Two-lever Drug Discrimination Paradigm Research Articles

Mechanisms of imidazoline I2 receptor agonist-induced antinociception in rats: involvement of monoaminergic neurotransmission.

Although the antinociceptive efficacies of imidazoline I2 receptor agonists have been established, the exact post-receptor mechanisms remain unknown. This study tested the hypothesis that monoaminergic transmission is critical for I2 receptor agonist-induced antinociception. von Frey filaments were used to assess antinociceptive effects of two I2 receptor agonists, 2-BFI and CR4056 on chronic constriction injury (CCI)-induced neuropathic pain or complete Freund's adjuvant (CFA)-induced inflammatory pain in rats. Rectal temperature was measured to assess hypothermic effects of 2-BFI. A two-lever drug discrimination paradigm in which rats were trained to discriminate 5.6mg·kg-1 2-BFI (i.p.) from its vehicle was used to examine the discriminative stimulus effects of 2-BFI. In each experiment, pharmacological mechanisms were investigated by combining 2-BFI or CR4056 with various pharmacological manipulations of the monoaminergic system including selective reuptake inhibition, monoamine depletion and monoamine receptor antagonism. In the CCI model, selective reuptake inhibitors of 5-HT (fluoxetine) or noradrenaline (desipramine), but not dopamine (GBR12909), enhanced 2-BFI-induced antinociception. Selective depletion of 5-HT or noradrenaline almost abolished 2-BFI-induced antinociception. 5-HT1A , 5-HT2A and α1 -adrenoceptor antagonists, but not other monoaminergic antagonists, attenuated 2-BFI and CR4056-induced antinociception in CCI and/or CFA models. However, none of these monoamine receptor antagonists significantly altered 2-BFI-induced hypothermia or discriminative stimulus effects. Antinociception induced by I2 receptor agonists was mediated by serotonergic and noradrenergic mechanisms with 5-HT1A , 5-HT2A and α1 -adrenoceptor being particularly important. In contrast, the hypothermic and discriminative stimulus effects of I2 receptor agonists were mediated by distinct, independent mechanisms.

Generalization tests using different dosing routes from those of drug discrimination training in rats.

The purpose of this study was to investigate the discriminative stimulus properties of morphine and codeine using different administration routes to that used at drug discrimination training. Rats were trained to discriminate morphine at 3 mg/kg from saline by the intraperitoneal route in a standard two-lever drug discrimination paradigm. Generalization of morphine by the subcutaneous and the oral routes, and codeine by the intraperitoneal and the oral routes to the discriminative stimulus properties of the morphine training dose were investigated. Morphine at 3 mg/kg by the subcutaneous route generalized to the morphine training dose and 10 of 12 rats showed 80% or more morphine-lever responses. In the administration of morphine by the oral route, morphine at 30 mg/kg generalized to the morphine training dose and all rats showed 80% or more morphine-lever responses within the range of 3 to 30 mg/kg. In the administration of codeine by the intraperitoneal route, codeine at 20 mg/kg generalized to the morphine training dose and 14 of the 15 animals showed 80% or more morphine-lever responses within the range of 3 to 20 mg/kg. In the administration of codeine by the oral route, codeine at 60 mg/kg generalized to the morphine training dose and 14 of the 15 animals showed 80% or more morphine-lever responses within the range of 10 to 60 mg/kg. Thus, the discriminative stimulus properties of morphine and codeine were comparable when using different administration routes to those at discrimination training.

Role of intracellular Ca2+ signaling in the antinociceptive and discriminative stimulus effects of the imidazoline I2 receptor agonist 2-BFI in rats.

Recent research has established the imidazoline I2 receptor as a promising target for the development of novel analgesics. However, despite an increasing understanding of imidazoline I2 receptor-mediated behavioral effects, little is known about post-I2-receptor signaling mechanisms. This study examined the effects of several inhibitors of Ca2+ signaling mechanisms on two behavioral effects of the prototypical imidazoline I2 receptor ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI). The von Frey filament test was used to examine the antinociceptive effects of 2-BFI in complete Freund's adjuvant (CFA)-induced inflammatory pain in rats. A two-lever drug discrimination paradigm in which rats were trained to discriminate 5.6mg/kg (intraperitoneally) 2-BFI from its vehicle was used to examine the discriminative stimulus effects of 2-BFI. The L-type Ca2+ channel blockers verapamil and nimodipine, the calmodulin antagonist W-7, and the internal Ca2+ release inhibitor ryanodine all attenuated the antinociceptive effects of 2-BFI. Oxycodone- and acetaminophen-induced antinociception was unaffected by pretreatment with the Ca2+ channel blockers. Rats learned to reliably discriminate 5.6mg/kg 2-BFI from saline. The I2 receptor agonists BU224, RS45041, tracizoline, and CR4056 all fully substituted for 5.6mg/kg 2-BFI while idazoxan, S22687, 2,5-dimethoxy-4-methylamphetamine (DOM), and phenyzoline produced partial or no substitution. Verapamil, nimodipine, and W-7 did not alter the discriminative stimulus effects of 2-BFI. These results indicate that the antinociceptive effects of 2-BFI involve intracellular Ca2+ elevation and/or downstream Ca2+/calmodulin signaling, whereas the discriminative stimulus effects of 2-BFI are mediated by a distinct, independent mechanism.

Differential discriminative-stimulus effects of cigarette smoke condensate and nicotine in nicotine-discriminating rats

Although it is widely accepted that nicotine plays a key role in tobacco dependence, nicotine alone cannot account for all of the pharmacological effects associated with cigarette smoke found in preclinical models. Thus, the present study aimed to determine the differential effects of the interoceptive cues of nicotine alone versus those of cigarette smoke condensate (CSC) in nicotine-trained rats. First, the rats were trained to discriminate nicotine (0.4mg/kg, subcutaneous [s.c.]) from saline in a two-lever drug discrimination paradigm. Then, to clarify the different neuropharmacological mechanisms underlying the discriminative-stimulus effects in the nicotine and CSC in nicotine-trained rats, either the α4β2 nicotinic acetylcholine receptor (nAChR) antagonist dihydro-β-erythroidine (DHβE; 0.3–1.0mg/kg, s.c.) or the α7 nAChR antagonist methyllycaconitine citrate (MLA; 5–10mg/kg, intraperitoneal [i.p.]) was administered prior to the injection of either nicotine or CSC. Separate set of experiments was performed to compare the duration of action of the discriminative-stimulus effects of CSC and nicotine. CSC exhibited a dose-dependent nicotine generalization, and interestingly, 1.0mg/kg of DHβE antagonized the discriminative effects of nicotine (0.4mg/kg) but not CSC (0.4mg/kg nicotine content). However, pretreatment with MLA had no effect. In the time-course study, CSC had a relatively longer half-life in terms of the discriminative-stimulus effects compared with nicotine alone. Taken together, the present findings indicate that CSC has a distinct influence on interoceptive effects relative to nicotine alone and that these differential effects might be mediated, at least in part, by the α4β2, but not the α7, nAChR.

Discriminative-stimulus effects of NS9283, a nicotinic α4β2* positive allosteric modulator, in nicotine-discriminating rats

Neuronal α4β2* nicotinic acetylcholine receptors mediate cognition, pain, and the discriminative and reinforcing effects of nicotine. In addition to traditional orthosteric agonists, α4β2* positive allosteric modulators (PAMs) have recently been identified. With increased subtype selectivity relative to agonists, PAMs administered alone or in combination with low-dose α4β2* agonists may be used as powerful tools for increasing our understanding of α4β2* pharmacology. The present experiments tested the nicotine discriminative-stimulus effects of the α4β2* PAM NS9283 (A-969933) in the presence and absence of low-dose nicotine or nicotinic subtype-selective agonist. Rats were trained to discriminate 0.4mg/kg nicotine from saline in a two-lever drug discrimination paradigm. In subsequent generalization tests, rats were administered nicotine, the α4β2*-preferring agonist ABT-594, and NS9283, alone or in two-drug combinations. Nicotine and ABT-594 showed dose-dependent nicotine generalization. NS9283 alone resulted in a non-significant increase in nicotine-appropriate lever selection. Combination of non-effective doses of nicotine or ABT-594 with escalating doses of NS9283 resulted in a complete conversion to 100% nicotine-appropriate choice in the case of nicotine combination and incomplete, though significant, generalization for ABT-594. The α4β2* PAM NS9283 alone did not produce nicotine-like discriminative effects, but did demonstrate dose-related increases in nicotine lever choice when combined with a non-effective dose of nicotine or the α4β2* agonist ABT-594. This finding provides confirmation of the positive allosteric modulating effect of NS9283 in a functional in vivo paradigm. NS9283 is a potentially valuable tool for studying the role of α4β2* receptors in various nicotinic acetylcholine receptor-related functions.

‘Hybrid’ benzofuran–benzopyran congeners as rigid analogs of hallucinogenic phenethylamines

Phenylalkylamines that possess conformationally rigidified furanyl moieties in place of alkoxy arene ring substituents have been shown previously to possess the highest affinities and agonist functional potencies at the serotonin 5-HT 2A receptor among this chemical class. Further, affinity declines when both furanyl rings are expanded to the larger dipyranyl ring system. The present paper reports the synthesis and pharmacological evaluation of a series of ‘hybrid’ benzofuranyl–benzopyranyl phenylalkylamines to probe further the sizes of the binding pockets within the serotonin 5-HT 2A agonist binding site. Thus, 4( a– b), 5( a– b), and 6 were prepared as homologs of the parent compound, 8-bromo-1-(2,3,6,7-tetrahydrobenzo[1,2- b:4,5- b′]difuran-4-yl)-2-aminopropane 2, and their affinity, functional potency, and intrinsic activity were assessed using cells stably expressing the rat 5-HT 2A receptor. The behavioral pharmacology of these new analogs was also evaluated in the two-lever drug discrimination paradigm. Although all of the hybrid isomers had similar, nanomolar range receptor affinities, those with the smaller furanyl ring at the arene 2-position ( 4a– b) displayed a 4- to 15-fold greater functional potency than those with the larger pyranyl ring at that position ( 5a– b). When the furan ring of the more potent agonist 4b was aromatized to give 6, a receptor affinity similar to the parent difuranyl compound 2 was attained, along with a functional potency equivalent to 2, 4a, and 4b. In drug discrimination experiments using rats trained to discriminate LSD from saline, 4b was more than two times more potent than 5b, with the latter having a potency similar to the classic hallucinogenic amphetamine 1 (DOB).

Chronic Phenethylamine Hallucinogen Treatment Alters Behavioral Sensitivity to a Metabotropic Glutamate 2/3 Receptor Agonist

Recent clinical studies in schizophrenic patients show that a selective agonist of group II metabotropic glutamate (mGlu) receptors has robust efficacy in treating positive and negative symptoms. Group II mGlu receptor agonists also modulate the in vivo activity of psychotomimetic drugs, reducing the ability of psychotomimetic hallucinogens to increase glutamatergic transmission. The use of mouse models provides an opportunity to investigate the dynamic action that mGlu2/3 receptors play in regulating the behavioral effects of hallucinogen-induced glutamatergic neurotransmission using genetic as well as pharmacological strategies. The current study sought to characterize the use of the two-lever drug discrimination paradigm in ICR (CD-1) mice, using the hallucinogenic 5-HT2A/2C receptor agonist (-)-2,5-dimethoxy-4-bromoamphetamine [(-)-DOB)] as a stimulus-producing drug. The (-)-DOB discriminative stimulus was dose-dependent, generalized to the hallucinogen lysergic acid diethylamide, and was potently blocked by the 5-HT2A receptor antagonist M100907. However, contrary to our prediction, the hallucinogen-induced discriminative stimulus was not regulated by mGlu2/3 receptors. In a series of follow-up studies using hallucinogen-induced head twitch response and phencyclidine-induced hyperlocomotion, it was additionally discovered that the repeated dosing regimen required for discrimination training attenuated the behavioral effects of the mGlu2/3 receptor agonist LY379268. Furthermore chronic studies, using a 14 day (-)-DOB treatment, confirmed that repeated hallucinogen treatment causes a loss of behavioral activity of mGlu2/3 receptors, likely resulting from persistent activation of mGlu2/3 receptors by a hallucinogen-induced hyperglutamatergic state.

Effect of the 5-HT 6 serotonin antagonist MS-245 on the actions of (−)nicotine

The 5-HT 6 serotonin receptor antagonist MS-245 neither substitutes for nor antagonizes the discriminative stimulus effects of (−)nicotine. However, MS-245 was shown to enhance the potency of (−)nicotine in Sprague–Dawley rats trained to discriminate 0.6 mg/kg of (−)nicotine from saline vehicle in a typical two-lever drug discrimination paradigm such that a combination of MS-245 (5.0 mg/kg) plus the ED 50 dose of (−)nicotine caused the animals to respond as if they had received the training dose of (−)nicotine. MS-245 also potentiated the hypolocomotor actions, but not the antinociceptive effects, of (−)nicotine in mice. The results suggest possible involvement of serotonin-regulated signaling mechanisms in certain behavioral effects of nicotine.

Behavioral Tolerance to Lysergic Acid Diethylamide is Associated with Reduced Serotonin-2A Receptor Signaling in Rat Cortex

Tolerance is defined as a decrease in responsiveness to a drug after repeated administration. Tolerance to the behavioral effects of hallucinogens occurs in humans and animals. In this study, we used drug discrimination to establish a behavioral model of lysergic acid diethylamide (LSD) tolerance and examined whether tolerance to the stimulus properties of LSD is related to altered serotonin receptor signaling. Rats were trained to discriminate 60 microg/kg LSD from saline in a two-lever drug discrimination paradigm. Two groups of animals were assigned to either chronic saline treatment or chronic LSD treatment. For chronic treatment, rats from each group were injected once per day with either 130 microg/kg LSD or saline for 5 days. Rats were tested for their ability to discriminate either saline or 60 microg/kg LSD, 24 h after the last chronic injection. Rats receiving chronic LSD showed a 44% reduction in LSD lever selection, while rats receiving chronic vehicle showed no change in percent choice on the LSD lever. In another group of rats receiving the identical chronic LSD treatment, LSD-stimulated [35S]GTPgammaS binding, an index of G-protein coupling, was measured in the rat brain by autoradiography. After chronic LSD, a significant reduction in LSD-stimulated [35S]GTPgammaS binding was observed in the medial prefrontal cortex and anterior cingulate cortex. Furthermore, chronic LSD produced a significant reduction in 2,5-dimethoxy-4-iodoamphetamine-stimulated [35S]GTPgammaS binding in medial prefrontal cortex and anterior cingulate cortex, which was blocked by MDL 100907, a selective 5-HT2A receptor antagonist, but not SB206553, a 5-HT2C receptor antagonist, indicating a reduction in 5-HT2A receptor signaling. 125I-LSD binding to 5-HT2A receptors was reduced in cortical regions, demonstrating a reduction in 5-HT2A receptor density. Taken together, these results indicate that adaptive changes in LSD-stimulated serotonin receptor signaling may mediate tolerance to the discriminative stimulus effects of LSD.

Role of Acetaldehyde in the Discriminative Stimulus Effects of Ethanol

Acetaldehyde has been suggested to mediate some of the effects of ethanol. Acetaldehyde can be produced by the enzyme catalase within the brain after ethanol administration. The catalase inhibitor 3-amino-1,2,4-triazole (AT) reduces the production of acetaldehyde, and AT administration can reduce a number of ethanol-induced behavioral effects; this suggests the involvement of acetaldehyde in these behaviors. However, a role for acetaldehyde in mediating the discriminative stimulus effects of ethanol remains unclear. The contribution of acetaldehyde to the discriminative stimulus effects of ethanol was investigated by use of a two-lever drug discrimination paradigm with food reinforcement. Male Long-Evans rats were trained to discriminate water from either 1.0 or 2.0 g/kg ethanol. Stimulus substitution tests were conducted with ethanol (0-2.5 g/kg by gavage) and acetaldehyde (0-300 mg/kg intraperitoneally). A cumulative dose-response procedure was then used to investigate the effects of pretreatments with AT (0.5 and 1.0 g/kg intraperitoneally) on ethanol discrimination. Acetaldehyde up to doses that decreased response rates (300 mg/kg) did not substitute for the discriminative stimulus effects of 1.0 or 2.0 g/kg ethanol. In addition, AT pretreatment did not affect the dose-response curves for ethanol discrimination. These results show that exogenous acetaldehyde administration does not produce discriminative stimulus effects that are similar to those of ethanol. Also, pretreatment with the catalase inhibitor did not affect the dose-response curve for ethanol discrimination, and this suggests that endogenously produced acetaldehyde does not contribute to the discriminative stimulus effects of ethanol. Together these results suggest that acetaldehyde does not mediate the discriminative stimulus effects of 1.0 to 2.0 g/kg ethanol.

Attenuation of the amphetamine discriminative cue in rats with the atypical antipsychotic olanzapine

Sixteen male Sprague–Dawley rats were trained to discriminate between saline and amphetamine injections (1.0 mg/kg ip) using a standard two-lever (FR10) drug discrimination paradigm. A baseline dose–effect curve was generated for amphetamine administration alone, using doses both above and below the training dose (0.0–2.2 mg/kg ip). Once completed, a single dose of olanzapine (OLZ; 1.5 mg/kg sc) was tested for its ability to attenuate the amphetamine cue. OLZ pretreatment (60 min) successfully interfered with an animal's ability to discriminate amphetamine injections across various doses. The percentage of correct responding on the amphetamine lever and rate of responding were both significantly decreased across some but not all of the amphetamine doses. Therefore, we believe that this preliminary investigation has successfully shown that an OLZ dose of 1.5 mg/kg sc at 60 min can interfere with an animal's ability to detect some subjective cue(s) associated with amphetamine administration.

Effect of ring fluorination on the pharmacology of hallucinogenic tryptamines.

A series of fluorinated analogues of the hallucinogenic tryptamines N,N-diethyltryptamine (DET), 4-hydroxy-N,N-dimethyltryptamine (4-OH-DMT, psilocin), and 5-methoxy-DMT was synthesized to investigate possible explanations for the inactivity of 6-fluoro-DET as a hallucinogen and to determine the effects of fluorination on the molecular recognition and activation of these compounds at serotonin receptor subtypes. The target compounds were evaluated using in vivo behavioral assays for hallucinogen-like and 5-HT(1A) agonist activity and in vitro radioligand competition assays for their affinity at 5-HT(2A), 5-HT(2C), and 5-HT(1A) receptor sites. Functional activity at the 5-HT(2A) receptor was determined for all compounds. In addition, for some compounds functional activity was determined at the 5-HT(1A) receptor. Hallucinogen-like activity, evaluated in the two-lever drug discrimination paradigm using LSD-trained rats, was attenuated or abolished for all of the fluorinated analogues. One of the tryptamines, 4-fluoro-5-methoxy-DMT (6), displayed high 5-HT(1A) agonist activity, with potency greater than that of the 5-HT(1A) agonist 8-OH-DPAT. The ED(50) of 6 in the two-lever drug discrimination paradigm using rats trained to discriminate the 5-HT(1A) agonist LY293284 was 0.17 micromol/kg, and the K(i) at [(3)H]8-OH-DPAT-labeled 5-HT(1A) receptors was 0.23 nM. The results indicate that fluorination of hallucinogenic tryptamines generally has little effect on 5-HT(2A/2C) receptor affinity or intrinsic activity. Affinity at the 5-HT(1A) receptor was reduced, however, in all but one example, and all of the compounds tested were full agonists but with reduced functional potency at this serotonin receptor subtype. The one notable exception was 4-fluoro-5-methoxy-DMT (6), which had markedly enhanced 5-HT(1A) receptor affinity and functional potency. Although it is generally considered that hallucinogenic activity results from 5-HT(2A) receptor activation, the present results suggest a possible role for involvement of the 5-HT(1A) receptor with tryptamines.

Evaluation of the reinforcing properties and phencyclidine-like discriminative stimulus effects of dextromethorphan and dextrorphan in rats and rhesus monkeys.

Dextromethorphan (DXM) and its metabolite, dextrorphan (DXO) have neuroprotective and anticonvulsant properties through their activity as N-methyl-D-aspartate (NMDA) receptor channel blockers. Based on this receptor activity, coupled with reports of DXM abuse, both were evaluated for abuse potential and phencyclidine (PCP)-like behavioral effects in two animal models. The discriminative stimulus properties of DXO and DXM were tested in rats (3-56 mg/kg DXM, i.p. and 2.2-40.9 mg/kg DXO, i.p.) and rhesus monkeys (0.3-10 mg/kg DXM, i.m. and 0.25-8.0 mg/kg DXO, i. m.) trained to discriminate PCP from saline using a standard two-lever drug-discrimination paradigm under a fixed-ratio (FR) schedule of food reinforcement. In a second set of experiments, i.v. self-administration of DXO (10-100 microg/kg/infusion) and DXM (10-1000 microg/kg/infusion) were tested under a FR schedule of reinforcement in monkeys trained to lever press for infusions of PCP during daily 1-h sessions. In rats, both DXM and DXO produced a dose-dependent substitution for PCP. When tested in monkeys, DXM yielded partial (1 monkey) and full (2 monkeys) substitution for PCP, while DXO substituted fully for PCP in all four subjects tested. In the self-administration study, in five of the six subjects, at least one dose of DXM served as a positive reinforcer, maintaining infusion rates above those for saline. For DXO, at least one dose maintained infusion numbers well above mean saline infusion numbers in all subjects. Taken together, these data show that DXM has some PCP-like effects in rats and monkeys, but that they are more reliably produced by its metabolite, DXO. Thus, high doses of DXM may have some PCP-like abuse potential in humans but this potential may be associated with, or enhanced by, metabolism of DXM to DXO.

Thieno[3,2-b]- and thieno[2,3-b]pyrrole bioisosteric analogues of the hallucinogen and serotonin agonist N,N-dimethyltryptamine.

The synthesis and biological activity of 6-[2-(N, N-dimethylamino)ethyl]-4H-thieno[3,2-b]pyrrole (3a) and 4-[2-(N, N-dimethylamino)ethyl]-6H-thieno[2,3-b]pyrrole (3b), thienopyrroles as potential bioisosteres of N,N-dimethyltryptamine (1a), are reported. Hallucinogen-like activity was evaluated in the two-lever drug discrimination paradigm using LSD- and DOI-trained rats. Neither 3a nor 3b substituted for LSD or DOI up to doses of 50 micromol/kg. By comparison, 1a fully substituted in LSD-trained rats. However, 3a and 3b fully substituted for the 5-HT1A agonist LY293284 ((-)-(4R)-6-acetyl-4-(di-n-propylamino)-1,3,4, 5-tetrahydrobenz[c,d]indole). Both 3a and 3b induced a brief "serotonin syndrome" and salivation, an indication of 5-HT1A receptor activation. At the cloned human 5-HT2A receptor 3b had about twice the affinity of 3a. At the cloned human 5-HT2B and 5-HT2C receptors, however, 3a had about twice the affinity of 3b. Therefore, thiophene lacks equivalence as a replacement for the phenyl ring in the indole nucleus of tryptamines that bind to 5-HT2 receptor subtypes and possess LSD-like behavioral effects. Whereas both of the thienopyrroles had lower affinity than the corresponding 1a at 5-HT2 receptors, 3a and 3b had significantly greater affinity than 1a at the 5-HT1A receptor. Thus, thienopyrrole does appear to serve as a potent bioisostere for the indole nucleus in compounds that bind to the serotonin 5-HT1A receptor. These differences in biological activity suggest that serotonin receptor isoforms are very sensitive to subtle changes in the electronic character of the aromatic systems of indole compounds.

Role of D1 and D2 dopamine receptors in the discriminative stimulus properties of the atypical antipsychotic clozapine in rats

The purpose of the present study was to assess the role of dopamine D1 and D2 receptors in the discriminative stimulus properties of the atypical antipsychotic clozapine (CLZ). Two groups of rats were trained to discriminate either a moderate dose of clozapine (5.0 mg/kg) from vehicle or a high dose of clozapine (10.0 mg/kg) from vehicle in a two-lever drug discrimination paradigm. Generalization testing with clozapine yielded an ED50 of 0.9 mg/kg (95% confidence limits = 0.5–2.0 mg/kg) for the 5.0 CLZ group and 2.0 mg/kg (95% confidence limits = 1.4–2.8 mg/kg) for the 10.0 CLZ group. Substitution testing with the D1 antagonist SCH 23390 and the D2 dopamine antagonist haloperidol failed to produce clozapine-appropriate responding for either of the clozapine training doses. The antipsychotic drug thioridazine (which binds to a number of neurotransmitters in addition to dopamine) produced partial substitution (64.5% drug lever responding) in the 5.0 CLZ group at the 5.0 mg/kg dose. These results suggest that antagonism of D1 and D2 dopamine receptors alone is not sufficient to produce clozapine-appropriate responding, even with the higher training dose of 10.0 mg/kg. Drug Dev. Res. 46:139–147, 1999. © 1999 Wiley-Liss, Inc.

Role of D1 and D2 dopamine receptors in the discriminative stimulus properties of the atypical antipsychotic clozapine in rats

The purpose of the present study was to assess the role of dopamine D1 and D2 receptors in the discriminative stimulus properties of the atypical antipsychotic clozapine (CLZ). Two groups of rats were trained to discriminate either a moderate dose of clozapine (5.0 mg/kg) from vehicle or a high dose of clozapine (10.0 mg/kg) from vehicle in a two-lever drug discrimination paradigm. Generalization testing with clozapine yielded an ED50 of 0.9 mg/kg (95% confidence limits = 0.5–2.0 mg/kg) for the 5.0 CLZ group and 2.0 mg/kg (95% confidence limits = 1.4–2.8 mg/kg) for the 10.0 CLZ group. Substitution testing with the D1 antagonist SCH 23390 and the D2 dopamine antagonist haloperidol failed to produce clozapine-appropriate responding for either of the clozapine training doses. The antipsychotic drug thioridazine (which binds to a number of neurotransmitters in addition to dopamine) produced partial substitution (64.5% drug lever responding) in the 5.0 CLZ group at the 5.0 mg/kg dose. These results suggest that antagonism of D1 and D2 dopamine receptors alone is not sufficient to produce clozapine-appropriate responding, even with the higher training dose of 10.0 mg/kg. Drug Dev. Res. 46:139–147, 1999. © 1999 Wiley-Liss, Inc.

Evidence for overshadowing by components of the heterogeneous discriminative stimulus effects of ethanol

The present study used a drug discrimination paradigm to characterize the contribution of separate receptor systems to the stimulus effects of different training doses of ethanol. In a two-lever drug discrimination paradigm two groups of adult male Long-Evans rats ( n=8 per group) were trained to discriminate either 1.0 g/kg ethanol from water or 2.0 g/kg ethanol from water, administered intragastrically (i.g.), 30 min prior to the start of daily sessions in which responding was maintained under a fixed ratio 20 schedule of food presentation. Following training, cumulative dosing substitution tests were conducted with the GABA A positive modulator pentobarbital (1–17 mg/kg, i.p.), the uncompetitive NMDA antagonist dizocilpine (0.01–0.3 mg/kg, i.p.) and the 5-HT 1B/2C agonist m-trifluoromethylphenylpiperazine (TFMPP 0.17–1.7 mg/kg, i.p.). Next, the rats initially trained at 1.0 g/kg ethanol were retrained to discriminate 2.0 g/kg ethanol from water, and the rats initially trained at 1.0 g/kg were retrained to discriminate 2.0 g/kg ethanol from water. Both groups were then re-tested with the same ligands. Regardless of training history, animals currently discriminating 1.0 g/kg were more sensitive to the ethanol-like effects of TFMPP and pentobarbital compared to rats discriminating 2.0 g/kg ethanol. However, no difference in sensitivity to the ethanol-like effects of dizocilpine based on ethanol training dose was detected. These results support the view that ethanol is a heterogeneous discriminative stimulus comprised of GABA A, NMDA and 5-HT 1B/2C receptor-mediated activity. Furthermore, changes in sensitivity to GABA A and 5-HT ligands as a function of training dose could be indicative of overshadowing by other components of ethanol's heterogeneous cue. Finally, it appears that the current profile of ethanol's heterogeneous stimulus effects, rather than an interaction with ethanol training history, determines the substitution pattern of specific receptor ligands.

Phencyclidine-like discriminative stimulus effects of polyamine modulators of N-methyl- d-aspartate receptor activity in rats

Excessive N-methyl- d-aspartate (NMDA) receptor activation has been implicated in many acute and chronic neuropathologies. NMDA antagonists might prove to be useful treatments, unfortunately, some can produce phencyclidine (PCP)-like side effects. The polyamine-site modulators, spermine (SPM) and spermidine (SPD), produce dose related biphasic modulation of NMDA channel currents while another polyamine, arcaine (ARC), produces only negative modulatory effects. The PCP-like effects of these compounds were tested in rats trained to discriminate PCP from saline in a standard two-lever drug discrimination paradigm under a fixed ratio schedule of food reinforcement. SPM, SPD and ARC occasioned little, if any, responding on the PCP-associated lever, even at response rate suppressing doses. The results provide further evidence that differences exist between the discriminative stimulus effects produced by drugs active at different sites on the NMDA receptor and suggest that the polyamine modulatory site should be a good target for development of NMDA antagonist medications with a reduced propensity for PCP-like acute behavioral effects.

Substituted naphthofurans as hallucinogenic phenethylamine-ergoline hybrid molecules with unexpected muscarinic antagonist activity.

A series of substituted racemic naphthofurans were synthesized as "hybrid" molecules of the two major prototypical hallucinogenic drug classes, the phenethylamines and the tryptamines/ergolines. Although it was hypothesized that these new agents might possess high affinity for the serotonin 5-HT2A/2C receptor subtypes, unexpected affinity for muscarinic receptors was observed. The compounds initially synthesized for this study were (+/-)-anti- and syn-4-amino-6-methoxy-2a,3,4,5-tetrahydro-2H-naphtho[1,8-bc]furan (4a,b), respectively, and their 8-bromo derivatives 4c,d, respectively. The brominated primary amines 4c,d were assayed initially for activity in the two-lever drug discrimination (DD) paradigm in rats trained to discriminate saline from LSD tartrate (0. 08 mg/kg). Also, 4c,d were evaluated for their ability to compete against agonist and antagonist radioligands at cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptors. After the syn diastereomers were found to have the highest activity in these preliminary assays, the N-alkylated analogues syn-N,N-dimethyl-4-amino-6-methoxy-2a,3,4, 5-tetrahydro-2H-naphtho[1,8-bc]furan (4e) and syn-N, N-dipropyl-4-amino-6-methoxy-2a,3,4,5-tetrahydro-2H-naphtho[1, 8-bc]furan (4f) were prepared and assayed for their affinities at [3H]ketanserin-labeled 5-HT2A and [3H]-8-OH-DPAT-labeled 5-HT1A sites. All of the molecules tested had relatively low affinity for serotonin receptors, yet a preliminary screen indicated that compound 4d had affinity for muscarinic receptors. Thus, 4b,d,e were evaluated for their affinity at muscarinic M1-M5 receptors and also assessed for their functional characteristics at the M1 and M2 isoforms. Compound 4d had affinities of 12-33 nM at all of the muscarinic sites, with 4b,e having much lower affinity. All three compounds fully antagonized the effects of carbachol at the M1 receptor, while only 4d completely antagonized carbachol at the M2 receptor. The fact that the naphthofurans lack LSD-like activity suggests that they do not bind to the serotonin receptor in a way such that the tricyclic naphthofuran nucleus is bioisosteric with, and directly superimposable upon, the A, B, and C rings of LSD. This also implies, therefore, that the hallucinogenic phenethylamines cannot be directly superimposed on LSD in a common binding orientation for these two chemical classes, contrary to previous hypotheses.

A Study Examining Intravenous Ethanol‐Conditioned Place Preference in C57BL/6J Mice

The reinforcing effects of intravenous (I.V.) ethanol were examined in C57BL/6J (C57) mice with a conditioned-place-preference (CPP) paradigm. Before CPP testing, adult mice underwent jugular catheterization. On the following day, subjects were acclimated to a two-compartment CPP chamber. A 15-min nondrug pretest was conducted to determine compartment preference. For the treatment group, I.V. ethanol [30% (v/v), 3.4 microl/min, 25 min] was paired with the nonpreferred compartment, whereas I.V. saline was paired with the preferred compartment. The control group received I.V. saline in both compartments. Two conditioning sessions were conducted per day (0900 and 1500), and the order of the infusions was counterbalanced across subjects. The drug-free posttest was identical to the pretest, except that it occurred on the day after the final drug/compartment pairing. The entire procedure required 6 days. After just two pairings with ethanol, with a cumulative ethanol dose of only 0.82 g/kg/day, significant CPP was noted in the treatment group, whereas no change in compartment preference was noted for the control group. A separate group of C57 mice were trained to discriminate intraperitoneal ethanol (1.5 g/kg) from saline using a two-lever drug discrimination paradigm. After training was complete, these mice also underwent jugular catheterization. Substitution testing was conducted with I.V. ethanol [30% (v/v), 6.4 microl/min, 12 min] and saline. The results indicate that the subjective effects of ethanol did not differ according to the route of administration. Together, these experiments provide evidence that ethanol is rewarding for C57 mice, as indexed by ethanol CPP, and that the subjective effects of intravenously and intraperitoneally administered ethanol are similar.