlierand later-trained animals were shown not to be significantly
Requests for reprints should be addressed to Dr. Martin D. Schechter, Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272. 117
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different. More importantly, after "phase generalization" testing, i.e., testing the 6-rain PI-trained animals at 30 min and the 30-rain PI-trained animals at 6 rain, the rats trained at 30 rain maintained criterion discrimination when tested at either 6, 15, or 30 rain. In contrast, the rats trained at the 6-rain PI discriminated ethanol at a significantly reduced level when tested at the 30-rain PI. These results reiterated the suggestion that the nature of the discriminable stimuli produced by a low dose of ethanol are different at 6 and 30 rain postadministration. Furthermore, the animals trained at 30 min have the ability to discriminate the 6-min stimuli, because when trained at 30 min they experienced the full range of effects, i.e., they perceived the effects of ethanol, from the moment of injection through the beginning of the 30-min PI trial. In contrast, those animals who were trained at 6 rain and tested at 30 min had never experienced the interoceptive stimuli that exist 30 min after ethanol injection and, indeed, may only be able to discriminate the earlier (presumably excitatory) effects of ethanol. These cited studies were all conducted using heterogeneously bred Sprague-Dawley rats, i.e., rats neither chosen nor bred for different sensitivities to the effects of ethanol. One laboratory has selectively bred rats for differences in sensitivity to the sedative effects of ethanol (11,20) measured as the interval from loss of righting reflex to its recovery following.an hypnotic (3.0-3.6 g/kg) dose of ethanol. This became an accepted method for determining differences in ethanol sensitivity, and this measure has been successfully used to distinguish rats who have high alcohol sensitivity (HAS) from those who have a lesser sleeping time (LAS). The purpose of the present experiment was to use these HAS/LAS selectively bred rats to test their ability to discriminate 600 mg/kg ethanol at both an early PI of 6 rain and at a later PI (30 rain) in an effort to see if the learning rates or reactivity to lower doses of ethanol would differ. In addition, the effects of testing phase generalization of PIs in animals trained at these two times is investigated. METHOD
Subjects Twenty male rats were received from the Alcohol Research Center at the University of Colorado Health Science Center, which is a site where the selectively bred rats are developed as to their different phenotypes, i.e., low alcohol sensitivity (LAS) and high alcohol sensitivity (HAS). The animals constitute the fourteenth generation selectively bred from the original N/Nih heterogenous stock. On receipt, the animals were individually housed in suspended metal cages with wire floors and were allowed free access to commercial rat chow and water ad lib. The animals were kept in a vivarium facility with an ambient temperature of 20-220C on a 12-h (0600-1800) light/dark cycle. At arrival, the mean weight (+_ SD) of the LAS group was 235.4 (37.0) g and for the HAS group was 247.9 (39.3) g. Animals were kept at free-feeding weights to conduct a previously reported study (15) and then were reduced to, and maintained at, approximately 85070 of their free-feeding weights by daily rationing of commercial rat chow; this was done to motivate discriminative learning for food reward.
Apparatus Twelve standard rodent operant chambers (Lafayette Instruments Corp., Lafayette, IN), each containing two levers
situated 7 cm apart and 7 cm above a metal grid floor, were used as the environmental space. Equidistance between the levers was placed a food receptacle that delivered 45-rag Noyes food pellets. Each operant chamber was enclosed in a soundattenuated cubicle with an exhaust fan and a 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.
Shaping to Lever-Press Each of the food-deprived rats was trained to respond on one of two levers (randomly selected) in the first training session by employing "autoshaping," i.e., the rat was placed into the operant chamber and allowed access to the levers until it pressed one lever. Once the rat had pressed on a CRF (continuous reinforcement schedule; FRI), it was placed, on the next day's training session, on an FR1 schedule on that lever at either 3 or 27 min after the intraperitoneal (i.p.) administration of a constant volume (6 ml/kg) of distilled water (vehicle). The entire training period for both the "6-min rats" and the "30-rain rats" was 6 min in duration such that the former group was placed into the operant chamber at 3 min postinjection and removed at the tenth min, whereas the latter group was placed at 27 min postinjection and removed at the thirtyfourth min. The fixed ratio (FR) schedule was made gradually more difficult, i.e., increasing from an FRI to an FRI0 schedule, with each increment triggered when an animal, in its daily training session, received a minimum of 20 reinforcements according to the FR schedule of that day. Thus, e.g., when a rat pressed the correct lever 60 times while on the FR3 schedule, the next daily session was a FR4 schedule which, in turn, required 80 responses prior to increasing to an FR5 schedule, and so on. Once any individual rat attained the first session on the FRI0 schedule, that rat was given two additional sessions after vehicle administration on the FRI0 schedule of reinforcement. It was, subsequently, placed into its home cage without any further training upon the first lever. The animal's weight was, however, continued at approximately 85°70 of its free-feeding weight during this period. Those rats that did not rapidly attain the FRI0 schedule on the first lever were continued in their training until all animals reached this reinforcement schedule. At that time, i.e., when the last rat reached the FR10 schedule for the third time, all rats (including those who had been in their home cage without continued training) were given three sessions each on the FR 10 schedule. On the next training day, the first lever was deactivated and the second lever activated on an FRI schedule. The "6-min rats" and the "30-min rats" were once again trained beginning at either 3 or 27 min postinjection of a similar volume of vehicle. On that initial session, only presses on the second lever resulted in reinforcement on the FR1 schedule, whereas presses on the previously activated (first) lever produced no programmed consequence. The animals were trained daily, using the same 20 reinforcement criterion prior to incrementation of the FR schedule, until all rats attained the FRI0 schedule on the second lever. Those animals that attained/performed the FR10 schedule (for two sessions) more rapidly than others were returned to their home cage without any continued training so as to ensure that all rats had exactly five FR10 training sessions on the second lever. Once again, when all animals had attained that criterion, the next phase of discrimination training was begun. After the FRI0 schedule was attained by all rats on the
ETHANOL DISCRIMINATION IN HAS/LAS RATS second lever, the drug (ethanol) condition was introduced to the training regimen. An equal volume of ethanol (10070 w/v) yielding a dose of 600 mg/kg i.p. was administered and, at either 3 or 27 min postinjection, the rat was placed into the operant chamber and required to depress the second lever ten times in order to receive reinforcement. This FRI0 schedule after ethanol administration on the second-trained lever, was continued for five consecutive dally sessions.
Drug Discrimination Training The next phase constituted the drug-discrimination training and employed the biweekly, repeating schedule of V, D, D, V, V; D, V, V, D, D; where D = 600 mg/kg ethanol and V = an equal volume of vehicle (distilled water). The vehiclecorrect lever was, in reality, the animals first trained lever, whereas the ethanol-correct lever was the second lever conditioned to be pressed on the FR10 schedule. During drug-discrimination training, the rats were administered either vehicle or ethanol once per day and placed into the operant chamber for a 3-min period, commencing at either 6 or 30 min postadministration. Thus, the early group was removed after 9 min postinjection, whereas the later group were removed after 33 min postadministration of either ethanol or vehicle. Training was continued using the biweekly administration/training schedule until all rats attained the criterion of eight first choice correct responses (state-appropriate lever pressed ten times first) in ten consecutive daily trials. This first 8/10 criterion was called "session-to-criterion 1" (STC #1). To ensure accurate discriminative performance, a second 8/10 STC was required (STC #2).
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 from each of the two groups were tested at the PI used in their training after administration of lower (300 and 450 mg/
119 kg) doses of ethanol. Each dose was tested twice, once following an ethanol maintenance session and once following a vehicle maintenance session. This counterbalancing controlled possible residual influence from the previous day's maintenance session. The rat was removed immediately following ten presses on either lever without receiving reinforcement. If at any time during testing, a rat's discrimination on maintenance sessions fell below the 80070 criterion, data on that animal was dropped from the results. In addition, if an animal did not choose a lever by pressing it ten times in the restricted (3 min) time period, the results on that animal were not used. These factors, as well as the death of two animals for reasons unrelated to the experimentation, decreased the "N" of the 6-min PI group to three for each of the LAS and HAS animals.
Phase-Generalization Testing Discrimination of the training dose and the two lower doses of 300 and 450 mg/kg of ethanol, as well as the vehicle control, was tested at the novel time of 30 min in rats trained to discriminate 600 mg/kg ethanol at the 6-min PI. Likewise, each treatment was tested at 6 rain postinjection in those animals trained to discriminate between ethanol and its vehicle at the 30-min PI. Each novel time point was tested on two occasions, once following an ethanol and once following a vehicle maintenance session at the appropriate PI used to train the particular group.
Measurements and Statistics 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 preference prior to reinforcement. The quantal measurement is the percentage of animals that select the ethanolappropriate lever as their "selected" lever, i.e., the lever to first accumulate ten presses. The quantitative measurement is the number of responses on the ethanol lever divided by the
TABLE 1 DISCRIMINATIVEPERFORMANCEOF LAS AND HAS RATSTRAINEDAT 6 MIN POSTINJECTION OF 600 mg/kg ETHANOLAND TESTEDWITHLOWERDOSESOF ETHANOLAT 6 AND 30 MIN Dose Response EtOH (mg/kg)
at 6 min
e/n/N*
Quantal
at 30min Quant.
e/n/N
LAS (n = 3). STC(SD)--#1, 11.0 (+ 11.5), range 2-24; #2, 21.3 (+ 11.4), range 12-34 600 15/16/18 93.8 80.7 2/6/6 450 3/5/6 60.0 53.6 0/5/6 300 1/3/6 33.3 47.4 1/6/6 0(veh.) 2/15/18 13.3 19.4 0/6/6 EDs0 367.2 (95°70 CL) (258.0-522.7) HAS (n = 3). STC (SD)-#1, 6.0 (+ 4.6), range 1-10; #2, 17.0 (+ 3.6), range 13-20 600 17/21/24 81.0 66.4 2/6/6 450 4/6/6 66.7 55.9 2/6/6 300 2/6/6 33.3 39.5 1/5/6 0(veh.) 0/24/24 0.0 2.0 0/6/6 ED~o 371.0 (96°70CL) (288.3-477.5)
Quantal
Quant.
33.3 0.0 16.7 0.0
40.2 12.2 40.9 3.2
33.3 33.3 20.0 0.0
42.2 40.2 21.2 0.0
*Number of first choices (10 times pressed first) ethanol-lever selection/number of rats responding within 3-min time period allowed/number of experiments conducted.
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total number of responses on both the ethanol and the vehicle lever at the time that ten 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 measure of the magnitude, as well as the direction, of lever preference. The LitchfieldWilcoxon procedure (8), which employs probits versus log dose effects, was used to generate EDs0 values from the ethanol quantal dose-response data in each of the PI groups, when available. A computer-based program of this procedure was used (22). RESULTS
The learning rates, as indicated by sessions-to-criterion (STC #1 and #2), as well as the results of dose-response and phase generalization experiments in both LAS and HAS rats trained at the 6-min PI, are represented as Table 1. Referring to the STCs, it appears that the HAS animals learned the discrimination between 600 mg/kg ethanol and its vehicle at a slightly faster rate than did the LAS rats; however, this difference was not statistically significant. The LAS animals trained at the 6-min PI with 600 mg/kg ethanol were seen to respond in maintenance sessions, within the 3-min time limit, on 16 of the 18 trials and, on 15 of those 16 they chose the ethanolappropriate lever ( " e / n / N " of Table 1). In vehicle maintenance sessions, they responded, within 3 min, on 15 of 18 trials and chose the ethanol-appropriate lever on two of those trials (i.e., they chose the vehicle-appropriate lever on 13 of 15 trials in which a lever accumulated ten presses in 3 min). The decreasing doses of ethanol produced a progressive decrease in discriminative performance, and when the ethanol doses were subjected to analysis (8) the EDs0 value was 367.2 mg/kg ethanol. Similar results were seen (lower part of Table 1) with the HAS rats' dose-response relationship and an almost identical calculated EDs0 value of 371.0 mg/kg. When the 6-min PI LAS or HAS rats were tested, on two occasions each, with either the training dose or lower doses of ethanol or vehicle, there was a lack of generalization, i.e., the rats trained at the 6-min PI correctly discriminated ethanol when
tested at the 30-min PI on only 0-33.3% of the trials in which they responded. Table 2 represents the same measurements in LAS and HAS rats trained at the 30-min PI, tested with various doses of ethanol at that PI and at 6 min postadministration. The STCs for both the LAS and HAS animals were significantly (p < 0.05) higher than those shown for the LAS and HAS animals trained at the 6-rain PI. For both groups, administration and testing of decreased doses of ethanol produced decreasing discriminative performance with the EDs0 value at the 30-rain PI of 416.4 mg/kg for the LAS rats and a very similar ED~0 value for each of the two groups of animals. The EDs0 value of 409.0 mg/kg for the HAS animals. When each of the two groups of animals were tested on two occasions each with 0.0 (vehicle), 300, 450, and 600 mg/kg ethanol at 6 min postadministration, there was a generalization of ethanol discrimination; this allowed for generation of EDs0 value for ethanol at the 6-min PI in the LAS animals trained at 30 min was 396.3 mg/kg, whereas for the HAS animals it was 367.8 mg/kg; not significantly different (22) from each other or from any of the other cited EDs0 values. DISCUSSION
Various measurements can be taken to determine how discriminable a drug is and how sensitive animals are to that drug. One way to quantify discriminability is to determine the speed of acquisition of the drug versus vehicle discrimination. This is readily accomplished by measuring the number of training sessions given prior to the beginning of criterion performance, when criterion is defined as the number of sessions needed out of the number of consecutive sessions in which a correct choice must be made, e.g., eight correct first lever selections made in ten consecutive daily sessions. This has been called sessions-to-criterion (STC; 10). Another indication of the effectiveness of a drug's capability of controlling differential responding is to determine its dose-effect relationship. Thus, subsequent to criterion training, decreasing doses of the training drug can be administered, and in test sessions (i.e., sessions conducted under conditions of extinction) the ani-
TABLE 2 DISCRIMINATIVEPERFORMANCEOF LAS AND HAS RATSTRAINEDAT 30 MIN POSTINJECTION OF 600 mg/kg ETHANOLAND TESTEDWITH LOWERDOSESAT 30 AND 6 MIN at 30 min Dose Response EtOH (mg/kg)
e/n/N
Quantal
LAS (n = 5). STC (SD)-#1, 38.2 (_+ 22.6), range 9-61; 600 9/10/20 90.0 450 6/9/10 66.7 300 1/10/10 10.0 0(veh.) 2/15/15 13.3 EDs0 416.4 (95% CL) (325.6-532.5)
at 6 min Quant.
e/n/N
#2, 51.0 (+ 20.2), range 27-71 78.3 7/9/10 61.1 4/10/10 20.2 3/7/10 15.9 1/9/10
HAS (n = 5). STC (SD)-# 1, 27.4 (+_ 20.6), range 11-59; #2, 46.8 (+ 18.1), range 26-69 600 12/14/20 85.7 77.8 9/9/10 450 5/9/10 55.6 51.5 6/10/10 300 2/9/10 22.2 25.0 3/9/10 0(veh.) 2/13/15 15.4 16.8 0/9/10 EDs0 (95% CL)
409.0 (319.1-524.2)
Quantal
Quant.
77.8 40.0 42.9 11.1 396.3 (258.2-608.3)
70.9 51.6 47.7 21.6
100.0 60.0 33.3 0.0
71.4 62.0 44.6 12.6
367.8 (291.3-464.3)
ETHANOL DISCRIMINATION IN HAS/LAS RATS mals' discriminative performance can be determined. Generally, as the dose of the training drug decreases, so does responding on the drug-appropriate lever; the EDso value can then be determined. Thus, sessions-to-criterion and doseresponsiveness have been frequently studied to describe the strength of the discriminability of a drug. In contrast, the role of the interval between the administration of the drug and the start of the training and/or test session (here called the "postadministration interval" or PI) has been less fully studied (21) and drugs may have different profiles of effect at different times after they are administered. This possibility may be an important consideration when drugs are observed to possess biphasic action over time. Ethanol is such a drug, as it has been reported to produce a transient state of excitation prior to the onset of its depressant effects, as documented both in animals (12) and humans (19,23). The present study used the fourteenth generation of rats bred for differential sensitivity to an hypnotic dose of ethanol in an effort to see if the selective breeding for this action of ethanol carried over to their ability to discriminate the interoceptive stimuli produced by ethanol at two different postadministration intervals. The results indicated that neither the discrimination learning rate (as indicated by sessions-to-criterion) nor the sensitivity to ethanol (as indicated by the EDs0 values generated by the dose-response experiments) differed between the LAS and HAS rats. This confirms previous work (6) using the tenth generation of these animals and the same training dose of ethanol but a different schedule of reinforcement, viz., a variable ratio (VR5) schedule. In that earlier reported dose-response experimentation, EDs0 values did not differ between these two groups of rats after extensive training/testing (50-60 sessions). Because there were no differences between the HAS and LAS animals at either of the two postadministration intervals, the remainder of the discussion focuses on the differences in both groups at each of these two times, as well as to the phase-generalization results. It appears that the animals trained at the 30-min PI required a significantly greater number of training sessions to attain the discriminative performance criterion. The most parsimonious explanation for this observation is one of pharmacokinetics, in that the amount of ethanol present in the blood/ brain appears to peak at 7.5 min after i.p. administration of a similar dose of ethanol with a decrease at the 30 min PI (5,7). It has been suggested that there is a different relationship between the dose administered and subsequent brain concentration and the stimulus strength of a drug (9). Thus, the average number of training sessions to establish discriminative criteria decreases with increasing training doses and, therefore, with increasing amounts of ethanol to the brain (1). Previous work in this laboratory (13) using the same dose of ethanol and a more usual PI of 15 min indicated an average of 37 training sessions to attain the same discriminative criterion. This STC value is closer to that seen in the animals trained at the 30-min PI, and indicated that the animals trained at the earlier PI learned the discrimination at a faster rate. In fact,
121 when rats were trained to discriminate 1.2 g/kg ethanol at a PI of 15 min, their discriminative performance was maximal when they were tested at a 7.5-min PI, and fell 30% when they were tested at 30 min postinjection (5). Although the present study used selectively bred animals, another study, designed to correlate ethanol sensitivity and discriminative performance, separated female Wistar rats as to their drinking behavior with 6% w/v solution of ethanol over a 4-week period (24). It was established that one group, designated ethanol-preferring, derived approximately 60% of its total fluid from ethanol, whereas a second group, designated ethanol nonpreferring, derived approximately 200/0 from alcohol. When each of these groups were trained to discriminate the effects of 780 mg/kg ethanol i.p. (with a 20-min PI) in a shock-motivated T-maze apparatus, the two groups were shown to learn the discrimination at the same rate. Subsequent work by the same investigator (25) employed the selectively bred lines of alcohol-accepting (AA) and alcohol nonaccepting (ANA) rats (3) and trained these two groups to discriminate 1.0 g/kg ethanol. The nonpreferring rats were reported to achieve discrimination criterion performance faster than the alcohol-accepting animals. This suggested that the animals with low ethanol drinking preference, i.e., the ANA group, may have a greater sensitivity for the perception of the ethanol discriminative cue. The present work, using a different line of selectively bred rats, does not confirm this experiment in that it suggests drug discrimination is not based on the depressant effects of ethanol. Nevertheless, it must be kept in mind that, although the A A / A N A rats were bred for ethanol preference, the present rats were bred for difference in sensitivity to ethanol's hypnotic effects, and previous work has indicated that rats that prefer alcohol are less sensitive to the hypnotic and depressive effects than are those animals who do not prefer alcohol (4). The phase generalization results confirm an earlier report (14) that indicated asymmetrical generalization in heterogeneously bred rats, using only the training dose of 600 mg/kg. In the present study, doses of 300, 450, and 600 mg/kg ethanol were employed in the phase-generalization study and the different effects of ethanol at the 6- and 30-min PI were observed once again. Thus, it appears that the earlier ethanol discriminative stimuli differ from those stimuli produced when rats are trained at the latter PI. The possibility exists that the biphasic action of ethanol may have an earlier excitatory, reinforcing phase as the blood alcohol concentrations rise and a later depressive (both on locomotor activity and neurobehavioral reward) stage as the blood levels fall (7). ACKNOWLEDGEMENTS I would like to express my gratitude to Drs. Richard A. Deitrich and Laura J. Draski for supplying the LAS/HAS rats; to Denise McBurney for her continuing technical expertise; to Marty Hilgert and Karen Kelley for word processing; to Dr. Edward C. Krimmer for his advice on this manuscript. This research was funded entirely by NIAAA Grant #8598.
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Ethanol-Produced Interoceptive Stimuli Are Time Dependent in Selectively Bred HAS and LAS Rats M A R T I N D. S C H E C H T E R
Department o f Pharmacology, Northeastern Ohio Universities College o f Medicine, Rootstown, O H 44272 Received 16 M a y 1991; Accepted 30 A u g u s t 1991 SCHECHTER, M. D. Ethanol-produced interoceptive stimuli are time dependent in selectively bred HAS and LAS rats. ALCOHOL 9(2) 117-122, 1992.- Fourteenth generation high alcohol-sensitive (HAS) and low alcohol-sensitive (LAS) rats were trained to discriminate the effects of 600 mg/kg intraperitoneally administered ethanol from its vehicle at 6 and 30 min postadministration. Each of the earlier- and later-trained animals were given lower doses of ethanol and EDso values at their trained postadministration interval were found to be nonsignificantly different. Thus, there was no difference between HAS and LAS animals as to their sensitivity to the discriminative effects of ethanol. Phase-generalization studies, where rats trained at 6 min postadministration were tested with the drug at 30 rain postadministration were shown not to generalize, whereas the animals trained at 30 min postadministration and tested at 6 min postinjection were shown to readily discriminate the discriminative stimuli. This asymmetrical generalization lends evidence to the biphasic action of ethanol, and suggests that the earlier phase is quantitatively different than the latter phase. The similarity in sensitivity of the LAS and HAS animals, furthermore, suggests that the discrimination of ethanol is not based on its hypnotic effects. Ethanol
Drug discrimination
HAS
LAS
Biphasiceffects
A paradigm that is particularly well suited for assessing the subjective effects of drugs that act on the central nervous system is the drug discrimination procedure. Discriminative control of differential response has been observed to be the property of virtually every psychoactive drug tested to date, with ethanol being the first drug so employed (2). 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 on 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 chamber, 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°7o saline). The animal's differential response is made contingent on the interoceptive stimuli that it perceives after a particular dose (the training dose) of the drug at a specific postadministration time. The drug, at that time, functions as a discriminative stimulus or "cue" that signals the availability of reinforcement contingent on the occurrence of an appropriate choice response. The discriminability of a drug can be measured by
Rats
how many training sessions it takes before the animals learn to discriminate that particular drug, as well as the dose necessary to produce reliable discriminative performance. The drug-discrimination technique has been used to train rats to discriminate 1.0 g/kg intraperitoneally administered ethanol at two different postinjection intervals, i.e., at 6 and 30 min postinjection (16-18). These investigators reported that subsequent to ethanol training at the earlier time, which they call the excitatory phase, rats did not associate the drug state when tested at a 30-min postinjection interval (PI). Likewise, rats trained at the 30-rain PI (a group trained at what was termed the sedative phase) (17) could not accurately discriminate ethanol when tested at the 6-rain PI. These observations led to the suggestion that the interoceptive stimuli that formed the basis for the ethanol discriminative cue at 6-rain PI are qualitatively different from those that are utilized for differential responding at the 30-min PI. More recent work from this laboratory (14), using a lower training dose (600 mg/kg) of ethanol to train one group of animals at 6 min PI and a second group at 30 min PI, expanded on the Altshuler group's work in that each of the two groups received different doses of ethanol at the trained PI, and the EDso values between earlierand later-trained animals were shown not to be significantly
Requests for reprints should be addressed to Dr. Martin D. Schechter, Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272. 117
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different. More importantly, after "phase generalization" testing, i.e., testing the 6-rain PI-trained animals at 30 min and the 30-rain PI-trained animals at 6 rain, the rats trained at 30 rain maintained criterion discrimination when tested at either 6, 15, or 30 rain. In contrast, the rats trained at the 6-rain PI discriminated ethanol at a significantly reduced level when tested at the 30-rain PI. These results reiterated the suggestion that the nature of the discriminable stimuli produced by a low dose of ethanol are different at 6 and 30 rain postadministration. Furthermore, the animals trained at 30 min have the ability to discriminate the 6-min stimuli, because when trained at 30 min they experienced the full range of effects, i.e., they perceived the effects of ethanol, from the moment of injection through the beginning of the 30-min PI trial. In contrast, those animals who were trained at 6 rain and tested at 30 min had never experienced the interoceptive stimuli that exist 30 min after ethanol injection and, indeed, may only be able to discriminate the earlier (presumably excitatory) effects of ethanol. These cited studies were all conducted using heterogeneously bred Sprague-Dawley rats, i.e., rats neither chosen nor bred for different sensitivities to the effects of ethanol. One laboratory has selectively bred rats for differences in sensitivity to the sedative effects of ethanol (11,20) measured as the interval from loss of righting reflex to its recovery following.an hypnotic (3.0-3.6 g/kg) dose of ethanol. This became an accepted method for determining differences in ethanol sensitivity, and this measure has been successfully used to distinguish rats who have high alcohol sensitivity (HAS) from those who have a lesser sleeping time (LAS). The purpose of the present experiment was to use these HAS/LAS selectively bred rats to test their ability to discriminate 600 mg/kg ethanol at both an early PI of 6 rain and at a later PI (30 rain) in an effort to see if the learning rates or reactivity to lower doses of ethanol would differ. In addition, the effects of testing phase generalization of PIs in animals trained at these two times is investigated. METHOD
Subjects Twenty male rats were received from the Alcohol Research Center at the University of Colorado Health Science Center, which is a site where the selectively bred rats are developed as to their different phenotypes, i.e., low alcohol sensitivity (LAS) and high alcohol sensitivity (HAS). The animals constitute the fourteenth generation selectively bred from the original N/Nih heterogenous stock. On receipt, the animals were individually housed in suspended metal cages with wire floors and were allowed free access to commercial rat chow and water ad lib. The animals were kept in a vivarium facility with an ambient temperature of 20-220C on a 12-h (0600-1800) light/dark cycle. At arrival, the mean weight (+_ SD) of the LAS group was 235.4 (37.0) g and for the HAS group was 247.9 (39.3) g. Animals were kept at free-feeding weights to conduct a previously reported study (15) and then were reduced to, and maintained at, approximately 85070 of their free-feeding weights by daily rationing of commercial rat chow; this was done to motivate discriminative learning for food reward.
Apparatus Twelve standard rodent operant chambers (Lafayette Instruments Corp., Lafayette, IN), each containing two levers
situated 7 cm apart and 7 cm above a metal grid floor, were used as the environmental space. Equidistance between the levers was placed a food receptacle that delivered 45-rag Noyes food pellets. Each operant chamber was enclosed in a soundattenuated cubicle with an exhaust fan and a 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.
Shaping to Lever-Press Each of the food-deprived rats was trained to respond on one of two levers (randomly selected) in the first training session by employing "autoshaping," i.e., the rat was placed into the operant chamber and allowed access to the levers until it pressed one lever. Once the rat had pressed on a CRF (continuous reinforcement schedule; FRI), it was placed, on the next day's training session, on an FR1 schedule on that lever at either 3 or 27 min after the intraperitoneal (i.p.) administration of a constant volume (6 ml/kg) of distilled water (vehicle). The entire training period for both the "6-min rats" and the "30-rain rats" was 6 min in duration such that the former group was placed into the operant chamber at 3 min postinjection and removed at the tenth min, whereas the latter group was placed at 27 min postinjection and removed at the thirtyfourth min. The fixed ratio (FR) schedule was made gradually more difficult, i.e., increasing from an FRI to an FRI0 schedule, with each increment triggered when an animal, in its daily training session, received a minimum of 20 reinforcements according to the FR schedule of that day. Thus, e.g., when a rat pressed the correct lever 60 times while on the FR3 schedule, the next daily session was a FR4 schedule which, in turn, required 80 responses prior to increasing to an FR5 schedule, and so on. Once any individual rat attained the first session on the FRI0 schedule, that rat was given two additional sessions after vehicle administration on the FRI0 schedule of reinforcement. It was, subsequently, placed into its home cage without any further training upon the first lever. The animal's weight was, however, continued at approximately 85°70 of its free-feeding weight during this period. Those rats that did not rapidly attain the FRI0 schedule on the first lever were continued in their training until all animals reached this reinforcement schedule. At that time, i.e., when the last rat reached the FR10 schedule for the third time, all rats (including those who had been in their home cage without continued training) were given three sessions each on the FR 10 schedule. On the next training day, the first lever was deactivated and the second lever activated on an FRI schedule. The "6-min rats" and the "30-min rats" were once again trained beginning at either 3 or 27 min postinjection of a similar volume of vehicle. On that initial session, only presses on the second lever resulted in reinforcement on the FR1 schedule, whereas presses on the previously activated (first) lever produced no programmed consequence. The animals were trained daily, using the same 20 reinforcement criterion prior to incrementation of the FR schedule, until all rats attained the FRI0 schedule on the second lever. Those animals that attained/performed the FR10 schedule (for two sessions) more rapidly than others were returned to their home cage without any continued training so as to ensure that all rats had exactly five FR10 training sessions on the second lever. Once again, when all animals had attained that criterion, the next phase of discrimination training was begun. After the FRI0 schedule was attained by all rats on the
ETHANOL DISCRIMINATION IN HAS/LAS RATS second lever, the drug (ethanol) condition was introduced to the training regimen. An equal volume of ethanol (10070 w/v) yielding a dose of 600 mg/kg i.p. was administered and, at either 3 or 27 min postinjection, the rat was placed into the operant chamber and required to depress the second lever ten times in order to receive reinforcement. This FRI0 schedule after ethanol administration on the second-trained lever, was continued for five consecutive dally sessions.
Drug Discrimination Training The next phase constituted the drug-discrimination training and employed the biweekly, repeating schedule of V, D, D, V, V; D, V, V, D, D; where D = 600 mg/kg ethanol and V = an equal volume of vehicle (distilled water). The vehiclecorrect lever was, in reality, the animals first trained lever, whereas the ethanol-correct lever was the second lever conditioned to be pressed on the FR10 schedule. During drug-discrimination training, the rats were administered either vehicle or ethanol once per day and placed into the operant chamber for a 3-min period, commencing at either 6 or 30 min postadministration. Thus, the early group was removed after 9 min postinjection, whereas the later group were removed after 33 min postadministration of either ethanol or vehicle. Training was continued using the biweekly administration/training schedule until all rats attained the criterion of eight first choice correct responses (state-appropriate lever pressed ten times first) in ten consecutive daily trials. This first 8/10 criterion was called "session-to-criterion 1" (STC #1). To ensure accurate discriminative performance, a second 8/10 STC was required (STC #2).
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 from each of the two groups were tested at the PI used in their training after administration of lower (300 and 450 mg/
119 kg) doses of ethanol. Each dose was tested twice, once following an ethanol maintenance session and once following a vehicle maintenance session. This counterbalancing controlled possible residual influence from the previous day's maintenance session. The rat was removed immediately following ten presses on either lever without receiving reinforcement. If at any time during testing, a rat's discrimination on maintenance sessions fell below the 80070 criterion, data on that animal was dropped from the results. In addition, if an animal did not choose a lever by pressing it ten times in the restricted (3 min) time period, the results on that animal were not used. These factors, as well as the death of two animals for reasons unrelated to the experimentation, decreased the "N" of the 6-min PI group to three for each of the LAS and HAS animals.
Phase-Generalization Testing Discrimination of the training dose and the two lower doses of 300 and 450 mg/kg of ethanol, as well as the vehicle control, was tested at the novel time of 30 min in rats trained to discriminate 600 mg/kg ethanol at the 6-min PI. Likewise, each treatment was tested at 6 rain postinjection in those animals trained to discriminate between ethanol and its vehicle at the 30-min PI. Each novel time point was tested on two occasions, once following an ethanol and once following a vehicle maintenance session at the appropriate PI used to train the particular group.
Measurements and Statistics 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 preference prior to reinforcement. The quantal measurement is the percentage of animals that select the ethanolappropriate lever as their "selected" lever, i.e., the lever to first accumulate ten presses. The quantitative measurement is the number of responses on the ethanol lever divided by the
TABLE 1 DISCRIMINATIVEPERFORMANCEOF LAS AND HAS RATSTRAINEDAT 6 MIN POSTINJECTION OF 600 mg/kg ETHANOLAND TESTEDWITHLOWERDOSESOF ETHANOLAT 6 AND 30 MIN Dose Response EtOH (mg/kg)
at 6 min
e/n/N*
Quantal
at 30min Quant.
e/n/N
LAS (n = 3). STC(SD)--#1, 11.0 (+ 11.5), range 2-24; #2, 21.3 (+ 11.4), range 12-34 600 15/16/18 93.8 80.7 2/6/6 450 3/5/6 60.0 53.6 0/5/6 300 1/3/6 33.3 47.4 1/6/6 0(veh.) 2/15/18 13.3 19.4 0/6/6 EDs0 367.2 (95°70 CL) (258.0-522.7) HAS (n = 3). STC (SD)-#1, 6.0 (+ 4.6), range 1-10; #2, 17.0 (+ 3.6), range 13-20 600 17/21/24 81.0 66.4 2/6/6 450 4/6/6 66.7 55.9 2/6/6 300 2/6/6 33.3 39.5 1/5/6 0(veh.) 0/24/24 0.0 2.0 0/6/6 ED~o 371.0 (96°70CL) (288.3-477.5)
Quantal
Quant.
33.3 0.0 16.7 0.0
40.2 12.2 40.9 3.2
33.3 33.3 20.0 0.0
42.2 40.2 21.2 0.0
*Number of first choices (10 times pressed first) ethanol-lever selection/number of rats responding within 3-min time period allowed/number of experiments conducted.
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total number of responses on both the ethanol and the vehicle lever at the time that ten 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 measure of the magnitude, as well as the direction, of lever preference. The LitchfieldWilcoxon procedure (8), which employs probits versus log dose effects, was used to generate EDs0 values from the ethanol quantal dose-response data in each of the PI groups, when available. A computer-based program of this procedure was used (22). RESULTS
The learning rates, as indicated by sessions-to-criterion (STC #1 and #2), as well as the results of dose-response and phase generalization experiments in both LAS and HAS rats trained at the 6-min PI, are represented as Table 1. Referring to the STCs, it appears that the HAS animals learned the discrimination between 600 mg/kg ethanol and its vehicle at a slightly faster rate than did the LAS rats; however, this difference was not statistically significant. The LAS animals trained at the 6-min PI with 600 mg/kg ethanol were seen to respond in maintenance sessions, within the 3-min time limit, on 16 of the 18 trials and, on 15 of those 16 they chose the ethanolappropriate lever ( " e / n / N " of Table 1). In vehicle maintenance sessions, they responded, within 3 min, on 15 of 18 trials and chose the ethanol-appropriate lever on two of those trials (i.e., they chose the vehicle-appropriate lever on 13 of 15 trials in which a lever accumulated ten presses in 3 min). The decreasing doses of ethanol produced a progressive decrease in discriminative performance, and when the ethanol doses were subjected to analysis (8) the EDs0 value was 367.2 mg/kg ethanol. Similar results were seen (lower part of Table 1) with the HAS rats' dose-response relationship and an almost identical calculated EDs0 value of 371.0 mg/kg. When the 6-min PI LAS or HAS rats were tested, on two occasions each, with either the training dose or lower doses of ethanol or vehicle, there was a lack of generalization, i.e., the rats trained at the 6-min PI correctly discriminated ethanol when
tested at the 30-min PI on only 0-33.3% of the trials in which they responded. Table 2 represents the same measurements in LAS and HAS rats trained at the 30-min PI, tested with various doses of ethanol at that PI and at 6 min postadministration. The STCs for both the LAS and HAS animals were significantly (p < 0.05) higher than those shown for the LAS and HAS animals trained at the 6-rain PI. For both groups, administration and testing of decreased doses of ethanol produced decreasing discriminative performance with the EDs0 value at the 30-rain PI of 416.4 mg/kg for the LAS rats and a very similar ED~0 value for each of the two groups of animals. The EDs0 value of 409.0 mg/kg for the HAS animals. When each of the two groups of animals were tested on two occasions each with 0.0 (vehicle), 300, 450, and 600 mg/kg ethanol at 6 min postadministration, there was a generalization of ethanol discrimination; this allowed for generation of EDs0 value for ethanol at the 6-min PI in the LAS animals trained at 30 min was 396.3 mg/kg, whereas for the HAS animals it was 367.8 mg/kg; not significantly different (22) from each other or from any of the other cited EDs0 values. DISCUSSION
Various measurements can be taken to determine how discriminable a drug is and how sensitive animals are to that drug. One way to quantify discriminability is to determine the speed of acquisition of the drug versus vehicle discrimination. This is readily accomplished by measuring the number of training sessions given prior to the beginning of criterion performance, when criterion is defined as the number of sessions needed out of the number of consecutive sessions in which a correct choice must be made, e.g., eight correct first lever selections made in ten consecutive daily sessions. This has been called sessions-to-criterion (STC; 10). Another indication of the effectiveness of a drug's capability of controlling differential responding is to determine its dose-effect relationship. Thus, subsequent to criterion training, decreasing doses of the training drug can be administered, and in test sessions (i.e., sessions conducted under conditions of extinction) the ani-
TABLE 2 DISCRIMINATIVEPERFORMANCEOF LAS AND HAS RATSTRAINEDAT 30 MIN POSTINJECTION OF 600 mg/kg ETHANOLAND TESTEDWITH LOWERDOSESAT 30 AND 6 MIN at 30 min Dose Response EtOH (mg/kg)
e/n/N
Quantal
LAS (n = 5). STC (SD)-#1, 38.2 (_+ 22.6), range 9-61; 600 9/10/20 90.0 450 6/9/10 66.7 300 1/10/10 10.0 0(veh.) 2/15/15 13.3 EDs0 416.4 (95% CL) (325.6-532.5)
at 6 min Quant.
e/n/N
#2, 51.0 (+ 20.2), range 27-71 78.3 7/9/10 61.1 4/10/10 20.2 3/7/10 15.9 1/9/10
HAS (n = 5). STC (SD)-# 1, 27.4 (+_ 20.6), range 11-59; #2, 46.8 (+ 18.1), range 26-69 600 12/14/20 85.7 77.8 9/9/10 450 5/9/10 55.6 51.5 6/10/10 300 2/9/10 22.2 25.0 3/9/10 0(veh.) 2/13/15 15.4 16.8 0/9/10 EDs0 (95% CL)
409.0 (319.1-524.2)
Quantal
Quant.
77.8 40.0 42.9 11.1 396.3 (258.2-608.3)
70.9 51.6 47.7 21.6
100.0 60.0 33.3 0.0
71.4 62.0 44.6 12.6
367.8 (291.3-464.3)
ETHANOL DISCRIMINATION IN HAS/LAS RATS mals' discriminative performance can be determined. Generally, as the dose of the training drug decreases, so does responding on the drug-appropriate lever; the EDso value can then be determined. Thus, sessions-to-criterion and doseresponsiveness have been frequently studied to describe the strength of the discriminability of a drug. In contrast, the role of the interval between the administration of the drug and the start of the training and/or test session (here called the "postadministration interval" or PI) has been less fully studied (21) and drugs may have different profiles of effect at different times after they are administered. This possibility may be an important consideration when drugs are observed to possess biphasic action over time. Ethanol is such a drug, as it has been reported to produce a transient state of excitation prior to the onset of its depressant effects, as documented both in animals (12) and humans (19,23). The present study used the fourteenth generation of rats bred for differential sensitivity to an hypnotic dose of ethanol in an effort to see if the selective breeding for this action of ethanol carried over to their ability to discriminate the interoceptive stimuli produced by ethanol at two different postadministration intervals. The results indicated that neither the discrimination learning rate (as indicated by sessions-to-criterion) nor the sensitivity to ethanol (as indicated by the EDs0 values generated by the dose-response experiments) differed between the LAS and HAS rats. This confirms previous work (6) using the tenth generation of these animals and the same training dose of ethanol but a different schedule of reinforcement, viz., a variable ratio (VR5) schedule. In that earlier reported dose-response experimentation, EDs0 values did not differ between these two groups of rats after extensive training/testing (50-60 sessions). Because there were no differences between the HAS and LAS animals at either of the two postadministration intervals, the remainder of the discussion focuses on the differences in both groups at each of these two times, as well as to the phase-generalization results. It appears that the animals trained at the 30-min PI required a significantly greater number of training sessions to attain the discriminative performance criterion. The most parsimonious explanation for this observation is one of pharmacokinetics, in that the amount of ethanol present in the blood/ brain appears to peak at 7.5 min after i.p. administration of a similar dose of ethanol with a decrease at the 30 min PI (5,7). It has been suggested that there is a different relationship between the dose administered and subsequent brain concentration and the stimulus strength of a drug (9). Thus, the average number of training sessions to establish discriminative criteria decreases with increasing training doses and, therefore, with increasing amounts of ethanol to the brain (1). Previous work in this laboratory (13) using the same dose of ethanol and a more usual PI of 15 min indicated an average of 37 training sessions to attain the same discriminative criterion. This STC value is closer to that seen in the animals trained at the 30-min PI, and indicated that the animals trained at the earlier PI learned the discrimination at a faster rate. In fact,
121 when rats were trained to discriminate 1.2 g/kg ethanol at a PI of 15 min, their discriminative performance was maximal when they were tested at a 7.5-min PI, and fell 30% when they were tested at 30 min postinjection (5). Although the present study used selectively bred animals, another study, designed to correlate ethanol sensitivity and discriminative performance, separated female Wistar rats as to their drinking behavior with 6% w/v solution of ethanol over a 4-week period (24). It was established that one group, designated ethanol-preferring, derived approximately 60% of its total fluid from ethanol, whereas a second group, designated ethanol nonpreferring, derived approximately 200/0 from alcohol. When each of these groups were trained to discriminate the effects of 780 mg/kg ethanol i.p. (with a 20-min PI) in a shock-motivated T-maze apparatus, the two groups were shown to learn the discrimination at the same rate. Subsequent work by the same investigator (25) employed the selectively bred lines of alcohol-accepting (AA) and alcohol nonaccepting (ANA) rats (3) and trained these two groups to discriminate 1.0 g/kg ethanol. The nonpreferring rats were reported to achieve discrimination criterion performance faster than the alcohol-accepting animals. This suggested that the animals with low ethanol drinking preference, i.e., the ANA group, may have a greater sensitivity for the perception of the ethanol discriminative cue. The present work, using a different line of selectively bred rats, does not confirm this experiment in that it suggests drug discrimination is not based on the depressant effects of ethanol. Nevertheless, it must be kept in mind that, although the A A / A N A rats were bred for ethanol preference, the present rats were bred for difference in sensitivity to ethanol's hypnotic effects, and previous work has indicated that rats that prefer alcohol are less sensitive to the hypnotic and depressive effects than are those animals who do not prefer alcohol (4). The phase generalization results confirm an earlier report (14) that indicated asymmetrical generalization in heterogeneously bred rats, using only the training dose of 600 mg/kg. In the present study, doses of 300, 450, and 600 mg/kg ethanol were employed in the phase-generalization study and the different effects of ethanol at the 6- and 30-min PI were observed once again. Thus, it appears that the earlier ethanol discriminative stimuli differ from those stimuli produced when rats are trained at the latter PI. The possibility exists that the biphasic action of ethanol may have an earlier excitatory, reinforcing phase as the blood alcohol concentrations rise and a later depressive (both on locomotor activity and neurobehavioral reward) stage as the blood levels fall (7). ACKNOWLEDGEMENTS I would like to express my gratitude to Drs. Richard A. Deitrich and Laura J. Draski for supplying the LAS/HAS rats; to Denise McBurney for her continuing technical expertise; to Marty Hilgert and Karen Kelley for word processing; to Dr. Edward C. Krimmer for his advice on this manuscript. This research was funded entirely by NIAAA Grant #8598.
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