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Brain self-stimulation, locomotor activity and tissue concentrations of ethanol in male rats
Drug
67
ano! Alcohol
Elsevier
Dependence, 21 (19881 M-i’5 Scientific Publishers Ireland Ltd.
Brain self-stimulation, locomotor activity and tissue concentrations of ethanol in male rats* Gerald J. Schaefer**,
Department
of Psychiatry,
Emory
William R. Richardson, Richard P. Michael
University
Robert W. Bonsall and
School of Medicine, The Georgia Mental Health Institute, N.E., Atlanta, GA 30.3’06 IU.S.A.I
1256 Briarcliff
Rd.,
(Received July lOth, 198’71 These,studies were aimed at correlating the effects of ethanol on operant behavior and on locomotor activity with its distribution in selected tissues in the body. One group of male rats was trained on a continuous reinforcement schedule for intracranial self-stimulation (ICSS) with electrodes in the lateral hypothalamus. Another group was studied in a locomotor activity apparatus, and both groups were given ethanol intraperitoneally over the dose-range 0.3- 1.7 g/kg. Urine was collected 15 min and 60 min after ethanol administration and samples of blood, brain, heart, lung, liver, muscle and testis were obtained at both time points. Depressions of ICSS and of locomotor activity occurred, and these changes in behavior were correlated with increasing concentrations of ethanol in blood, urine and tissue. Thus, the disrupting effects of ethanol on behavior which oecurred shortly after its acute administration were closely linked to its concentrations throughout the body. Key words: ethanol; brain self-stimulation;
locomotor activity
Introduction When ethanol is administered to rats intraperitoneally (i.p.1, it usually produces a graded decrease in operant behaviors that employ positive reinforcements, such as lever-pressing for food [1,2], and it also decreases locomotor activity [3-71. This depressant effect has been shown to correlate with blood ethanol concentrations [3,8]. There have also been several studies in which ethanol was administered to rats trained to lever-press for brain self-stimulation (ICSS). Although both an increase and no change in lever-press rates have been reported with low to moderate doses [9,10], those studies using doses above 1.2 g/kg, have usually reported a decrease [ll- 151. There is little information on the time-course of ethanol-induced changes in ICSS response rates or on the rela*Supported by the Georgia Department of Human Resources. **To whom correspondence and reprint requests should be sent.
tionship between the behavioral changes and the concentrations of ethanol in body tissues. The aims of this study were, first, to examine the dose-response and time-course relations between ethanol and lever-pressing for ICSS, second, to examine the associated changes in locomotor activity and, third, to examine the relation between behavior and ethanol concentrations in blood, urine and in the following tissues - brain, heart, lung, liver, muscle and testis. A systematic analysis that correlates levels of ethanol in body tissues with locomotor activity and responding for ICSS has not been conducted previously and will add information on how ethanol may influence behavior.
Materials
aud Methods
Subjects
Male Sprague - Dawley rats (n = ‘741bred in our laboratory from stock purchased from Charles River (Wilmington, MA) were used.
68
The animals in the brain stimulation experiments (n = 101 and those used for blood/urine collections (n = 161 were approximately 150 days old (450- 730 gl at the beginning of the experiment. The animals in the locomotor activity and blood/tissue concentration experiments (n = 48) were 55-96 days old (270-600 gl. Other than during tests, all animals were maintained in group cages (2- 4lcagel housed in a colony room with lights on from 07:00-19:OO with free access to food and water.
Apparatus
Brain stimulation tests were conducted in an operant chamber constructed in this laboratory [16]. Briefly, the chamber (31 x 30 x 29 cm high) was equipped with a single, conventional lever (Model G6312, Gerbrands, Arlington, MA) on a wall 10 cm above the grid floor. Electrical pulses were generated by a constant-current, biphasic stimulator [17] and consisted of a 200 ms train of square-wave pulses at 100 Hz with a pulse duration of 0.5 ms. The median current intensity was 50 PA with a range of 20 - 325 PA. Pulses were delivered to the animals’ brains through a two-channel commutator (Model 590, Mercotac, San Diego, CA) via a shielded cable that allowed the animal to move freely about the chamber. Motor activity was tested in a Digiscan RXY activity monitor (Omnitech, Columbus, OH) interfaced with a Behavioral Control Unit [18]. This device measured total horizontal activity by summing all infrared beam interruptions. It also measured ambulatory activity by a circuit which ignored repetitive interruptions of the same photobeam, thereby measuring movement of the animal about the box. In addition, the time that the animal was at rest or was moving at one of three speeds was recorded. These values were displayed in four different channels as time in seconds summed over the test session. The activity monitor containing the test box (39.4 X 39.4 X 30.5 cm high, inside dimensions) was housed inside a sound attenuating cabinet equipped with a fan and a 25-W red light bulb.
Surgery
and histology
Animals in the brain stimulation experiments were implanted with bipolar platinum electrodes (tip diameter = 0.125 mm, Plastic Products, Roanoke, VA) aimed at the medial forebrain bundle-lateral hypothalamus using coordinates: AP 5.2, L 1.7, H 2.2 [19]. Surgery was performed under sodium pentobarbital anesthesia (50 mg/kg, IP), and animals were given atropine sulfate (0.25 mg, SC) to minimize respiratory discomfort. The electrode was secured in place with crania-plastic cement which was applied to the top of the electrode assembly and to the 4 - 5 stainless steel screws which were fixed to the skull. This produced a secure anchor for the electrode and the commutator cable. At the completion of surgery, animals were administered 100 000 units of benzathine penicillin G and procaine penicillin G intramuscularly. At the conclusion of the brain stimulation experiment, animals were overdosed with sodium pentobarbital and perfused via the heart with 10% formol-saline. After fixation, 50-pm sections were cut, and alternate sections were stained with cresyl violet and Weil’s stain, The stained sections were then viewed under a microprojector to identify the precise location of the electrode tips. Chemical methods
Urine and blood samples from the second group of animals (see below) were assayed for ethanol content by a standard enzymatic procedure [20] which measured the conversion of ethanol to acetaldehyde (Procedure No 332-UV, Sigma Diagnostics, St. Louis, MO). Blood and tissue samples from the third group of animals (see below) were analyzed simultaneously by the enzymatic procedure and by gas chromatography. For the latter method, l-ml samples of whole blood were added to 1 ml of cold 10% trichloroacetic acid (TCA), vortexed and placed in crushed ice. Samples of brain, heart, lung, liver, testis and muscle (right quadriceps) were collected and l-g samples of each tissue were added to 1 ml of cold distilled water, cooled in ice and homogenized using a Polytron
69
(Brinkman Instruments, Westburg, NY). Homogenates were then mixed with 0.2 ml 50% TCA and precipitated proteins were removed from blood and tissue samples by centrifugation at 1500 X g for 20 min. Duplicate 0.2-d aliquots were injected into a glass-lined injection port at 25OOC using a split ratio of 1:50 (Perkin-Elmer Sigma 1, Norwalk, CN) and chromatographed with a 30 m x 0.25 mm Durawax DX-4 fused silica capillary column with a 1 pm film thickness (J & W Scientific Inc., Rancho Cordova, CA) using a temperature gradient of 55O - 100 OC at 20 OC/min. The carrier gas was hydrogen, and peaks were detected by flame ionization and integrated using a Perkin-Elmer Sigma 15 data station. The TCA peak served as an internal standard, and the method was calibrated with external standards prepared by adding ethanol to aliquots of blood and tissue from untreated animals. The overall coefficient of variation (duplicate samples1 was 17.5% (< 10% for values > 0.5 mg/ml), and the sensitivity of the method (mean level for zero standards + 2 S.D.1 was 0.005 mglml. Results from the gas chromatographic and enzymatic procedures were highly correlated (r = 0.991, and the former results are presented here. Procedure
Animals in the first group (n = 10) were implanted with stimulating electrodes and allowed at least 1 week to recover from surgery. They were then trained to press the lever in the operant chamber for brain stimulation. Each lever-press produced one stimulus (continuous reinforcement schedule). When the rate of lever-pressing had stabilized, testing with ethanol began. Animals were administered either saline (Monday and Thursday) or ethanol (Tuesday and Friday), and after 15 or 60 min had elapsed, they were placed in the test chamber for 20 min and the number of lever presses was recorded. Both saline and ethanol were administered IP in a volume of 10 ml/kg body weight. Absolute ethyl alcohol was diluted with 0.9% saline to 30% w/v to produce a stock solution, and further dilutions were made by the ad-
dition of 0.9% saline to aliquots of the stock solution. By this means the volume injected remained constant and the ethanol concentrations were varied. Animals received doses of 1.7,l.O or 0.3 g/kg ethanol. Animals in the second group (n = 16) were not implanted with electrodes nor given leverpressing experience, but in all other respects were treated similarly to the animals in the first group. However, at either 15 or 60 min after injection, they were placed in metabolism cages for 20 min for urine collection. When the collection of urine was concluded at the 60-min time point (80 min after ethanol or saline administration), the animals were killed by decapitation, and trunk blood was collected in heparinized tubes for analysis of ethanol content. Animals in the third group (n = 48) were tested for changes in locomotor activity after ethanol administration, and were also used to determine blood and tissue ethanol concentrations. On day 1, the experimentally naive animals were placed in the activity monitor for 12 min. and then removed. On day 2 all animals were injected with saline IP and either 15 or 60 min later they were placed in the activity monitor. Days 1 and 2 were used to habituate the animals to the apparatus and injection procedure. On day 3, the routine of day 2 was repeated, but animals were injected either with saline or ethanol (0.3, 1.0 or 1.7 g/kg). At the end of the 12-min activity session, animals were and decapitated, removed immediately Trunk blood was collected in heparinized tubes for analysis of ethanol content. Data analysis
The total number of lever-presses/20 min provided the data for the brain self-stimulation experiment. In tests of locomotor activity, data for the first 2 min in the apparatus were discarded and the number of infrared beam interruptions during the remaining 10 min were used for analysis. The time elapsing in each of the four channels of the Behavioral Control Unit was also recorded. All locomotor activity data were converted to percentages of
70
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cn w rn v) W a a a W >
l
W -I
l
Sal. DOSE
OF
ETHANOL
ADMINISTERED
0.3
I 0
I.7
(g/kg)
Panel A: effects of saline and graded doses of ethanol on the rate of lever-pressing for brain self-stimulation during Fig. 1. 20-min test sessions. Saline or ethanol were administered either 15 min ( 0) or 60 min (0) before the session. The 10 animals used in this experiment received all doses of ethanol at both treatment times. Vertical lines give the standard errors of means in this and subsequent figures. Panel B: effects of saline and graded doses of ethanol on the concentration of ethanol in urine obtained during two 20-min sampling periods beginning 15 min (0) and 60 min (0) after the administration of ethanol. Groups of 4 animals were used for saline and for each ethanol dose. After the second sampling period, animals were killed and trunk blood was collected (A). *Significantly different from saline P < 0.01.
the scores after saline injections. Analyses of variance were used to test the main effects of dose and time, as well as their interaction effects (SPSWPC + , SPSS Inc. Information Analysis Systems, Chicago, IL). Post hoc testing was performed using Dunnett’s procedure (2tailed). Similar analyses were also used to assess the significance of changes in the urine, blood and tissue concentrations of ethanol. Correlation coefficients were performed to assess the relationship between motor activity patterns and blood and tissue concentrations of ethanol.
O.OOl], but neither the effect of time nor the dose x time interaction was significant. At the 1.0 g/kg dose a small decrease in lever-pressing occurred, but at the 1.7 g/kg dose there was a 90% decrease in lever-pressing. The effects were closely similar 15 and 60 min after ethanol administration. In a similar group of rats, over the dose range 0.3 - 1.7 g/kg there was a significant increase in ethanol levels in urine at both time points [F(3,241 = 72.5, P < O.OOl]. This corresponded with the significant increase in blood levels in samples obtained 60 min after ethanol administration [F(3,121 = 326.2, P < O.OOl].
Results
Figure 1 shows the effects of graded doses of ethanol on the rate of lever-pressing for brain self-stimulation (Fig. lA1, together with levels of ethanol in blood at 60 min and in urine at 15 min and 60 min after its administration (Fig. 1B). A highly significant main effect of dose was found for the ICSS data [F(3,271 = 45.2, P <
Figure 2 shows the effects of ethanol on locomotor activity (Fig. 2A) together with changes in the blood ethanol levels in these animals (Fig. 2Bl. At 15 min after ethanol administration, there was a clear, dose-related decrease in locomotor activity, but this pattern was different at 60 min when a decrease in activity was apparent only at the highest dosage. The main effect of dose was significant [F(3,401 =
71
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Panel A: effects of saline and graded doses of ethanol on total horizontal activity (beam interruptions) during IO-min test sessions. Saline or ethanol was administered either 15 min (0) of 60 min (01 before the session. Data have been converted to percentages of saline scores and the horizontal interrupted line gives mean values when saline was administered. These were 830 f 78 at 15 min, and 592 + 113 at 60 min (beam interruption410 mini. Animals were used only once, and each data point is the mean of six animals. Panel B: effects of saline and graded doses of ethanol on the concentrations of ethanol in trunk blood of the same animals. Immediately after the end of the activity test animals were killed and trunk blood was collected at 15 min (Al and at 60 min (Al. *Significantly different from saline,P < 0.01.
16.0, P < O.OOl] and the dose x time interaction approached significance [P(3,401 = 2.8, P = 0.0531. There were marked increases in blood levels of ethanol after its administration, and there was a significant main effect of dose [H3,401 = 326.8, P < O.OOl] and a significant dose x time interaction [F(3,401 = 3.6, P < 0.021. In addition, 15 min after ethanol administration there was a high correlation (T = 0.86, P < 0.01, n = 241 between locomotor activity and ethanol blood concentration; at 60 min the correlation was lower (r = 0.57, P < 0.01, n = 241. The concentrations of ethanol in each of the six tissues are shown in Fig. 3. Results were similar to those found in blood. For example, in brain there was a significant main effect of dose [F(3,401 = 216.5, P < O.OOl] while the interaction between dose and time approached significance [F(3,401 = 2.45, P = 0.081. There was also a high correlation between blood and brain levels of ethanol: at 15 min: r = 0.99, P < 0.01, n = 24, at 60 min: r = 0.99, P < 0.01, n = 24. In heart, there was a significant effect of
dose [H3,401 = 282.1, P < O.OOl], and a significant dose x time interaction [F(3,401 = 3.3, P = 0.0291, due to higher levels at 60 min with the 1.7 g/kg dose. In lung, only the dose-effect was significant [F(3,40) = 211.1, P < 0.011, and this was also the case in liver [H3,401 = 195.3, P < O.OOl]. In muscle, both the main effect of dose [F(3,401 = 146.9, P < O.OOl]and the dose x time interaction were significant [F(3,401 = 3.6, P = 0.0221. Finally, in testis, only the main effect of dose was significant [N3,401 = 230.2, P < O.OOl], and the concentrations of ethanol in testis and brain were similar. Table I gives more detailed information about the pattern of changes in locomotor activity following ethanol administration. These data were derived from the different channels of the locomotor activity Behavioral Control Unit. There was a graded decrease in activity when ethanol was administered 15 min before the test session, and animals were almost inactive at the highest dose. The drug produced a relatively greater depression of fast activity (chan-
72
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DOSE
OF
ETHANOL
(g/kg)
ADMINISTERED
Fig..3. Effects of saline and graded doses of ethanol on the concentrations of ethanol in brain, heart, lung, liver, muscle and testis at 15 min (0 1and 6Omin(01 after treatment. These tissues were from the same animals whose behavioral and blood data are shown in fig. 2. *Significantly different from saline, P < 0.01.
Table I. Ambulation, time spent at rest, and time spent moving at different speeds after the administration of saline or ethanol. Ten-minute tests in the activity monitor. Ethanol data expressed as a percentage of the saline data. N = 6 at each dose- and time-interval. (Means f S.E.Ms). *P < 0.05 compared with saline: **P < 0.01 compared with saline.
Ambulation ChannelI Time at rest Channel2 Slow activity Channel3 Medium activity Channel4 Fast activity
Injection-test Interval (mini
Saline (time in sl
15 60 15 60 15 60 15 60 15 60
365 + 35 244, f 54 382.5 f 28.2 431.5 f 31.9 128.2 + 14.7 109.5 -c 18.6 74.4 f 9.5 48.2 f 11.3 17.0 + 4.3 10.8 * 2.7
Ethanol (I of saline scores) 0.3 g/kg 96.8 f 77.5 + 99.7 f 105.0 -c 98.8 + 94.7 + 101.8 + 80.7 * 96.5 + 41.5 +
15.0 6.4 7.2 2.3 10.2 6.5 17.9 8.4 16.8 8.6
1.0 g/kg
1.7 g/kg
40.8 f 6.9++ 99.2 2 29.9 127.5 -e 5.2* 101.5 f 8.6 66.7 f 11.4 91.0 f 16.9 31.2 f 6.5** 106.0 f 32.5 19.3 f 6.6 ** 104.3 -t 41.8
11.2 f 21.0 f 146.3 f 130.2 -c 26.3 f: 28.3 + 7.5 f 15.0 f 8.0 f 3.3 f
2.7** 3.7** 2.6** l.l** 6.6+* 3.0** 2.3** 3.4+* 0.4+* 1.4*
73
nel 41 than of slow activity (channel 21. Conversely, there was a graded increase with dose in time spent at rest (channel 11. The data from channel 1 showed a large inverse correlation with the activity data in Fig. 2B, at 15 min: T= -0.98, P < 0.01, n = 24. When ethanol was administered 60 min before testing, the effects on activity were less marked, although animals were still significantly depressed at the 1.7 g/kg dose and a large inverse correlation between horizontal activity and time spent at rest was found, r = -0.99,P< O.Ol,n = 24. Histology
Nine of the ten electrode tips terminated in the lateral hypothalamus with an anterior-posterior distribution from 5.6 - 4.4 mm anterior to the vertical zero plane [19]. Three of the nine electrodes also touched the zona incerta and one touched the internal capsule. A tenth electrode terminated entirely in the zona incerta. There were no motor effects in animals whose electrodes were slightly misplaced, and there were no differences in the responses to ethanol as a function of tip location. The data from all 10 animals were therefore used in the analysis. Discussion
These studies have extended previous work by demonstrating that both locomotor activity and the more complex conditioned behavior associated with ICSS changed in a similar way to a graded increase in ethanol dosage. Furthermore, when behavior became disrupted there were high concentrations of ethanol in blood and also in representative tissues throughout the body. The Sprague - Dawley derived rats used here showed a dose-dependent decrease in lever-pressing for brain selfstimulation when tested either 15 or 60 min after ethanol administration, results which were in good agreement with earlier reports on the effects of ethanol on ICSS and on food-reinforced behavior [1,2,11- 151. These data were also in agreement with recent work employing the auto-titration procedure which measures both rates of responding and reinforcement
thresholds [21]. Using the same dose-range and injection-test intervals as those in the current report, ethanol produced a dose-dependent decrease in response rates without altering reinforcement thresholds. When the injectiontest interval was extended from 15 to 180 min, there was a time-dependent attenuation of the decrease in response rates, but no changes in threshold [22]. In the present experiments, a group of non-implanted rats otherwise treated similarly to implanted rats showed a graded increase in both urine and blood levels of ethanol. Marked changes in behavior occurred only when blood and urine levels were above 1.0 mg/ ml. Since animals were well trained and habituated to the lever-pressing task, it appeared that at the intermediate dose of 1 g/kg they were able to overcome ethanol’s intoxicating effects to some degree and this may have been due to the development of behavioral tolerance [23]. It should be emphasized, however, that different groups of animals were used for the behavior and the biochemistry. Thus, we did not attempt to determine if performing the operant task itself altered the effects of ethanol. Locomotor activity showed a graded decrease with increasing dosage which correlated highly with the increase in blood levels in these same animals 15 min after ethanol administration. At 60 min the effects were somewhat different. The intermediate dose no longer produced a significant decrease in activity and, although blood ethanol levels were significantly above baseline, they were lower than in animals tested at 15 min. It seems unlikely, however, that the slight decrease in blood levels 1 h after ethanol administration would account for the lack of effect on motor activity at this time point. Clearly, the differences in behavior were not due to a practice effect, since these animals were only used for a single ethanol test. Rather, these data pointed to the rapid development of tolerance to ethanol [24], and this was most evident with the intermediate dose of 1.0 g/kg. These results are reminiscent of previous work in which rapid tolerance occurred to the deficit in performance of a moving belt test [25]. With the highest dose, however, when
74
blood ethanol levels were also high, dramatic and significant decreases in motor activity were seen at both test intervals. Consistent with the ICSS data, there were no increases in locomotor activity. Both total activity (Fig. 2A1 and ambulation scores (Table 11were decreased with increasing concentrations of ethanol; animals also spent more time resting as tissue concentrations increased. A more detailed analysis of activity revealed that at 15 min. movement at high speed was reduced to a greater extent than that at low speed. Our results complement those of Kulig et al. [7] who examined more complex coordinated handlimb movements in rats after ethanol administration. The animals used for ICSS were highly trained whereas those tested for locomotor activity were given an opportunity for some habituation. We now have data showing that when these animals are habituated to the test environment for 30 min before injection, a low dose of ethanol (0.1 g/kg) can produce increases in motor activity [26]. The tissue analyses showed that ethanol was present in all areas studied approximately 30 and 75 min after its administration, and levels correlated with decreases in motor activity measures. The lack of any increases in locomotor activity may have been due to the injection-test intervals used here. Perhaps increases might have occurred if the animals were habituated for a longer time and tested immediately after administration of a low ethanol dose [26,27]. A similar case could be made for brain self-stimulation. Again, with our testing conditions, only decreases in leverpressing were observed and this was consistent with response-rate data in the auto-titration procedure [21]. It has been proposed that increases in locomotor activity are positively correlated with the reinforcing effect of ethanol [28]. Rats that drank ethanol showed an increase in motor activity with IP ethanol injecIn our study, there was close tion. correspondence between the changes in motor activity and the changes in ICSS rates. In both paradigms only dose-dependent decreases were observed, suggesting that under present conditions positive reinforcement would not be ob-
served; yet another example of the enigma surrounding ethanol’s effects on behavior and of the difficulty in demonstrating its reinforcing properties [29]. These same data, however, emphasize the possibility that the intoxicating properties of ethanol may result from its actions at many sites including those in the peripheral and central nervous system. Acknowledgment
We thank Ms. Matty Lane of the Central Pharmacy-Toxicology Laboratory for performing the enzymatic ethanol assays. References 1 D.C.Bird et al., Psychopharmacology,87 (1985) 414. 2 F.A. Holloway and D.R. Vardiman, Psychon. Sci., 24 (19711218. 3 G.D. Frye and G.R. Breese, Psychopharmacology,75 (1981) 372.
4 E.P. Riley et al., J. Stud. Alcohol, 37 (1976) 1535. 5 G.P. Hunt and D.H. Overstreet, Psychopharmacology, 55 (1977) 75.
6 J. Brick et al., Alcohol: Clin. Exp. Res., 8 (1984) 204. 7 B.M. Kulig et al., Toxicol. Appl. Pharmacol., 80 (1985) 1. 8 R. Smoothy and M.S. Berry, Psychopharmacology, 85 (1985) 57.
9 S.A. Lorens and S.M. Sainati. Life Sci., 23 (1978) 1359. 10 R.H. Carlson and R. Lydic, Psychopharmacology, 50 (1976) 61.
11 P. Vrtunski et al., Q.J. Stud. Alcohol, 34 (1973) 718. 12 13 14 15 16 17 18 19 20 21 22 23
24
R.W. Flanagan, Dis. Abstr. Int., 36 (1976) 4746-B. D.J. Magnuson and L.D. Reid, Bull. Psychon. Sot., 10 (1977) 364. A. Routtenberg, Ann. N.Y. Acad. Sci., 362 (1981) 60. P. De Witte and M.F. Bada, Exp. Neural., 82 (1983) 675. G.J. Schaefer and R.P. Michael, Psychopharmacology, 74 (1981) 17. G.J. Schaefer et al., Physiol. Behav., 29 (1982) 163. G.J. Schaefer et al., Physiol. Behav., 37 (1986) 181. L.J. Pellegrino et al., A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York, 1979. F. Lundquist, in: D. Glick (Ed.), Methods of Biochemical Analysis, Interscience, New York, 1957. G.J. Schaefer and R.P. Michael, Alcohol, 4 (1987) 209. G.J. Schaefer and R.P. Michael, Sot. Neurosci. Abstr.. 12 (1986) 53. B. Tabakoff, in: K. Eriksson et al. (Eds.), Animal Models in Alcohol Research, Academic Press, New York, 1980. J.M. Littleton, in: H. Rigter and J.C. Crabbe (Eds.),
75
25 26 27
Alcohol Tolerance and Dependence, ElsevierlNorth Holland Biomedical Press, 1980. A.E. LeBlanc et al. Psychopharmacologia, 4lt1975143. G.J. Schaefer and R.P. Michael, Sot. Neurosci. Abstr., 13 (198’71339. G. Di Chiara and A. Imperato, Eur. J. Pharmacol., 115 (1985) 131.
28 29
M.B. Waller et al., Pharmacol. 11986161’7. B. Tabakoff and P.L. Hoffman, Oreland (Eds.1, Brain Reward Raven Press, New York, 1987.
Biochem.
Behav.,
in: J. Engel Systems and
24
and L. Abuse,
67
ano! Alcohol
Elsevier
Dependence, 21 (19881 M-i’5 Scientific Publishers Ireland Ltd.
Brain self-stimulation, locomotor activity and tissue concentrations of ethanol in male rats* Gerald J. Schaefer**,
Department
of Psychiatry,
Emory
William R. Richardson, Richard P. Michael
University
Robert W. Bonsall and
School of Medicine, The Georgia Mental Health Institute, N.E., Atlanta, GA 30.3’06 IU.S.A.I
1256 Briarcliff
Rd.,
(Received July lOth, 198’71 These,studies were aimed at correlating the effects of ethanol on operant behavior and on locomotor activity with its distribution in selected tissues in the body. One group of male rats was trained on a continuous reinforcement schedule for intracranial self-stimulation (ICSS) with electrodes in the lateral hypothalamus. Another group was studied in a locomotor activity apparatus, and both groups were given ethanol intraperitoneally over the dose-range 0.3- 1.7 g/kg. Urine was collected 15 min and 60 min after ethanol administration and samples of blood, brain, heart, lung, liver, muscle and testis were obtained at both time points. Depressions of ICSS and of locomotor activity occurred, and these changes in behavior were correlated with increasing concentrations of ethanol in blood, urine and tissue. Thus, the disrupting effects of ethanol on behavior which oecurred shortly after its acute administration were closely linked to its concentrations throughout the body. Key words: ethanol; brain self-stimulation;
locomotor activity
Introduction When ethanol is administered to rats intraperitoneally (i.p.1, it usually produces a graded decrease in operant behaviors that employ positive reinforcements, such as lever-pressing for food [1,2], and it also decreases locomotor activity [3-71. This depressant effect has been shown to correlate with blood ethanol concentrations [3,8]. There have also been several studies in which ethanol was administered to rats trained to lever-press for brain self-stimulation (ICSS). Although both an increase and no change in lever-press rates have been reported with low to moderate doses [9,10], those studies using doses above 1.2 g/kg, have usually reported a decrease [ll- 151. There is little information on the time-course of ethanol-induced changes in ICSS response rates or on the rela*Supported by the Georgia Department of Human Resources. **To whom correspondence and reprint requests should be sent.
tionship between the behavioral changes and the concentrations of ethanol in body tissues. The aims of this study were, first, to examine the dose-response and time-course relations between ethanol and lever-pressing for ICSS, second, to examine the associated changes in locomotor activity and, third, to examine the relation between behavior and ethanol concentrations in blood, urine and in the following tissues - brain, heart, lung, liver, muscle and testis. A systematic analysis that correlates levels of ethanol in body tissues with locomotor activity and responding for ICSS has not been conducted previously and will add information on how ethanol may influence behavior.
Materials
aud Methods
Subjects
Male Sprague - Dawley rats (n = ‘741bred in our laboratory from stock purchased from Charles River (Wilmington, MA) were used.
68
The animals in the brain stimulation experiments (n = 101 and those used for blood/urine collections (n = 161 were approximately 150 days old (450- 730 gl at the beginning of the experiment. The animals in the locomotor activity and blood/tissue concentration experiments (n = 48) were 55-96 days old (270-600 gl. Other than during tests, all animals were maintained in group cages (2- 4lcagel housed in a colony room with lights on from 07:00-19:OO with free access to food and water.
Apparatus
Brain stimulation tests were conducted in an operant chamber constructed in this laboratory [16]. Briefly, the chamber (31 x 30 x 29 cm high) was equipped with a single, conventional lever (Model G6312, Gerbrands, Arlington, MA) on a wall 10 cm above the grid floor. Electrical pulses were generated by a constant-current, biphasic stimulator [17] and consisted of a 200 ms train of square-wave pulses at 100 Hz with a pulse duration of 0.5 ms. The median current intensity was 50 PA with a range of 20 - 325 PA. Pulses were delivered to the animals’ brains through a two-channel commutator (Model 590, Mercotac, San Diego, CA) via a shielded cable that allowed the animal to move freely about the chamber. Motor activity was tested in a Digiscan RXY activity monitor (Omnitech, Columbus, OH) interfaced with a Behavioral Control Unit [18]. This device measured total horizontal activity by summing all infrared beam interruptions. It also measured ambulatory activity by a circuit which ignored repetitive interruptions of the same photobeam, thereby measuring movement of the animal about the box. In addition, the time that the animal was at rest or was moving at one of three speeds was recorded. These values were displayed in four different channels as time in seconds summed over the test session. The activity monitor containing the test box (39.4 X 39.4 X 30.5 cm high, inside dimensions) was housed inside a sound attenuating cabinet equipped with a fan and a 25-W red light bulb.
Surgery
and histology
Animals in the brain stimulation experiments were implanted with bipolar platinum electrodes (tip diameter = 0.125 mm, Plastic Products, Roanoke, VA) aimed at the medial forebrain bundle-lateral hypothalamus using coordinates: AP 5.2, L 1.7, H 2.2 [19]. Surgery was performed under sodium pentobarbital anesthesia (50 mg/kg, IP), and animals were given atropine sulfate (0.25 mg, SC) to minimize respiratory discomfort. The electrode was secured in place with crania-plastic cement which was applied to the top of the electrode assembly and to the 4 - 5 stainless steel screws which were fixed to the skull. This produced a secure anchor for the electrode and the commutator cable. At the completion of surgery, animals were administered 100 000 units of benzathine penicillin G and procaine penicillin G intramuscularly. At the conclusion of the brain stimulation experiment, animals were overdosed with sodium pentobarbital and perfused via the heart with 10% formol-saline. After fixation, 50-pm sections were cut, and alternate sections were stained with cresyl violet and Weil’s stain, The stained sections were then viewed under a microprojector to identify the precise location of the electrode tips. Chemical methods
Urine and blood samples from the second group of animals (see below) were assayed for ethanol content by a standard enzymatic procedure [20] which measured the conversion of ethanol to acetaldehyde (Procedure No 332-UV, Sigma Diagnostics, St. Louis, MO). Blood and tissue samples from the third group of animals (see below) were analyzed simultaneously by the enzymatic procedure and by gas chromatography. For the latter method, l-ml samples of whole blood were added to 1 ml of cold 10% trichloroacetic acid (TCA), vortexed and placed in crushed ice. Samples of brain, heart, lung, liver, testis and muscle (right quadriceps) were collected and l-g samples of each tissue were added to 1 ml of cold distilled water, cooled in ice and homogenized using a Polytron
69
(Brinkman Instruments, Westburg, NY). Homogenates were then mixed with 0.2 ml 50% TCA and precipitated proteins were removed from blood and tissue samples by centrifugation at 1500 X g for 20 min. Duplicate 0.2-d aliquots were injected into a glass-lined injection port at 25OOC using a split ratio of 1:50 (Perkin-Elmer Sigma 1, Norwalk, CN) and chromatographed with a 30 m x 0.25 mm Durawax DX-4 fused silica capillary column with a 1 pm film thickness (J & W Scientific Inc., Rancho Cordova, CA) using a temperature gradient of 55O - 100 OC at 20 OC/min. The carrier gas was hydrogen, and peaks were detected by flame ionization and integrated using a Perkin-Elmer Sigma 15 data station. The TCA peak served as an internal standard, and the method was calibrated with external standards prepared by adding ethanol to aliquots of blood and tissue from untreated animals. The overall coefficient of variation (duplicate samples1 was 17.5% (< 10% for values > 0.5 mg/ml), and the sensitivity of the method (mean level for zero standards + 2 S.D.1 was 0.005 mglml. Results from the gas chromatographic and enzymatic procedures were highly correlated (r = 0.991, and the former results are presented here. Procedure
Animals in the first group (n = 10) were implanted with stimulating electrodes and allowed at least 1 week to recover from surgery. They were then trained to press the lever in the operant chamber for brain stimulation. Each lever-press produced one stimulus (continuous reinforcement schedule). When the rate of lever-pressing had stabilized, testing with ethanol began. Animals were administered either saline (Monday and Thursday) or ethanol (Tuesday and Friday), and after 15 or 60 min had elapsed, they were placed in the test chamber for 20 min and the number of lever presses was recorded. Both saline and ethanol were administered IP in a volume of 10 ml/kg body weight. Absolute ethyl alcohol was diluted with 0.9% saline to 30% w/v to produce a stock solution, and further dilutions were made by the ad-
dition of 0.9% saline to aliquots of the stock solution. By this means the volume injected remained constant and the ethanol concentrations were varied. Animals received doses of 1.7,l.O or 0.3 g/kg ethanol. Animals in the second group (n = 16) were not implanted with electrodes nor given leverpressing experience, but in all other respects were treated similarly to the animals in the first group. However, at either 15 or 60 min after injection, they were placed in metabolism cages for 20 min for urine collection. When the collection of urine was concluded at the 60-min time point (80 min after ethanol or saline administration), the animals were killed by decapitation, and trunk blood was collected in heparinized tubes for analysis of ethanol content. Animals in the third group (n = 48) were tested for changes in locomotor activity after ethanol administration, and were also used to determine blood and tissue ethanol concentrations. On day 1, the experimentally naive animals were placed in the activity monitor for 12 min. and then removed. On day 2 all animals were injected with saline IP and either 15 or 60 min later they were placed in the activity monitor. Days 1 and 2 were used to habituate the animals to the apparatus and injection procedure. On day 3, the routine of day 2 was repeated, but animals were injected either with saline or ethanol (0.3, 1.0 or 1.7 g/kg). At the end of the 12-min activity session, animals were and decapitated, removed immediately Trunk blood was collected in heparinized tubes for analysis of ethanol content. Data analysis
The total number of lever-presses/20 min provided the data for the brain self-stimulation experiment. In tests of locomotor activity, data for the first 2 min in the apparatus were discarded and the number of infrared beam interruptions during the remaining 10 min were used for analysis. The time elapsing in each of the four channels of the Behavioral Control Unit was also recorded. All locomotor activity data were converted to percentages of
70
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l
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ETHANOL
ADMINISTERED
0.3
I 0
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Panel A: effects of saline and graded doses of ethanol on the rate of lever-pressing for brain self-stimulation during Fig. 1. 20-min test sessions. Saline or ethanol were administered either 15 min ( 0) or 60 min (0) before the session. The 10 animals used in this experiment received all doses of ethanol at both treatment times. Vertical lines give the standard errors of means in this and subsequent figures. Panel B: effects of saline and graded doses of ethanol on the concentration of ethanol in urine obtained during two 20-min sampling periods beginning 15 min (0) and 60 min (0) after the administration of ethanol. Groups of 4 animals were used for saline and for each ethanol dose. After the second sampling period, animals were killed and trunk blood was collected (A). *Significantly different from saline P < 0.01.
the scores after saline injections. Analyses of variance were used to test the main effects of dose and time, as well as their interaction effects (SPSWPC + , SPSS Inc. Information Analysis Systems, Chicago, IL). Post hoc testing was performed using Dunnett’s procedure (2tailed). Similar analyses were also used to assess the significance of changes in the urine, blood and tissue concentrations of ethanol. Correlation coefficients were performed to assess the relationship between motor activity patterns and blood and tissue concentrations of ethanol.
O.OOl], but neither the effect of time nor the dose x time interaction was significant. At the 1.0 g/kg dose a small decrease in lever-pressing occurred, but at the 1.7 g/kg dose there was a 90% decrease in lever-pressing. The effects were closely similar 15 and 60 min after ethanol administration. In a similar group of rats, over the dose range 0.3 - 1.7 g/kg there was a significant increase in ethanol levels in urine at both time points [F(3,241 = 72.5, P < O.OOl]. This corresponded with the significant increase in blood levels in samples obtained 60 min after ethanol administration [F(3,121 = 326.2, P < O.OOl].
Results
Figure 1 shows the effects of graded doses of ethanol on the rate of lever-pressing for brain self-stimulation (Fig. lA1, together with levels of ethanol in blood at 60 min and in urine at 15 min and 60 min after its administration (Fig. 1B). A highly significant main effect of dose was found for the ICSS data [F(3,271 = 45.2, P <
Figure 2 shows the effects of ethanol on locomotor activity (Fig. 2A) together with changes in the blood ethanol levels in these animals (Fig. 2Bl. At 15 min after ethanol administration, there was a clear, dose-related decrease in locomotor activity, but this pattern was different at 60 min when a decrease in activity was apparent only at the highest dosage. The main effect of dose was significant [F(3,401 =
71
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Panel A: effects of saline and graded doses of ethanol on total horizontal activity (beam interruptions) during IO-min test sessions. Saline or ethanol was administered either 15 min (0) of 60 min (01 before the session. Data have been converted to percentages of saline scores and the horizontal interrupted line gives mean values when saline was administered. These were 830 f 78 at 15 min, and 592 + 113 at 60 min (beam interruption410 mini. Animals were used only once, and each data point is the mean of six animals. Panel B: effects of saline and graded doses of ethanol on the concentrations of ethanol in trunk blood of the same animals. Immediately after the end of the activity test animals were killed and trunk blood was collected at 15 min (Al and at 60 min (Al. *Significantly different from saline,P < 0.01.
16.0, P < O.OOl] and the dose x time interaction approached significance [P(3,401 = 2.8, P = 0.0531. There were marked increases in blood levels of ethanol after its administration, and there was a significant main effect of dose [H3,401 = 326.8, P < O.OOl] and a significant dose x time interaction [F(3,401 = 3.6, P < 0.021. In addition, 15 min after ethanol administration there was a high correlation (T = 0.86, P < 0.01, n = 241 between locomotor activity and ethanol blood concentration; at 60 min the correlation was lower (r = 0.57, P < 0.01, n = 241. The concentrations of ethanol in each of the six tissues are shown in Fig. 3. Results were similar to those found in blood. For example, in brain there was a significant main effect of dose [F(3,401 = 216.5, P < O.OOl] while the interaction between dose and time approached significance [F(3,401 = 2.45, P = 0.081. There was also a high correlation between blood and brain levels of ethanol: at 15 min: r = 0.99, P < 0.01, n = 24, at 60 min: r = 0.99, P < 0.01, n = 24. In heart, there was a significant effect of
dose [H3,401 = 282.1, P < O.OOl], and a significant dose x time interaction [F(3,401 = 3.3, P = 0.0291, due to higher levels at 60 min with the 1.7 g/kg dose. In lung, only the dose-effect was significant [F(3,40) = 211.1, P < 0.011, and this was also the case in liver [H3,401 = 195.3, P < O.OOl]. In muscle, both the main effect of dose [F(3,401 = 146.9, P < O.OOl]and the dose x time interaction were significant [F(3,401 = 3.6, P = 0.0221. Finally, in testis, only the main effect of dose was significant [N3,401 = 230.2, P < O.OOl], and the concentrations of ethanol in testis and brain were similar. Table I gives more detailed information about the pattern of changes in locomotor activity following ethanol administration. These data were derived from the different channels of the locomotor activity Behavioral Control Unit. There was a graded decrease in activity when ethanol was administered 15 min before the test session, and animals were almost inactive at the highest dose. The drug produced a relatively greater depression of fast activity (chan-
72
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ETHANOL
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Fig..3. Effects of saline and graded doses of ethanol on the concentrations of ethanol in brain, heart, lung, liver, muscle and testis at 15 min (0 1and 6Omin(01 after treatment. These tissues were from the same animals whose behavioral and blood data are shown in fig. 2. *Significantly different from saline, P < 0.01.
Table I. Ambulation, time spent at rest, and time spent moving at different speeds after the administration of saline or ethanol. Ten-minute tests in the activity monitor. Ethanol data expressed as a percentage of the saline data. N = 6 at each dose- and time-interval. (Means f S.E.Ms). *P < 0.05 compared with saline: **P < 0.01 compared with saline.
Ambulation ChannelI Time at rest Channel2 Slow activity Channel3 Medium activity Channel4 Fast activity
Injection-test Interval (mini
Saline (time in sl
15 60 15 60 15 60 15 60 15 60
365 + 35 244, f 54 382.5 f 28.2 431.5 f 31.9 128.2 + 14.7 109.5 -c 18.6 74.4 f 9.5 48.2 f 11.3 17.0 + 4.3 10.8 * 2.7
Ethanol (I of saline scores) 0.3 g/kg 96.8 f 77.5 + 99.7 f 105.0 -c 98.8 + 94.7 + 101.8 + 80.7 * 96.5 + 41.5 +
15.0 6.4 7.2 2.3 10.2 6.5 17.9 8.4 16.8 8.6
1.0 g/kg
1.7 g/kg
40.8 f 6.9++ 99.2 2 29.9 127.5 -e 5.2* 101.5 f 8.6 66.7 f 11.4 91.0 f 16.9 31.2 f 6.5** 106.0 f 32.5 19.3 f 6.6 ** 104.3 -t 41.8
11.2 f 21.0 f 146.3 f 130.2 -c 26.3 f: 28.3 + 7.5 f 15.0 f 8.0 f 3.3 f
2.7** 3.7** 2.6** l.l** 6.6+* 3.0** 2.3** 3.4+* 0.4+* 1.4*
73
nel 41 than of slow activity (channel 21. Conversely, there was a graded increase with dose in time spent at rest (channel 11. The data from channel 1 showed a large inverse correlation with the activity data in Fig. 2B, at 15 min: T= -0.98, P < 0.01, n = 24. When ethanol was administered 60 min before testing, the effects on activity were less marked, although animals were still significantly depressed at the 1.7 g/kg dose and a large inverse correlation between horizontal activity and time spent at rest was found, r = -0.99,P< O.Ol,n = 24. Histology
Nine of the ten electrode tips terminated in the lateral hypothalamus with an anterior-posterior distribution from 5.6 - 4.4 mm anterior to the vertical zero plane [19]. Three of the nine electrodes also touched the zona incerta and one touched the internal capsule. A tenth electrode terminated entirely in the zona incerta. There were no motor effects in animals whose electrodes were slightly misplaced, and there were no differences in the responses to ethanol as a function of tip location. The data from all 10 animals were therefore used in the analysis. Discussion
These studies have extended previous work by demonstrating that both locomotor activity and the more complex conditioned behavior associated with ICSS changed in a similar way to a graded increase in ethanol dosage. Furthermore, when behavior became disrupted there were high concentrations of ethanol in blood and also in representative tissues throughout the body. The Sprague - Dawley derived rats used here showed a dose-dependent decrease in lever-pressing for brain selfstimulation when tested either 15 or 60 min after ethanol administration, results which were in good agreement with earlier reports on the effects of ethanol on ICSS and on food-reinforced behavior [1,2,11- 151. These data were also in agreement with recent work employing the auto-titration procedure which measures both rates of responding and reinforcement
thresholds [21]. Using the same dose-range and injection-test intervals as those in the current report, ethanol produced a dose-dependent decrease in response rates without altering reinforcement thresholds. When the injectiontest interval was extended from 15 to 180 min, there was a time-dependent attenuation of the decrease in response rates, but no changes in threshold [22]. In the present experiments, a group of non-implanted rats otherwise treated similarly to implanted rats showed a graded increase in both urine and blood levels of ethanol. Marked changes in behavior occurred only when blood and urine levels were above 1.0 mg/ ml. Since animals were well trained and habituated to the lever-pressing task, it appeared that at the intermediate dose of 1 g/kg they were able to overcome ethanol’s intoxicating effects to some degree and this may have been due to the development of behavioral tolerance [23]. It should be emphasized, however, that different groups of animals were used for the behavior and the biochemistry. Thus, we did not attempt to determine if performing the operant task itself altered the effects of ethanol. Locomotor activity showed a graded decrease with increasing dosage which correlated highly with the increase in blood levels in these same animals 15 min after ethanol administration. At 60 min the effects were somewhat different. The intermediate dose no longer produced a significant decrease in activity and, although blood ethanol levels were significantly above baseline, they were lower than in animals tested at 15 min. It seems unlikely, however, that the slight decrease in blood levels 1 h after ethanol administration would account for the lack of effect on motor activity at this time point. Clearly, the differences in behavior were not due to a practice effect, since these animals were only used for a single ethanol test. Rather, these data pointed to the rapid development of tolerance to ethanol [24], and this was most evident with the intermediate dose of 1.0 g/kg. These results are reminiscent of previous work in which rapid tolerance occurred to the deficit in performance of a moving belt test [25]. With the highest dose, however, when
74
blood ethanol levels were also high, dramatic and significant decreases in motor activity were seen at both test intervals. Consistent with the ICSS data, there were no increases in locomotor activity. Both total activity (Fig. 2A1 and ambulation scores (Table 11were decreased with increasing concentrations of ethanol; animals also spent more time resting as tissue concentrations increased. A more detailed analysis of activity revealed that at 15 min. movement at high speed was reduced to a greater extent than that at low speed. Our results complement those of Kulig et al. [7] who examined more complex coordinated handlimb movements in rats after ethanol administration. The animals used for ICSS were highly trained whereas those tested for locomotor activity were given an opportunity for some habituation. We now have data showing that when these animals are habituated to the test environment for 30 min before injection, a low dose of ethanol (0.1 g/kg) can produce increases in motor activity [26]. The tissue analyses showed that ethanol was present in all areas studied approximately 30 and 75 min after its administration, and levels correlated with decreases in motor activity measures. The lack of any increases in locomotor activity may have been due to the injection-test intervals used here. Perhaps increases might have occurred if the animals were habituated for a longer time and tested immediately after administration of a low ethanol dose [26,27]. A similar case could be made for brain self-stimulation. Again, with our testing conditions, only decreases in leverpressing were observed and this was consistent with response-rate data in the auto-titration procedure [21]. It has been proposed that increases in locomotor activity are positively correlated with the reinforcing effect of ethanol [28]. Rats that drank ethanol showed an increase in motor activity with IP ethanol injecIn our study, there was close tion. correspondence between the changes in motor activity and the changes in ICSS rates. In both paradigms only dose-dependent decreases were observed, suggesting that under present conditions positive reinforcement would not be ob-
served; yet another example of the enigma surrounding ethanol’s effects on behavior and of the difficulty in demonstrating its reinforcing properties [29]. These same data, however, emphasize the possibility that the intoxicating properties of ethanol may result from its actions at many sites including those in the peripheral and central nervous system. Acknowledgment
We thank Ms. Matty Lane of the Central Pharmacy-Toxicology Laboratory for performing the enzymatic ethanol assays. References 1 D.C.Bird et al., Psychopharmacology,87 (1985) 414. 2 F.A. Holloway and D.R. Vardiman, Psychon. Sci., 24 (19711218. 3 G.D. Frye and G.R. Breese, Psychopharmacology,75 (1981) 372.
4 E.P. Riley et al., J. Stud. Alcohol, 37 (1976) 1535. 5 G.P. Hunt and D.H. Overstreet, Psychopharmacology, 55 (1977) 75.
6 J. Brick et al., Alcohol: Clin. Exp. Res., 8 (1984) 204. 7 B.M. Kulig et al., Toxicol. Appl. Pharmacol., 80 (1985) 1. 8 R. Smoothy and M.S. Berry, Psychopharmacology, 85 (1985) 57.
9 S.A. Lorens and S.M. Sainati. Life Sci., 23 (1978) 1359. 10 R.H. Carlson and R. Lydic, Psychopharmacology, 50 (1976) 61.
11 P. Vrtunski et al., Q.J. Stud. Alcohol, 34 (1973) 718. 12 13 14 15 16 17 18 19 20 21 22 23
24
R.W. Flanagan, Dis. Abstr. Int., 36 (1976) 4746-B. D.J. Magnuson and L.D. Reid, Bull. Psychon. Sot., 10 (1977) 364. A. Routtenberg, Ann. N.Y. Acad. Sci., 362 (1981) 60. P. De Witte and M.F. Bada, Exp. Neural., 82 (1983) 675. G.J. Schaefer and R.P. Michael, Psychopharmacology, 74 (1981) 17. G.J. Schaefer et al., Physiol. Behav., 29 (1982) 163. G.J. Schaefer et al., Physiol. Behav., 37 (1986) 181. L.J. Pellegrino et al., A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York, 1979. F. Lundquist, in: D. Glick (Ed.), Methods of Biochemical Analysis, Interscience, New York, 1957. G.J. Schaefer and R.P. Michael, Alcohol, 4 (1987) 209. G.J. Schaefer and R.P. Michael, Sot. Neurosci. Abstr.. 12 (1986) 53. B. Tabakoff, in: K. Eriksson et al. (Eds.), Animal Models in Alcohol Research, Academic Press, New York, 1980. J.M. Littleton, in: H. Rigter and J.C. Crabbe (Eds.),
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Alcohol Tolerance and Dependence, ElsevierlNorth Holland Biomedical Press, 1980. A.E. LeBlanc et al. Psychopharmacologia, 4lt1975143. G.J. Schaefer and R.P. Michael, Sot. Neurosci. Abstr., 13 (198’71339. G. Di Chiara and A. Imperato, Eur. J. Pharmacol., 115 (1985) 131.
28 29
M.B. Waller et al., Pharmacol. 11986161’7. B. Tabakoff and P.L. Hoffman, Oreland (Eds.1, Brain Reward Raven Press, New York, 1987.
Biochem.
Behav.,
in: J. Engel Systems and
24
and L. Abuse,