JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 JPET FastThis Forward.article has not Published been copyedited on and December formatted. The 8, final 2005 version as DOI:10.1124/jpet.105.094003may differ from this version.

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Title Page

Comparative cue generalisation profiles of L-838,417, SL651498,

zolpidem, CL218,872, ocinaplon, bretazenil, zopiclone and various

benzodiazepines in chlordiazepoxide and zolpidem drug Downloaded from

discrimination

jpet.aspetjournals.org

Mirza NR, Rodgers RJ and Mathiasen LS

NeuroSearch A/S, 93 Pederstrupvej, Ballerup, DK-2750, Denmark (NRM,

LSM); Behavioural Neuroscience Laboratory, Institute of Psychological at ASPET Journals on October 1, 2021

Sciences, University of Leeds, Leeds LS2 9JT, England (JRR, LSM)

1

Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 This article has not been copyedited and formatted. The final version may differ from this version.

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Running Title Page: Subtype-selective GABAA receptor modulators in drug discrimination

Contact and address for correspondence:

Naheed R. Mirza

Department of In-vivo Pharmacology

NeuroSearch A/S

93 Pederstrupvej Downloaded from DK-2750 Ballerup

Denmark

Tele: +45 44 60 82 71 jpet.aspetjournals.org FAX: +45 44 60 80 80

E-mail: [email protected]

at ASPET Journals on October 1, 2021 Number of text pages: 19

Number of tables: 0

Number of figures: 4

Number of references: 35

Number of words in Abstract: 252

Number of words in Introduction: 1028

Number of words in Discussion: 1598

Non-standard abbreviations: None

Preferred section assignment: Behavioural Pharmacology

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Abstract

The zolpidem discriminative cue is mediated by GABAA-α1 receptors, whereas the chlordiazepoxide cue may be mediated via non-α1 GABAA receptors, since compounds with selective affinity for GABAA-α1 receptors fully generalise to the former cue. We predicted that L-

838,417, a partial agonist at non-α1 GABAA receptors and an antagonist at GABAA-α1 receptors, would generalise to the chlordiazepoxide but not the zolpidem discriminative cue. SL651498 is a full agonist at GABAA-α2 receptors, with lower efficacy at GABAA-α3 receptors, and least efficacy Downloaded from at GABAA-α1 and GABAA-α5 receptors. Since SL651498 has efficacy at GABAA-α1 receptors, we anticipated it would generalise to both discriminative cues. Rats were trained to discriminate either

zolpidem (3 mg/kg) or chlordiazepoxide (5 mg/kg) from vehicle using a two lever operant jpet.aspetjournals.org procedure. The generalisation profiles of L-838,417 and SL651498 were compared with non- selective full agonists, GABAA-α1-selective ligands zolpidem and CL218,872, the non-selective partial agonist bretazenil, and the novel anxioselective drug ocinaplon. A non-selective partial at ASPET Journals on October 1, 2021 agonist was included since L-838,417 and SL651498 are partial agonists at some GABAA receptors, and this property may influence their generalisation profiles. All non-selective full agonists and ocinaplon fully generalised to both cues. CL218,872 and zolpidem generalised to zolpidem only, whereas L-838,417 fully generalised to chlordiazepoxide only. SL651498 fully generalised to chlordiazepoxide and occasioned significant zolpidem-appropriate responding. Bretazenil was similar to SL651498. In conclusion, at this training dose, the chlordiazepoxide discriminative stimulus is mediated primarily via non-α1 GABAA receptors, and the generalisation profiles of the ligands tested appears to correspond with their in vitro profiles at GABAA receptor subtypes.

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Introduction

The majority of GABAA receptors in the mammalian brain contain at least one α, β, and γ subunit

(Barnard et al., 1998). The likely stoichiometry is two α, two β and one γ subunit arranged pentamerically around a central pore through which Cl- ions flow when the receptor is activated.

Classical benzodiazepines modulate GABAA neurotransmission by binding to a site that traverses an α and γ subunit of α1, α2, α3 or α5 containing GABAA receptors (Sieghart, 1995). Mice with a point mutation of either α1 (Rudolph et al., 1999; McKernan et al., 2000) or α2 (Low et al., 2000) Downloaded from subunits are insensitive to the motor-impairing and anxiolytic-like effects of diazepam, respectively, suggesting different functional roles for GABAA- α1 and α2 receptors.

jpet.aspetjournals.org

Pharmacological evidence has also alluded to different functional roles for GABAA receptor subtypes (e.g., Griebel et al., 1999a, Chen et al., 1996). For example, the triazolopyridazine CL-

218,872 was found to bind with different affinity to two populations of benzodiazepine receptor, at ASPET Journals on October 1, 2021 originally designated type I (α1 containing GABAA receptors) and II (α2, α3 or α5 containing

GABAA receptors) receptors (Klepner et al., 1979; Squires et al., 1979). Furthermore, the benzodiazepine receptor antagonist β-carboline-3-carboxylate t-butyl ester (β-CCT) selectively antagonised the anticonvulsant and anxiolytic, but not the myorelaxant, effects of diazepam, whereas flumazenil blocked all three effects (Shannon et al., 1984). Further studies confirmed β-

CCT was more selective than flumazenil in antagonising the behavioural effects of benzodiazepine site ligands in vivo (Griebel et al., 1999b; Paronis et al., 2001; Rowlett et al., 2005b). In vitro studies showed that β-CCT had selective affinity for GABAA-α1 receptors (Huang et al., 1999), and was more potent at antagonising zolpidem potentiation of GABA’s effect at GABAA-α1 receptors than diazepam potentiation of GABA at GABAA-α3 or GABAA-α5 receptors (Griebel et al., 1999b).

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The discriminative stimulus effects of benzodiazepines have been widely described, and this tool has been employed to determine receptor selectivity and efficacy of benzodiazepine site ligands

(see review by Lelas et al., 2000). For example, in squirrel monkeys trained to discriminate zolpidem at low (1 mg/kg) and high doses (>3 mg/kg), generalisation to non-selective benzodiazepines occurred in the former but not latter set of animals (Rowlett et al., 1999; Rowlett et al., 2000). Moreover, in animals discriminating high doses of zolpidem, generalisation to the

GABAA-α1 selective drug zaleplon occured. Thus, at certain training doses, the zolpidem Downloaded from discriminative stimulus is mediated via GABAA-α1 receptors. In rats the zolpidem discriminative stimulus might also be mediated via GABAA-α1 receptors, whereas the chlordiazepoxide

α discriminative stimulus might be mediated by non- 1 GABAA receptors (Sanger and Benavides, jpet.aspetjournals.org

1993; Sanger et al., 1999). This is based on the finding that drugs with affinity-selectivity for

GABAA-α1 receptors (e.g., zolpidem, CL218,872) fully generalised to the zolpidem but not the

chlordiazepoxide cue. at ASPET Journals on October 1, 2021

In this study we tested the hypothesis that the zolpidem and chlordiazepoxide discriminative stimuli in rats are mediated predominantly via GABAA-α1 and non-α1 GABAA receptors, respectively, by testing novel compounds not previously available as well as well-characterised pharmacological tools. The compounds tested fell in to various categories on the basis of affinity and efficacy: (1) non-selective full agonists, modulators which potentiate the effect of GABA at GABAA- α1, α2, α3 and α5 receptors equivalently (Sieghart, 1995; Hadingham et al., 1993; Faure-Halley et al., 1993).

This category included benzodiazepines and zopiclone, largely overlapping with compounds tested previously (Sanger and Benavides, 1993; Sanger et al., 1999); (2) selective positive benzodiazepine site modulators, which were subdivided in to two groups: (2a) compounds with selective affinity, including the imidazopyridine zolpidem and triazolopyridazine CL218,872, ligands that selectively bind to GABAA-α1 receptors. These compounds selectively potentiate the effect of GABA at

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GABAA-α1 receptors, although this is concentration-dependent; i.e., they only show 10-20 fold affinity selectivity for GABAA-α1 receptors over GABAA-α2 or α3 receptors, despite >1000-fold selectivity over GABAA-α5 receptors (Sieghart, 1995; Hadingham et al., 1992; 1993; Faure-Halley et al., 1993). To add to the complexity, zolpidem has high intrinsic efficacy at GABAA-α1 receptors, whereas CL218,872 displays reduced efficacy at this receptor, i.e., CL218,872 is a partial agonist at

GABAA-α1 receptors. (2b) Functionally (or efficacy) selective compounds include L-838,417

(McKernan et al., 2000) and SL651,498 (Griebel et al., 2001). These compounds bind with similar Downloaded from affinity to α1, α2, α3 or α5 containing GABAA receptors, but are able to differentially modulate these receptor subtypes leading to selectivity. Thus, L-838,417 does not potentiate the effect of jpet.aspetjournals.org GABA at GABAA-α1 receptors, but does so at GABAA- α2, α3 and α5 receptors. Moreover, L-

838,417 is a partial agonist at these latter receptor subtypes. SL651498 differs in that it fully potentiates the effect of GABA at GABAA-α2 receptors, has marginally lower efficacy at GABAA- at ASPET Journals on October 1, 2021 α3 receptors, with least efficacy (partial agonist profile) at GABAA- α1 and α5 receptors.

We predicted that zolpidem and CL218,872 would generalise fully to the zolpidem but not chlordiazepoxide cue, whereas an opposite profile would be seen with L-838,417 and, possibly

SL651498 (see below). However, since a number of the selective positive modulators described above have partial agonist profiles, in addition to showing either affinity or functional selectivity, any difference in behavioural profile could potentially be attributed to their partial agonism rather than selectivity properties (Lelas et al., 2000). Therefore we included a (3) non-selective partial agonists, such as bretazenil, modulators which potentiates the effect of GABA equally at GABAA-

α1, α2, α3 and α5 receptors, albeit with reduced efficacy compared to a full agonist (Haefely et al.,

1990; Pieri et al., 1988). Finally, we predicted that since SL651498 has appreciably greater efficacy at GABAA-α1 receptors than L-838,417 (Atack, 2003), it would show correspondingly greater zolpidem-like effects.

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A number of compounds with selectivity for non-α1 GABAA receptors are currently in clinical trials, including SL651498. However, compounds that modulate GABAA receptors with different profiles are also being tested in the clinic. For example, the pyrazolopyrimidine ocinaplon has a anxioselective profile in man, although Phase III clinical trials for the treatment of generalised anxiety disorder have been halted due to potential liver toxicity risks (Lippa et al., 2005; Basile et al., 2004; www.bioworld.com). Ocinaplon is a low affinity benzodiazepine site ligand with modest selectivity for GABAA-α1 receptors (Lippa et al., 2005). We predicted that ocinaplon would Downloaded from generalise similarly to both discriminative cues. jpet.aspetjournals.org at ASPET Journals on October 1, 2021

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Methods

Animals

Male PVG hooded rats were used (M&B Breeding & Research Centre, Denmark, 200-250 g).

Animals were housed and habituated for at least 7 days in Macrolon III cages (20 x 40 x 18 cm, 2 rats/cage; Scanbur BK A/S, Ejby, Denmark) before operant conditioning commenced. Food

 (Altromin , Brogaarden A/S, Buddinge, Denmark) was available ad libitum, whereas water was available 10 minutes every day after drug discrimination training sessions (see below) and ad Downloaded from libitum at weekends. Animals were housed on a 12-hour light/dark cycle (lights on at 7 a.m. and off at 7 p.m.). All testing procedures were in accordance with “Methods and Welfare Considerations in

Behavioral Research with Animals” (NIH Publication No. 02-5083, March 2002) and the Danish jpet.aspetjournals.org

Committee for Experiments on Animals.

Drugs and solutions at ASPET Journals on October 1, 2021

Diazepam, midazolam, alprazolam, lorazepam, triazolam, chlordiazepoxide, zopiclone and

CL218,872 were all purchased from various commercial sources (Sigma-Aldrich, RBI or Tocris).

Zolpidem, ocinaplon, SL651498 and L-838,417 were all synthesized by the medicinal chemistry department, NeuroSearch A/S. Bretazenil and flumazenil were gifts from Roche (Switzerland). All drugs were prepared in 5% cremophor and administered i.p. in a volume of 1 ml/kg, The majority of drugs were administered 30 min prior to drug discrimination test sessions. However, zolpidem, midazolam and zopiclone were administered 15 min and triazolam 10 min prior to test sessions.

Drug Discrimination Protocol

For discrimination training, standard operant chambers (Coulbourn Instruments, PA, USA) equipped with 2 levers and a water-spout located equidistant between the two levers were used. The operant chambers were enclosed within sound isolation cubicles, and behavioural contingencies,

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JPET #94003 reinforcement scheduling and data collection as outlined below were controlled by a MedState

Notation (version 2.0) WMPCTM software package (MED Associates Inc., VT, USA).

Basic training. Animals were water deprived for 24 hours prior to the first training session. Rats were shaped to lever press for water on a schedule where one water reward (0.2 ml) was delivered every 60 sec and one lever press on either left or right lever was reinforced with an additional water delivery. Animals were kept on this schedule until they operated both levers to obtain water Downloaded from rewards. Rats that did not operate levers at all after 5 days were excluded from further training.

Once adequate lever responding was attained rats were switched to a FR1 schedule (i.e., water was delivered each time a lever was pressed) without any supplemental water every 60 sec. Animals jpet.aspetjournals.org were then progressed rapidly to a FR10 schedule (i.e. water was delivered only after 10 consecutive presses were made on a single lever). Rats were trained to reliably press both levers on the FR10 schedule for water reward before commencing discrimination training. Further training was given at ASPET Journals on October 1, 2021 for individual rats if necessary, e.g., if a rat had a non-preferred lever, it was maintained on this lever for 5 days until it was able to reliably complete the FR10 schedule.

Discrimination training. In total 24 animals were trained to discriminate chlordiazepoxide (5 mg/kg) from vehicle and a different group of 24 animals were trained to discriminate zolpidem (3 mg/kg) from vehicle, although not all animals were trained in the same time-frame. For each discrimination half the rats were trained so that the left lever was the drug-associated lever, and for the other half the right lever was the drug-associated lever. In all cases, the opposite lever was the vehicle-associated lever. Drug (D) or vehicle (V) injections were given before training sessions according to the following pseudorandom 2-weekly sequence (Monday-Friday): DVVDDVDDVV.

Training sessions lasted for 15 minutes and only the lever appropriate to the injection schedule above was active in a given session (i.e., resulted in water delivery on completion of an FR10).

Each rat was trained to a criterion of making the first 10 consecutive responses on the correct lever,

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JPET #94003 appropriate for drug or vehicle injection, when first placed in the chamber and the session initiated.

Moreover, animals had to make ≥80% of response on the correct lever, appropriate for drug or vehicle injection, in a session. This level of performance had to be maintained for two weeks prior to drug testing. Drug discrimination training took ~3 months. Initial studies showed that flumazenil

(10 mg/kg, i.p.) could antagonise both discriminative cues (data not shown).

Generalisation Tests. The testing schedule differed from the training schedule in that both levers Downloaded from resulted in water delivery on a FR10 schedule and the session lasted 5 minutes. Test sessions with experimental drugs (see Drugs and solutions) were run on Tuesdays and Thursdays, with baseline

sessions according to the above mentioned pseudorandom injection sequence on other days. If at jpet.aspetjournals.org any time during baseline training sessions a rat selected the wrong lever, it was retained on training until for three consecutive sessions it was able to: (i) make the first 10 consecutive responses on the

correct lever (appropriate for drug or vehicle injection) when first placed in the chamber; (ii) make at ASPET Journals on October 1, 2021

≥80% responses on the lever appropriate to the injection protocol (i.e., drug or vehicle injection).

This criterion in addition to the fact that not all 24 animals from each of the two discriminations were available at the same time means the N for each drug tested differs (see Figure legends).

Data Analyses. Two measures of generalisation potential of a test drug to the training drug are presented (see Stolerman, 1993): (i) the percentage of responses on the drug-associated lever (drug lever responses/total responses x 100); and (ii) the number of rats correctly selecting the drug appropriate lever (i.e., the first 10 consecutive responses on the drug lever when first placed in the chambers and the session initiated). An animal had to make at least 10 responses on one of the levers to be included in either index of discrimination. A test compound was deemed to show full generalisation to the training drug when ≥80% responding was on the drug lever and/or ≥80% of animals selected the drug-associated lever. Response rate (responses/sec) were analysed by one-way

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Results

1. Non-selective full agonists

Chlordiazepoxide discrimination. All compounds in figure 1 resulted in a dose-dependent increase in percentage drug-lever appropriate responding, with rats making >90% drug-lever responses after administration of the highest dose of each drug tested (Figs. 1.1 and 1.2, upper panels). The only exception was triazolam, with rats making >90% responses on the drug lever at all doses tested, and with no clear dose-dependency. Similarly, more animals selected the drug lever (i.e., the first 10 Downloaded from consecutive responses were on the drug lever) with increasing dose of each drug, with 90-100% of animals selecting the drug lever at some dose of each drug (see fractions in Figs. 1.1 and 1.2, upper

panels). jpet.aspetjournals.org

With respect to effects on response rate, zopiclone (F[3,63]=0.9), chlordiazepoxide (F[3,44]=1.7),

diazepam (F[3,28]=0.8) and midazolam (F[3,42]=1.0) had no significant effect on this measure at ASPET Journals on October 1, 2021 even at doses exceeding those giving >90% responses on the drug lever (Fig. 1.1, upper panels). For these compounds almost all animals met the inclusion criteria of completing at least 10 responses on one of the levers. By contrast, triazolam (F[5,103]=9.8, P<0.001), alprazolam (F[5,79]=10.9,

P<0.001) and lorazepam (F[3,31]=6.7, P<0.001) significantly and dose-dependently reduced response rates (Fig. 1.2, upper panels).

Zolpidem discrimination. In the zolpidem cue condition, full generalisation was seen with all non- selective full agonists, i.e., rats made ≥80% of responses on the drug lever at some dose of each of the drugs (Figs. 1.1 and 1.2, lower panels). Furthermore, the number of animals selecting the drug lever (i.e., the first 10 consecutive responses were on the drug lever) increased as a function of dose

(as seen for the chlordiazepoxide cue), with >80% of animals selecting the zolpidem lever at some dose of each drug (see fractions in Figs. 1.1 and 1.2, lower panels).

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Regarding response rates, all compounds significantly reduced response rate at some dose (Figs. 1.1 and 1.2, lower panels). Thus, in contrast to their lack of significant effect on response rate in chlordiazepoxide trained animals, in zolpidem trained animals zopiclone (F[5,91]=8.4, P<0.001), chlordiazepoxide (F[3,26]=21.6, P<0.001), diazepam (F[4,76]=9.9, P<0.001) and midazolam

(F[3,30]=24.2, P<0.001) all significantly reduced response rate (Fig. 1.1, lower panels). Triazolam

(F[5,66]=29.4, P<0.001), alprazolam (F[5,66]=14.8, P<0.001) and lorazepam (F[3,25]=8.2, Downloaded from P<0.001) all significantly and dose-dependently reduced response rate in zolpidem trained animals

(Fig. 1.2, lower panels), as they did in chlordiazepoxide trained animals. Of particular interest, for the majority of drugs tested, rats only made ≥80% responses on the zolpidem lever at doses that also jpet.aspetjournals.org significantly reduced response rate. Exceptions were lorazepam (Fig. 1.2, panel F) and zopiclone

(Fig. 1.1, panel H), where full generalisation was seen at doses of 1.0 and 10 mg/kg, respectively, with no significant reduction in response rate. However, at higher doses, both lorazepam (3.0 at ASPET Journals on October 1, 2021 mg/kg) and zopiclone (30 mg/kg) significantly reduced response rates. This was particularly evident at 3 mg/kg lorazepam, leading to a disruption of discriminative control.

2. Selective positive benzodiazepine site modulators

Chlordiazepoxide discrimination. Neither zolpidem nor CL218,872, generalised to the chlordiazepoxide cue whether measured as the percentage of drug lever responses made by rats or number of animals selecting the drug lever (i.e., the first 10 consecutive responses were on the drug lever, see Figs. 2A and 2B). A maximum of 3/11 zolpidem and 3/9 CL218,872 treated rats selected the drug lever at any one dose (see fractions in Figs. 2A and 2B). Both zolpidem (F[4,45]=9.8,

P<0.001) and CL218,872 (F[4,48]=2.9, P<0.03) significantly reduced response rate. This was particularly marked at 10 mg/kg zolpidem, with only 2 animals attaining the inclusion criterion.

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In contrast, both L-838,417 (Fig. 2C) and SL651498 (Fig. 2D) dose-dependently increased chlordiazepoxide-appropriate responding, with rats making >90% responses on the drug lever after administration of the highest dose of either drug. Furthermore the number of animals selecting the drug lever (i.e., the first 10 consecutive responses were on the drug lever) increased as a function of dose, with 90-100% of animals selecting the chlordiazepoxide lever at the highest dose of L-

838,417 and SL651498 (see fractions in Figs. 2C and 2D). SL651498 had no significant effect on response rate (F[3,59]=0.2) over the dose range tested. By contrast, the effect of L-838,417 on Downloaded from response rate was marginally significant (F[4,29]=2.54, P<0.06). Whilst not significant this finding is worth noting since it would appear that L-838,417 had a tendency to increase response rate in chlordiazepoxide trained animals (Fig. 2C). jpet.aspetjournals.org

Zolpidem discrimination. A very different, and to some extent opposite, pattern of results was seen with the four test drugs in this discriminative cue condition. Firstly, both zolpidem (Fig. 2E) and at ASPET Journals on October 1, 2021 CL218,872 (Fig. 2F) fully generalised to this cue, with rats making >90% of responses on the drug- lever at doses of 1.0 and 3.0 mg/kg, respectively. At these doses of zolpidem and CL218,872, 100% of animals selected the drug lever (i.e., the first 10 consecutive responses were on the drug lever, see fractions in Figs. 2E and 2F). As in chlordiazepoxide trained animals, zolpidem significantly

(F[3,39]=3.7, P<0.02) reduced response rate (Fig. 2E). In contrast, CL218,872 had no effect

(F[3,59]=1.3), although doses higher than 3 mg/kg were not tested as in chlordiazepoxide trained animals (Fig. 2F).

In contrast to its full generalisation to the chlordiazepoxide cue, L-838,417 resulted in rats making a maximum of ~50% responses on the zolpidem-associated lever (Fig. 2G). This was also reflected in a maximum of only 2/8 animals selecting the zolpidem lever (i.e., the first 10 consecutive responses were on the drug lever) at any given dose (see fractions in Fig. 2G). However, as was the case for the chlordiazepoxide cue, L-838,417 (up to 30 mg/kg) had no significant effect on response rate

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(F[4,39]=1.2) and all animals tested attained the response criterion. By contrast, SL651498 dose- dependently generalised to the zolpidem cue leading rats to make ~80% drug lever appropriate responses at 10 mg/kg (Fig. 2H). At this same dose 5/7 (71%) animals selected the zolpidem lever

(i.e., the first 10 consecutive responses were on the drug lever), compared to 13/14 (91%) selecting the chlordiazepoxide lever at this dose. As with its lack of effect on response rate in chlordiazepoxide trained animals, SL651498 had no significant effect (F[3,29]=0.6) on response rate in zolpidem trained animals. Downloaded from

3. The partial positive allosteric modulator bretazenil and the novel anxioselective agent ocinaplon jpet.aspetjournals.org Chlordiazepoxide discrimination. The partial agonist bretazenil dose-dependently occasioned drug appropriate responding with rats making 100% of responses on the drug associated lever at 10 mg/kg (Fig. 3A). There was no effect on response rate after bretazenil (F[4,53]=0.4) across the dose at ASPET Journals on October 1, 2021 range tested and, at the highest dose, all animals selected the drug lever (i.e., the first 10 consecutive responses were on the drug lever, see fractions in Fig. 3A). The dose response curve for ocinaplon was steep, with rats fully generalising to the drug associated lever after being administered the highest dose of 10 mg/kg (Fig. 3B), and with all animals at this dose selecting the drug lever (i.e., the first 10 consecutive responses were on the drug lever, see fractions in Fig. 3B). At no dose did ocinaplon reduce response rate in these animals (F[3,30]=2.2).

Zolpidem discrimination. Bretazenil also showed appreciable generalisation to the zolpidem discriminative cue (Fig. 3C). However, the dose-response curve in the zolpidem-trained animals was shallow compared to that in chlordiazepoxide-trained animals, although bretazenil did occasion

~70% drug-lever appropriate responding by rats at doses between 3-30 mg/kg. Moreover, 8/10

(80%) animals selected the drug lever (i.e., the first 10 consecutive responses were on the drug lever) at 10 mg/kg bretazenil in the zolpidem cue. No dose of bretazenil affected response rate in

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JPET #94003 these animals (F[5,65]=1.2). Ocinaplon had a very similar profile in zolpidem-trained animals as it did in chlordiazepoxide-trained animals, i.e., a steep dose-response curve with rats making ≥80% responses on the drug associated lever at 10 mg/kg, most animals (7/8) selecting the drug-lever (i.e., the first 10 consecutive responses were on the drug lever), and no effect on response rate

(F[3,31]=0.8) at the doses tested (Fig. 3D). Downloaded from jpet.aspetjournals.org at ASPET Journals on October 1, 2021

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Discussion

The data generated from animals trained to discriminate either zolpidem or chlordiazepoxide indicate that drug discrimination may be a useful tool for further in vivo characterisation of subtype selective GABAA receptor positive modulators. Full generalisation to chlordiazepoxide by the non- selective full agonists lorazepam, midazolam, diazepam, triazolam, alprazolam and zopiclone agrees with previous reports (Sanger et al., 1987; Sanger and Benavides, 1993). Likewise we have shown that these compounds also all fully generalise to the zolpidem cue. The full generalisation to Downloaded from both discriminative cues can probably be attributed to the fact that these compounds do not differ substantially in terms of affinity or efficacy at α1, α2, α3 or α5 containing GABAA receptors

(Sieghart, 1995; Hadingham et al., 1993; Faure-Halley et al., 1993). jpet.aspetjournals.org

Interestingly, previous studies have shown that chlordiazepoxide only partially generalised to the zolpidem cue in rats and squirrel monkeys (Depoortere et al., 1986; Rowlett et al., 1999). In the at ASPET Journals on October 1, 2021 present study, chlordiazepoxide at 30 mg/kg fully generalised to the zolpidem cue, although it is possible that this level of generalisation may reflect the limited number of animals (N=4) attaining the inclusion criteria at this dose. However, with most non-selective full agonists, maximum generalisation to the zolpidem cue occurred at doses significantly reducing the response rate, whereas full generalisation to the chlordiazepoxide was not concurrent with a reduction in response rate. This dissociation in the effect on response rate between the two cues was not an artefact of a difference in baseline responding between animals trained on the two discriminations (compare upper and lower panels of all figures). The basis for this dissociation are not clear, although it may be that chlordiazepoxide-discriminating rats have some cross-tolerance to the rate-decreasing effects of non-selective full agonists (compare response rate across the top and bottom panels of

Fig. 1.1).

16 JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 This article has not been copyedited and formatted. The final version may differ from this version.

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In contrast to the non-selective modulators described above, zolpidem and CL218,872 which both show affinity selectivity for GABAA-α1 receptors, showed very limited generalisation to the chlordiazepoxide cue. By contrast, zolpidem and CL218,872 fully and dose-dependently generalised to the zolpidem cue. However, ~60-70% generalisation to the chlordiazepoxide discriminative cue has previously been demonstrated with these two GABAA-α1 selective compounds (Sanger et al., 1987; Sanger and Benavides, 1993). Whether our data differ from previous reports due to differences in methodological procedures or potentially differences in the Downloaded from strain of rat employed by us and others is unclear. However, the rate decreasing effects of zolpidem and CL218,872 in chlordiazepoxide discriminating animals (Figs. 2A and 2B) suggests that dose limitation cannot easily account for the lack of generalisation to the chlordiazepoxide cue. Rather, jpet.aspetjournals.org since zolpidem and CL218,872 show affinity selectivity for GABAA-α1 receptors, the lack of significant generalisation to the training dose of chlordiazepoxide suggests that this cue is mediated

primarily via non-α1 GABAA receptors, whereas the zolpidem cue is primarily mediated by at ASPET Journals on October 1, 2021

GABAA-α1 receptors. Even though CL218,872 and zolpidem differ in some respects, namely that

CL218,872 is generally 10-fold less potent at the GABAA-α1 receptor and has lower intrinsic efficacy at this receptor subtype than zolpidem (Sieghart, 1995), the two compounds have the same selective discriminative profile in the present study suggesting that selective affinity for the

GABAA-α1 receptor is the key determinant of the profile of both compounds.

However, concluding that the chlordiazepoxide cue is primarily mediated via non-α1 GABAA receptors on the basis of a null effect with zolpidem and CL218,872 is a weak argument, and would be strengthened if it were based on a positive data set. In this regard, L-838,417, a compound with functional selectivity for GABAA- α2, α3 and α5 receptors over GABAA-α1 receptors, has a generalisation pattern in the two discriminative cue conditions opposite to that seen with zolpidem and CL218,872. Thus, L-838,417 dose-dependently and fully generalised to the chlordiazepoxide

17 JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #94003 cue (Fig. 2C), whereas it occasioned only a maximum of ~50% zolpidem-appropriate responding

(Fig. 2G). Also, whereas 100% of animals selected the chlordiazepoxide lever at 30 mg/kg L-

838,417, only 25% selected the zolpidem lever at this same dose. At the doses tested, L-838,417 had no effect on response rate in either set of discriminating animals. Since L-838,417 has no intrinsic efficacy at GABAA-α1 receptors (McKernan et al., 2000), this property would appear to explain its lack of generalisation to the zolpidem discriminative cue, argued to be mediated by

GABAA-α1 receptors above. However, whilst this is the most parsimonious conclusion, particularly Downloaded from if the percentage drug-lever response data and the number of animals selecting the drug lever are considered together, it is arguable that L-838,417 does induce some zolpidem appropriate responding if the former measure is taken in isolation (i.e., ~50% drug lever responses, see Fig. jpet.aspetjournals.org

2G). One possibility for this apparent zolpidem-like property of L-838,417 is that the zolpidem discriminative stimulus is not exclusively mediated via GABAA-α1 receptors but also has GABAA-

α2 and α3 receptor components, possibly consistent with the modest 10-20 fold affinity selectivity at ASPET Journals on October 1, 2021 of zolpidem for the former compared to the latter receptor subtypes (Sieghart, 1995). Future studies assessing the influence of different training doses of zolpidem may shed light on this issue.

Recently, Rowlett et al. (2005a) showed that zolpidem and diazepam fully generalised to the triazolam cue in squirrel monkeys whereas L-838,417 showed no generalisation up to 10 mg/kg.

Although it was suggested that the reason for this was that the triazolam cue was mediated via

GABAA-α1 receptors at which L-838,417 has no efficacy (McKernan et al., 2000), assessing the discriminative profile of L-838,417 in animals trained to discriminate zolpidem may have been more conclusive (Rowlett et al., 1999; 2000). Nonetheless, our data with L-838,417 concur with the conclusions of Rowlett et al. (2005a), and demonstrate that its selective generalisation to the chlordiazepoxide cue over the zolpidem cue mimics the selectivity seen with this compound in vitro

(McKernan et al., 2000).

18 JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 This article has not been copyedited and formatted. The final version may differ from this version.

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Like L-838,417, bretazenil fully generalised to the chlordiazepoxide cue. However, in contrast to L-

838,417, bretazenil showed more robust zolpidem lever responding (~70%) and, moreover, 80% of animals selected the zolpidem lever at 10 mg/kg bretazenil, whereas a maximum of 25% of L-

838,417 treated animals selected the zolpidem lever at any dose (compare Figs. 2G and 3C). Thus despite bretazenil (like L-838,417) having low efficacy at GABAA- α2, α3 and α5 receptors, its weak intrinsic efficacy at GABAA-α1 receptors (unlike L-838,417) (Atack, 2003), appears key to Downloaded from occasioning zolpidem-like discriminative effects. Interestingly, the profile of SL651498 was similar to that of bretazenil: (i) SL651498 fully generalised to the chlordiazepoxide cue at 10 mg/kg, (ii) showed appreciable (~80%) zolpidem appropriate responding; and (iii) ~70% of animals jpet.aspetjournals.org administered SL651498 (3-10 mg/kg) selected the zolpidem lever. Thus, despite SL651498 and L-

838,417 both possessing in vitro functional selectivity profiles (see Introduction), the greater

α absolute efficacy of SL651498 at GABAA- 1 receptors compared to L-838,417 (Griebel et al., at ASPET Journals on October 1, 2021

2001; McKernan et al., 2000), results in zolpidem-like discriminative stimulus properties. Recently,

Licata et al. (2005) demonstrated that SL651498 partially generalised to the triazolam discriminative cue in squirrel monkeys, and that this effect could be blocked by the GABAA-α1 selective antagonist β-CCT. By contrast, as discussed above, L-838,417 does not generalise to the triazolam cue in squirrel monkeys (Rowlett et al., 2005a).

Ocinaplon apparently shows anxioselectivity in man (Lippa et al., 2005) and generalised fully to both discriminative cues. However, ocinpalon has weak µM affinity for the [3H]flunitrazepam binding site (Chilman-Blair et al., 2003) and has a complex electrophysiology profile, whereby it shows equivalent efficacy to diazepam at GABAA receptors composed of α1β2γ2 subunits but lower efficacy when the α1 subunit is replaced by a α2, α3 or α5 subunit (most notably if these latter subunits are combined with a γ3 subunit; Lippa et al., 2005). This in vitro profile would explain why

19 JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #94003 it generalises fully to both cues. However, the complex selectivity profile that ocinaplon possesses may impart an anxioselective profile in man which differs from the current focus on compounds with selectivity based upon the α-subunit of GABAA receptors, and suggests that the γ subunit may be an important determinant for generating anxioselective agents. Indeed, mice heterozygous for the

γ2 subunit of the GABAA receptor show reduced synaptic clustering and an enhanced anxiety-like behavioural phenotype (Crestani et al., 1999).

Downloaded from In conclusion, the current study shows that the relative generalisation patterns of benzodiazepine site ligands to chlordiazepoxide and zolpidem discriminative cues generally corresponds with the in vitro profiles of these compounds at different GABA receptor subtypes described in the literature. A jpet.aspetjournals.org

In particular, newly available ligands such as L-838,417 with functional selectivity for α2, α3 or α5 containing GABAA receptors, and no intrinsic efficacy at GABAA-α1 receptors, generalise fully to

the chloridazepoxide but not the zolpidem cue. This profile is opposite to that seen with zolpidem at ASPET Journals on October 1, 2021 and CL218,872 which have selective affinity for GABAA-α1 receptors and which generalise fully to the zolpidem but not the chlordiazepoxide cue. Since the non-selective partial agonist bretazenil shows greater zolpidem-like discriminative effects compared to L-838,417, the in vivo profile of L-

838,417 differs from that of a partial agonist in rodents. Furthermore, another functionally selective agonist, SL651498, with greater efficacy at GABAA-α1 receptors compared to L-838,417 occasions appreciable zolpidem-like discriminative effects, suggesting that absolute efficacy at GABAA-α1 receptors rather than functional selectivity is the important determinant in generating zolpidem-like effects. However, future studies should consider the issue of training dose, as we already allude to above, to confirm our findings. Moreover, the use of selective antagonists to qualify the conclusions we draw here is important. However, as β-CCT (see Introduction) only has a modest 20-fold affinity selectivity for GABAA-α1 receptors over GABAA- α2 or α3 receptors, better subtype

20 JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #94003 selective antagonists are needed to fully explore the pharmacological basis of the zolpidem and chlordiazepoxide discriminative cues (Rowlett et al., 2005b). Downloaded from jpet.aspetjournals.org at ASPET Journals on October 1, 2021

21 JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 This article has not been copyedited and formatted. The final version may differ from this version.

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Legends for Figures

Figure 1.1 Upper panels (chlordiazepoxide cue) – degree of generalisation (percentage of responses on the drug-lever, ●) and effect on response rate (responses/sec, ○) at various doses of: A. chlordiazepoxide (N=12); B. diazepam (N=8); C. midazolam (N=11); and D. zopiclone (N=16), in rats trained to discriminate chlordiazepoxide (5 mg/kg, i.p) from vehicle. Lower panels (zolpidem cue) – degree of generalisation (percentage of responses on the drug-lever, ●) and effect on response rate (responses/sec, ○) at various doses of: E. chlordiazepoxide (N=8); F. diazepam Downloaded from (N=16); G. midazolam (N=8); and H. zopiclone (N=16), in rats trained to discriminate zolpidem (3 mg/kg, i.p) from vehicle. The lower number of each fraction alongside each “●” symbol indicates the number of animals making at least 10 responses on one of the two levers during the test session, jpet.aspetjournals.org whereas the upper number indicates the number of these animals selecting the drug lever (i.e., the first 10 consecutive responses were on the drug lever, see Materials and Methods). Response rate

(○): *P<0.05 versus vehicle response rate. at ASPET Journals on October 1, 2021

Figure 1.2 Upper panels (chlordiazepoxide cue) – degree of generalisation (percentage of responses on the drug-lever, ●) and effect on response rate (responses/sec, ○) at various doses of: A. triazolam

(N=20); B. alprazolam (N=15); and C. lorazepam (N=8), in rats trained to discriminate chlordiazepoxide (5 mg/kg, i.p) from vehicle. Lower panels (zolpidem cue) – degree of generalisation (percentage of responses on the drug-lever, ●) and effect on response rate

(responses/sec, ○) at various doses of: D. triazolam (N=16); E. alprazolam (N=12); and F. lorazepam (N=8), in rats trained to discriminate zolpidem (3 mg/kg, i.p) from vehicle. The lower number of each fraction alongside each “●” symbol indicates the number of animals making at least

10 responses on one of the two levers during the test session, whereas the upper number indicates the number of these animals selecting the drug lever (i.e., the first 10 consecutive responses were on the drug lever, see Materials and Methods). Response rate (○): *P<0.05 versus vehicle response rate.

27 JPET Fast Forward. Published on December 8, 2005 as DOI: 10.1124/jpet.105.094003 This article has not been copyedited and formatted. The final version may differ from this version.

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Figure 2 Upper panels (chlordiazepoxide cue) – degree of generalisation (percentage of responses on the drug-lever, ●) and effect on response rate (responses/sec, ○) at various doses of: A. zolpidem

(N=11); B. CL218,872 (N=11); C. SL651498 (N=16); and D. L-838,417 (N=6), in rats trained to discriminate chlordiazepoxide (5 mg/kg, i.p) from vehicle. Lower panels (zolpidem cue) – degree of generalisation (percentage of responses on the drug-lever, ●) and effect on response rate

(responses/sec, ○) at various doses of: E. zolpidem (N=11); F. CL218,872 (N=11); G. SL651498 Downloaded from (N=8); and H. L-838,417 (N=8), in rats trained to discriminate zolpidem (3 mg/kg, i.p) from vehicle. The lower number of each fraction alongside each “●” symbol indicates the number of animals making at least 10 responses on one of the two levers during the test session, whereas the jpet.aspetjournals.org upper number indicates the number of these animals selecting the drug lever (i.e., the first 10 consecutive responses were on the drug lever, see Materials and Methods). Response rate (○):

*P<0.05 versus vehicle response rate. at ASPET Journals on October 1, 2021

Figure 3 Upper panels (chlordiazepoxide cue) – degree of generalisation (percentage of responses on the drug-lever, ●) and effect on response rate (responses/sec, ○) at various doses of: A. ocinaplon (N=8); and B. bretazenil (N=11), in rats trained to discriminate chlordiazepoxide (5 mg/kg, i.p) from vehicle. Lower panels (zolpidem cue) – degree of generalisation (percentage of responses on the drug-lever, ●) and effect on response rate (responses/sec, ○) at various doses of: C. ocinaplon (N=8) and D. bretazenil (N=11), in rats trained to discriminate zolpidem (3 mg/kg, i.p) from vehicle. The lower number of each fraction alongside each “●” symbol indicates the number of animals making at least 10 responses on one of the two levers during the test session, whereas the upper number indicates the number of these animals selecting the drug lever (i.e., the first 10 consecutive responses were on the drug lever, see Materials and Methods). Response rate (○):

*P<0.05 versus vehicle response rate.

28 Chlordiazepoxide Diazepam Midazolam Zopiclone

1.4 1.4 1.4 1.4 100 AB12/12 8/8 100 C10/11 100 D16/16 10/11 15/16 1.2 5/6 1.2 9/11 1.2 1.2 80 80 80 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 1.0 1.0 1.0 1.0 60 60 60 0.8 0.8 0.8 0.8 JPET FastForward.PublishedonDecember8,2005asDOI:10.1124/jpet.105.094003 4/11 40 0.6 0.6 40 0.6 40 0.6

3/10 3/7 Response Rate 20 0.4 0.4 20 0.4 20 0.4 2/16 0/12 0.2 0.2 0/10 0.2 0/16 0.2 0 0/8 0 0 Chlordiazepoxide-lever responses, % Chlordiazepoxide-lever

VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0

100 E 4/4 1.4 100 FGH1.4 100 14/16 1.4 12/13 1.2 1.2 1.2 13/16 80 80 80 7/8 14/15 5/7 1.0 1.0 5/8 14/16 1.0 9/16 60 60 60 0.8 0.8 0.8 * 40 0.6 40 0.6 40 12/16 0.6 * *

2/8 6/16 Response Rate 0.4 0.4 1/8 * 20 0.4 20 * 20 * 6/13 0.2 0.2 0.2 Zolpidem-lever responses, % 0/8 0/16 0/7 0 0 0 0/16

VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 Chlordiazepoxide (mg/kg) Diazepam (mg/kg) Midazolam (mg/kg) Zopicolone (mg/kg)

Figure 1.1

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on October 1, 2021 1, October on Journals ASPET at Triazolam Alprazolam Lorazepam

14/15 1.4 1.4 1.4 100 ABC17/19 100 12/12 9/11 100 15/15 11/12 8/8 15/15 1.2 1.2 1.2 80 80 80 15/20 6/8 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 1.0 7/15 1.0 1.0 60 60 8/15 60 0.8 0.8 0.8 JPET FastForward.PublishedonDecember8,2005asDOI:10.1124/jpet.105.094003 5/8 40 0.6 40 0.6 40 0.6 * * Response Rate 20 * * 0.4 20 0.4 20 0.4 * 1/15 1/20 0.2 * 0.2 0.2 0 0 0 0/8 Chlordiazepoxide-lever responses, % Chlordiazepoxide-lever responses, VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.1 0.3 1 3 10

100 DEF6/7 1.4 9/12 1.4 8/8 1.4 13/15 1.2 1.2 6/7 1.2 80 6/6 9/12 1.0 1.0 5/7 1.0 60 8/16 6/7 6/12 3/4 0.8 0.8 0.8

40 0.6 0.6 0.6

* Response Rate 0.4 0.4 0.4 20 5/12 1/7 * 0.2 * 0.2 0.2 Zolpidem-lever responses, % 0/16 1/12 * 0 * * *

VEH 0.1 0.3 1 3 10 VEH 0.1 0.3 1 3 10 VEH 0.1 0.3 1 3 10 Triazolam (mg/kg) Alprazolam (mg/kg) Lorazepam (mg/kg)

Figure 1.2

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1.4 100 1.4 1.4 100 AB100 C 6/6 100 D 13/14 1.2 1.2 1.2 80 80 80 4/6 80 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 1.0 1.0 4/6 1.0 60 2/11 60 60 60 0.8 0.8 0.8 3/11 JPET FastForward.PublishedonDecember8,2005asDOI:10.1124/jpet.105.094003 40 40 40 0.6 3/9 0.6 40 0.6 0/2 Response Rate 0.4 1/6 20 0.4 20 20 20 4/15 0.4 0/9 1/11 2/15 0/11 0.2 0.2 0.2 0/11 * * 0 2/16 0 1/11 1/9 * 0 0/6 0 Chlordiazepoxide-lever responses, % Chlordiazepoxide-lever VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0

1.4 1.4 1.4 100 EF10/10 100 100 G H 8/9 1.2 15/15 1.2 1.2 80 80 80 5/7 1.0 1.0 5/7 1.0 60 60 60 0.8 0.8 2/8 0.8 2/8 7/15 40 40 0.6 40 0.6 1/8 0.6

3/10 Response Rate 0.4 20 0.4 20 1/15 0.4 20 1/15 0/8 1/8 0.2 0.2 0.2 Zolpidem-lever responses, % Zolpidem-lever responses, 0/8 0 1/11 0 0 0/8

VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 Zolpidem (mg/kg) CL218,872 (mg/kg) L838,417 (mg/kg) SL651498 (mg/kg)

Figure 2

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100 1.4 A 11/11 100 B 7/7 9/10 1.2

80 80 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 8/11 1.0 60 60

5/8 JPET FastForward.PublishedonDecember8,2005asDOI:10.1124/jpet.105.094003 0.8

40 3/11 40 0.6 Response Rate 20 20 0.4

0/11 0.2 0 0 0/8

Chlordiazepoxide-leverresponses, % Chlordiazepoxide-leverresponses, 0/8

VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0

100 100 1.4 C 8/10 D 7/8 1.2 80 7/11 80 1.0 6/11 5/8 60 60 1/11 0.8

40 40 0.6 Response Rate 20 3/12 20 0.4

Zolpidem-leverresponses, % 0/11 0/8 0.2 0 0 0/8

VEH 0.3 1.0 3.0 10.0 30.0 VEH 0.3 1.0 3.0 10.0 30.0 Bretazenil (mg/kg) Ocinaplon (mg/kg)

Figure 3

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