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Relative reinforcing effects of different benzodiazepine doses for rhesus monkeys
Drug and Alcohol Dependence 68 (2002) 275 /283 www.elsevier.com/locate/drugalcdep
Relative reinforcing effects of different benzodiazepine doses for rhesus monkeys Thomas H. Gomez, John D. Roache, Richard A. Meisch * Substance Abuse Research Center, Department of Psychiatry and Behavioral Sciences, Medical School, University of Texas-Houston Health Science Center, 1300 Moursund Street, Houston, TX 77030-3497, USA Received 5 February 2002; received in revised form 1 July 2002; accepted 3 July 2002
Abstract The relative reinforcing effects of different doses of benzodiazepines were determined by giving rhesus monkeys concurrent access to different diazepam and midazolam concentrations. For each monkey a dose response function was obtained using three drug concentrations: low (L), intermediate (I), and high (H). The benzodiazepine and the water vehicle were concurrently available under independent fixed-ratio (FR) schedules. After establishing that each concentration was a reinforcer in comparison to vehicle, relative preference for the different concentrations was examined by making pairs of concentrations concurrently available under independent FR schedules. Three pairs were studied (H vs. L, H vs. I, and I vs. L). With both drugs, higher concentrations maintained greater response rates than lower concentrations. Thus, relative reinforcing effects increased with dose. These findings are similar to those obtained with other reinforcing drugs and provide further evidence that benzodiazepines share significant characteristics with other drug reinforcers. Importantly, absolute response rates (responses per session) obtained when only one drug dose was present were not reliably predictive of subsequent preferences for the dose. Both benzodiazepines served as effective reinforcers in that consistent levels of responding were maintained across doses and above vehicle levels under concurrent FR 32 schedules. As with other reinforcing drugs, the reinforcing effects of benzodiazepines increase with increases in dose over a broad range of values. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Benzodiazepines; Self-administration; Reinforcing effects; Concurrent schedules; Dose preference; Oral route; Rhesus monkeys
1. Introduction Benzodiazepines are widely prescribed drugs that are relatively safe and effective medications for a number of medical problems (Hollister et al., 1993). Benzodiazepines are also occasionally abused (Woods et al., 1992), and most abused drugs serve as reinforcers for laboratory animals (Johanson and Balster, 1978). However, in studies with laboratory animals it has been difficult to consistently demonstrate that benzodiazepines can act as effective reinforcers (for a review of benzodiazepine self-administration studies, see Griffiths and Weerts, 1997). Most studies report that only some of the animals in the study will self-administer benzodiazepines, or that the self-administration occurs only under a narrow set
* Corresponding author. Tel.: /1-713-500-2863; fax: /1-713-5002849 E-mail address: [email protected] (R.A. Meisch).
of conditions. In contrast, in a study with squirrel monkeys intravenous delivery of midazolam maintained high and consistent rates of responding (Munzar et al., 2001). Also, in two studies from our laboratory, orally delivered benzodiazepines served as effective reinforcers for rhesus monkeys when taken by the oral route, as they maintained high and consistent response rates over a range of concentrations and schedule sizes (Gomez et al., 2002; Stewart et al., 1994). With most reinforcing drugs, relative reinforcing effects increase with increases in dose (Meisch, 2000). If benzodiazepines share important features with other reinforcing drugs, then their relative reinforcing effects should also increase as the dose becomes larger. In the absence of specific training procedures, it has been difficult to demonstrate that orally delivered drugs will serve as reinforcers. The three principal problems in establishing orally delivered drugs as reinforcers have been the aversive taste of most drug solutions, the
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consumption of only small volumes of drug solution, and the delay between the behavior of drinking the drug solution and the onset of CNS effects. Procedures have been developed that result in the establishment of orally delivered drugs as reinforcers for laboratory animals (Meisch, 2001). For example, the potent opioid etonitazene can come to serve as a reinforcer by adding increasing amounts of the drug to a dilute ethanol solution and subsequently decreasing the ethanol concentration to zero (Meisch, 1995). With rhesus monkeys, most types of abused drugs can now be established as reinforcers via the oral route (Meisch, 2001). In particular, it is now possible to obtain behavior that is maintained by oral delivery of the benzodiazepines midazolam, alprazolam, triazolam, and diazepam (Gomez et al., 2002; Stewart et al., 1994). With orally self-administered benzodiazepines, the relation between dose and response rate is usually an inverted U-shaped function (Stewart et al., 1994). The ascending portion of the dose response function is attributed to increases in reinforcing effects with increases in drug dose. However, there is controversy regarding the relation between dose and reinforcing effects on the descending portion of the dose response function, and several explanations have been put forth (Bickel et al., 2000). One strategy for further examination of the entire dose-response function is to use choice procedures to compare preferences for different drug concentrations. In earlier studies with rhesus monkeys, a marked preference was established for benzodiazepines over their vehicle (Gomez et al., 2002; Stewart et al., 1994). The purpose of the present study was to go beyond comparisons of drug and vehicle, and instead examine choices between different drug concentrations to determine their relative reinforcing effects.
2. Materials and methods 2.1. Subjects The subjects were five adult male rhesus monkeys (Macaca mulatta). All of the monkeys had extensive experience with oral drug self-administration. Four (MP1, M-PL, M-GD, M-LA) of the monkeys had histories of ethanol and pentobarbital self-administration. M-TK had experience with ethanol self-administration, and both M-P1 and M-TK had served in an earlier benzodiazepine self-administration study (Stewart et al., 1994). All five monkeys were subjects in a more recent benzodiazepine self-administration study (Gomez et al., 2002). The monkeys were housed individually in their chambers in a climate controlled room (:/22 8C) in which a 12-h light and dark cycle was in effect (lights on at 7:00 AM). At the beginning of the study, the weights of the five monkeys were as follows: M-GD, 7.5
kg; M-LA, 9.1 kg; M-P1, 6.8 kg; M-PL, 9.3 kg; and MTK, 7.8 kg. Subjects were maintained at 85 /90% of their normal weights through daily feeding with a measured amount of commercially available chow (Lab DietTM High Protein Monkey Diet #5045, PMI Nutrition International, Brentwood, MO), plus fresh fruit and vegetables. The amount of food was sufficient to maintain stable body weights, and the monkeys’ health and appearance were good. Food restriction has been shown to enhance drug self-administration (Carroll and Meisch, 1984), prevent obesity (Meisch and Lemaire, 1989), and increase life span and general health (Masoro, 1985; Pugh et al., 1999). Animal care was in accordance with the regulations of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (1996), and all procedures were approved by the Institutional Animal Use and Care Committee of the University of Texas Houston Health Science Center. 2.2. Apparatus Each subject was individually housed 24 h a day in a stainless-steel primate cage (Lab Products, Sanford, DE), which also served as the experimental chamber. Each cage had three solid walls and one barred wall. Cage dimensions were 76 /102/81 cm3. A liquiddelivery apparatus panel was attached to the outside of one side wall, and spouts and stimulus lights protruded into the cage through holes cut in that wall. Attached to the back of the apparatus panel was a Tshaped bar; on each limb of this bar was fastened a stainless-steel reservoir covered with a lid. Liquids contained in each reservoir passed through polyethylene tubing to a solenoid-operated valve at the rear of one of the two brass spouts. These spouts (1.2 cm outside diameter, 0.2 cm inside diameter) extended 2 cm into the cage, 64 cm above the floor and 15.5 cm either side of the midline. The spouts were embedded in PlexiglasTM disks that covered the 7-cm diameter holes in the cage wall through which they entered. At each spout, two 1.1 W lights, one located 2.5 cm on either side of the spout and visible through the PlexiglasTM, were aligned diagonally; these ‘spout lights’ were capped with green translucent lenses. Another two 1.1 W spout lights, one located 2.5 cm on either side of the spout, were aligned on the opposite diagonal, and were capped with white translucent lenses. Thus, each spout was in the center of a square pattern of four spout lights, two green and two white. The electronic components for the drinkometer circuit were housed in an enclosure at the rear of the spout. The liquid-delivery apparatus has been described extensively elsewhere (Gieske, 1978; Henningfield and Meisch, 1976). A 2.5 cm (diameter), cluster of green light-emitting diodes was located 11.5 cm directly above each brass spout. The programming of experimental
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events and the recording of behavior were accomplished with a DEC PDP-11 computer and SKED† software (State Systems II, Kalamazoo, MI). This equipment was located in a room near the rooms containing the experimental chambers. 2.3. Procedure 2.3.1. During sessions Experimental sessions were 3 h in length (from 1100 to 1400 h) and were conducted 7 days per week. The stimulus lights above each spout blinked at a rate of 10 Hz. Identical discriminative stimuli were used for both spouts to control for differential responding that might otherwise result from the presence of dissimilar exteroceptive visual stimuli. Each mouth contact with a spout illuminated the green-lensed pair of spout lights for the duration of the response. Deliveries of liquids were contingent upon completion of a fixed-ratio schedule. The final response in the schedule requirement initiated the liquid flow of :/0.65 ml of the appropriate solution. To further reduce the possible differential responding that might be caused by a monkey’s preference for a particular spout, the drug solution and vehicle were alternated between spouts each session. 2.3.2. Between sessions A timeout period was in effect during the hour immediately before the session (1000 /1100 h). During this period the number of water deliveries and the volume of water consumed since the last experimental session were recorded and liquids appropriate for the next session were placed in the two reservoirs. Each system was primed to ensure that the appropriate solution was present on the first delivery. Liquid volumes were measured after flushing to obtain the exact volume in the reservoirs at each session’s onset. For 1 h immediately following the session (1400 /1500 h) another timeout period was in effect during which data from the session were recorded and water was placed into one reservoir and flushed through the tubing to the spout. Water was then available under a fixed-ratio (FR) 1 schedule from one spout from 1500 until 1600 h. A final timeout period was in effect from 1600 until 1700, at the beginning of which the monkeys’ maintenance feeding was placed in the food hopper attached to the cage. Finally, from 1700 until 1000 h of the next day, water was available under a FR 1 schedule from one spout. The spout from which water was available between sessions alternated every other day. Between sessions, water availability was signaled by steady illumination of the appropriate green stimulus light, and each mouth contact was signaled by illumination of the white-lensed pair of spout lights.
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2.3.3. Establishment of benzodiazepine reinforcing effects: ethanol fading procedure Initially, midazolam or diazepam was established as an orally delivered reinforcer by use of an ethanol fading procedure. In the first phase, increasing concentrations of drug were added across sessions to a 1 or 2% (w/v) ethanol solution that was already serving as a reinforcer. Each concentration was present for at least six sessions and until the number of responses was stable. The concentration was increased until there were clear signs of pharmacological effects such as ataxia, a slumped posture, muscle relaxation, and diminished motor activity. In the next phase the ethanol concentration was decreased in steps until it was absent. A successful outcome was the persistence of drug self-administration at levels greater than vehicle values. For two monkeys, M-P1 and M-TK, the specific steps in the fading procedure have been published (Stewart et al., 1994). The procedures used with the other monkeys were similar and have been described (Gomez et al., 2002). 2.3.4. Concentration response functions After the drug had been established as a reinforcer, drug concentrations were presented in a descending sequence with water concurrently available from the alternate spout. After the block of sessions at the lowest concentration, a second determination was made at the highest concentration. On changing from the lowest to the highest concentration, two sessions at the intermediate concentration were conducted to avoid any disruption that might occur with an abrupt transition from the lowest to the highest concentration. Changes from one concentration to another were made only after behavior was stable for six sessions, without an upward or downward trend in the number of deliveries for each liquid. The FR size for each monkey was the FR value that resulted in clear differences between drug and vehicle maintained responding. The number of deliveries and intake of drugs (mg of drug/kg of body weight/3-h session) for the last six sessions of stable behavior were expressed as the mean9/SEM. 2.3.5. Comparisons between concentrations With midazolam and diazepam three comparisons were conducted under concurrent FR schedules in a fixed sequence: (1) highest concentration versus the lowest concentration, (2) highest concentration versus the intermediate concentration, and (3) intermediate concentration versus the lowest concentration. Following the comparisons of the intermediate versus lowest concentrations, a second determination was made with two pairs of concentrations, and these were presented in a sequence that counterbalanced the first two comparisons. Thus, the fourth comparison repeated the second comparison (highest concentration versus the intermediate concentration), and the last comparison repeated the
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first comparison (highest concentration versus lowest concentration). Changes from one concentration to another were made after behavior was stable for six sessions, that is, without an upward or downward trend in the number of deliveries for each liquid. With two monkeys, M-LA and M-GD, the FR size was increased during the comparisons of diazepam concentrations in order to produce a clear difference in response rates maintained by the two concentrations. Additional choice comparisons between concentrations were conducted with midazolam. Two monkeys (M-TK and MP1) served as subjects. The pairs of concentrations were studied in the sequence: 0 vs. 0.025; 0.025 vs. 0.05; 0.05 vs. 0.1; 0.1 vs. 0.2; 0.2 vs. 0.4. Subsequently, the concentration pairs were tested a second time in a sequence that was counterbalanced: 0.1 vs. 0.2; 0.05 vs. 0.1; 0.025 vs. 0.05; 0.025 vs. 0. 2.4. Drugs Concentrated stock solutions (0.8 mg/ml) of diazepam and midazolam were prepared weekly in dilute HCl and refrigerated. Because of the limited solubility of benzodiazepines in water, dilute acid was added to form the HCl salt. The concentrations of HCl used were: 0.01 N, diazepam, and 0.0025 N, midazolam. Daily working solutions were prepared at room temperature by appropriate dilutions of the stock solution with deionized water approximately 2 h before use. Freebases of benzodiazepines act as a buffer; therefore, for each drug the pH remained fairly constant across a range of concentrations (:/4.2 for midazolam and 3.2 for diazepam). The stability of the acidic benzodiazepine stock solutions was verified by gas chromatography. 2.5. Statistical analysis To assess the relation between relative and absolute response rates a Spearman coefficient of rank correlation was calculated separately for midazolam and diazepam. Two ranks were assigned to each drug concentration based on the absolute and relative response rate maintained by that concentration.
3. Results
Fig. 1. Deliveries of midazolam and the water vehicle as a function of drug concentration for three monkeys, M-P1, M-TK, and M-GD. Circles are drug deliveries, and triangles are vehicle deliveries. Each point represents the mean of 6 consecutive sessions of stable behavior, and the brackets show the standard error of the mean. Absence of brackets indicates that they fell within the area of the plotted symbol. At the 0.4 mg/ml concentration, the points to the right are the results of the initial block of sessions at 0.4 mg/ml, and the adjacent points are the results of a second determination conducted after completion of the block of sessions at the lowest concentration. The points giving the results of the second determination at 4.0 mg/ml were shifted slightly to the left for clarity.
except for M-GD, where the retest value was lower. Drug intake (mg of drug/kg of body weight/3-h session) increased as concentration increased (Table 1).
3.1. Midazolam: concentration response function 3.2. Midazolam: comparisons between concentrations Fig. 1 shows that deliveries of midazolam systematically exceeded deliveries of the water vehicle. In most cases the range of drug deliveries was above the range of water deliveries, and thus the concentrations were serving as reinforcers. When a second determination was made at the 0.4 mg/ml concentration, the values for both drug and water were similar to the initial values
In the choice phase the three drug concentrations were studied in pairs, under concurrent FR schedules in a counter-balanced sequence. Fig. 2 shows that the higher concentration was preferred to the lower concentration, and usually the preference was large. However, in two of the 15 comparisons, the higher
T.H. Gomez et al. / Drug and Alcohol Dependence 68 (2002) 275 /283 Table 1 Midazolam intake (mg/kg/3-h session) as a function of concentration in three rhesus monkeys M-GD
M-P1
M-TK
mg/ml
Intake
mg/ml
Intake
mg/ml
Intake
0.4a 0.2 0.1 0.4
1.52 0.61 0.24 1.48
0.4 0.1 0.025 0.4
2.16 1.27 0.20 2.12
0.4 0.1 0.025 0.4
1.57 0.55 0.17 1.67
a
Concentrations are listed in the order in which they were studied.
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ence for one concentration over another when the 0.2 and 0.1 mg/ml concentrations were studied. With monkeys M-TK and M-P1 comparisons of midazolam concentrations were also conducted by presenting concentrations in an ascending and then descending sequence. For each monkey the higher concentration was preferred to the lower concentration in 8 of 9 comparisons (data not shown). With each monkey a lack of preference was seen at one of the two comparisons of the lowest two concentrations, 0.05 and 0.025 mg/ml. 3.3. Relation between response rates when only one midazolam concentration was present to response rates when two concentrations were present There was no reliable correspondence between rates of drug-maintained behavior obtained in the concentration response series and the concentration choice series. For example, in the choice phase, the most preferred concentration was 0.4 mg/ml (Fig. 2). However, for two monkeys (M-P1 and M-TK), the 0.4 mg/ml concentration maintained lower response rates than did the 0.025 and 0.1 mg/ml concentrations when the concentrations were presented sequentially (Fig. 1). Additionally, with monkey M-TK the lowest concentration, 0.025 mg/ml, supported higher rates than the 0.1 and 0.4 mg/ml concentrations when concentrations were presented sequentially (Fig. 1). However, it was the least preferred concentration in the choice series (Fig. 2). The Spearman coefficient of rank correlation relating absolute and relative rate was /0.244 (P /0.5). 3.4. Diazepam: concentration response functions Fig. 3 shows that deliveries of a range of diazepam concentrations consistently exceeded deliveries of water and that the differences between drug and water values usually were large. With three of the four monkeys, a second determination was made at the 0.4 mg/ml concentration and the values were similar to the initial values. Drug intake increased with increases in concentration (Table 2). 3.5. Diazepam: comparisons between concentrations
Fig. 2. Deliveries of midazolam solutions (mg/ml) under conditions of concurrent access to two drug solutions for three monkeys. Each bar represents the mean of six consecutive sessions of stable behavior, and the brackets show the standard error of the mean. Note that in most comparisons the higher concentration was preferred.
concentration was not preferred. For M-P1 the initial comparison of the 0.4 and 0.1 mg/ml concentrations revealed no preference; however, the second comparison of these concentrations showed a clear preference for the higher concentration. For M-GD there was no prefer-
When pairs of diazepam concentrations were studied, the higher concentration was preferred in 19 of 20 comparisons (Fig. 4). For two of the monkeys (M-LA and M-TK) preference for the higher concentration was uniformly large. With a third monkey (M-GD) there was a consistent preference for the higher concentrations but the differences were less than seen with M-LA and M-TK. Although M-PL preferred 0.2 and 0.4 mg/ml to the lowest concentration, 0.1 mg/ml, the 0.4 mg/ml concentration was slightly preferred over the 0.2 mg/ml
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Fig. 3. Deliveries of diazepam and the water vehicle as a function of drug concentration for four monkeys, M-LA, M-GD, M-PL, and M-TK. Circles are drug deliveries, and triangles are vehicle deliveries. Each point represents the mean of 6 consecutive sessions of stable behavior, and the brackets show the standard error of the mean. Absence of brackets indicates that they fell within the area of the plotted symbol. At the 0.4 mg/ml concentration, the points to the right are the results of the initial block of sessions at 0.4 mg/ml, and the adjacent points are the results of a second determination conducted after completion of the block of sessions at the lowest concentration. The points giving the results of the second determination at 4.0 mg/ml were shifted slightly to the left for clarity.
concentration on the first comparison and was not preferred on the second comparison (Fig. 4). 3.6. Relation between response rates when only one diazepam concentration was present to response rates when two concentrations were present Across the four monkeys, different diazepam concentration response functions were seen (Fig. 3). However, all four monkeys had similar patterns of preference in that higher concentrations were preferred to lower concentrations (Fig. 4). Thus, as was found with
midazolam, there was not a reliable correspondence between response rate when a single concentration was present and response rate when two concentrations were available concurrently. The Spearman coefficient of rank correlation relating absolute and relative rate was 0.272 (P /0.35). 3.7. Time course and pattern of responding As in other oral drug self-administration studies the highest rate of responding was at the beginning of the session (Meisch, 2001). Subsequently, following a pause,
Table 2 Diazepam intake (mg/kg/3-h session) as a function of concentration in four rhesus monkeys M-GD mg/ml a
0.4 0.2 0.1 0.4
a
M-LA
M-PL
M-TK
Intake
mg/ml
Intake
mg/ml
Intake
mg/ml
Intake
1.74 1.34 1.09 2.29
0.4 0.2 0.1 0.4
4.84 1.91 0.35 4.89
0.4 0.2 0.1 0.4
1.94 1.38 0.70 1.83
0.4 0.1 0.025 0.4
3.07 0.90 0.15 2.99
Concentrations are listed in the order in which they were studied.
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Fig. 4. Deliveries of diazepam solutions (mg/ml) under conditions of concurrent access to two solutions for four monkeys. Each bar represents the mean of six consecutive sessions of stable behavior, and the brackets show the standard error of the mean. Several comparisons were conducted at FR 16 as indicated in the figure. Note that in most comparisons the higher concentration was preferred.
there were one or more bouts later in the session. The pattern was characteristic of responding seen with FR schedules. At high drug concentrations signs of intoxication were observed, such as decreased motor activity, slumping, ataxia, and incoordination.
4. Discussion The principal finding was that higher concentrations of two benzodiazepines were preferred to lower concentrations. Thus, over the range of values studied, reinforcing effects increased with increases in concentration. These findings are consistent with many studies conducted with the oral and IV routes and with a wide range of abused drugs. Such studies have used either measures of choice or measures of relative persistence of responding across increases in schedule size (Meisch, 2000). In spite of the many studies demonstrating increases in reinforcing effects with increases in drug dose, some investigators maintain that reinforcing effects do not increase directly with drug dose (Norman and Tsibulsky, 2001). Their reasoning is based on the false premise that absolute response rate directly reflects the magnitude of reinforcing effects. They note that, in many cases, absolute response rates go down with increases in drug dose and therefore conclude that higher doses produce less, not more, reinforcement. In
the present study, absolute response rates obtained when each concentration was studied alone, were not predictive of preferences measured under conditions of choice. For example, Figs. 1 and 3 show different dose response functions for each monkey, with increasing, decreasing, and inverted U-shape patterns. These absolute response rates clearly did not correspond with preferences for higher concentrations observed with concurrent access to two concentrations. Similar results were obtained in a study of the reinforcing effects of cocaine /methadone combinations and the component drugs (Wang et al., 2001). In that study drug combinations and the component drugs were studied under both sequential and concurrent access. Under sequential access, response rates maintained by the combination and the component drugs were often similar. In contrast, under concurrent access response rates for the drug combinations were higher than rates for the component drugs. Thus, in both studies absolute response rates were not a reliable indicator of relative reinforcing effects. The findings with benzodiazepines are congruent with the results of other oral self-administration studies that examined choices between either drug concentrations or drug volumes. Such studies have been conducted with cocaine (Meisch and Stewart, 1995), ethanol (Stewart et al., 2002), methadone (Meisch et al., 1996), pentobarbital (Meisch et al., 1992; Meisch and Lemaire, 1988, 1989; Meisch and Spiga, 1998), and phencyclidine
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(Carroll, 1987). In all cases, higher doses were preferred to lower doses. Choices between drug doses also have been examined in IV self-administration studies, and the results reveal a preference for larger doses over lower doses (Iglauer et al., 1975; Iglauer and Woods, 1974; Johanson and Schuster, 1975; Manzardo et al., 2001; Woolverton and Johanson, 1984). Thus, the results of studies of choice between different drug doses show that over a broad range of values, reinforcing effects increase with increases in dose. That the reinforcing effects of benzodiazepines increase as a function of dose is further evidence that the benzodiazepines share important features with other reinforcing drugs. What remains to be determined is why the benzodiazepines did not function as effective reinforcers in most self-administration studies. An understanding of the conditions under which benzodiazepines do and do not serve as reinforcers will aid our comprehension of the circumstances under which these drugs might be abused. Choice between drug and vehicle (or placebo) is a powerful technique for assessing drug reinforcing effects (Spiga and Roache, 1997), and the present study demonstrates that choice between two concurrently available drug doses is an effective procedure for determining relative reinforcing effects of drug doses.
Acknowledgements This study was supported by NIDA grant DA 08913. Richard A. Meisch is the recipient of a NIDA Research Scientist Award (DA 00159). We thank Dr Howard M. Rhoades for help with the statistical analysis and Mr. Oscar Brown for his professional animal care.
References Bickel, W.K., Marsch, L.A., Carroll, M.E., 2000. Deconstructing relative reinforcing efficacy and situating the measures of reinforcement within behavioral economics: a theoretical proposal. Psychopharmacology 153, 44 /56. Carroll, M.E., 1987. Concurrent access to two concentrations of orally delivered phencyclidine: effects of feeding conditions. J. Exp. Anal. Behav. 47, 347 /362. Carroll, M.E., Meisch, R.A., 1984. Increased drug-reinforced behavior due to food deprivation. In: Thompson, T., Dews, P.B., Barrett, J.E. (Eds.), Advances in Behavioral Pharmacology, vol. 4. Academic Press, Orlando, FL, pp. 47 /88. Gieske, D., 1978. Integrated drinking device for monkeys. Reports from the Research Laboratories of the Department of Psychiatry, University of Minnesota, Minneapolis, MN (PR-78-1). Gomez, T.H., Roache, J.D., Meisch, R.A., 2002. Orally delivered alprazolam, diazepam, and triazolam as reinforcers in rhesus monkeys. Psychopharmacology 161, 86 /94. Griffiths, R.R., Weerts, E.M., 1997. Benzodiazepine self-administration in humans and laboratory animals */implications for problems of long-term use and abuse. Psychopharmacology 134, 1 /37.
Henningfield, J.E., Meisch, R.A., 1976. Drinking device for rhesus monkeys. Pharmacol. Biochem. Behav. 4, 609 /610. Hollister, L.E., Muller-Oerlinghausen, B., Rickels, K., Shader, R.I., 1993. Clinical uses of benzodiazepines. J. Clin. Psychopharmacol. 13, 1S /169S. Iglauer, C., Llewellyn, M.E., Woods, J.H., 1975. Concurrent schedules of cocaine injection in rhesus monkeys: dose variations under independent and non-independent variable-interval procedures. Pharmacol. Rev. 27, 367 /383. Iglauer, C., Woods, J.H., 1974. Concurrent performances: reinforcement by different doses of intravenous cocaine in rhesus monkeys. J. Exp. Anal. Behav. 22, 179 /196. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996. Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC. Johanson, C.E., Balster, R.L., 1978. A summary of the results of a drug self-administration study using substitution procedures in rhesus monkeys. Bull. Narc. 30, 43 /54. Johanson, C.E., Schuster, C.R., 1975. A choice procedure for drug reinforcers: cocaine and methylphenidate in the rhesus monkey. J. Pharmacol. Exp. Ther. 193, 676 /688. Manzardo, A.M., Del Rio, J.A., Stein, L., Belluzzi, J.D., 2001. Rats choose cocaine over dopamine agonists in a two-lever self-administration preference test. Pharmacol. Biochem. Behav. 70, 257 /265. Masoro, E.J., 1985. Nutrition and aging */a critical review. J. Nutr. 115, 842 /848. Meisch, R.A., 1995. Oral self-administration of etonitazene in rhesus monkeys: use of a fading procedure to establish etonitazene as a reinforcer. Pharmacol. Biochem. Behav. 50, 571 /580. Meisch, R.A., 2000. Relative persistence of behavior: a fundamental measure of relative reinforcing effects. Exp. Clin. Psychopharm. 8, 333 /349. Meisch, R.A., 2001. Oral drug self-administration: an overview of laboratory animal studies. Alcohol 24, 117 /128. Meisch, R.A., Lemaire, G.A., 1988. Oral self-administration of pentobarbital by rhesus monkeys: relative reinforcing effects under concurrent fixed-ratio schedules. J. Exp. Anal. Behav. 50, 75 /86. Meisch, R.A., Lemaire, G.A., 1989. Oral self-administration of pentobarbital by rhesus monkeys: maintenance of behavior by different concurrently available volumes of drug solution. J. Exp. Anal. Behav. 52, 111 /126. Meisch, R.A., Lemaire, G.A., Cutrell, E.B., 1992. Oral self-administration of pentobarbital by rhesus monkeys: relative reinforcing effects under concurrent signalled differential-reinforcement-oflow-rates schedules. Drug Alcohol Depend. 30, 215 /225. Meisch, R.A., Spiga, R., 1998. Matching under nonindependent variable-ratio schedules of drug reinforcement. J. Exp. Anal. Behav. 70, 23 /34. Meisch, R.A., Stewart, R.B., 1995. Relative reinforcing effects of different doses of orally delivered cocaine. Drug Alcohol Depend. 37, 141 /147. Meisch, R.M., Stewart, R.S., Wang, N.-S., 1996. Orally delivered methadone as a reinforcer for rhesus monkeys. The relation between drug concentration and choice. Pharmacol. Biochem. Behav. 54, 547 /554. Munzar, P., Yasar, S., Redhi, G.H., Justinova, Z., Goldberg, S.R., 2001. High rates of midazolam self-administration in squirrel monkeys. Behav. Pharm. 12, 257 /265. Norman, A.B., Tsibulsky, V.L., 2001. Satiety threshold regulates maintained self-administration: comment on Lynch and Carroll (2001). Exp. Clin. Psychopharm. 9, 151 /154. Pugh, T.D., Klopp, R.G., Weindruch, R., 1999. Controlling caloric consumption protocols for rodents and rhesus monkeys. Neurobiol. Aging 20, 157 /165. Spiga, R., Roache, J.D., 1997. Human drug self-administration: a review and methodological critique. In: Johnson, B.A., Roache,
T.H. Gomez et al. / Drug and Alcohol Dependence 68 (2002) 275 /283 J.D. (Eds.), Drug Addiction and its Treatment: Nexus of Neuroscience and Behavior. Lippincott-Raven, Philadelphia, pp. 39 / 71. Stewart, R.B., Lemaire, G.A., Roache, J.D., Meisch, R.A., 1994. Establishing benzodiazepines as oral reinforcers: midazolam and diazepam self-administration in rhesus monkeys. J. Pharmacol. Exp. Ther. 271, 200 /211. Stewart, R.B., Wang, N.-S., Bass, A.A., Meisch, R.A., 2002. Relative reinforcing effects of different oral ethanol doses in rhesus monkeys. J. Exp. Anal. Behav. 77, 49 /64.
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Wang, N.S., Brown, V.L., Grabowski, J., Meisch, R.A., 2001. Reinforcement by orally delivered methadone, cocaine, and methadone-cocaine combinations in rhesus monkeys: are the combinations better reinforcers? Psychopharmacology 156, 63 /72. Woods, J.H., Katz, J.L., Winger, G., 1992. Benzodiazepines: use, abuse, and consequences. Pharm. Rev. 44, 151 /234. Woolverton, W.L., Johanson, C.E., 1984. Preference in rhesus monkeys given a choice between cocaine and D,L /cathinone. J. Exp. Anal. Behav. 41, 35 /43.
Relative reinforcing effects of different benzodiazepine doses for rhesus monkeys Thomas H. Gomez, John D. Roache, Richard A. Meisch * Substance Abuse Research Center, Department of Psychiatry and Behavioral Sciences, Medical School, University of Texas-Houston Health Science Center, 1300 Moursund Street, Houston, TX 77030-3497, USA Received 5 February 2002; received in revised form 1 July 2002; accepted 3 July 2002
Abstract The relative reinforcing effects of different doses of benzodiazepines were determined by giving rhesus monkeys concurrent access to different diazepam and midazolam concentrations. For each monkey a dose response function was obtained using three drug concentrations: low (L), intermediate (I), and high (H). The benzodiazepine and the water vehicle were concurrently available under independent fixed-ratio (FR) schedules. After establishing that each concentration was a reinforcer in comparison to vehicle, relative preference for the different concentrations was examined by making pairs of concentrations concurrently available under independent FR schedules. Three pairs were studied (H vs. L, H vs. I, and I vs. L). With both drugs, higher concentrations maintained greater response rates than lower concentrations. Thus, relative reinforcing effects increased with dose. These findings are similar to those obtained with other reinforcing drugs and provide further evidence that benzodiazepines share significant characteristics with other drug reinforcers. Importantly, absolute response rates (responses per session) obtained when only one drug dose was present were not reliably predictive of subsequent preferences for the dose. Both benzodiazepines served as effective reinforcers in that consistent levels of responding were maintained across doses and above vehicle levels under concurrent FR 32 schedules. As with other reinforcing drugs, the reinforcing effects of benzodiazepines increase with increases in dose over a broad range of values. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Benzodiazepines; Self-administration; Reinforcing effects; Concurrent schedules; Dose preference; Oral route; Rhesus monkeys
1. Introduction Benzodiazepines are widely prescribed drugs that are relatively safe and effective medications for a number of medical problems (Hollister et al., 1993). Benzodiazepines are also occasionally abused (Woods et al., 1992), and most abused drugs serve as reinforcers for laboratory animals (Johanson and Balster, 1978). However, in studies with laboratory animals it has been difficult to consistently demonstrate that benzodiazepines can act as effective reinforcers (for a review of benzodiazepine self-administration studies, see Griffiths and Weerts, 1997). Most studies report that only some of the animals in the study will self-administer benzodiazepines, or that the self-administration occurs only under a narrow set
* Corresponding author. Tel.: /1-713-500-2863; fax: /1-713-5002849 E-mail address: [email protected] (R.A. Meisch).
of conditions. In contrast, in a study with squirrel monkeys intravenous delivery of midazolam maintained high and consistent rates of responding (Munzar et al., 2001). Also, in two studies from our laboratory, orally delivered benzodiazepines served as effective reinforcers for rhesus monkeys when taken by the oral route, as they maintained high and consistent response rates over a range of concentrations and schedule sizes (Gomez et al., 2002; Stewart et al., 1994). With most reinforcing drugs, relative reinforcing effects increase with increases in dose (Meisch, 2000). If benzodiazepines share important features with other reinforcing drugs, then their relative reinforcing effects should also increase as the dose becomes larger. In the absence of specific training procedures, it has been difficult to demonstrate that orally delivered drugs will serve as reinforcers. The three principal problems in establishing orally delivered drugs as reinforcers have been the aversive taste of most drug solutions, the
03765-8716/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 6 - 8 7 1 6 ( 0 2 ) 0 0 2 2 2 - 3
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consumption of only small volumes of drug solution, and the delay between the behavior of drinking the drug solution and the onset of CNS effects. Procedures have been developed that result in the establishment of orally delivered drugs as reinforcers for laboratory animals (Meisch, 2001). For example, the potent opioid etonitazene can come to serve as a reinforcer by adding increasing amounts of the drug to a dilute ethanol solution and subsequently decreasing the ethanol concentration to zero (Meisch, 1995). With rhesus monkeys, most types of abused drugs can now be established as reinforcers via the oral route (Meisch, 2001). In particular, it is now possible to obtain behavior that is maintained by oral delivery of the benzodiazepines midazolam, alprazolam, triazolam, and diazepam (Gomez et al., 2002; Stewart et al., 1994). With orally self-administered benzodiazepines, the relation between dose and response rate is usually an inverted U-shaped function (Stewart et al., 1994). The ascending portion of the dose response function is attributed to increases in reinforcing effects with increases in drug dose. However, there is controversy regarding the relation between dose and reinforcing effects on the descending portion of the dose response function, and several explanations have been put forth (Bickel et al., 2000). One strategy for further examination of the entire dose-response function is to use choice procedures to compare preferences for different drug concentrations. In earlier studies with rhesus monkeys, a marked preference was established for benzodiazepines over their vehicle (Gomez et al., 2002; Stewart et al., 1994). The purpose of the present study was to go beyond comparisons of drug and vehicle, and instead examine choices between different drug concentrations to determine their relative reinforcing effects.
2. Materials and methods 2.1. Subjects The subjects were five adult male rhesus monkeys (Macaca mulatta). All of the monkeys had extensive experience with oral drug self-administration. Four (MP1, M-PL, M-GD, M-LA) of the monkeys had histories of ethanol and pentobarbital self-administration. M-TK had experience with ethanol self-administration, and both M-P1 and M-TK had served in an earlier benzodiazepine self-administration study (Stewart et al., 1994). All five monkeys were subjects in a more recent benzodiazepine self-administration study (Gomez et al., 2002). The monkeys were housed individually in their chambers in a climate controlled room (:/22 8C) in which a 12-h light and dark cycle was in effect (lights on at 7:00 AM). At the beginning of the study, the weights of the five monkeys were as follows: M-GD, 7.5
kg; M-LA, 9.1 kg; M-P1, 6.8 kg; M-PL, 9.3 kg; and MTK, 7.8 kg. Subjects were maintained at 85 /90% of their normal weights through daily feeding with a measured amount of commercially available chow (Lab DietTM High Protein Monkey Diet #5045, PMI Nutrition International, Brentwood, MO), plus fresh fruit and vegetables. The amount of food was sufficient to maintain stable body weights, and the monkeys’ health and appearance were good. Food restriction has been shown to enhance drug self-administration (Carroll and Meisch, 1984), prevent obesity (Meisch and Lemaire, 1989), and increase life span and general health (Masoro, 1985; Pugh et al., 1999). Animal care was in accordance with the regulations of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (1996), and all procedures were approved by the Institutional Animal Use and Care Committee of the University of Texas Houston Health Science Center. 2.2. Apparatus Each subject was individually housed 24 h a day in a stainless-steel primate cage (Lab Products, Sanford, DE), which also served as the experimental chamber. Each cage had three solid walls and one barred wall. Cage dimensions were 76 /102/81 cm3. A liquiddelivery apparatus panel was attached to the outside of one side wall, and spouts and stimulus lights protruded into the cage through holes cut in that wall. Attached to the back of the apparatus panel was a Tshaped bar; on each limb of this bar was fastened a stainless-steel reservoir covered with a lid. Liquids contained in each reservoir passed through polyethylene tubing to a solenoid-operated valve at the rear of one of the two brass spouts. These spouts (1.2 cm outside diameter, 0.2 cm inside diameter) extended 2 cm into the cage, 64 cm above the floor and 15.5 cm either side of the midline. The spouts were embedded in PlexiglasTM disks that covered the 7-cm diameter holes in the cage wall through which they entered. At each spout, two 1.1 W lights, one located 2.5 cm on either side of the spout and visible through the PlexiglasTM, were aligned diagonally; these ‘spout lights’ were capped with green translucent lenses. Another two 1.1 W spout lights, one located 2.5 cm on either side of the spout, were aligned on the opposite diagonal, and were capped with white translucent lenses. Thus, each spout was in the center of a square pattern of four spout lights, two green and two white. The electronic components for the drinkometer circuit were housed in an enclosure at the rear of the spout. The liquid-delivery apparatus has been described extensively elsewhere (Gieske, 1978; Henningfield and Meisch, 1976). A 2.5 cm (diameter), cluster of green light-emitting diodes was located 11.5 cm directly above each brass spout. The programming of experimental
T.H. Gomez et al. / Drug and Alcohol Dependence 68 (2002) 275 /283
events and the recording of behavior were accomplished with a DEC PDP-11 computer and SKED† software (State Systems II, Kalamazoo, MI). This equipment was located in a room near the rooms containing the experimental chambers. 2.3. Procedure 2.3.1. During sessions Experimental sessions were 3 h in length (from 1100 to 1400 h) and were conducted 7 days per week. The stimulus lights above each spout blinked at a rate of 10 Hz. Identical discriminative stimuli were used for both spouts to control for differential responding that might otherwise result from the presence of dissimilar exteroceptive visual stimuli. Each mouth contact with a spout illuminated the green-lensed pair of spout lights for the duration of the response. Deliveries of liquids were contingent upon completion of a fixed-ratio schedule. The final response in the schedule requirement initiated the liquid flow of :/0.65 ml of the appropriate solution. To further reduce the possible differential responding that might be caused by a monkey’s preference for a particular spout, the drug solution and vehicle were alternated between spouts each session. 2.3.2. Between sessions A timeout period was in effect during the hour immediately before the session (1000 /1100 h). During this period the number of water deliveries and the volume of water consumed since the last experimental session were recorded and liquids appropriate for the next session were placed in the two reservoirs. Each system was primed to ensure that the appropriate solution was present on the first delivery. Liquid volumes were measured after flushing to obtain the exact volume in the reservoirs at each session’s onset. For 1 h immediately following the session (1400 /1500 h) another timeout period was in effect during which data from the session were recorded and water was placed into one reservoir and flushed through the tubing to the spout. Water was then available under a fixed-ratio (FR) 1 schedule from one spout from 1500 until 1600 h. A final timeout period was in effect from 1600 until 1700, at the beginning of which the monkeys’ maintenance feeding was placed in the food hopper attached to the cage. Finally, from 1700 until 1000 h of the next day, water was available under a FR 1 schedule from one spout. The spout from which water was available between sessions alternated every other day. Between sessions, water availability was signaled by steady illumination of the appropriate green stimulus light, and each mouth contact was signaled by illumination of the white-lensed pair of spout lights.
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2.3.3. Establishment of benzodiazepine reinforcing effects: ethanol fading procedure Initially, midazolam or diazepam was established as an orally delivered reinforcer by use of an ethanol fading procedure. In the first phase, increasing concentrations of drug were added across sessions to a 1 or 2% (w/v) ethanol solution that was already serving as a reinforcer. Each concentration was present for at least six sessions and until the number of responses was stable. The concentration was increased until there were clear signs of pharmacological effects such as ataxia, a slumped posture, muscle relaxation, and diminished motor activity. In the next phase the ethanol concentration was decreased in steps until it was absent. A successful outcome was the persistence of drug self-administration at levels greater than vehicle values. For two monkeys, M-P1 and M-TK, the specific steps in the fading procedure have been published (Stewart et al., 1994). The procedures used with the other monkeys were similar and have been described (Gomez et al., 2002). 2.3.4. Concentration response functions After the drug had been established as a reinforcer, drug concentrations were presented in a descending sequence with water concurrently available from the alternate spout. After the block of sessions at the lowest concentration, a second determination was made at the highest concentration. On changing from the lowest to the highest concentration, two sessions at the intermediate concentration were conducted to avoid any disruption that might occur with an abrupt transition from the lowest to the highest concentration. Changes from one concentration to another were made only after behavior was stable for six sessions, without an upward or downward trend in the number of deliveries for each liquid. The FR size for each monkey was the FR value that resulted in clear differences between drug and vehicle maintained responding. The number of deliveries and intake of drugs (mg of drug/kg of body weight/3-h session) for the last six sessions of stable behavior were expressed as the mean9/SEM. 2.3.5. Comparisons between concentrations With midazolam and diazepam three comparisons were conducted under concurrent FR schedules in a fixed sequence: (1) highest concentration versus the lowest concentration, (2) highest concentration versus the intermediate concentration, and (3) intermediate concentration versus the lowest concentration. Following the comparisons of the intermediate versus lowest concentrations, a second determination was made with two pairs of concentrations, and these were presented in a sequence that counterbalanced the first two comparisons. Thus, the fourth comparison repeated the second comparison (highest concentration versus the intermediate concentration), and the last comparison repeated the
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first comparison (highest concentration versus lowest concentration). Changes from one concentration to another were made after behavior was stable for six sessions, that is, without an upward or downward trend in the number of deliveries for each liquid. With two monkeys, M-LA and M-GD, the FR size was increased during the comparisons of diazepam concentrations in order to produce a clear difference in response rates maintained by the two concentrations. Additional choice comparisons between concentrations were conducted with midazolam. Two monkeys (M-TK and MP1) served as subjects. The pairs of concentrations were studied in the sequence: 0 vs. 0.025; 0.025 vs. 0.05; 0.05 vs. 0.1; 0.1 vs. 0.2; 0.2 vs. 0.4. Subsequently, the concentration pairs were tested a second time in a sequence that was counterbalanced: 0.1 vs. 0.2; 0.05 vs. 0.1; 0.025 vs. 0.05; 0.025 vs. 0. 2.4. Drugs Concentrated stock solutions (0.8 mg/ml) of diazepam and midazolam were prepared weekly in dilute HCl and refrigerated. Because of the limited solubility of benzodiazepines in water, dilute acid was added to form the HCl salt. The concentrations of HCl used were: 0.01 N, diazepam, and 0.0025 N, midazolam. Daily working solutions were prepared at room temperature by appropriate dilutions of the stock solution with deionized water approximately 2 h before use. Freebases of benzodiazepines act as a buffer; therefore, for each drug the pH remained fairly constant across a range of concentrations (:/4.2 for midazolam and 3.2 for diazepam). The stability of the acidic benzodiazepine stock solutions was verified by gas chromatography. 2.5. Statistical analysis To assess the relation between relative and absolute response rates a Spearman coefficient of rank correlation was calculated separately for midazolam and diazepam. Two ranks were assigned to each drug concentration based on the absolute and relative response rate maintained by that concentration.
3. Results
Fig. 1. Deliveries of midazolam and the water vehicle as a function of drug concentration for three monkeys, M-P1, M-TK, and M-GD. Circles are drug deliveries, and triangles are vehicle deliveries. Each point represents the mean of 6 consecutive sessions of stable behavior, and the brackets show the standard error of the mean. Absence of brackets indicates that they fell within the area of the plotted symbol. At the 0.4 mg/ml concentration, the points to the right are the results of the initial block of sessions at 0.4 mg/ml, and the adjacent points are the results of a second determination conducted after completion of the block of sessions at the lowest concentration. The points giving the results of the second determination at 4.0 mg/ml were shifted slightly to the left for clarity.
except for M-GD, where the retest value was lower. Drug intake (mg of drug/kg of body weight/3-h session) increased as concentration increased (Table 1).
3.1. Midazolam: concentration response function 3.2. Midazolam: comparisons between concentrations Fig. 1 shows that deliveries of midazolam systematically exceeded deliveries of the water vehicle. In most cases the range of drug deliveries was above the range of water deliveries, and thus the concentrations were serving as reinforcers. When a second determination was made at the 0.4 mg/ml concentration, the values for both drug and water were similar to the initial values
In the choice phase the three drug concentrations were studied in pairs, under concurrent FR schedules in a counter-balanced sequence. Fig. 2 shows that the higher concentration was preferred to the lower concentration, and usually the preference was large. However, in two of the 15 comparisons, the higher
T.H. Gomez et al. / Drug and Alcohol Dependence 68 (2002) 275 /283 Table 1 Midazolam intake (mg/kg/3-h session) as a function of concentration in three rhesus monkeys M-GD
M-P1
M-TK
mg/ml
Intake
mg/ml
Intake
mg/ml
Intake
0.4a 0.2 0.1 0.4
1.52 0.61 0.24 1.48
0.4 0.1 0.025 0.4
2.16 1.27 0.20 2.12
0.4 0.1 0.025 0.4
1.57 0.55 0.17 1.67
a
Concentrations are listed in the order in which they were studied.
279
ence for one concentration over another when the 0.2 and 0.1 mg/ml concentrations were studied. With monkeys M-TK and M-P1 comparisons of midazolam concentrations were also conducted by presenting concentrations in an ascending and then descending sequence. For each monkey the higher concentration was preferred to the lower concentration in 8 of 9 comparisons (data not shown). With each monkey a lack of preference was seen at one of the two comparisons of the lowest two concentrations, 0.05 and 0.025 mg/ml. 3.3. Relation between response rates when only one midazolam concentration was present to response rates when two concentrations were present There was no reliable correspondence between rates of drug-maintained behavior obtained in the concentration response series and the concentration choice series. For example, in the choice phase, the most preferred concentration was 0.4 mg/ml (Fig. 2). However, for two monkeys (M-P1 and M-TK), the 0.4 mg/ml concentration maintained lower response rates than did the 0.025 and 0.1 mg/ml concentrations when the concentrations were presented sequentially (Fig. 1). Additionally, with monkey M-TK the lowest concentration, 0.025 mg/ml, supported higher rates than the 0.1 and 0.4 mg/ml concentrations when concentrations were presented sequentially (Fig. 1). However, it was the least preferred concentration in the choice series (Fig. 2). The Spearman coefficient of rank correlation relating absolute and relative rate was /0.244 (P /0.5). 3.4. Diazepam: concentration response functions Fig. 3 shows that deliveries of a range of diazepam concentrations consistently exceeded deliveries of water and that the differences between drug and water values usually were large. With three of the four monkeys, a second determination was made at the 0.4 mg/ml concentration and the values were similar to the initial values. Drug intake increased with increases in concentration (Table 2). 3.5. Diazepam: comparisons between concentrations
Fig. 2. Deliveries of midazolam solutions (mg/ml) under conditions of concurrent access to two drug solutions for three monkeys. Each bar represents the mean of six consecutive sessions of stable behavior, and the brackets show the standard error of the mean. Note that in most comparisons the higher concentration was preferred.
concentration was not preferred. For M-P1 the initial comparison of the 0.4 and 0.1 mg/ml concentrations revealed no preference; however, the second comparison of these concentrations showed a clear preference for the higher concentration. For M-GD there was no prefer-
When pairs of diazepam concentrations were studied, the higher concentration was preferred in 19 of 20 comparisons (Fig. 4). For two of the monkeys (M-LA and M-TK) preference for the higher concentration was uniformly large. With a third monkey (M-GD) there was a consistent preference for the higher concentrations but the differences were less than seen with M-LA and M-TK. Although M-PL preferred 0.2 and 0.4 mg/ml to the lowest concentration, 0.1 mg/ml, the 0.4 mg/ml concentration was slightly preferred over the 0.2 mg/ml
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280
Fig. 3. Deliveries of diazepam and the water vehicle as a function of drug concentration for four monkeys, M-LA, M-GD, M-PL, and M-TK. Circles are drug deliveries, and triangles are vehicle deliveries. Each point represents the mean of 6 consecutive sessions of stable behavior, and the brackets show the standard error of the mean. Absence of brackets indicates that they fell within the area of the plotted symbol. At the 0.4 mg/ml concentration, the points to the right are the results of the initial block of sessions at 0.4 mg/ml, and the adjacent points are the results of a second determination conducted after completion of the block of sessions at the lowest concentration. The points giving the results of the second determination at 4.0 mg/ml were shifted slightly to the left for clarity.
concentration on the first comparison and was not preferred on the second comparison (Fig. 4). 3.6. Relation between response rates when only one diazepam concentration was present to response rates when two concentrations were present Across the four monkeys, different diazepam concentration response functions were seen (Fig. 3). However, all four monkeys had similar patterns of preference in that higher concentrations were preferred to lower concentrations (Fig. 4). Thus, as was found with
midazolam, there was not a reliable correspondence between response rate when a single concentration was present and response rate when two concentrations were available concurrently. The Spearman coefficient of rank correlation relating absolute and relative rate was 0.272 (P /0.35). 3.7. Time course and pattern of responding As in other oral drug self-administration studies the highest rate of responding was at the beginning of the session (Meisch, 2001). Subsequently, following a pause,
Table 2 Diazepam intake (mg/kg/3-h session) as a function of concentration in four rhesus monkeys M-GD mg/ml a
0.4 0.2 0.1 0.4
a
M-LA
M-PL
M-TK
Intake
mg/ml
Intake
mg/ml
Intake
mg/ml
Intake
1.74 1.34 1.09 2.29
0.4 0.2 0.1 0.4
4.84 1.91 0.35 4.89
0.4 0.2 0.1 0.4
1.94 1.38 0.70 1.83
0.4 0.1 0.025 0.4
3.07 0.90 0.15 2.99
Concentrations are listed in the order in which they were studied.
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281
Fig. 4. Deliveries of diazepam solutions (mg/ml) under conditions of concurrent access to two solutions for four monkeys. Each bar represents the mean of six consecutive sessions of stable behavior, and the brackets show the standard error of the mean. Several comparisons were conducted at FR 16 as indicated in the figure. Note that in most comparisons the higher concentration was preferred.
there were one or more bouts later in the session. The pattern was characteristic of responding seen with FR schedules. At high drug concentrations signs of intoxication were observed, such as decreased motor activity, slumping, ataxia, and incoordination.
4. Discussion The principal finding was that higher concentrations of two benzodiazepines were preferred to lower concentrations. Thus, over the range of values studied, reinforcing effects increased with increases in concentration. These findings are consistent with many studies conducted with the oral and IV routes and with a wide range of abused drugs. Such studies have used either measures of choice or measures of relative persistence of responding across increases in schedule size (Meisch, 2000). In spite of the many studies demonstrating increases in reinforcing effects with increases in drug dose, some investigators maintain that reinforcing effects do not increase directly with drug dose (Norman and Tsibulsky, 2001). Their reasoning is based on the false premise that absolute response rate directly reflects the magnitude of reinforcing effects. They note that, in many cases, absolute response rates go down with increases in drug dose and therefore conclude that higher doses produce less, not more, reinforcement. In
the present study, absolute response rates obtained when each concentration was studied alone, were not predictive of preferences measured under conditions of choice. For example, Figs. 1 and 3 show different dose response functions for each monkey, with increasing, decreasing, and inverted U-shape patterns. These absolute response rates clearly did not correspond with preferences for higher concentrations observed with concurrent access to two concentrations. Similar results were obtained in a study of the reinforcing effects of cocaine /methadone combinations and the component drugs (Wang et al., 2001). In that study drug combinations and the component drugs were studied under both sequential and concurrent access. Under sequential access, response rates maintained by the combination and the component drugs were often similar. In contrast, under concurrent access response rates for the drug combinations were higher than rates for the component drugs. Thus, in both studies absolute response rates were not a reliable indicator of relative reinforcing effects. The findings with benzodiazepines are congruent with the results of other oral self-administration studies that examined choices between either drug concentrations or drug volumes. Such studies have been conducted with cocaine (Meisch and Stewart, 1995), ethanol (Stewart et al., 2002), methadone (Meisch et al., 1996), pentobarbital (Meisch et al., 1992; Meisch and Lemaire, 1988, 1989; Meisch and Spiga, 1998), and phencyclidine
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(Carroll, 1987). In all cases, higher doses were preferred to lower doses. Choices between drug doses also have been examined in IV self-administration studies, and the results reveal a preference for larger doses over lower doses (Iglauer et al., 1975; Iglauer and Woods, 1974; Johanson and Schuster, 1975; Manzardo et al., 2001; Woolverton and Johanson, 1984). Thus, the results of studies of choice between different drug doses show that over a broad range of values, reinforcing effects increase with increases in dose. That the reinforcing effects of benzodiazepines increase as a function of dose is further evidence that the benzodiazepines share important features with other reinforcing drugs. What remains to be determined is why the benzodiazepines did not function as effective reinforcers in most self-administration studies. An understanding of the conditions under which benzodiazepines do and do not serve as reinforcers will aid our comprehension of the circumstances under which these drugs might be abused. Choice between drug and vehicle (or placebo) is a powerful technique for assessing drug reinforcing effects (Spiga and Roache, 1997), and the present study demonstrates that choice between two concurrently available drug doses is an effective procedure for determining relative reinforcing effects of drug doses.
Acknowledgements This study was supported by NIDA grant DA 08913. Richard A. Meisch is the recipient of a NIDA Research Scientist Award (DA 00159). We thank Dr Howard M. Rhoades for help with the statistical analysis and Mr. Oscar Brown for his professional animal care.
References Bickel, W.K., Marsch, L.A., Carroll, M.E., 2000. Deconstructing relative reinforcing efficacy and situating the measures of reinforcement within behavioral economics: a theoretical proposal. Psychopharmacology 153, 44 /56. Carroll, M.E., 1987. Concurrent access to two concentrations of orally delivered phencyclidine: effects of feeding conditions. J. Exp. Anal. Behav. 47, 347 /362. Carroll, M.E., Meisch, R.A., 1984. Increased drug-reinforced behavior due to food deprivation. In: Thompson, T., Dews, P.B., Barrett, J.E. (Eds.), Advances in Behavioral Pharmacology, vol. 4. Academic Press, Orlando, FL, pp. 47 /88. Gieske, D., 1978. Integrated drinking device for monkeys. Reports from the Research Laboratories of the Department of Psychiatry, University of Minnesota, Minneapolis, MN (PR-78-1). Gomez, T.H., Roache, J.D., Meisch, R.A., 2002. Orally delivered alprazolam, diazepam, and triazolam as reinforcers in rhesus monkeys. Psychopharmacology 161, 86 /94. Griffiths, R.R., Weerts, E.M., 1997. Benzodiazepine self-administration in humans and laboratory animals */implications for problems of long-term use and abuse. Psychopharmacology 134, 1 /37.
Henningfield, J.E., Meisch, R.A., 1976. Drinking device for rhesus monkeys. Pharmacol. Biochem. Behav. 4, 609 /610. Hollister, L.E., Muller-Oerlinghausen, B., Rickels, K., Shader, R.I., 1993. Clinical uses of benzodiazepines. J. Clin. Psychopharmacol. 13, 1S /169S. Iglauer, C., Llewellyn, M.E., Woods, J.H., 1975. Concurrent schedules of cocaine injection in rhesus monkeys: dose variations under independent and non-independent variable-interval procedures. Pharmacol. Rev. 27, 367 /383. Iglauer, C., Woods, J.H., 1974. Concurrent performances: reinforcement by different doses of intravenous cocaine in rhesus monkeys. J. Exp. Anal. Behav. 22, 179 /196. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996. Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC. Johanson, C.E., Balster, R.L., 1978. A summary of the results of a drug self-administration study using substitution procedures in rhesus monkeys. Bull. Narc. 30, 43 /54. Johanson, C.E., Schuster, C.R., 1975. A choice procedure for drug reinforcers: cocaine and methylphenidate in the rhesus monkey. J. Pharmacol. Exp. Ther. 193, 676 /688. Manzardo, A.M., Del Rio, J.A., Stein, L., Belluzzi, J.D., 2001. Rats choose cocaine over dopamine agonists in a two-lever self-administration preference test. Pharmacol. Biochem. Behav. 70, 257 /265. Masoro, E.J., 1985. Nutrition and aging */a critical review. J. Nutr. 115, 842 /848. Meisch, R.A., 1995. Oral self-administration of etonitazene in rhesus monkeys: use of a fading procedure to establish etonitazene as a reinforcer. Pharmacol. Biochem. Behav. 50, 571 /580. Meisch, R.A., 2000. Relative persistence of behavior: a fundamental measure of relative reinforcing effects. Exp. Clin. Psychopharm. 8, 333 /349. Meisch, R.A., 2001. Oral drug self-administration: an overview of laboratory animal studies. Alcohol 24, 117 /128. Meisch, R.A., Lemaire, G.A., 1988. Oral self-administration of pentobarbital by rhesus monkeys: relative reinforcing effects under concurrent fixed-ratio schedules. J. Exp. Anal. Behav. 50, 75 /86. Meisch, R.A., Lemaire, G.A., 1989. Oral self-administration of pentobarbital by rhesus monkeys: maintenance of behavior by different concurrently available volumes of drug solution. J. Exp. Anal. Behav. 52, 111 /126. Meisch, R.A., Lemaire, G.A., Cutrell, E.B., 1992. Oral self-administration of pentobarbital by rhesus monkeys: relative reinforcing effects under concurrent signalled differential-reinforcement-oflow-rates schedules. Drug Alcohol Depend. 30, 215 /225. Meisch, R.A., Spiga, R., 1998. Matching under nonindependent variable-ratio schedules of drug reinforcement. J. Exp. Anal. Behav. 70, 23 /34. Meisch, R.A., Stewart, R.B., 1995. Relative reinforcing effects of different doses of orally delivered cocaine. Drug Alcohol Depend. 37, 141 /147. Meisch, R.M., Stewart, R.S., Wang, N.-S., 1996. Orally delivered methadone as a reinforcer for rhesus monkeys. The relation between drug concentration and choice. Pharmacol. Biochem. Behav. 54, 547 /554. Munzar, P., Yasar, S., Redhi, G.H., Justinova, Z., Goldberg, S.R., 2001. High rates of midazolam self-administration in squirrel monkeys. Behav. Pharm. 12, 257 /265. Norman, A.B., Tsibulsky, V.L., 2001. Satiety threshold regulates maintained self-administration: comment on Lynch and Carroll (2001). Exp. Clin. Psychopharm. 9, 151 /154. Pugh, T.D., Klopp, R.G., Weindruch, R., 1999. Controlling caloric consumption protocols for rodents and rhesus monkeys. Neurobiol. Aging 20, 157 /165. Spiga, R., Roache, J.D., 1997. Human drug self-administration: a review and methodological critique. In: Johnson, B.A., Roache,
T.H. Gomez et al. / Drug and Alcohol Dependence 68 (2002) 275 /283 J.D. (Eds.), Drug Addiction and its Treatment: Nexus of Neuroscience and Behavior. Lippincott-Raven, Philadelphia, pp. 39 / 71. Stewart, R.B., Lemaire, G.A., Roache, J.D., Meisch, R.A., 1994. Establishing benzodiazepines as oral reinforcers: midazolam and diazepam self-administration in rhesus monkeys. J. Pharmacol. Exp. Ther. 271, 200 /211. Stewart, R.B., Wang, N.-S., Bass, A.A., Meisch, R.A., 2002. Relative reinforcing effects of different oral ethanol doses in rhesus monkeys. J. Exp. Anal. Behav. 77, 49 /64.
283
Wang, N.S., Brown, V.L., Grabowski, J., Meisch, R.A., 2001. Reinforcement by orally delivered methadone, cocaine, and methadone-cocaine combinations in rhesus monkeys: are the combinations better reinforcers? Psychopharmacology 156, 63 /72. Woods, J.H., Katz, J.L., Winger, G., 1992. Benzodiazepines: use, abuse, and consequences. Pharm. Rev. 44, 151 /234. Woolverton, W.L., Johanson, C.E., 1984. Preference in rhesus monkeys given a choice between cocaine and D,L /cathinone. J. Exp. Anal. Behav. 41, 35 /43.