Acquisition and maintenance of intravenous cocaine self-administration in Lewis and Fischer inbred rat strains1

Brain Research 778 Ž1997. 418–429

Interactive report

Acquisition and maintenance of intravenous cocaine self-administration in Lewis and Fischer inbred rat strains 1 Therese A. Kosten

a, )

, Mindy J.D. Miserendino a , Colin N. Haile a , Jenine L. DeCaprio a , Peter I. Jatlow b , Eric J. Nestler a

a

b

Department of Psychiatry, Yale UniÕersity School of Medicine, and Connecticut Mental Health Center, New HaÕen, CT 06508, USA Department of Laboratory Medicine, Yale UniÕersity School of Medicine, and Connecticut Mental Health Center, New HaÕen, CT 06508, USA Accepted 16 September 1997

Abstract Lewis and Fischer inbred rat strains differ in behavioral and biochemical responses to psychoactive drugs: Lewis rats show greater behavioral responses to psychoactive drugs than Fischer rats and they fail to show biochemical adaptations in the mesolimbic dopamine system after chronic drug exposure, in contrast to Fischer and outbred rats. This suggests that Fischer and Lewis rats may differ in the initial, reinforcing effects of psychoactive drugs, but not in responses seen after the exposure that occurs with maintenance of drug-reinforced behavior. Thus, the present study tested whether these strains differ in acquisition or maintenance of intravenous cocaine self-administration. Acquisition of cocaine self-administration was examined in separate groups that were allowed 15 days to acquire the operant at one of three cocaine doses Ž0.25, 0.5, or 1.0 mgrkgrinfusion.. Compared to Fischer rats, Lewis rats acquired cocaine self-administration after fewer training trials and at lower doses. After maintenance, both strains showed characteristic extinction responding with saline substitution and dose-related responding to cocaine, although Fischer rats tended to show higher response rates. Finally, cocaine plasma levels, obtained after an intravenous cocaine infusion Ž1.0 mgrkg., showed no strain differences suggesting that the strain difference in acquisition was not due to cocaine pharmacokinetics. These strain differences in acquisition of cocaine self-administration may be related to reported strain differences in the mesolimbic dopamine system. Further, because acquisition of drug self-administration is an animal model of vulnerability to drug addiction, these inbred strains may be useful to study factors underlying such vulnerability. q 1997 Elsevier Science B.V. Keywords: Pharmacogenetics; Inbred strains; Operant responding; Cocaine plasma levels; Drug reinforcement; Vulnerability to drug abuse

1. Introduction Genetic factors may be involved in drug and alcohol addiction in humans w9,21,38,41x. How genetic factors contribute to psychoactive drug effects can be examined using inbred rat strains which, in contrast to outbred rats, provide a stable genotype and are a valuable tool to assess mechanisms related to drug-related behaviors w10,15,32,41x. Two inbred strains that differ in behavioral responses to psychoactive drugs are Lewis ŽLEW. and Fischer 344 ŽF344. rats. LEW rats, compared to F344 rats, show greater behavioral responses to several drugs across many behavioral procedures, including oral self-administration w14,44–47x, acquisition of intravenous morphine self-ad) Corresponding author: Tel.: q1 Ž203. 789-7090X353; Fax: q1 Ž203. 562-7079; E-mail: [email protected] 1 First published on the World Wide Web on 4 November 1997.

0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 2 0 5 - 5

ministration w1x, place conditioning w23,31x, locomotor sensitization w7,31x, and cannabinoid facilitation of intracranial self-stimulation w13x. However, neither strain readily acquires intravenous nicotine self-administration, in contrast to Long-Evans and Sprague-Dawley rats w42x. LEW and F344 rat strains also differ in properties of the mesolimbic dopamine ŽDA. system. This system consists of dopaminergic neurons in the ventral tegmental area ŽVTA. and their projections to the nucleus accumbens ŽNAc. and other forebrain structures and is an important neural substrate for the behavioral effects of psychoactive drugs w30,53x. LEW rats have lower basal extracellular DA metabolite levels in the NAc w7,43x and lower numbers of spontaneously active DA neurons in the VTA w34x, compared to F344 rats. Acute cocaine increases DA levels in both strains with LEW rats showing a more prolonged elevation w7,43x. LEW and F344 rats differ in biochemical characteristics of this system which resemble differences

T.A. Kosten et al.r Brain Research 778 (1997) 418–429

between drug-exposed versus drug-naive outbred rats w3,4,48x. Specifically, LEW rats show: Ž1. higher levels of tyrosine hydroxylase in the VTA; Ž2. lower levels of three neurofilament proteins in the VTA; Ž3. higher levels of adenylyl cyclase and cyclic AMP-dependent protein kinase activity in the NAc; and Ž4. lower levels of Gi a in the NAc, compared to F344 rats. Moreover, chronic morphine exposure regulates these proteins in outbred and F344 rats, but not in LEW rats w2,23,24x. The strain differences seen in the mesolimbic DA system, coupled with observations that chronic drug exposure regulates biochemical parameters in this system in F344 rats to levels seen in drug-naive LEW rats, lead to the following prediction: the initial behavioral responses to psychoactive drugs may differ between these two strains, but not those seen after prolonged drug exposure. The initial reinforcing effects of a drug can be compared between strains using acquisition of drug self-administration. In contrast, maintenance of self-administration reflects the reinforcing properties of a drug after drug exposure. The present study examined whether LEW and F344 inbred rat strains differ in acquisition or in maintenance of cocaine self-administration. Moreover, cocaine plasma levels were examined for potential strain differences in pharmacokinetics. 2. Materials and methods 2.1. General methods 2.1.1. Animals and housing Male LEW and F344 rats ŽHarlan, Indianapolis, IN., weighing 280–360 g, were used in these studies. Food ŽPurina chow. and tap water were available ad libitum. Rats were housed individually in hanging, wire-mesh cages in a temperature-controlled colony room with a 12:12 h lightrdark cycle Žlights on at 7:00 am. and were trained and tested between 8:00 and 11:00 am. The facilities were accredited by the American Association of the Accreditation of Laboratory Animal Care and all procedures were approved by the institutional Animal Care and Use Committee in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. 2.1.2. Surgery Rats were implanted with chronically indwelling catheters made of Silastic tubing using a method modified from Weeks w50x. Catheters were implanted into the right jugular vein under Na pentobarbital anesthesia ŽNembutol 50 mgrkg; Abbott Laboratory, Chicago IL.. The catheters were passed under the skin to exit at the nape of the neck through a cannula Ž22 gauge, curved metal tube. that was attached to the skull with jeweler’s screws and dental acrylic. A minimum of four days of recovery was allowed before initiation of the experiments. Catheters were flushed once daily with a heparin-saline solution and catheter

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patency was tested at the end of the experiment by the ability of a Brevital ŽLilly Co., Indiananopolis, IN. injection Ž0.2 ml of a 10 mgrml solution. to cause loss of consciousness within 1–2 s. 2.1.3. Drugs Cocaine HCl Žprovided by the National Institute on Drug Abuse, Research Triangle Institute, Research Triangle Park, NC. was prepared in sterile saline. In some cases, the three cocaine doses used were set by varying the infusion duration times to 5 s for 0.25 mgrkgrinfusion, 10 s for 0.5 mgrkgrinfusion, and 20 s for 1.0 mgrkgrinfusion using a cocaine unit dose of 0.5 mgrkgrinfusion per unit volume of 100 ml. This results in unit volumes per infusion of 50, 100, and 200 ml, respectively by dose. In other cases, the cocaine unit dose was varied so that the three cocaine doses were each delivered in unit volumes of 100 ml over 10 s. For the cocaine plasma level study, an intravenous cocaine infusion of 1.0 mgrkg was delivered over 10 s. 2.1.4. Apparatus Rats were placed in standard operant chambers ŽCoulbourn Instruments, Allentown, PA. that were modified so that the floor space was 10Y L = 7.5Y W. The chambers were housed in ventilated, sound-attenuating cubicles ŽCoulbourn Instruments. equipped with fans to mask outside noise. The catheterrcannula system of each animal was attached to a syringe pump system, which consists of an infusion pump ŽRazel Model A. with a 20 ml glass syringe, connected by a swivel ŽStoelting a1. with Teflon tubing. The swivel was connected to the cannula assembly with tygon tubing protected by a metal spring and secured to a screw mounted on the animal’s head. On one wall of each chamber there were two response levers each located 1 inch from the side wall above which were three ‘cue’ lights. Minimal downward pressure Žabout 25 g. on one lever Žactive. resulted in a programmed consequence, whereas depression of the other lever Žinactive. had no programmed consequences. One depression of the active lever resulted in a cocaine infusion ŽFixed ratio 1; FR1., followed by a 5 s time out period during which another depression of the lever did not result in a cocaine delivery. During the infusion, the cue lights were illuminated. The house lights were turned off during the entire infusion and time out periods. Experimental parameters Ži.e., schedule of reinforcement, time period, etc.. were programmed using a software package ŽCoulbourn Instruments. installed on an IBM clone computer and data were tabulated using a Coulbourn designed software system. 2.2. Experiment 1: comparison of LEW and F344 rats in maintenance of cocaine self-administration This experiment tested whether LEW and F344 rats differ in the maintenance of cocaine self-administration.

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This was assessed first by examining dose-related responding to cocaine using both within- and between-session dose–response tests. Additional between-session tests with varied infusion duration times were performed in order to validate procedures to be employed in Experiment 2. A further test of whether cocaine maintained self-administration behavior in the two strains was the examination of the pattern of extinction responding seen after saline substitution w54x. 2.2.1. Procedure Rats were trained initially to press a lever for food pellets Ž45 mg Bio-Serv, Inc; Frenchtown, NJ. under an FR1 schedule of reinforcement. After lever press behavior was acquired, nine LEW and 12 F344 rats were implanted with jugular catheters. In most cases, rats were then trained to lever press for a 1.0 mgrkgrinfusion of cocaine under an FR1 schedule of reinforcement during daily, 2 h sessions. The training dose was lowered to 0.5 mgrkgrinfusion subsequently. In the other cases, 0.5 mgrkgrinfusion was the sole training dose of cocaine. Once stable response rates were obtained with the final training dose of cocaine Žstandard deviation of the mean for three consecutive days was less than 20% of the mean., tests for cocaine dose-related responding to 0.25, 0.5, and 1.0 mgrkgrinfusion were run. These cocaine doses represent the descending limb of the cocaine dose response function wherein rate of responding is expected to decrease from 0.25 to 1.0 mgrkgrinfusion doses under an FR1 schedule of reinforcement. Several test sessions were performed, some of which were used to confirm the validity of obtaining dose–response data by varying infusion times and using a withinsession procedure. Previous studies, employing primates w52x and rats w17x, suggested that such procedures lead to comparable dose–response data. The present study attempts to confirm and extend these findings utilizing LEW and F344 inbred rat strains. The following test sessions were executed with the order mixed across animals. First, dose-related responding to each dose was determined using a within-session procedure modified from Emmett-Oglesby et al. w12x. This 2 h session consisted of four 30 min epochs. In the first 30 min epoch, the 0.5 mgrkg cocaine dose was delivered. During the next three 30 min epochs, 1.0, 0.5, and 0.25 mgrkgrinfusion doses were delivered under the FR1 schedule of reinforcement, with dose varied by changing the infusion times to 20 s, 10 s, and 5 s, respectively. Second, three 2 h tests were performed in which the cocaine dose delivered was held constant for the session and was determined using the same infusion times as employed in the within-session test. Third, two 2 h tests were performed in which the 0.25 and 1.0 mgrkgrinfusion doses were delivered by varying the cocaine stock solution and using 10 s infusion times. Data from all 2 h sessions were averaged across the final three 30 min epochs in order to compare to data obtained from the

within-session test. Finally, extinction sessions were run for two consecutive days in which saline was substituted for cocaine after one cocaine priming infusion. Then, cocaine self-administration was reinitiated for two days w54x. Patterns of responding were examined; extinction was said to occur if rate of responding was high during the first 30 min and decreased to minimal responding during the last 30 min epoch. This pattern was compared to the two comparable 30 min epochs for the two days previous to and to the two days following the extinction sessions during which cocaine Ž0.5 mgrkgrinfusion. was available. 2.2.2. Data analysis Numbers of self-administered cocaine infusions during the within-session test were compared across strains using a two-way ANOVA with main effect of strain and repeated measure effect of dose. Next, data obtained from the within-session test were compared to data obtained from the 2 h tests in which the infusion times were varied in the manner used for the within-session test. In addition, data obtained from 2 h tests in which the infusion times were varied were compared to data obtained from 2 h tests in which the infusion times were held constant at 10 s. For the latter two comparisons, two-way ANOVAs with main effect of strain and repeated measure effect of test session were used. Finally, data from the extinction test were analyzed by comparing the numbers of self-administered infusions during the first 30 min epoch to the numbers of infusions made during the last 30 min epoch on the days when cocaine was available compared to the days when saline was substituted for cocaine. Data from three phases were used: Ž1. the two days prior to saline substitution were averaged over days and used as the measure of cocaine maintenance responding; Ž2. the two days of saline substitution were averaged over days and used as the measure of extinction responding; and Ž3. the two days subsequent to saline substitution in which cocaine was again made available were averaged and used the measure of reinstatement responding. These data were analyzed using a 2 = 3 = 2 ANOVA representing the main effect of strain and the repeated measures of phase and epoch. The Greenhouse–Geisser adjustment was used for ANOVAs that employed repeated measures w51x. 2.3. Experiment 2: comparison of LEW and F344 rats in acquisition of cocaine self-administration This experiment assessed whether there are strain differences in acquisition of cocaine self-administration. Three cocaine dose training conditions were used in separate groups of rats, including the dose used for maintenance of intravenous cocaine self-administration in Experiment 1 Ž0.5 mgrkgrinfusion. as well as one higher Ž1.0 mgrkg. dose and one lower Ž0.25 mgrkg. dose. There were no differences in response rate seen when cocaine dose was

T.A. Kosten et al.r Brain Research 778 (1997) 418–429

altered by varying the stock concentration versus varying the infusion times in the previous experiment; therefore, cocaine dose was manipulated by altering infusion times in this experiment. 2.3.1. Procedure Fifty-six LEW and F344 rats were assigned to one of three cocaine dose training groups: 0.25 Ž n s 18rstrain., 0.5 Ž n s 22rstrain., or 1.0 Ž n s 16rstrain. mgrkgrinfusion. Rats were placed in the operant chamber during daily, 2 h sessions given five to seven days per week for 15 days. Rats were placed near the levers and given one priming injection of cocaine on each training day. No other shaping procedure was used. Numbers of active and inactive lever presses as well as numbers of self-administered cocaine infusions were tabulated for each session. 2.3.2. Data analyses Acquisition of cocaine self-administration was defined in the following way, based on Horger et al. w27x. The means and standard deviations of the number of inactive lever presses were determined within each cocaine dose training group for each day. These data were used to determine the 99% confidence interval of this number for each day. When the number of self-administered cocaine infusions was above the 99% confidence interval of the number of inactive lever presses for three consecutive days and remained at this level for at least 75% of the subsequent days, the animal was said to have acquired the operant. If an animal showed an abnormally high number of presses on the inactive lever Žat least three standard deviations above the mean., its datum was eliminated from the confidence interval analyses. The percentages of rats that acquired self-administration on each day were analyzed using life table analysis w22x to determine possible strain and cocaine dose training group effects. Data on numbers of active and inactive lever presses as well as number of self-administered cocaine infusions were averaged across five, 3-day blocks of the 15 day acquisition period using median as the measure of central tendency. This data reduction procedure was used for ease of presentation and to avoid violating the assumptions of repeated measures analysis of variance ŽANOVA. with the use of 15 repeated measure data points w51x. These data were analyzed using a three-way ANOVA with main effects of strain and cocaine dose training group with repeated measure effect of day. Further analyses evaluated possible strain and cocaine dose training group differences on the differential numbers of active versus inactive lever presses over days using a four-way ANOVA with main effects of strain, cocaine dose training group, and lever type, with repeated measure effect of day. This latter analysis was performed to further evaluate acquisition of cocaine self-administration behavior by assessing differential lever selection over time. The Greenhouse–Geisser adjustment was used on all repeated measure effects w51x.

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2.4. Experiment 3: comparison of LEW and F344 rats in cocaine plasma leÕels Cocaine plasma levels, obtained from intravenous blood samples after an intravenous infusion of cocaine, were assessed in LEW and F344 rats. This experiment was conducted to determine whether the strain differences in acquisition of intravenous cocaine self-administration seen in the previous experiment might be due to strain differences in cocaine pharmacokinetics given that previous reports are conflicting. Previous studies, which used IP cocaine administration and analyzed trunk blood, report either no strain differences in cocaine plasma levels w23x or that LEW rats have higher levels w7x. 2.4.1. Procedure Five LEW and F344 rats were implanted with jugular catheters. Using a modified procedure of Nobiletti et al. w35x, an intravenous infusion of cocaine Ž1 mgrkg; 10 s infusion time. was administered. The catheters were flushed immediately with 0.2 ml of heparinized saline to wash out residual cocaine in the catheter. Blood samples Žapproximately 1 ml. were withdrawn at 5, 15, and 30 min post-infusion on the first day. Two days later, the procedure was repeated and blood samples were withdrawn at 10 and 20 min post-infusion. The first 200–300 ml of each sample was discarded and the remainder of the blood sample was placed in grey-stoppered vacutainer tubes containing sufficient sodium fluoride to prevent enzymatic hydrolysis of cocaine. Plasma was separated immediately and stored at y708C until assays were performed. Cocaine was assayed by reversed-phase high pressure liquid chromatography w28x. 2.4.2. Data analysis Plasma levels of cocaine Žngrml. at the five time points were analyzed using a two-way ANOVA with main effect of strain and repeated measure of time. Additional pharmacokinetic parameters were determined using established noncompartmental calculations w18x and evaluated for statistical significance using t-tests for independent samples. These parameters included total systematic clearance ŽCL; lrminrkg., peak plasma concentration Ž Cmax ., and area under the time-concentration curve ŽAUC., which was determined using the linear trapezoidal rule and extrapolated to infinity.

3. Results 3.1. Experiment 1: comparison of LEW and F344 rats in maintenance of cocaine self-administration This experiment investigated whether LEW and F344 rats show differences in the reinforcing effects of cocaine by examining maintenance of intravenous self-administra-

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Fig. 1. Self-administration response records for one representative LEW rat and one representative F344 rat are presented as a function of test phase. The two lines per test phase represent the records from the first 30 min and from the last 30 min epochs of a 2 h session. Data presented in part A depict records obtained during a phase of maintenance of cocaine Ž0.5 mgrkgrinfusion. self-administration. Data presented in part B depict records obtained when saline was substituted for cocaine Ži.e., extinction.. Data presented in part C depict records obtained after maintenance of cocaine Ž0.5 mgrkgrinfusion. self-administration was reinitiated. Numbers presented in parentheses are the total number of self-administered infusions per time block. The characteristic well-spaced responding is seen for both the LEW and F344 rats under the FR1 schedule of reinforcement when cocaine is available ŽA and C.. When saline is substituted for cocaine, the characteristic increase in rates of responding is seen during the first 30 min epoch whereas rates of responding decrease during the last 30 min epoch for both the LEW and F344 rats ŽB..

tion behavior. This was accomplished by comparing the strains in dose-related responding to cocaine and in extinction responding with saline substitution and the subsequent reinitiation of responding when cocaine was made available again.

Eight of the original nine LEW rats trained initially in cocaine self-administration acquired the operant, and nine of the original 12 F344 rats acquired the operant. At least five rats per strain were run in each of the following tests of cocaine reinforcement. Cocaine Ž0.5 mgrkgrinfusion.

Table 1 Mean Ž" S.E.M.. numbers of active and inactive bar presses during two days each of cocaine maintenance Ž0.5 mgrkgrinfusion., saline substitution, and cocaine reinitiation by strain Phase

Cocaine Maintenance Saline Substitution Cocaine Reinitiation

Day

1 2 1 2 1 2

LEW Rats Ž n s 5.

F344 Rats Ž n s 5.

Active bar presses

Inactive bar presses

Active bar presses

Inactive bar presses

First 30 min

Last 30 min

First 30 min

Last 30 min

First 30 min

Last 30 min

First 30 min

Last 30 min

10.2 " 1.1 11.2 " 1.5 22.6 " 5.2 16.6 " 2.2 9.3 " 1.0 9.6 " 0.9

5.2 " 1.4 7.6 " 1.9 3.6 " 1.4 1.0 " 0.5 6.0 " 0.8 5.8 " 0.8

0.6 " 0.6 0"0 1 " 0.5 0.8 " 0.5 0.2 " 0.2 0.4 " 0.2

0"0 0"0 0.8 " 0.6 0.6 " 0.2 0"0 0"0

11.0 " 2.7 9.8 " 1.7 19.0 " 4.1 14.2 " 2.0 11.8 " 3.1 12.2 " 0.4

7.4 " 1.9 8.4 " 2.2 0.2 " 0.2 4.6 " 3.2 8.4 " 0.8 9.6 " 1.2

0"0 0"0 0"0 0.2 " 0.2 0"0 0"0

0"0 0.2 " 0.2 1.4 " 1.4 0.2 " 0.2 0"0 0.2 " 0.2

T.A. Kosten et al.r Brain Research 778 (1997) 418–429

maintained behavior in both LEW and F344 rats as shown in Fig. 1. This figure presents response records from one representative LEW rat and from one representative F344 rat. Data shown in Fig. 1A illustrate the number and pattern of responding during the first 30 min epoch and during the last 30 min epoch of a 2 hour session. As seen in Fig. 1A, both the LEW rat and the F344 rat showed the characteristic well-timed responding over the session. There were no strain differences in cocaine maintenance responding Že.g., numbers of self-administered cocaine infusions. across all LEW and F344 rats that had acquired the operant, as seen in Table 1 Ž P ) 0.10.. To examine whether cocaine was maintaining self-administration behavior, two days of extinction tests were performed in which saline was substituted for cocaine after one priming infusion Ž0.5 mgrkg. was given. Extinction responding is demonstrated typically by a burst of responding on the active lever followed by minimal responding. As seen in Fig. 1B, the response records from the representative LEW and F344 rats obtained on the first day of extinction show that the number of responses made on the active lever during the first 30 min epoch were quite large as compared to the small number of responses made during the last 30 min epoch. Following these saline substitution days, cocaine was again made available and responding on the active lever returned to the same level and characteristic pattern seen before saline substitution, as seen in Fig. 1C. The group differences in numbers of active lever presses between the two 30 min epochs were significant, as demonstrated by the significant effect of Epoch, F Ž1,22. s 91.87; P - 0.0001. Further, the differences between epochs differed by phase Že.g., cocaine maintenance, saline substitution, and reinitiation of cocaine maintenance., as supported by the significant interaction of Phase X Epoch, F Ž2,22. s 26.09; P - 0.0001. Specifically, response rates on the active lever were greater during the first 30 min epoch compared to the last 30 min epoch, as seen in Table 1. These epoch differences were significant for the cocaine maintenance phase, F Ž1,9. s 19.93; P - 0.005, and for the cocaine reinstatement phase, F Ž1,9. s 13.29; P - 0.01, which likely reflects a loading effect of the first 30 min epoch. However, the epoch difference was much greater during the saline substitution phase, F Ž1,9. s 76.52; P 0.0001, as seen in Table 1. There were no strain differences in extinction responding across all animals tested as seen in Table 1 Ž P ) 0.10.. Rates of responding on the inactive lever were minimal across phases for both strains, as seen in Table 1. However, greater numbers of inactive lever presses were seen during the saline substitution phase compared to the other two phases, as supported by the significant Phase effect, F Ž2,56. s 5.27; P - 0.01. This test demonstrates that cocaine is maintaining operant behavior in both LEW and F344 rats. A further test of whether cocaine maintained self-administration behavior was performed. Dose-related re-

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Fig. 2. Dose-related responding to cocaine is presented for LEW rats Žclosed symbols. and for F344 rats Žopen symbols.. Both strains show a decrease in responding as cocaine dose is increased, characteristic of responding under an FR1 schedule. F344 rats tended to show higher response rates compared to LEW rats. Data obtained from the within session test, in which dose was varied by altering infusion times Žcircles., do not differ from data obtained from between session tests in which dose was varied by altering concentrations Žsquares.. Additional between session tests of responding to doses of 0.25 and 1.0 mgrkgrinfusion were run in which dose was varied by altering infusion times in the same manner as was done in the within-session test Žtriangles.. These data do not differ from those obtained from the other between session tests.

sponding to cocaine was examined using both a within-session test w12x and between session tests. The results of these dose–response tests are presented in Fig. 2. As seen in Fig. 2, dose-related responding to cocaine was demonstrated in the within-session test, as supported by the significant Dose effect F Ž2,22. s 14.90; P - 0.002. The greatest numbers of self-administered cocaine infusions were seen with the 0.25 mgrkgrinfusion dose and the least numbers were seen with the 1.0 mgrkgrinfusion dose. This dose–response pattern was seen for both strains as supported by the lack of significance of the Strain by Dose interaction Ž P ) 0.10.. There were no strain differences in this test overall Ž P ) 0.1.; however, when each dose was analyzed separately, F344 rats showed significantly greater numbers of self-administered cocaine infusions at the 1.0 mgrkgrinfusion dose, F Ž1,12. s 9.62; P - 0.05. Dose-related responding to cocaine was also seen with the between-session tests when all doses were administered using 10 s infusion times, as demonstrated by the significant Dose effect, F Ž2,37. s 37.77; P - 0.0001. Again, the cocaine dose–response pattern of the greatest numbers of self-administered cocaine infusions were seen with the lowest cocaine dose and the least numbers were seen with the highest dose for both strains, as supported by the lack of significance of the Strain by Dose interaction Ž P ) 0.1.. There was a tendency for F344 rats to show greater numbers of self-administered cocaine infusions overall, F Ž1,37. s 3.32; P - 0.08. Finally, the numbers of inactive lever presses were minimal across tests for both strains; the means ranged from 0 to 0.6 Ž P X s ) 0.1. Dose-related responding to cocaine was seen with the between-session tests in which the infusion times were varied, as demonstrated by the significant Dose effect, F Ž2,41. s 45.40; P - 0.0001. Again, the cocaine dose–re-

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sponse pattern did not differ by strain Ž P ) 0.1., and there was a tendency for F344 rats to show greater numbers of self-administered cocaine infusions overall, F Ž1,41. s 3.98; P s 0.05. The data obtained from the within-session dose–response test do not differ from data obtained from the between-session tests using the varied infusion times. Incorporation of this Test condition factor into the ANOVA revealed no significant effect of Test condition nor any significant interactions Ž P X s ) 0.10.. Again, the Dose factor was highly significant, F Ž2,80. s 38.51; P - 0.0001 and, with this analysis, the Strain effect was significant, F Ž1,80. s 5.60; P - 0.05, but no interactions with Strain were significant. Finally, the numbers of self-administered cocaine infusions seen with the 0.25 mgrkgrinfusion dose did not differ whether the infusion time was 5 or 10 s nor did these numbers differ with the 1.0 mgrkgrinfusion dose under the 10 or 20 s infusion time conditions ŽPX s ) 0.1. as seen in Fig. 2. These tests demonstrate that dose-related responding occurs with cocaine and that response rates do not differ when the 0.25 and 1.0 mgrkg cocaine doses were presented using different infusion times. However, there was a trend for F344 rats to show greater numbers of self-administered cocaine infusions at the 0.25 mgrkgrinfusion dose under this analysis, F Ž1,25. s 4.18; P - 0.06, and a significant effect of Strain was seen with the 1.0 mgrkgrinfusion dose, F Ž1,27. s 8.5; P 0.008. Finally, the numbers of inactive lever presses were minimal across tests for both strains; the means ranged from 0 to 0.9 Ž P X s ) 0.1. 3.2. Experiment 2: strain comparisons of acquisition of cocaine self-administration This experiment investigated whether LEW and F344 rats show differences in the initial reinforcing effects of

cocaine by examining the acquisition of this behavior. This was accomplished by comparing numbers of training trials required to obtain self-administration in separate groups of rats from each strain that were trained at one of three doses Ž0.25, 0.5, and 1.0 mgrkgrinfusion.. Results demonstrate the LEW rats more readily acquire the operant after fewer training trials and at a lower dose. Of the original 56 F344 and 56 LEW rats assigned to this study, 45 F344 and 48 LEW rats completed the 15 days of acquisition training and had patent catheters Ži.e., tested positive in the Brevital test.. The eight LEW rats that did not complete the study included six with catheter problems and two deaths. The 11 F344 rats that did not complete the study included seven which had problems with their catheters and four deaths. The percentages of LEW and F344 rats that met acquisition criteria at each day of training are presented in Fig. 3 by cocaine dose training group. ŽNote that acquisition could occur on Day 3 at the earliest by definition as described in Section 2.1.. Acquisition more readily occurred for both strains at higher cocaine doses, as supported by the significant cocaine dose training group effect, x 2 Ž2. s 6.81; P - 0.01. As seen in Fig. 3B, a greater percentage of LEW rats acquired cocaine self-administration compared to F344 rats across the 15 days of training, as supported by the significant strain effect, x 2 Ž1. s 13.22; P - 0.001. The greatest strain difference was seen at the 0.5 mgrkgrinfusion cocaine training dose, with no strain differences seen at the other two training doses. LEW rats acquired cocaine self-administration after fewer training trials, compared to F344 rats, as seen in the life table presented as Fig. 3A. Specifically, the percentages of F344 rats acquiring cocaine self-administration were low until Day 12 at which time the group trained with 1.0 mgrkg cocaine began to show greater percent-

Fig. 3. This life table presents the percentages of rats that met acquisition criteria for cocaine self-administration across the 15 days of training for LEW Žclosed symbols. and F344 Žopen symbols. rats by cocaine dose training group in part A. Groups were trained with 0.25 Žsquares., 0.5 Žcircles., or 1.0 Žtriangles. mgrkgrinfusion in daily 2 h sessions. LEW rats more readily acquired the operant requiring fewer training trials, as compared to F344 rats, particularly at the 0.5 mgrkg dose. For both strains, acquisition occurred more readily at higher, as compared to lower, cocaine doses. Part B presents the percentages of LEW Žclosed bars. and F344 Žopen bars. rats that acquired cocaine self-administration by the end of the training period.

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Fig. 4. Numbers of active lever presses that result in cocaine infusions Žsquares. and inactive lever presses Žcircles. are presented for LEW Žclosed symbols. and F344 Žopen symbols. rats over the five 3-day blocks of acquisition training. Data in A are from the groups trained with 0.25 mgrkgrinfusion. Data in B are from groups trained with 0.5 mgrkg infusion and data in C are from groups trained with 1.0 mgrkgrinfusion. Overall, the numbers of active lever presses increased, whereas the numbers of inactive lever presses decreased over time. This differential lever selection occurred more quickly for LEW rats, compared to F344 rats, trained with 0.5 mgrkgrinfusion cocaine.

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ages of acquisition. In contrast, the percentages of LEW rats acquiring cocaine self-administration were greater earlier in the acquisition training period, especially for the 0.5 mgrkg dose group. In fact, the percentage of LEW rats trained with 0.5 mgrkg cocaine that acquired by the end of the study was similar to that of the 1.0 mgrkg cocaine dose group; however, acquisition occurred more quickly in the former cocaine training dose group. This is due to the high numbers of presses on the inactive lever for LEW rats in the 1.0 mgrkg group seen early in the acquisition period Žsee below.. The numbers of lever presses for self-administered cocaine infusions and inactive lever presses are presented over the five, 3-day blocks of acquisition training by strain and cocaine dose training group in Fig. 4. LEW rats exhibited greater numbers of lever presses overall compared to F344 rats and supported by the significant Strain effect, F Ž1,174. s 18.09; P - 0.0001. When each lever type was analyzed separately, both the numbers of lever presses for self-administered cocaine infusions and inactive lever presses were greater in LEW rats, compared to F344 rats, F X s Ž1,87. s 12.30; P - 0.001 Žcocaine infusions. and 5.83; P - 0.02 Žinactive lever presses.. Although the factor of cocaine dose training group did not affect numbers of lever presses Ž P X s ) 0.10., this tended to differ by strain, as supported by the trend for Strain = Dose, F Ž2,174. s 2.37; P - 0.10. Further, this interaction was significant for inactive lever presses, F Ž2,87. s 3.83; P - 0.02. The latter effect likely reflects that LEW rats trained with the highest cocaine dose showed very high inactive lever presses during the first two time blocks and that LEW rats trained with the lowest cocaine dose showed a greater number of inactive lever presses overall, compared to F344 rats. Differential lever selection, an indication of whether

Fig. 5. Plasma levels of cocaine Žngrml. assayed at five time points from 5 to 30 min after an intravenous cocaine infusion Ž1.0 mgrkg; 10 s duration. are presented for LEW Žclosed symbols. and F344 Žopen symbols. rats. Cocaine plasma levels decreased over time with no strain difference seen.

Table 2 Parameters of cocaine plasma level parameters in LEW and F344 rats

Cma x AUC Ž0–`, ng minrml. CL Žlrminrkg.

LEW

F344

P

296"27 8649"846

413"77 9980"1935

0.19 0.55

0.121"0.031

0.116"0.049

0.86

behavior was shaped by cocaine, was demonstrated by the significant effect of Lever type, F Ž1,174. s 39.41; P 0.0001. As seen in Fig. 4, numbers of active lever presses were greater than numbers of inactive lever presses. Moreover, active lever presses increased over time, whereas inactive lever presses decreased, as supported by the significant interaction of Lever type = Time Block, F Ž4,348. s 3.69; P - 0.01. This suggests that behavior was being shaped by cocaine. The numbers of inactive lever presses decreased over time blocks, as supported by the significant effect of Block, F Ž4,348. s 4.47; P - 0.005, particularly in the highest cocaine dose training groups, as supported by the significant Time Block = Dose interaction, F Ž8,348. s 2.50; P - 0.01. Cocaine dose training group also tended to have a differential effect on numbers of lever presses for cocaine infusions over time block, F Ž4,348. s 1.71; P - 0.10. The significant three-way interaction of Time Block = Strain = Dose, F Ž8,696. s 2.30; P - 0.02, may reflect that lever selection over time blocks differed by cocaine dose training group depending on the strain examined. 3.3. Experiment 3: strain comparisons of cocaine plasma leÕels Cocaine plasma levels were compared between LEW and F344 rats to investigate whether pharmacokinetic differences may explain the strain difference in acquisition of cocaine self-administration. The plasma concentrations Žngrml. of cocaine obtained between 5 and 30 min after an intravenous cocaine infusion Ž1 mgrkg; 10 s duration. are presented in Fig. 5. Cocaine plasma concentrations decreased over time, as supported by the significant time effect, F Ž4,24. s 21.97; P - 0.0001. The time course of the decrease in cocaine plasma levels seen in Fig. 5 shows that the half-life of cocaine is approximately 15 min; these levels are within the range reported previously w35x. There was no strain difference in plasma concentrations of cocaine and no strain differences were found with respect to the other pharmacokinetic parameters assessed, as seen in Table 2. These parameters included the markers of cocaine exposure, peak plasma concentration Ž Cmax . and area under the time–concentration curve ŽAUC., as well as total systemic clearance ŽCL., P X s ) 0.10.

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4. Discussion The present study shows that LEW rats more readily acquire intravenous cocaine self-administration compared to F344 rats: acquisition of this operant occurs with fewer training trials and at lower cocaine doses in LEW versus F344 rats. After acquisition, cocaine maintains behavior in both strains. The strain difference in acquisition is likely not due to cocaine pharmacokinetics because there are no strain differences in cocaine plasma levels after an intravenous cocaine infusion. Rather, this may reflect that LEW rats are more sensitive to the initial, reinforcing effects of cocaine than F344 rats. This strain difference in acquisition is consistent with previous research in which LEW rats, compared to F344 rats, exhibit greater behavioral responses to psychoactive drugs including procedures thought to reflect reinforcement Žsee Section 1.. For example, LEW rats, compared to F344 rats, show greater oral self-administration of cocaine w14x and ethanol w44x. In the latter study, however, LEW rats show greater self-administration of water making the conclusion that ethanol is more reinforcing in LEW rats, compared to F344 rats, tenuous. Moreover, orally administered cocaine fails to serve as a reinforcer in LEW rats under four different methods w5,6x. Further, oral drug self-administration may be compromised by potential strain differences in taste or bioavailability factors. Cocaine does serve as a potent reinforcer in rats via the intravenous route of administration w39,40x. Previous cocaine self-administration studies have utilized F344 rats, e.g., w11,12x demonstrating that this strain can acquire the behavior. The present study replicates this finding and extends it to LEW rats. This is supported by the data from the acquisition study showing that number of inactive lever presses decreases whereas number of active lever presses increases over time for both LEW and F344 rats, suggesting that behavior is shaped differentially due to the reinforcing effects of cocaine. Further, when saline is substituted for cocaine during maintenance, both strains show the characteristic pattern of extinction: a burst of responding followed by minimal responding, demonstrating that cocaine maintains the operant. Finally, dose-related responding to cocaine is seen during maintenance in both strains, although F344 rats tend to show higher response rates, compared to LEW rats. The results of the present study with cocaine are consistent with a recent study in which LEW rats more readily acquire intravenous morphine self-administration, compared to F344 rats w1x. Strain differences in acquisition of drug self-administration could be attributed to several factors, including differences in learning or performance abilities or to differences in sensitivity to the effects of cocaine in general or more specifically, to its reinforcing effects. Preliminary data suggest that there may be strain differences in learning or operant performance because LEW rats more readily acquire operant responding for food

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reinforcement w33x and then maintain higher rates of responding, compared to F344 rats ŽKosten et al., in preparation.. No strain differences are seen, however, in extinction responding or in responding under progressive ratio schedules of reinforcement for food. Whether these strain differences in acquisition and maintenance of operant responding for drug and food reinforcement reflect differences in learning, performance, or sensitivity to positive reinforcement effects in general cannot be distinguished at this time. F344 rats are capable of learning an association between an environmental stimulus and the cocaine stimulus, as evidenced from studies utilizing classical conditioning procedures. In contrast to LEW rats, which develop a conditioned place preference to cocaine, F344 rats show only a conditioned place avoidance to a moderately high dose of cocaine w31x. This result demonstrates the capacity of F344 rats to learn a response to cocaine, although it does not reflect the positive reinforcing effects of the drug. Further, using the same dose and treatment regimen, both strains exhibit conditioned taste aversion to cocaine w31x. However, in another report, LEW rats more readily acquire conditioned taste aversion to cocaine compared to F344 rats w19x. These authors suggest that LEW rats may be more sensitive to the noxious effects of cocaine compared to F344 rats. An explanation of strain differences in sensitivity to cocaine is consistent with much of the data obtained from the present study. That is, a decrease in sensitivity to cocaine for F344 rats, compared to LEW rats, could explain the need for a higher dose to acquire self-administration. Further, after cocaine maintains self-administration, F344 rats exhibit slightly higher levels of responding across doses which may reflect a somewhat lower sensitivity to cocaine, relative to LEW rats Ži.e., a relative rightward shift in the descending dose–response curve. or a decrease in potency or efficacy of cocaine. Yet, we found no strain differences in dose-related to cocaine after it maintains discrimination behavior ŽKosten et al., unpublished findings.. Examination of several behavioral effects across a wide range of cocaine doses may shed light on which factor or combination of factors may underlie the strain difference in acquisition of self-administration. The strain difference in acquisition of cocaine self-administration might reflect a greater locomotor stimulatory effect of cocaine in LEW rats. Indeed, lever press rate as a measure of reinforcement efficacy can be confounded by the activational effects of the reinforcer particularly under the fixed ratio schedule employed in the present experiment w29x. Yet, we do not find strain differences in the acute locomotor effects of cocaine w16,31x. However, sensitization to the locomotor effects of cocaine and methamphetamine occurs more readily in LEW rats, as compared to F344 rats w7,31x. In the acquisition study, a greater number of cocaine infusions are self-administered by LEW rats than F344 rats. In addition, the number of inactive

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lever presses is higher in LEW rats, compared to F344 rats, particularly during the initial training days for LEW rats in the highest cocaine dose training group. In fact, this high number of inactive lever presses in LEW rats interferes with acquisition of cocaine self-administration, because of the acquisition criterion of showing differential lever selection. The strain difference in acquisition of cocaine self-administration is likely not due to cocaine pharmacokinetics. In the present study, cocaine plasma levels do not differ between LEW and F344 rats from 5 to 30 min after an intravenous infusion of cocaine Ž1.0 mgrkg.. The plasma concentration sampling time of 30 minutes is approximately two half lives, which is less than optimal for confident determination of all pharmacokinetic parameters. However, following a single cocaine dose, area under the time–concentration curve, peak plasma concentration, and total systemic clearance do not differ between strains. This suggests that differences in cocaine exposure or cocaine accumulation following multiple doses are unlikely to explain the observed strain differences in acquisition of cocaine self-administration. We cannot rule out the possibility that strain differences may have been seen if other cocaine doses or different time periods were tested. The lack of strain difference in plasma cocaine levels in venous blood samples after an intravenous cocaine infusion in the present study is consistent with our previous report in which no strain differences were seen in cocaine levels in trunk blood after IP cocaine injections w23x. However, using procedures similar to Guitart et al., Camp et al. w7x report greater plasma cocaine levels in LEW rats. The discrepant findings in this latter study could be due, in part, to assessing plasma cocaine levels after IP versus after intravenous administration: there is much greater variability in plasma cocaine levels when cocaine is administered IP w35x. Enhanced acquisition of cocaine self-administration in LEW rats and the possible greater reinforcing effects of cocaine may be related to demonstrated strain differences in characteristics of the mesolimbic DA system Žsee Section 1.. Moreover, chronic drug exposure regulates specific proteins of the mesolimbic DA system in F344 and outbred rats to the levels seen in drug-naive LEW rats w3,4,23,24,48x. That cocaine maintains behavior in both strains to a fairly similar degree may be due to the biochemical adaptations that occurred in the mesolimbic DA system of the F344 rats as a result of the drug exposure that occurs during training. These data suggest that these inbred strains may provide a model to study genetic factors involved in vulnerability to drug abuse. Acquisition of drug self-administration has been shown to be enhanced by prior exposure to psychoactive drugs w25–27,36,37x, stressors w20,37x, and food-deprivation w49x, and attenuated by concurrent exposure to non-drug reinforcers w8x. Further research that explores the interactions between these environmental and genetic factors may pro-

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