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BEHAVIORAL PHARMACOLOGY

Contribution of ₁GABA_A and ₅GABA_A Receptor Subtypes to the Discriminative Stimulus Effects of Ethanol in Squirrel Monkeys

Harvard Medical School, New England Primate Research Center, Southborough, Massachusetts (D.M.P., A.D., R.D.S., J.K.R.); and Department of Chemistry, University of Wisconsin, Milwaukee, Wisconsin (J.M.C., X.L., W.Y.)

Received November 8, 2004; accepted January 12, 2005.

	Abstract

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Ethanol's ability to enhance GABA neurotransmission via GABA_A receptors has been implicated as an important mechanism underlying its discriminative stimulus (DS) effects in animals and subjective effects in humans. The present study assessed the contribution of ₁GABA_A and ₅GABA_A receptors to the DS effects of ethanol. Squirrel were monkeys trained to discriminate i.v. ethanol from saline under a fixed-ratio schedule of food delivery. Under test conditions, ethanol engendered a dose-dependent increase in drug-lever responding, reaching an average maximum of >80%. In substitution experiments, the ₁GABA_A agonists zolpidem, zaleplon, and CL 218,872 (3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine), the ₅GABA_A agonists QH-ii-066 (1-methyl-7-acetyleno-5-phenyl-1,3-dihydro-benzo[e]-1,4-diazepin-2-one) and panadiplon [3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)imidazo(1,5-a)quinoxalin-4(5H)-one], and representative nonselective agonists partially to fully reproduced the ethanol DS. In antagonism studies, the ₁GABA_A antagonist -carboline-t-butyl ester did not attenuate the DS effects of ethanol or the ethanol-like effects of zolpidem and zaleplon. In contrast, pretreatment with the ₅GABA_A inverse agonist L-655,708 (ethyl[S]-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxlate) dose-dependently attenuated the DS effects of ethanol and the ethanol-like effects of QH-ii-066. RY-23 (tert-butyl 8-[(trimethylsilyl)ethynyl]-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]-[1,4]benzodiazepine-3-carboxylate), another ₅GABA_A inverse agonist, similarly attenuated the ethanol-like DS effects of QH-ii-066. Antagonism of both QH-ii-066 and ethanol by the ₅GABA_A inverse agonists occurred at doses that did not alter the rate of responding suggesting that this blockade was pharmacologically specific and not the result of a nonspecific disruption of operant behavior. These findings suggest a key role for ₅GABA_A, but not ₁GABA_A, receptor mechanisms in the DS effects of ethanol and the ethanol-like DS effects of benzodiazepine agonists.

The ability of ethanol to modulate GABA receptors has been proposed as an important mechanism underlying its behavioral effects in humans (e.g., Korpi, 1994). Results from behavioral studies in animals support a key role for specific subtypes of the GABA_A receptor in the effects of ethanol related to its abuse. For example, self-administration of ethanol, but not saccharin or sucrose, can be reduced by 3-propoxy--carboline hydrochloride and -carboline-t-butyl ester (-CCt), antagonists with selectivity for GABA_A receptors containing ₁ subunits (₁GABA_A receptors; Harvey et al., 2002; Foster et al., 2004). Moreover, mice lacking the ₁ subunit of the GABA_A receptor exhibit a reduced preference for ethanol in a two-bottle choice procedure (Blednov et al., 2003b). GABA_A receptors expressing the ₅ subunit (₅GABA_A receptors) also may contribute importantly to the behavioral effects of ethanol. Intrahippocampal infusions of the selective ₅GABA_A receptor inverse agonist tert-butyl 8-[(trimethylsilyl)ethynyl]-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4]benzodiazepine-3-carboxylate (RY-23) or i.p. injections of another ₅GABA_A receptor inverse agonist RY-24 selectively decrease ethanol self-administration in rats at doses that do not disrupt self-administration of saccharin or water (June et al., 2001; McKay et al., 2004).

Drug discrimination procedures may provide a useful experimental model of the subjective effects of drugs in humans because there is a close correspondence between the classification of drugs based on their discriminative stimulus (DS) effects in monkeys and subjective effects in humans (Schuster and Johanson, 1988). For example, in nondrug-abusing humans, the barbiturate pentobarbital and the benzodiazepine receptor ligands triazolam and zolpidem produce sedative-like, subject-rated drug effects (Rush et al., 1997). In these same subjects, after being trained to discriminate pentobarbital from placebo, pentobarbital, triazolam, and zolpidem but not caffeine engendered pentobarbital-like DS effects (Rush et al., 1997). Similar findings were observed in a companion study with pentobarbital-trained rhesus monkeys (Rowlett and Woolverton, 1997). Drug discrimination also has emerged as a valuable in vivo technique for evaluating pharmacological mechanisms underlying ethanol's subjective effects (Grant, 1999). The purpose of the present study was to investigate the role of ₁GABA_A and ₅GABA_A receptor mechanisms in the subjective effects of ethanol by determining the extent to which selective GABA_A ligands mimic and/or modulate the DS effects of ethanol in monkeys. ₁GABA_A receptor mechanisms were studied by determining the extent to which the ₁GABA_A-preferring agonists zolpidem, zaleplon, and 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine (CL 218,872) mimic the DS effects of ethanol and the degree to which the ₁GABA_A-preferring antagonist -CCt blocks these effects. Zolpidem binds preferentially at ₁GABA_A receptors and has little or no affinity at ₅GABA_A receptors (Huang et al., 2000), and zaleplon displays 27-fold selectivity for ₁GABA_A receptors compared with ₅GABA_A receptors (Damgen and Luddens, 1999). In addition, both zolpidem and zaleplon have been shown to exhibit a unique profile of behavioral effects that appear to be mediated principally by stimulation of ₁GABA_A receptors (e.g., Crestani et al., 2000; Noguchi et al., 2002; Rowlett et al., 2003). CL 218,872 exhibits lower efficacy than either zolpidem or zaleplon but demonstrates high selectivity for ₁GABA_A receptors versus ₅GABA_A receptors (100-fold; Wafford et al., 1993; Atack et al., 1999). The ₁GABA_A antagonist -CCt exhibits >150-fold selectivity for ₁GABA_A receptors versus ₅GABA_A receptors (Huang et al., 2000) and selectively antagonizes benzodiazepine-induced ataxia, a putative ₁GABA_A-mediated effect, as well as the DS effects of zolpidem in zolpidem-trained monkeys (Platt et al., 2002; Rowlett et al., 2003).

₅GABA_A receptor mechanisms were investigated by determining the degree to which the ₅GABA_A-preferring agonists1-methyl-7-acetyleno-5-phenyl-1,3-dihydro-benzo[e]-1,4-diazepin-2-one (QH-ii-066) and panadiplon [3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl) imidazo(1,5-a)quinoxalin-4(5H)-one] mimic the DS effects of ethanol and the extent to which the DS effects of ethanol are attenuated by the ₅GABA_A-preferring inverse agonists RY-23 and ethyl [S]-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxlate (L-655,708). QH-ii-066 exhibits 11-fold selectivity for ₅GABA_A receptors versus ₁GABA_A receptors (Huang et al., 1996). Panadiplon exhibits lower efficacy than QH-ii-066 but retains high selectivity for ₅GABA_A receptors versus ₁GABA_A receptors (150-fold; Petke et al., 1992; Lameh et al., 2000). RY-23 displays 75-fold selectivity for ₅GABA_A receptors compared with ₁GABA_A receptors and reduces ethanol self-administration in rodents (Liu et al., 1996; June et al., 2001). L-655,708 displays >100-fold selectivity for ₅GABA_A receptors compared with receptors expressing ₁GABA_A subunits (Casula et al., 2001).

	Materials and Methods

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Subjects and Surgical Procedure. Five male squirrel monkeys (Saimiri sciureus), weighing 750 to 1050 g, were studied in daily experimental sessions (Monday to Friday). All subjects were experimentally naive at the beginning of the study. Between sessions, monkeys lived in individual home cages where they had unlimited access to water. Monkeys were maintained at 90 to 95% of their free-feeding body weight by adjusting their access to food in the home cage (Teklad Monkey Diet, supplemented with fresh fruit; Harlan Teklad, Madison, WI). All animals were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, Department of Health, Education, and Welfare Publication No. National Institutes of Health 85-23, revised 1996. Research protocols were approved by the Harvard Medical School Institutional Animal Care and Use Committee.

Monkeys were prepared with a chronic indwelling venous catheter (polyvinyl chloride; i.d., 0.38 mm; o.d., 0.76 mm) using the general surgical procedures described by Carey and Spealman (1998). Under isoflurane anesthesia and aseptic conditions, one end of a catheter was passed to the level of the right atrium by way of a femoral or jugular vein. The distal end of the catheter was passed s.c. and exited in the midscapular region. Catheters were flushed daily with saline and were sealed with stainless steel obturators when not in use. Monkeys wore custom-made nylon-mesh jackets (Lomir Biomedical, Toronto, ON, Canada) at all times to protect the catheter.

Apparatus. Experimental sessions were conducted in ventilated and sound-attenuating chambers. Monkeys were seated in primate chairs with two response levers mounted on the panel in front of the monkey. Each press of a lever with a minimum downward force of approximately 0.25 N produced an audible click and was recorded as a response. Colored lights mounted above the levers could be illuminated to serve as visual stimuli. Food pellets (Bioserve Precision pellets, Formula 0069, 190 mg; Bioserve Biotechnologies, Laurel, MD) could be delivered to a tray located between the levers.

Ethanol Discrimination Procedure. Monkeys initially were trained to respond on each of two levers under a 10-response fixed ratio (FR 10) schedule of food reinforcement. Once consistent lever pressing was established, the monkeys were implanted with i.v. catheters, and drug discrimination training was started 2 to 4 days after recovery from surgery. The training dose of ethanol was 1.0 g/kg administered i.v. The i.v. route of administration was chosen to avoid taste cues, which can interfere with the stimulus control of behavior by pharmacological cues (Duka et al., 1999). After an i.v. injection of ethanol, 10 consecutive responses on one lever produced a food pellet, whereas after an i.v. injection of saline, 10 consecutive responses on the other lever produced a pellet. For three of the monkeys, responding on the right lever after an injection of ethanol resulted in pellet delivery. For the other monkeys, responding on the left lever after injection of ethanol was reinforced. Delivery of each pellet was followed by a 10-s time-out period. Responses on the incorrect lever (e.g., the saline-appropriate lever after ethanol injection) reset the FR requirement.

Training sessions consisted of a variable number of components (n = 1–4) of the FR schedule. The number of components per session was randomized from day to day with the restriction that each number occurred equally often within a block of 20 sessions. Each component ended after 10 food pellets had been delivered or after 5 min had elapsed, whichever occurred first. A 10-min time-out period, during which the lights were off and responses had no programmed consequences, preceded each component. During most training sessions, saline was injected during time-out periods preceding the first n – 1 components, and ethanol was injected before the nth component of the session. Periodically, saline was injected before all components of a training session to prevent an invariant association between the fourth component and ethanol injection. Injections of ethanol or saline were administered from outside the chamber via a catheter extension during the 5th min of the 10-min time-out periods. Each injection was followed by a 2-ml infusion of saline to flush the catheter of any residual drug solution.

Drug Testing Procedure. Once consistent stimulus control was achieved, drug test sessions were conducted once or twice per week with training sessions scheduled on intervening days. Test sessions were conducted only if 80% of responses were made on the injection-appropriate lever during at least four of the preceding five training sessions. In general, test sessions consisted of four FR components, each preceded by a 10-min time-out period. During each component, completion of 10 consecutive responses on either lever produced food. Dose-response functions were determined for test drugs using a cumulative dosing procedure. The drugs studied using this procedure were ethanol, the ₁GABA_A ligands zolpidem, zaleplon, CL 218,872, and -CCt, the ₅GABA_A agonists QH-ii-066 and panadiplon, the nonselective benzodiazepine receptor ligands flunitrazepam, midazolam, diazepam, and flumazenil, and the reference compounds muscimol, baclofen, and morphine. Under the cumulative dosing procedure, incremental doses of each drug (one-fourth to one-half increments) were injected i.v. during time-out periods that preceded sequential FR components, permitting a four-point cumulative dose-response function to be determined in a single session. When warranted, five or more different doses of a drug were studied by administering overlapping ranges of cumulative doses during test sessions on different days. The effects of most doses were determined twice, although low doses that were found to be inactive and high doses that produced adverse effects usually were studied only once in each subject.

Antagonism studies were carried out with the ₁GABA_A-preferring antagonist -CCt, the ₅GABA_A-preferring inverse agonists L-655,708 and RY-23 and the nonselective benzodiazepine antagonist flumazenil in a subset of three monkeys. Studies with -CCt and flumazenil were conducted by administering -CCt (3.0 mg/kg i.v.) or flumazenil (1.0 mg/kg i.v.) immediately before the session, followed by cumulative doses of zaleplon, zolpidem, or ethanol as described above. The doses of -CCt and flumazenil were chosen on the basis of earlier studies that found them to be equieffective at antagonizing the DS effects of zolpidem in squirrel monkeys when administered using procedures identical to those described here (Rowlett et al., 1999, 2003; Lelas et al., 2002). Because of the possible proconvulsant properties of L-655,708 and RY-23, antagonism studies were conducted with these compounds by administering high doses of QH-ii-066 (1.8 mg/kg) or ethanol (1.0 g/kg) in the first component of a four component test session, followed by saline or cumulative doses of L-655,708 or RY-23 as described above. Doses of L-655,708 (1.0 mg/kg) and RY-23 (3.0 mg/kg) that significantly reduced the ethanol-like DS effects of QH-ii-066 also were tested in combination with a high dose of zolpidem (1.0 mg/kg).

Analysis of Drug Effects. Percentage of ethanol-lever responding was computed for individual subjects in each component of a test session by dividing the number of responses on the ethanol lever by the total number of responses on both levers and multiplying by 100. Percentage of ethanol-lever responding was calculated for an individual monkey only if the response rate was >0.1 responses/s during the component. Mean percentage of ethanol-lever responding and S.E.M. were then calculated for the group of monkeys at each dose. A drug was considered to substitute fully for ethanol if the maximum percentage of drug-lever responding was 80%. The doses of drug estimated to engender 50% ethanol-appropriate responding (ED₅₀) or the doses of the antagonists estimated to decrease ethanol-appropriate responding to 50% (A₅₀) were determined for individual subjects by linear regression analysis in cases where the log dose-response function was defined by at least three data points or by linear interpolation in cases where the dose-response function was defined best by two points.

The overall rate of responding in each component was computed by dividing the total number of responses in a component (regardless of lever) by the total component duration. Rate of responding was converted to percentage of control by dividing an individual animal's rate after drug by the animal's average response rate during the last two saline training components before the test and multiplying by 100. Mean response rate (percentage of control ± S.E.M.) was then calculated for the group at each dose.

The effects of each drug on response rate were analyzed by separate repeated measures ANOVAs. Further analysis was performed using Dunnett's q statistic, which compares the effects of different doses of each drug with vehicle control. The reliability of the -CCt-induced shifts in the zaleplon, zolpidem, and ethanol dose-response functions was evaluated using separate repeated-measures two-way ANOVAs followed by Bonferroni t tests. Finally, in the few instances when only two treatments were being compared (see Results), one-sample t tests were used. The level for all statistical tests was p < 0.05.

Drugs. Ethanol (95%) was purchased from Pharmco Products (Brookfield, CT). -CCt, QH-ii-066, and RY-23 were synthesized at the University of Wisconsin (Milwaukee) as described previously (Cox et al., 1995; Huang et al., 1996; Liu et al., 1996). Flunitrazepam base, diazepam base, midazolam maleate, CL 218,872, muscimol base, baclofen base, morphine sulfate, and L-655,708 base were purchased from Sigma/RBI (Natick, MA). Other drugs were gifts from the manufacturers, including zolpidem base (Merck Sharp and Dohme, Harlow, UK), zaleplon (Wyeth-Ayerst, Princeton, NJ), panadiplon (also U-78875; Pharmacia and Upjohn Co., Kalamazoo, MI), and flumazenil (also Ro15-1788; Roche Pharmaceuticals, Nutley, NJ). Ethanol (95%) was diluted with 0.9% saline solution to a concentration of 0.5 g/ml before injection. Muscimol, baclofen, and morphine were dissolved in 0.9% saline solution. All other drugs were dissolved in small amounts of 95% ethanol and/or 0.1 N HCl as required and then diluted to the desired concentrations in a 50% propylene glycol/50% saline solution.

	Results

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Intravenous Ethanol Discrimination. Monkeys acquired the i.v. ethanol discrimination after 47 to 120 sessions (mean ± S.E.M., 82 ± 15). During training sessions on days immediately before test sessions, individual monkeys made an average of 97 (±1) responses on the ethanol-associated lever after injections of ethanol and 2 (±1) responses on the ethanol-associated lever after injections of saline. Rates of responding during training sessions were 0.60 (±0.04) responses/s after injections of ethanol and 0.72 (±0.06) responses/s after injections of saline.

Under test conditions, increasing cumulative doses of ethanol (0.1–3.0 g/kg) engendered dose-dependent increases in the percentage of responses on the ethanol-associated lever (Fig. 1, top). Low doses of ethanol (0.1–0.3 g/kg) engendered little or no responding on the ethanol-associated lever, whereas doses of ethanol 1.0 g/kg elicited virtually exclusive responding on the ethanol-associated lever. The mean ED₅₀ for the DS effects of ethanol was 0.5 (±0.03) g/kg. As shown in the bottom panel of Fig. 1, the average response rate decreased with administration of increasing doses of ethanol. After administration of 1.0 or 3.0 g/kg ethanol, rates of responding differed reliably compared with administration of vehicle [F(4,16) = 21.8, p < 0.05; Dunnett's tests, p < 0.05].

View larger version (10K): [in this window] [in a new window]   Fig. 1. Percentage of ethanol-lever responding (top) and response rate (bottom) engendered by ethanol in squirrel monkeys trained to discriminate ethanol from vehicle. Points above V show the effects of saline administered in the first component followed by cumulative doses of ethanol. Data are means (±S.E.M.) in five subjects. *, p < 0.05, Dunnett's test.

Effects of Nonselective Benzodiazepine Receptor Ligands and Reference Drugs. Dose-related increases in ethanol-lever responding were observed after cumulative doses of the nonselective benzodiazepine receptor agonists flunitrazepam, midazolam, and diazepam (Fig. 2). All drugs fully reproduced the DS effects of ethanol, engendering 97, 99, and 97% ethanol-lever responding, respectively. Midazolam, but not flunitrazepam or diazepam, also reduced response rates to 75% of control rates at the highest dose tested [F(4,16) = 35.2, p < 0.05; Dunnett's tests, p < 0.05; data not shown].

View larger version (17K): [in this window] [in a new window]   Fig. 2. Percentage of ethanol-lever responding engendered by the nonselective benzodiazepine receptor agonists flunitrazepam, midazolam, and diazepam in squirrel monkeys trained to discriminate ethanol from vehicle. Data are means (±S.E.M.) in five subjects.

The reference compounds muscimol (a direct GABA_A agonist), baclofen (a GABA_B agonist), and morphine (a µ opioid agonist) did not engender a majority of responses on the ethanol-associated lever at any dose tested, producing approximately 10, 30, and 2% ethanol-lever responding, respectively. The highest dose of morphine virtually eliminated responding in all subjects, and the highest doses of muscimol and baclofen suppressed responding to 47 to 86% of control rates. Higher doses of muscimol and baclofen were not tested due to the potentially toxic effects in monkeys (seizures, emesis; Spealman, 1985; D. M. Platt, unpublished data).

Effects of the ₁GABA_A-Preferring Agonists Zaleplon, Zolpidem, and CL 218,872. Increasing cumulative doses of the ₁GABA_A agonist zaleplon engendered dose-related increases in the percentage of responses on the ethanol-associated lever, with full substitution for ethanol observed at doses that also reduced response rate [F(6,12) = 13.1, p < 0.05; Dunnett's tests, p < 0.05; Fig. 3, left]. Although the ₁GABA_A agonist zolpidem and the ₁GABA_A partial agonist CL 218,872 also produced ethanol-lever responding, the average maximum percent ethanol-lever responding engendered by these drugs usually fell short of the maximum engendered by ethanol (Fig. 3, middle and right). Zolpidem produced an average maximum of 48 to 57% ethanol-lever responding at doses that reliably reduced responding to approximately 69 to 23% of control rates [F(6,18) = 13.0, p < 0.05; Dunnett's tests, p < 0.05]. CL 218,872 engendered an average maximum of 70% ethanol-lever responding. Higher doses of this compound were not tested because hematuria was observed in three subjects.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Percentage of ethanol-lever responding (top) and response rate (bottom) engendered by the 1GABAA-preferring agonists zaleplon, zolpidem, and CL 218,872 in squirrel monkeys trained to discriminate ethanol from vehicle. Data are means (±S.E.M.) in five subjects. *, p < 0.05, Dunnett's test.

Effects of the ₁GABA_A-Preferring Antagonist -CCt. All doses (0.1–3.0 mg/kg) of the ₁GABA_A antagonist -CCt engendered primarily saline-lever responding (maximum percent ethanol-lever responding = 6.5 ± 6.5) and did not alter response rates in any subject. Subsequent experiments evaluated the ability of -CCt to antagonize the ethanol-like DS effects of zaleplon and zolpidem and the DS effects of ethanol itself. The degree of antagonism produced by -CCt was compared directly with the degree of antagonism produced by flumazenil, a nonselective benzodiazepine receptor antagonist. Alone, flumazenil (0.003–1.0 mg/kg) engendered primarily saline-lever responding (maximum percent ethanol-lever responding = 11.5 ± 11.5) and did not alter response rate regardless of dose.

Pretreatment with -CCt (3.0 mg/kg) attenuated the ethanol-like DS effects of zaleplon in a surmountable fashion, resulting in a slight shift to the right in the zaleplon dose-response function (Fig. 4, top left) and a 3-fold increase in ED₅₀ value [zaleplon alone = 0.4 mg/kg (±0.2), zaleplon + -CCt = 1.2 mg/kg (±0.4)]. At 1.0 mg/kg zaleplon, -CCt reliably reduced ethanol-lever responding (Bonferroni t test, p < 0.05). However, -CCt was virtually ineffective as an antagonist of the ethanol-like DS effects of zolpidem, producing little change in the zolpidem dose-response function (Fig. 4, top middle) and not altering the ED₅₀ value for zolpidem [0.2 mg/kg (±0.1)]. Flumazenil (1.0 mg/kg) was a more effective antagonist of zaleplon than -CCt, producing a larger rightward shift in the zaleplon dose-response function (Fig. 4, bottom left) and a greater than 25-fold increase in ED₅₀ value [zaleplon alone = 0.4 mg/kg (±0.2), zaleplon + flumazenil > 10.0 mg/kg]. At both 1.0 and 3.0 mg/kg zaleplon, flumazenil reliably reduced ethanol-lever responding (Bonferroni t tests, p < 0.05). Similarly, flumazenil attenuated the effects of zolpidem (Fig. 4, bottom middle), engendering at least a 2-fold increase in ED₅₀ value [zolpidem alone = 0.2 mg/kg (±0.1), zolpidem + flumazenil > 1.0 mg/kg]. At 0.1 and 1.0 mg/kg zolpidem, flumazenil reliably reduced ethanol-lever responding (Bonferroni t tests, p < 0.05). The ability of both zaleplon and zolpidem to surmount the effects of flumazenil could not be assessed because combinations of 1.0 mg/kg flumazenil with high doses of zaleplon (>5.6 mg/kg) and zolpidem (>1.0 mg/kg) engendered hematuria in all subjects. The dose-response functions for the effects of both zaleplon and zolpidem on rate of responding also appeared to be shifted to the right by -CCt and flumazenil (data not shown); however, in these monkeys, no dose of zaleplon or zolpidem reduced response rates to greater than 50% of control rates, and ED₅₀ values could not be calculated for any subject.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Percentage of ethanol-lever responding engendered by the 1GABAA-preferring agonists zaleplon (left), zolpidem (middle), and ethanol itself (right), alone and after pretreatment with the 1GABAA-preferring antagonist -CCt (3.0 mg/kg; top) or the nonselective benzodiazepine receptor antagonist flumazenil (1.0 mg/kg; bottom), in squirrel monkeys trained to discriminate ethanol from vehicle. Data are means (±S.E.M.) in a subset of three subjects. *, p < 0.05, Bonferroni t test.

Neither -CCt nor flumazenil antagonized the DS effects of ethanol itself (Fig. 4, right). The ED₅₀ values for the DS effects of ethanol in the presence of either antagonist were virtually identical to that of ethanol alone [ethanol alone = 0.5 g/kg (±0.03), ethanol + -CCt = 0.8 g/kg (±0.3), ethanol + flumazenil = 0.6 g/kg (±0.3)]. Although ethanol itself induced only a slight decrease in rate of responding (maximally reduced to 85% of control), pretreatment with neither -CCt nor flumazenil altered the ethanol-induced reduction in response rate (data not shown).

Effects of the ₅GABA_A-Preferring Agonists QH-ii-066 and Panadiplon. The ₅GABA_A agonist QH-ii-066 had DS effects that were qualitatively similar to those of ethanol (Fig. 5). Increasing cumulative doses of QH-ii-066 engendered dose-related increases in the percentage of responses on the ethanol-associated lever. Full substitution for ethanol was observed in each subject at a dose that did not significantly alter response rate (1.0 mg/kg). Similarly, the ₅GABA_A partial agonist panadiplon increased ethanol-lever responding, engendering an average maximum of approximately 96% ethanol-lever responding (Fig. 5). At this dose, response rates were reduced reliably to approximately 43% of control rates [F(5,10) = 45.5, p < 0.05; Dunnett's tests, p < 0.05].

View larger version (12K): [in this window] [in a new window]   Fig. 5. Percentage of ethanol-lever responding (top) and response rate (bottom) engendered by the 5GABAA-preferring agonists QH-ii-066 and panadiplon in squirrel monkeys trained to discriminate ethanol from vehicle. Data are means (±S.E.M.) in five subjects. *, p < 0.05, Dunnett's test.

Effects of the ₅GABA_A-Preferring Inverse Agonists L-655,708 and RY-23. When administered in the first component, a high dose of QH-ii-066 (1.8 mg/kg) engendered almost exclusive responding on the ethanol-paired lever (Fig. 6, top left, points over QH). When followed in subsequent components with saline injections, QH-ii-066 continued to engender a majority of responses on the ethanol-paired lever (data not shown). Administration of increasing doses of either L-655,708 or RY-23 attenuated the ethanol-like effects of QH-ii-066, reducing the percentage of responses on the ethanol-paired lever to approximately 16 and 11%, respectively [Fig. 6, top; L-655,708, F(4,8) = 4.0; RY-23, F(4,8) = 8.8, p < 0.05; Dunnett's tests, p < 0.05]. Based on A₅₀ values, L-655,708 was approximately three times more potent than RY-23 at decreasing the ethanol-like effects of QH-ii-066 [L-655,708 = 0.4 (±0.2) mg/kg, RY-23 = 1.4 (±0.5) mg/kg]. When tested alone, QH-ii-066 did not alter the average rate of responding (Fig. 6, bottom, points over QH), nor were response rates affected substantially by increasing doses of either inverse agonist (Fig. 6, bottom).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Percentage of ethanol-lever responding (top) and response rate (bottom) engendered by the 5GABAA-preferring agonist QH-ii-066, alone and after pretreatment with the 5GABAA-preferring inverse agonists L-655,708 and RY-23 in squirrel monkeys trained to discriminate ethanol from vehicle. Data are means (±S.E.M.) in a subset of three subjects. *, p < 0.05, Dunnett's test.

The degree to which the reduction of the ethanol-like DS effects of QH-ii-066 reflected inhibition of ₅GABA_A receptors was evaluated by administering L-655,708 and RY-23 with zolpidem, a ligand that does not have measurable affinity at ₅GABA_A receptors in vitro (Fig. 7). When administered in the first component, a high dose of zolpidem (1.8 mg/kg) engendered approximately 51% (±12) ethanol-lever responding. Administration of doses of L-655,708 (1.0 mg/kg) or RY-23 (3.0 mg/kg) that reliably reduced the ethanol-like DS effects of QH-ii-066 failed to attenuate the ethanol-like DS effects of zolpidem, with zolpidem engendering approximately 67% (±8) and 47% (±15) ethanol-lever responding following administration of L-655,708 and RY-23, respectively. Compared with vehicle, zolpidem reliably suppressed rate of responding [t(4) = 4.3, p < 0.05], and this reduction in rate was unaltered by either inverse agonist [L-655,708, t(4) = –1.5; RY-23, t(4) = –0.4, N.S.].

View larger version (13K): [in this window] [in a new window]   Fig. 7. Percentage of ethanol-lever responding (top) and response rate (bottom) engendered by the 1GABAA-preferring agonist zolpidem, alone and after pretreatment with the 5GABAA-preferring inverse agonists L-655,708 and RY-23 in squirrel monkeys trained to discriminate ethanol from vehicle. Data are means (±S.E.M.) in a subset of three subjects.

RY-23 administration resulted in seizure-like activity in one monkey; therefore, only L-655,708 was evaluated for its ability to attenuate the DS effects of ethanol itself. When administered in the first component, ethanol (1.0 g/kg) engendered exclusive responding on the ethanol-paired lever (Fig. 8, top, point over ETOH). When followed in subsequent components with saline injections, ethanol continued to engender virtually all responses on the ethanol-paired lever (data not shown). Administration of increasing doses of L-655,708 attenuated the DS effects of ethanol, reliably reducing the percentage of responses on the ethanol-paired lever to 0% [Fig. 8, top; F(5,10) = 8.1, p < 0.05; Dunnett's test, p < 0.05] and producing an A₅₀ value of 1.4 (±0.5) g/kg. Alone, ethanol did not alter substantially rate of responding, nor was response rate affected by increasing doses of L-655,708 (Fig. 8, bottom).

View larger version (13K): [in this window] [in a new window]   Fig. 8. Percentage of ethanol-lever responding (top) and response rate (bottom) engendered by ethanol, alone and after pretreatment with the 5GABAA-preferring inverse agonist L-655,708 in squirrel monkeys trained to discriminate ethanol from vehicle. Data are means (±S.E.M.) in a subset of three subjects. *, p < 0.05, Dunnett's test.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, the nonselective benzodiazepine agonists flunitrazepam, midazolam, and diazepam shared DS effects with ethanol in squirrel monkeys. These results are in agreement with other reports indicating that nonselective benzodiazepine site agonists mimic the DS effects of ethanol in rodents, pigeons, other monkey species, and humans (e.g., Grant, 1999; Kostowski and Bienkowski, 1999; Jackson et al., 2003). In contrast, the direct GABA_A agonist muscimol engendered primarily saline-lever responding up to doses that have been shown to produce convulsions in squirrel monkeys (Spealman, 1985). This finding also is consistent with earlier studies in rodents and monkeys in which muscimol was administered peripherally (Shelton and Balster, 1994; Grant et al., 2000). Neither the GABA_B agonist baclofen nor the µ opioid agonist morphine produced significant ethanol-appropriate responding at doses that either induced untoward side effects or eliminated responding. Together, these findings suggest that there is a significant role for positive allosteric modulation of GABA_A receptors in mediating the DS effects of ethanol. Moreover, despite a common ability to increase Cl^– conductance at GABA_A receptors, ethanol appears to produce a stimulus more similar to that of benzodiazepines rather than to that of the direct GABA_A agonist muscimol. This latter finding is not surprising given evidence that muscimol itself produces a distinct DS that does not overlap completely with other GABA_A agonists that do not interact directly and selectively with the GABA site on the GABA_A receptor (Grech and Balster, 1997; Jones and Balster, 1998).

Recent evidence suggests that stimulation of specific subtypes of GABA_A receptors may play different roles in mediating the abuse-related effects of ethanol (June et al., 2001; Harvey et al., 2002; Blednov et al., 2003b; McKay et al., 2004). In the present study, the ₁GABA_A agonists zolpidem, zaleplon, and CL 218,872 partially to fully reproduced the DS effects of ethanol. Likewise, zolpidem and zaleplon have been shown to partially reproduce the DS effects of ethanol in rodents (Bienkowski et al., 1997; Sanger, 1997). However, maximum substitution occurred at doses that either markedly reduced response rate or had other untoward side effects (e.g., hematuria). The ₁GABA_A antagonist -CCt failed to antagonize the DS effects of ethanol or the ethanol-like DS effects of zolpidem and zaleplon at a dose previously shown to antagonize the effects of zolpidem in both triazolam- and zolpidem-trained monkeys (i.e., produced an 11-fold rightward shift in the zolpidem dose-response function; Lelas et al., 2002; Rowlett et al., 2003). The lack of antagonism of zolpidem and zaleplon by -CCt likely was not due to a general inability to alter the agonist dose-response functions because the nonselective antagonist flumazenil attenuated the ethanol-like DS effects of both zaleplon and zolpidem. Rather, these findings suggest that ₁GABA_A receptors do not play a primary role in mediating the DS effects of ethanol and indicate that any ethanol-like DS effects of ₁GABA_A agonists, although benzodiazepine receptor-mediated, were not mediated at ₁GABA_A receptors.

The apparent lack of involvement of ₁GABA_A receptors in the DS effects of ethanol is in contrast to the prominent role this receptor subtype plays in other behavioral effects of ethanol. For example, knockout mice lacking the ₁ subunit are less likely to demonstrate a loss of righting reflex induced by ethanol compared with wild-type mice (Blednov et al., 2003a) and exhibit a reduced preference for ethanol in a two-bottle choice procedure (Blednov et al., 2003b). Moreover, self-administration of ethanol can be reduced by the ₁GABA_A receptor antagonists 3-propoxy--carboline hydrochloride and -CCt in rats (Harvey et al., 2002; Foster et al., 2004). It is possible that these diverging results reflect species and/or procedural differences (e.g., route of drug administration). An alternative conclusion that can be drawn from these observations is that the DS, reinforcing, and motor-impairing effects of ethanol likely involve incompletely overlapping neurobiological processes. Although stimulation of GABA_A receptors undoubtedly plays a key role in each of these behavioral effects, potentially important differences appear to exist in the relative contribution of GABA_A receptors containing the ₁ subunit.

The observation that zolpidem, a ligand that does not bind at ₅GABA_A receptors, produced only partial ethanol-like responding raises the intriguing possibility that ₅GABA_A receptors may be critical to the full expression of ethanol-like DS effects. In support of this hypothesis, the ₅GABA_A-preferring agonists QH-ii-066 and panadiplon substituted fully for the DS effects of ethanol. In contrast, in monkeys trained to discriminate the benzodiazepine agonist triazolam from vehicle, neither QH-ii-066 nor panadiplon substituted for the DS effects of triazolam, and in combination with triazolam, QH-ii-066 even at high doses failed to modulate the DS effects of triazolam to any meaningful extent (Lelas et al., 2002; D. M. Platt, unpublished data). These findings raise the possibility of a specific and unique role for ₅GABA_A receptor mechanisms in mediating the DS effects of ethanol but not those of conventional benzodiazepine receptor agonists.

In subsequent antagonism studies, the ₅GABA_A receptor inverse agonists L-655,708 and RY-23 completely attenuated the ethanol-like DS effects of QH-ii-066 at doses that did not alter the ethanol-like DS effects of zolpidem. In addition, L-655,708 was found to reverse completely the DS effects of ethanol itself at doses that did not disrupt responding, suggesting that this blockade was pharmacologically specific and not the result of a nonspecific disruption of behavior. These findings support the idea that stimulation of ₅GABA_A receptors underlies the shared DS effects of ethanol and QH-ii-066. Together with the observation that RY-23 and RY-24, another ₅GABA_A inverse agonist, selectively reduce ethanol self-administration compared with self-administration of either saccharin or water in rats (June et al., 2001; McKay et al., 2004), our results suggest a key role for ₅GABA_A receptors in both the DS and reinforcing effects of ethanol that may ultimately underlie its abuse.

₅GABA_A receptors comprise only a small population of GABA_A receptors and are found primarily in the hippocampus (Sur et al., 1999). The hippocampus has been shown to mediate at least in part declarative memory, which has been associated with the learning of affective states associated with drug intake (Berke and Eichenbaum, 2001). Moreover, projections from the CA1 and CA3 hippocampal fields have been shown to innervate putative ethanol reward substrates (Kelley and Domesick, 1982). These findings raise the possibility that the effectiveness of ₅GABA_A inverse agonists at blocking the DS effects of ethanol may be related to their ability to modulate affective states associated with alcohol use.

In summary, our findings suggest a prominent role for ₅GABA_A, but not ₁GABA_A, receptor mechanisms in the DS effects of ethanol and the ethanol-like DS effects of benzodiazepine agonists. The results implicate ₅GABA_A receptors as potential pharmacological targets for medications to blunt ethanol's subjective effects and reduce alcohol abuse.

	Acknowledgements

 
We thank S. Malinak and K. Bano for assistance with data collection.

	Footnotes

 
This research was supported by U.S. Public Health Service Grants AA12407, AA13850, DA11792, MH46851, and RR00168. Preliminary reports of these data were presented at the 2003 and 2004 annual meetings of the Research Society on Alcoholism.

doi:10.1124/jpet.104.080275.

ABBREVIATIONS: -CCt, -carboline-t-butyl ester; RY-23, tert-butyl 8-[(trimethylsilyl)ethynyl]-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]-benzodiazepine-3-carboxylate; DS, discriminative stimulus; CL 218,872, 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine; QH-ii-066, 1-methyl-7-acetyleno-5-phenyl-1,3-dihydro-benzo[e]-1,4-diazepin-2-one; panadiplon, 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)imidazo(1,5-a)quinoxalin-4(5H)-one; L-655,708, ethyl [S]-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxlate; FR, fixed ratio.

Address correspondence to: Dr. Donna M. Platt, Harvard Medical School/New England Primate Research Center, One Pine Hill Drive, Box 9102, Southborough, MA 01772-9102. E-mail: donna_platt{at}hms.harvard.edu

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