Effects of DN-2327, a new anxiolytic,diazepam and buspirone on exploratory activity of the rat in an elevated plus-maze

In the present study, the effects of a new anxiolytic, DN-2327, were compared to those of diazepam and buspirone in rats in the elevated plus-maze test. Two indices of anxiety were obtained in this test: the number of entries into the open arms expressed as a percentage of the total number of arm entries and the percentage of time spent on the open arms. Both a typical anxiolytic, diazepam, at 2.5, 5 and 10 mg/kg, PO and a new anxiolytic, DN-2327, at 2.5 and 5 mg/kg, PO dose-dependently increased the two indices: the percentage of time spent on the open arms and the percentage of open-arm entries. On the other hand, pentylenetetrazol (PTZ) at 10 and 20 mg/kg, IP decreased the two indices dose dependently as did yohimbine at 1.5 and 3 mg/kg, IP. DN-2327 at 2.5 and 5 mg/kg, PO and diazepam at 5 and 10 mg/kg, PO dose dependently and significantly increased the two indices that were suppressed following administration of PTZ at 10 mg/kg, IP. The effects of both DN-2327, 5 mg/kg, PO, and diazepam, 10 mg/kg, PO, on the two indices were significantly antagonized by the benzodiazepine (BZD) receptor antagonist flumazenil, 20 mg/kg, IP. Buspirone (2.5–20 mg/kg, PO) did not affect either of the two responses but dose dependently decreased the number of rearings, although in the Vogel conflict test, the anti-conflict activity of buspirone was equipotent to that of diazepam and DN-2327 at the minimum effective dose (10 mg/kg, PO) of each drug. In conclusion, the present experiment revealed that the anxiolytic effect of DN-2327 in this test was clear, whereas buspirone showed no apparent effect.     

Effect of a new anxiolytic, DN-2327, on learning and memory in rats.

The effects of a new anxiolytic, (2-(7-chloro-1,8-naphthyridin-2-yl)-3- [(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-carbonylmethyl] isoindolin-1-one (DN-2327), on the execution of step-through passive avoidance and delayed spontaneous alternation tasks were assessed and compared with those of diazepam (DZP) and buspirone. DN-2327 and buspirone (both 10 and 20 mg/kg, PO) impaired performance in the 48-h passive avoidance recall test when given prior to the test session, but not when given before the training trial. DZP impaired the performance at doses of more than 5 and more than 10 mg/kg PO when given prior to the test session and when given before the training trial, respectively. The action of DZP (10 mg/kg PO) when given before the training trial was antagonized by flumazenil (20 mg/kg, IP) and tended to be antagonized by DN-2327 (10 and 30 mg/kg, PO), but was not affected by buspirone. No evidence for possible amnesic effects of DN-2327 or buspirone on working memory was found in the delayed spontaneous alternation task, but DZP (3 and 10 mg/kg, PO) caused significant impairment of working memory. Electroshock sensitivities detected by flinch, jump, and vocalization thresholds were not influenced significantly by DN-2327 (30 and 100 mg/kg, PO), DZP (10 and 30 mg/kg, PO) or buspirone (30 and 100 mg/kg, PO).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacologic profile of a new anxiolytic,DN-2327: effect of Ro15-1788 and interaction with diazepam in rodents

In order to characterize the pharmacologic profile of DN-2327, an isoindoline benzodiazepine (BZD) receptor ligand, its interactions with Ro15-1788 and diazepam were analyzed in rodents. The anti-conflict action of DN-2327 in two conflict tests using rats, the punished water-lick conflict (Vogel conflict) and the punished bar-pressing conflict test, was completely attenuated by treatment with Ro15-1788. The anti-convulsive (pentylenetetrazol [PTZ] induced convulsion) effect of DN-2327 was also reduced by Ro15-1788. These results suggest that the anti-conflict and anti-convulsive actions of DN-2327 may be mediated via BZD receptors. On the other hand, DN-2327 only slightly affected the motor coordination in mice and rats, as estimated by the inclined screen test and the climbing test, respectively; however, the compound attenuated the motor incoordination produced by diazepam. Furthermore, the pentobarbital potentiating effect of diazepam was reduced by pretreatment with DN-2327 in mice. In the Vogel conflict test, additive effects were observed upon the conflict test, additive effects were observed upon the concomitant administration of subeffective doses (5 mg/kg, PO) of DN-2327 and diazepam. DN-2327 at 20 mg/kg, PO, did not reduce but slightly potentiated the anti-conflict effect of the maximum effective dose of diazepam. For PTZ-induced convulsions, DN-2327, 0.5 and 20 mg/kg, PO, doses which produced partial and complete anti-convulsive effects, respectively, in rats did not reduce but increased additively the effects of diazepam. DN-2327 at 10 and 20 mg/kg, PO, doses which both produced partial anti-convulsive effects in mice, showed an additive effect with the partial effects of diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discriminative stimulus properties of β-carbolines characterized as agonists and inverse agonists at central benzodiazepine receptors

The discriminative stimulus properties of three -carboline derivatives were studied in three groups of rats trained, respectively, to discriminate diazepam (2.5 mg/kg IP), chlordiazepoxide (CDP, 5 mg/kg IP) or pentylenetetrazol (PTZ, 15 mg/kg IP) from saline in standard procedures employing two-lever operant chambers. Two -carbolines, ZK 91296 and ZK 93423, substituted for the benzodiazepines in both CDP- and diazepam-trained rats. The neutral benzodiazepine antagonist Ro 15-1788 blocked the diazepam discriminative stimulus and the ability of ZK 91296 to substitute for diazepam. A third -carboline, FG 7142, was not identified as benzodiazepine-like in generalization tests in either diazepam- or CDP-trained rats, but when administered together with CDP antagonized the benzodiazepine discriminative stimulus. In rats trained to discriminate PTZ from saline (a discrimination which is thought to depend on the anxiogenic properties of PTZ) the PTZ cue was antagonized by diazepam and ZK 93423, and partially antagonized by ZK 91296. The PTZ cue generalized to FG 7142 and this generalization was partially antagonized by Ro 15-1788. These results suggest that the three -carbolines provide more than one kind of discriminative stimulus, consistent with the classification of ZK 93423 as an agonist at central benzodiazepine receptors, with ZK 91 296 as a partial agonist, and with FG 7142 as an inverse agonist. Pharmacologically, ZK 93 423 and ZK 91 296 may exhibit anxiolytic qualities, whereas FG 7142 produces anxiogenic effects.     

Cholinergic mediation of the discriminative stimulus properties of clozapine

Rats were trained to discriminate clozapine (CLZ; 5.76 mg/kg, IPt-30 min) in a two-lever operant task in which responding on the correct lever was reinforced with water under a fixed ratio 32 schedule. The ED₅₀ of CLZ was 1.1 mg/kg. The CLZ cue was generalised to atropine (ED₅₀=8.7 mg/kg), scopolamine (ED₅₀=0.37 mg/kg) and fluperlapine (ED₅₀=4.0 mg/kg), but not to non-cholinergic compounds, i.e. buspirone, diazepam, ketanserin, prazosin or SCH 23390. The peripherally-acting muscarinic antagonist methylscopolamine did not substitute for CLZ. Furthermore, the CLZ cue was marginally attenuated byd-amphetamine; a high dose of oxotremorine (1 mg/kg) appeared to block the CLZ cue (to 22%). However, this effect could not be evaluated statistically due to an insufficient number of animals responding. These results may indicate that the discriminative stimulus effects of CLZ primarily involve antagonism of central muscarinic acetylcholine receptors.     

Discriminative stimulus properties of midazolam: comparison with other benzodiazepines

Rats (N=12) were trained to discriminate midazolam (1 mg/kg, IP) from vehicle in a food reinforced operant conditioning procedure. Midazolam, flunitrazepam, diazepam, chlordiazepoxide and pentobarbital showed dose-dependent substitution for midazolam. Buspirone and Ro 15-1788 did not substitute for midazolam. The midazolam cue was dose-dependently antagonized by Ro 15-1788. In rats (N=12) trained to discriminate chlordiazepoxide (3 mg/kg, IP) from vehicle midazolam, flunitrazepam, diazepam and chlordiazepoxide substituted completely and dose dependently for chlordiazepoxide. The relative potency of chlordiazepoxide and diazepam was three times less in the midazolam-trained animals than in the chlordiazepoxide-trained animals. Response rate and latency data further support the main finding that the midazolam cue is similar, but not identical to the cue of classical benzodiazepines.     

The discriminative stimulus properties of buspirone involve dopamine-2 receptor antagonist activity

To investigate a dopaminergic component in the discriminative stimulus properties of buspirone, rats were trained to discriminate 2.5 mg/kg buspirone from saline, using a two lever, food-rewarded, fixed ratio 10 operant procedure. To test the dopamine-2(D₂) antagonist action of buspirone, a second group of rats was trained to discriminate 0.16 mg/kg apomorphine from saline. In addition to a complete generalization to 8-OH-DPAT, the D₂ antagonists haloperidol, R 79598 and sulpiride showed a partial generalization to buspirone. The benzodiazepine ligands chlordiazepoxide and bretazenil did not generalize to the buspirone cue. Buspirone (2.0 mg/kg) completely blocked the apomorphine cue in the apomorphine trained rats. Haloperidol, R 79895 and sulpiride also blocked the apomorphine cue, although at doses much smaller than the doses needed to evoke buspirone responding in the buspirone trained group. 8-OH-DPAT did not antagonize apomorphine. It was concluded that the D₂ action of buspirone partially contributes to its discriminative stimulus properties. Mediation of the buspirone cue by 5-HT_1a receptor activation seemed predominant. Further, buspirone can act as a full D₂ antagonist in drug discrimination. A model was proposed suggesting a compound discriminative stimulus complex of buspirone with a dominant 5-HT_1a component that overshadows a less pronounced D₂ component.     

Discriminative stimulus properties of pentylenetetrazol and bemegride: Some generalization and antagonism tests

In an operant procedure of lever pressing on a FR 10 schedule of food reinforcement, male hooded rats were trained to respond with a lever on one side of a food cup following a drug injection, and to respond with a lever on the alternate side following a 1ml/kg saline injection. All of 14 subjects learned to discriminate reliably between the effects of 20 mg/kg pentylene-tetrazol (PTZ) and saline. Seven of eight rats learned to discriminate between the effects of bemegride (5 mg/kg) and saline. None of 14 rats learned to discriminate between 5 mg/kg PTZ and saline. The bemegride discriminative stimulus generalized to PTZ (20 mg/kg) and was antagonized by chlordiazepoxide (10 mg/kg). Chlordiazepoxide, diazepam, flurazepam, clobazam, and meprobamate were all effective antagonists of PTZ in a dose-dependent manner. Bemegride and cocaine generalized to the PTZ discriminative stimulus in a dose-dependent manner, but d-amphetamine, methylphenidate, and nicotine did not. Since bemegride and PTZ are convulsants at higher doses, the discriminative stimulus properties of these drugs might be based on a subtle convulsive brain state. The anxiolytic properties of benzodiazepines and meprobamate suggest that the discriminative stimulus produced by these convulsants is related to an anxiety-inducing action.     

Discriminative stimulus effects of tizanidine hydrochloride: Studies with rats trained to discriminate either tizanidine,clonidine, diazepam,fentanyl, or cocaine

Male Sprague Dawley rats were trained in a two-lever food-reinforced procedure to discriminate between the effects of saline and either tizanidine hydrochloride, clonidine hydrochloride, diazepam, fentanyl, or cocaine hydrochloride. Tizanidine-trained rats dose-dependently generalized the effects of tizanidine and clonidine but not pentobarbital, diazepam, morphine, or cocaine. Clonidine-trained rats dose-dependently generalized the effects of clonidine and tizanidine but not pentobarbital, diazepam, or morphine. Diazepam-trained rats dose-dependently generalized the effects of diazepam but did not generalize tizanidine. Fentanyl-trained rats dose-dependently generalized the effects of fentanyl but did not generalize tizanidine. Cocaine-trained rats did not generalize the effects of tizanidine to the cocaine discriminative stimulus. Yohimbine hydrochloride but not naloxone hydrochloride dose-dependently antagonized the discriminative stimuli produced by both tizanidine and clonidine. These data demonstrate that tizanidine shares discriminative stimulus properties with clonidine but not with pentobarbital, diazepam, fentanyl, morphine, or cocaine. The discriminative stimuli produced by tizanidine and clonidine are mediated via an agonistic interaction with alpha₂-adrenergic receptors and not via an agonistic interaction with opioid receptors.     

Withdrawal from chronic nicotine substitutes partially for the interoceptive stimulus produced by pentylenetetrazol (PTZ)

Rats were trained on an FR10 schedule of food reinforcement to press one lever after pentylenetetrazol (PTZ), 20 mg/kg, IP, and an alternate lever after saline. After acute nicotine, 0.64 mg/kg, SC, 35% of the rats pressed the PTZ-lever. Diazepam, 5 mg/kg, IP, blocked the stimulus produced by PTZ, and mecamylamine, 5 mg/kg, IP, blocked the stimulus produced by nicotine. Training was then suspended and rats were treated with nicotine, at 8-h intervals, 0.64 mg/kg on the 1st day, and 1.25 mg/kg on subsequent days, for 21 days. To determine whether nicotine withdrawal substitutes for the stimulus produced by PTZ, rats were tested with saline at various times after chronic nicotine injections. Data from this part of the study were replicated in another group given nicotine for 15 days. Saline at 8 h after nicotine (five determinations each group) produced a small but stable degree of PTZ lever selection (35±4%). At 48 h after termination of nicotine treatment, the percentage of rats selecting the PTZ lever (50%) was greater than that in a control group tested after an equivalent period without training. The PTZ-like stimulus detected after chronic nicotine was not altered by mecamylamine, was additive with PTZ, and was blocked by diazepam. These data suggest that withdrawal from chronic nicotine produces a weak PTZ-like stimulus, which can be antagonized by an anxiolytic drug.     

Quantal detection and homogeneous sensitivity in a pentylenetetrazol discrimination

Rats were trained to discriminate pentylenetetrazol (PTZ, 20 mg/kg) from saline in a two-lever operant task. Correct lever presses were reinforced with food under the control of a fixed ratio 10 schedule. In tests of the effect of PTZ dose on level selection, rats selected the PTZ lever in a dose-dependent manner, with peak latency at the approximate ED₅₀ dose (10 mg/kg). Rats usually pressed only the selected lever, regardless of dose, indicating that lever selection was a quantal (or bimodal) function of stimulus intensity. Lever biases observed during training sessions did not predict the performance of individual rats in tests with the ED₅₀ dose. In three independent trials with this intermediate dosage, the rats selecting the PTZ lever varied from trial to trial, suggesting that rats detecting this dose did not form a stable subgroup. The pattern of lever selections across these three trials was not significantly different from that predicted by a model in which all subjects shared the same probability for detecting the drug stimulus. These results demonstrate that lever selection in a two-lever drugdiscrimination task can be quantal in nature, and suggest that rats trained with PTZ, 20 mg/kg, are homogeneous in sensitivity to this stimulus. Offprint requests to: M.W. Emmett-Oglesby     

Rolipram forms a potent discriminative stimulus in drug discrimination experiments in rats

Long Evans rats were trained to discriminate 0.2 mg/kg IP (±)-rolipram from vehicle injection in a food-motivated two-lever operant task. Eight out of nine rats acquired the discrimination after an average of 91 sessions (min 65, max 137). The ED₅₀ of (±)-rolipram was 0.06 mg/kg IP. Generalization tests with (–)- and (+)-rolipram showed that the (–)-isomer was 8 times more active than (+)-rolipram with an ED₅₀ of 0.06 and 0.4 mg/kg IP respectively. The phosphodiesterase inhibitor RO 20-1724 partially (83%) generalized to (±)-rolipram in doses of 0.6 and 1.0 mg/kg IP. IBMX 5 mg/kg IP showed 63% generalization. Tests with imipramine and the (+)- and (–)-isomer of the noradrenaline uptake inhibitor oxaprotiline suggest that NA-uptake inhibiting drugs do not form an interoceptive cue which is (±)-rolipram-like. dbcAMP 12.5 mg/kg SC and 100 mg/kg SC dbcGMP did not generalize to the training drug. The nature of the discriminative stimulus produced by this dose of (±)-rolipram in rats remains to be elucidated. Offprint requests to: R. Ortman     

Drug discrimination based on the competitive N-methyl-d-aspartate antagonist,NPC 12626

The discriminative stimulus effects of the N-methyl-d-aspartate (NMDA) antagonists 3-([+/-]-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and phencyclidine were assessed in a drug discrimination based on the competitive NMDA antagonist 2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid (NPC 12626). Adult male Sprague-Dawley rats were trained to discriminate NPC 12626 from saline using a standard two-lever fixed ratio 32 schedule of food reinforcement. NPC 12626 dose-dependently substituted for the training dose (20 mg/kg IP) with an ED₅₀ of 9.5 mg/kg. The competitive NMDA antagonist CPP completely substituted for NPC 12626 (ED₅₀=1.4 mg/kg IP). The non-competitive NMDA antagonist phencyclidine, as well as pentobarbital and NMDA, failed to substitute completely for NPC 12626, even at doses of these drugs that reduced response rates. These data indicate that the discriminative stimulus properties of NPC 12626 are selective and shared by CPP but not by phencyclidine, pentobarbital or NMDA. The emerging evidence for differences in the discriminative stimulus effects of competitive NMDA antagonists and phencyclidine suggests that competitive antagonists such as NPC 12626 and CPP may not have phencyclidine-like abuse liability.     

Discriminative stimulus properties of a small dose of cocaine

This study characterized the interoceptive discriminative stimulus (IDS) produced by a small dose of cocaine. Rats were trained to use a dose of cocaine of 1.25 mg/kg vs saline as the basis for choosing one of two levers for food reinforcement on a fixed ratio 10 schedule. The discrimination was acquired over approx. 60 training sessions. d-Amphetamine generalized to cocaine with approximately equal potency (ED₅₀'s for cocaine and d-amphetamine were 0.07 and 0.06 mg/kg, respectively); 20 mg/kg cocaine and 10 mg/kg methylphenidate also generalized to the cocaine lever. Pentylenetetrazol, 20 mg/kg, did not generalize to the cocaine lever, and diazepam, 10 mg/kg, did not block the 1.25 mg/kg cocaine discrimination. These data indicate that when a small dose of cocaine is used as the basis of discrimination training, the discriminative stimulus that it produces is qualitatively and quantitatively similar to that produced by small doses of amphetamine, is still discriminated with a large dose of cocaine, and is dissimilar to the discriminative stimulus produced by pentylenetetrazol.     

Characteristics of pentobarbital discrimination in the gerbil: Transfer and antagonism

Experiment 1. Gerbils were trained in a T-shaped maze to discriminate the effects produced by pentobarbital (P-barb. 15 mg/kg, i.p.) and the effects of saline. The response, a left or right turn in the maze, was thus contingent upon the prevailing training condition (P-barb. or saline). The criterion of performing 8 correct first trial choices in 10 consecutive sessions was reached within 20 training sessions. Tests with descending doses of P-barb. yielded an ED₅₀ of 9 mg/kg. Tests with phenobarbital (40 mg/kg) or diazepam (2 and 4 mg/kg) solely maintained the drug response. P-barb. discrimination was reversed by megimide (ED₅₀: 8.5–9.6 mg/kg) and metrazol (ED₅₀: 24.9–27.9 mg/kg). Thus megimide was approximately 3 times more effective than metrazol. Metrazol (40 and 80 mg/kg) also counteracted the phenobarbital and diazepam response. Picrotoxin (2.5 and 5 mg/kg) was less effective whereas caffeine (100 mg/kg) and piracetam (100–1000 mg/kg) did not upset P-barb. discrimination. Experiment 2. Naive gerbils had to discriminate mixtures of P-barb. (15 mg/kg) plus either 40 or 80 mg/kg of metrazol from saline already at the start of the discriminative training. The drug combinations produced discriminable effects since most gerbils reached the acquisition criterion (8/10), although more slowly than gerbils trained with P-barb. solely. Gerbils trained without a drug stimulus (saline vs. saline) never attained the criterion during 60 consecutive sessions. In conclusion, reversal of established discrimination (Expt. 1) does not necessarily mean that the same drug combination lacks discriminable effects as demonstrated in Experiment 2.     

A behavioural and pharmacological evaluation of the discriminative stimulus induced by pentylenetetrazole in the pig

The anxiogenic nature of the interoceptive discriminative stimulus induced by pentylenetetrazole (PTZ) was investigated by examining the discriminatory behaviour of PTZ conditioned pigs during a conditioned emotional response (CER). A CER was induced in a non-operant situation, by pairing a tone stimulus with the application of a mild, non-injurious electric shock. Subsequent presentation of the conditioned tone stimulus alone produced a generalisation to the PTZ cue. This generalisation of the conditioned emotional state (CES) to the PTZ cue was antagonised by pretreatment with diazepam (0.5 mg/kg, PO; 30 min). The PTZ stimulus was also antagonised by diazepam (0.5 mg/kg, PO; 30 min) but not by an anticonvulsant dose of ethosuximide (30 mg/kg, PO; 1–3 h), providing further confirmation of the anxiogenic nature of the PTZ cue. Our results demonstrate the validity of the PTZ discrimination paradigm in pigs as a test of anxiety.     

Lack of tolerance development to benzodiazepines in antagonism of the pentylenetetrazol discriminative stimulus.

In an operant procedure of lever pressing on FR 10 schedule of food reinforcement male hooded rats were trained to respond on a lever on one side of a food cup following a 20 mg/kg pentylenetetrazol (PTZ) injection and to respond on a lever on the alternate side following a 1 ml/kg saline injection. Upon acquisition of the PTZ-saline discrimination, diazepam and chlordiazepoxide were tested and found to antagonize the PTZ discriminative stimulus. The animals were then injected with 10 mg/kg diazepam or chlordiazepoxide for ten consecutive days. New dose-response curves obtained following this treatment indicated that tolerance did not develop to the antagonism of the PTZ discriminative stimulus by these benzodiazepines.     

5-HT2C receptors are involved in the discriminative stimulus effects of citalopram in rats

 Rats were trained on a fixed ratio 10, food-reinforced schedule to recognize a discriminative stimulus (DS) elicited by the selective serotonin (5-HT) reuptake inhibitor (SSRI), citalopram (2.5 mg/kg, IP). The preferential, high efficacy agonist at 5-HT_2C receptors, Ro60-0175, dose-dependently generalized to citalopram with an ED₅₀ of 0.3 mg/kg, IP. Further, the selective 5-HT_2C receptor antagonist, SB242,084, dose-dependently (ED₅₀=0.1 mg/kg, IP) blocked the citalopram DS. These data suggest that 5-HT_2C receptors are involved in the DS properties of the SSRI, citalopram, in rats. They do not, however, exclude a potential role of other 5-HT receptor types. Received: 15 October 1998 / Final version: 10 December 1998     

Behavioral evidence for the role of noradrenaline in putative anxiolytic and sedative effects of benzodiazepines

The effects of clonidine on the antianxiety and sedation of benzodiazepines (BZD) were assessed respectively in rats trained in a two-lever diazepam cue discrimination procedure and in single-lever fixed-ratio (FR) water-reinforced performance. Clonidine (10–60 g/kg) significantly shifted to the left the dose-effect curves of diazepam in the discrimination paradigm. This treatment also shifted generalization dose-effect curves of the diazepam cue to chlordiazepoxide and CL 218872 to the left in the drug discrimination procedure. The diazepam cue was antagonized in a dose-related manner by Ro 15-1788, but not by bicuculline. Clonidine also potentiated the rate-decreasing effects of diazepam on the FR schedule when the dose of diazepam was increased to 0.3 mg/kg, but not the milder rate-decreasing effect of CL 218872 until the dose of CL 218872 was increased to 10 mg/kg. The potentiating effects of clonidine on the stimulus control and depression of diazepam were antagonized by yohimbine. Yohimbine (1.0 mg/kg) also significantly shifted the dose-effect curve of diazepam cue to the right. Bicuculline (3 mg/kg) completely antagonized the rate-decreasing effect of diazepam (1 mg/kg), but significantly potentiated the rate-suppressant effect of clonidine (10 g/kg). These results suggest that the central noradrenaline (NA) system may be involved not only in the antianxiety, but also the sadative action of BZD. The nature of NA involvement in relation to the different subtypes of BZD receptors requires further exploration.     

Characterization of the scopolamine stimulus in rats

The discriminative stimulus properties of scopolamine, a potent antagonist at muscarinic receptors, were used for testing the discriminative effects of drugs known to act on cholinergic transmission. Rats were trained in a standard two-bar operant conditioning procedure with food as the reinforcer, according to a FR10 schedule. The training dose of scopolamine was progressively reduced from 0.25 mg/kg SC to the low dose of 0.062 mg/kg SC. Scopolarmine yielded an accurate discrimination in all the six rats tested. The generalization gradient resulted in an ED₅₀ of 0.027 mg/kg. The scopolamine cue lasted for 1 h and was of central origin, since it was not mimicked by scopolamine methylbromide. The scopolamine stimulus generalized to atropine and trihexyphenidyl (respective ED₅₀ values 2.20 and 0.21 mg/kg SC). Atropine depressed rate of responding, while trihexyphenidyl did not. Antagonism experiments with both direct agonists at the muscarinic receptor (arecoline and oxotremorine) and indirect agonists, i.e., inhibitors of the acetylcholine esterase [physostigmine and tetrahydroaminoacridine (THA)], led to inconsistent results. Increasing the doses of the agonists in order to block the scopolamine cue may be limited by their rate suppressant effect on responding. Based upon previously published results, it is suggested that the muscarinic agonist cue is more useful than the antagonist cue for investigating muscarinic transmission.