James E. Barrett

5-HT receptors as targets for the development of novel anxiolytic drugs: Models, mechanisms and future directions.

The introduction of buspirone for the treatment of anxiety, together with the eventual suggestion of a mode of action involving the serotonin (5-HT)1A receptor subtype, has generated considerable research activity and renewed interest in the potential role of 5-HT in anxiety. The further identification of multiple 5-HT1 receptors, coupled with the possibility that these subtypes potentially are involved in discrete biobehavioral regulation and pathophysiological conditions, has greatly expanded the search for tools capable of probing these receptors and has raised hopes for a new generation of more specific compounds to treat other disorders associated with the 5-HT system such as depression, aggression, and sleep and eating disturbances. The involvement of 5-HT in anxiety has prompted a careful reevaluation of several traditional areas of research. This has included those methods used in the in vivo evaluation of drugs in preclinical animal test procedures used to assess potential anxiolytic activity, as well as the mechanisms associated with adaptive changes occurring during long-term drug administration. The proliferation of various procedures for studying the anxiolytic effects of 5-HT drugs has not always been accompanied by systematic behavioral and pharmacological validation. At the present time, this area of research is characterized by numerous inconsistent findings. Procedures that are objective and impartial to the behavioral effects of drugs provide distinct advantages for addressing some of these issues, as will the results from carefully controlled clinical studies. The main objective of this article is to provide an overview of the recent developments in research involving the 5-HT system and anxiety. The emphasis will be on the 5-HT1 receptor system and a review of the results in the predominant animal models used to evaluate these drugs, as well as an overview of the mechanisms currently believed to be responsible for the therapeutic activity of this class of compounds. Studies with the pigeon are reviewed, since this species appears distinctly sensitive to the anxiolytic-like effects of 5-HT1A drugs in conflict procedures. Although chronic administration of 5-HT1A drugs appears necessary for clinical anxiolytic and antidepressant activity, the most noteworthy neuropharmacological effects in animals seem to occur in 5-HT2 and, perhaps, 5-HT3 receptors which are downregulated. Studies summarizing the activity of drugs interacting with 5-HT1C/2 and 5-HT3 receptor sites are also discussed as they too may be involved in anxiety or the actions of anxiolytic drugs. The growing evidence suggesting an interaction between 5-HT receptor types, particularly between 5-HT1A and 5-HT1C/2 receptors, is reviewed, since drugs with these combined properties appear to be particularly efficacious in animal models of anxiety and warrant further detailed analyses. The development of drugs targeted specifically at multiple receptors may provide distinct therapeutic advantages for disorders such as anxiety and depression that appear to involve multiple neurotransmitter systems.

The discriminative stimulus effects of methamphetamine in pigeons

This experiment was designed to elucidate the neurotransmitter systems that mediate the discriminative stimulus effects of methamphetamine. Four pigeons were trained to peck one key following saline injections and a second key following methamphetamine injections (1.0 or 1.7 mg/kg, IM). Substitution tests revealed drug-appropriate responding following administration of the psychomotor stimulants methamphetamine, amphetamine and cocaine, the dopamine (DA) reuptake inhibitor bupropion, norepinephrine (NE) reuptake inhibitors imipramine and tomoxetine, and the serotonin (5-HT) releaser fenfluramine. Salinekey responding occurred following administration of the D1 agonist SKF-38393, the D1 antagonist SCH-23390, the α2 receptor agonist clonidine, the α1 antagonist prazosin, a nonselective β-antagonist propranolol and the selective 5-HT reuptake inhibitor fluoxetine. The D2/D3 agonist quinpirole produced drug-appropriate responding in two pigeons and partial substitution in the remaining two pigeons. The 5HT1A agonist 8-OH-DPAT produced drug-appropriate responding at higher doses (0.3–1.0 mg/kg), whereas much lower doses (0.003–0.1 mg/kg) antagonized the methamphetamine stimulus. The stimulus effects of methamphetamine were attenuated by pretreatment with prazosin, SCH-23390 and eticlopride, whereas pretreatment with propranolol and the 5-HT3 antagonist, MDL 72222, failed reliably to attenuate drug key responding. These results suggest that NE and DA reuptake inhibition and 5-HT release mediate the discriminative stimulus effects of methamphetamine as do the 5-HT1A and DA D1 and D2 receptors.

Evaluation of the discriminative stimulus effects of the novel sedative-hypnotic CL 284,846.

CL 284,846,N-[3-(3-cyanopyrazolo[1, 5-a]pyrimidin-7-yl)phenyl)]-N-ethylacetamide, is a novel non-benzodiazepine sedative-hypnotic with benzodiazepine-like sedative effects, but with less apparent liability for accompanying undesired side effects. In an effort to further characterize its pharmacological activity, CL 284,846 (3.0 mg/kg, IP, 30 min pretreatment) was established as a discriminative stimulus (DS) in rats (n=7). CL 284,846 (0.3–10.0 mg/kg) showed a dose-related increase in drug-appropriate responding up to the training dose and a dose-related decrease in response rate. The benzodiazepine agonist triazolam (0.1–1.0 mg/kg), the benzodiazepine partial agonist Ro 17-1812 (0.3–3.0 mg/kg) and the triazolopyridazine CL 218,872 (1.0–3.0 mg/kg) substituted for CL 284,846 in all rats, whereas the imidazopyridines zolpidem (3.0–10.0 mg/kg) and alpidem (10.0–30.0 mg/kg), the benzodiazepine partial agonist bretazenil (0.03–10.0 mg/kg) and the novel putative anxiolytic CL 273,547 (10.0–56.0 mg/kg) substituted in most, but not all, rats. Ro 17-1812, bretazenil, and CL 218,872 had no effect on response rate while the other drugs showed a concomitant decrease in rate. The 5-HT1A agonist buspirone (1.0–10.0 mg/kg) and the barbiturate pentobarbital (3.0–17.0 mg/kg) failed to substitute for CL 284,846 up to rate-decreasing doses. The benzodiazepine antagonist flumazenil (3.0–10.0 mg/kg) blocked the DS effects of CL 284,846 in most rats with no effect on response rate. Taken together, these results suggest that the DS effects of CL 284,846 are mediated via benzodiazepine receptors; however, the DS profile of CL 284,846 remains distinct from both benzodiazepine and non-benzodiazepine sedative-hypnotic drugs.

Discriminative stimulus effects of 8-OH-DPAT in pigeons: antagonism studies with the putative 5-HT1A receptor antagonists BMY 7378 and NAN-190.

Pigeons were trained to discriminate 0.3 mg/kg of the 5-HT1A receptor agonist 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) from saline. RU 24969 (5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole), at doses of 5.6-10 mg/kg, and eltoprazine (5.6 mg/kg), both mixed 5-HT1A/B agonists, substituted completely for 8-OH-DPAT, whereas 3.0-10 mg/kg of the 5-HT1B/C agonist TFMPP (1-(m-trifluromethylphenyl)piperazine) and 0.1-3.0 of the 5-HT3 antagonist MDL 72222 (3-tropanyl-3,5-dichlorobenzoate) yielded only saline-appropriate responses. Substitution for 8-OH-DPAT by eltoprazine and RU 24969, which does not occur in rats, provides in vivo support for the suggestion that the absence of a 5-HT1B receptor in the pigeon allows more complete expression of 5-HT1A-mediated effects. BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)]8-azaspirol-[4.5]- decane-7,9-dione) attenuated the 8-OH-DPAT stimulus at doses from 1.0 to 10 mg/kg but, when administered alone, also resulted in approximately 40% 8-OH-DPAT-appropriate responding at the highest dose. NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phthalamido)butyl)-piperazine (0.3-3.0 mg/kg) produced a dose-dependent and complete antagonism of the 8-OH-DPAT-discriminative stimulus; administered alone NAN-190 resulted only in saline-key responding. NAN-190 also reversed the rate-decreasing effects of higher doses of 8-OH-DPAT. The beta-adrenoceptor antagonist (+/-)-pindolol (5.6-17 mg/kg) antagonized the discriminative stimulus effects of lower 8-OH-DPAT doses but was unable to block the effects of higher doses of 8-OH-DPAT. Prazosin (1.0-10 mg/kg), which like NAN-190, is an alpha 1-antagonist, neither substituted for nor blocked the discriminative stimulus effects of 8-OH-DPAT. These results suggest that NAN-190 is an effective 5-HT1A receptor antagonist in this procedure with pigeons, with no indication of agonist actions, whereas BMY 7378 and pindolol are best characterized as partial 5-HT1A receptor agonists.

Anxiolytic and antidepressant mechanisms of 5-HT1A drugs in the pigeon : contributions from behavioral studies

The discovery that compounds acting through 5-hydroxytryptamine (5-HT) receptor subtypes can produce anxiolytic and/or antidepressant therapeutic effects in humans has resulted in considerable interest in the role of the 5-HT receptor system in both anxiety and depressive disorders. Because many of the clinically efficacious 5-HT1A anxiolytic drugs are either ineffective or produce inconsistent results in traditional or standard types of preclinical punishment or conflict procedures with rodents and other nonhuman mammals, there is considerable need for alternative behavioral assays sensitive to and selective for these compounds. In contrast to data with nonhuman mammals, 5-HT1A drugs are quite effective in pigeons studied under a punishment procedure. This paper reviews the use of the pigeon conflict procedure as a method for the detection and analysis of potential anxiolytic drugs acting through 5-HT1A receptors. Additionally, recent studies, also with the pigeon, have indicated that, in contrast to the rat, it is possible to establish an antidepressant such as imipramine as a discriminative stimulus, and then to use this procedure to evaluate the neuropharmacological bases for the behavioral and, presumably, therapeutic actions of these drugs. Using the drug discrimination procedure, it has been possible to examine a number of selective compounds that substitute for imipramine, thereby clarifying specific substrates for the antidepressant activity of this and related drugs. The pigeon promises to be a useful species in the pharmacological analyses of novel anxiolytic drugs and provides new approaches to the analysis and understanding of traditional as well as the more recently introduced antidepressant drugs.

K-Channel blockers attenuate the presynaptic effects of the D2D3 agonist quinpirole in monkeys

The present study investigated whether potassium channel blockade could modify the behavioral effects of the dopamine D2/D3 receptor agonist quinpirole in squirrel monkeys. The duration of immobility and/or unusual postures indicative of catalepsy-associated behavior or the duration of scratching, known to be related to the effects of low and high doses, respectively, of quinpirole, were scored during 5-min observation periods in three squirrel monkeys. Saline or incremental doses of quinpirole were administered 10 min before each observation period. Administration of saline did not increase the durations of catalepsy-associated behavior (8% of the observational period) or scratching (< 1% of the observational period). Low doses of quinpirole (0.003-0.03 mg/kg) dose dependently increased the duration of catalepsy-associated behavior to approximately 54% of the observational periods. Higher doses of quinpirole (0.1-0.3 mg/kg) did not increase the duration of catalepsy; rather, these doses increased the duration of scratching to approximately 57% of the observational periods. The differential induction of catalepsy-associated behavior or scratching is believed to be related to, respectively, pre- and postsynaptic activity of quinpirole on dopamine D2/D3 receptors. Pretreatment with the potassium channel blockers apamin, 4-aminopyridine, and amodiaquin attenuated the effects of quinpirole (0.03 mg/kg) on catalepsy-associated behavior, with cataleptic postures maintained for 27, 21, and 24% of the observational periods, respectively. In contrast, pretreatment with potassium channel blockers did not consistently affect the scratching induced by quinpirole. In addition, apamin did not attenuate the catalepsy-associated behavior induced by the postsynaptic D2 receptor antagonist haloperidol (0.01-0.1 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT3 Receptor-independent Inhibition of the Depolarization-induced 86Rb Efflux from Human Neuroblastoma Cells, TE671, by Ondansetron

The 5‐HT3‐receptor antagonist, ondansetron, has been shown to have positive effects in selected in‐vivo models of memory impairment and anxiety. The exact mechanisms underlying such bioactivities are unknown. In the present work, an 86Rb efflux bioassay was used to show that ondansetron has a unique ability to block voltage‐gated potassium channels in TE671 human neuroblastoma cells.

Use of cultured human neuroblastoma cells in rapid discovery of the voltage-gated potassium-channel blockers

Abstract— Depolarization of human neuroblastoma cells by high concentrations of extracellular potassium ions, leads to the activation of the voltage‐gated potassium channels. The activity of such potassium channels can be effectively and rapidly monitored by tracking the efflux of 86Rb from pre‐loaded target cells in response to the depolarizing stimulus. The inclusion of compounds with unknown activity in the assay medium, can result in the identification of novel blockers of the voltage‐gated potassium channels. Since this functional assay is performed in 96‐well microtitre plates, it represents a rapid and high‐volume primary screening method for the detection and identification of the voltage‐gated potassium‐channel blockers, which may have therapeutic utility in several indications including memory degeneration and cardiac arrhythmias.

Shock postponement reverses the effects of cocaine on the punished pecking of pigeons

The effects of cocaine on punished and unpunished key peck responding of pigeons was examined before and after a history of treadle pressing maintained by shock postponement. In one schedule component, the first peck after 3 min produced grain. The alternate component was similar, but every 30 responses was also punished by a mild shock. Punished responding occurred at approximately 25% of the rate of unpunished responding. Cocaine (0.1-10.0 mg/kg IM) did not affect or decrease punished responding; unpunished responding was not systemically affected. Next, a foot treadle was installed and treadle presses postponed shocks for 25 s; shocks occurred every 5 s in the absence of pressing. The treadle was removed when shocks were reliably postponed. Next, the multiple schedule of key pecking was reinstated. At least one dose of cocaine now increased punished pecking; unpunished responding was not systematically altered. These results complement related findings with monkeys and show that pigeons are suitable subjects for studying the reversal of the effects of cocaine on punished responding by a history of postponing shock.