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The zebrafish is an ideally suited vertebrate animal model for large-scale genetic screens and is emerging as a model organism in pharmacological and behavioral research. We investigated the effects of sedative hypnotics commonly used in humans on zebrafish locomotor activity and identified the corresponding genomic and receptor binding targets.We studied radioreceptor binding and behavioral responses to compounds with known sedative hypnotic properties representing multiple pharmacological classes. These included GABAergic hypnotics such as benzodiazepines, barbiturates, and baclofen; α-2 adrenergic agonists; and histaminergic H1 antagonists. An automated system was used to quantify behavioral effects. Zebrafish homologs of histamine receptor H1, γ-amino-n-butyric acid type A (α-subunit), and γ-amino-n-butyric acid type B (1 and 2) receptor genes were identified through translating queries of the zebrafish Zv4 database with human receptor protein sequences. A pilot screen of 154 N-ethyl-N-nitroso-urea-mutagenized F2 families was conducted with pentobarbital, flurazepam and mepyramine.Radioreceptor binding studies revealed high affinity binding sites for known γ-amino-n-butyric acid type A, γ-amino-n-butyric acid type B, and histaminergic ligands. Drug immersion of 5–7-day-old larvae reduced mobility and, in some cases, produced a complete state of unresponsive immobility similar to anesthesia. These effects were dose-dependent and rapidly reversible in water. As established in mammals, (R)-baclofen was more active behaviorally and had higher affinity in binding studies when compared with (S)-baclofen. In this model, (S)-baclofen only partially reduced activity at high dose and blocked (R)-baclofen behavioral hypnotic effects. Genomic sequences with high similarity to the corresponding pharmacological targets were identified, but no mutants were found in the pilot screen.These results demonstrate conservation of gene, protein and function for many established sedative hypnotic pathways. The results indicate feasibility of conducting large-scale pharmacogenomic screens to isolate novel proteins modulating susceptibility to hypnotic compounds in a vertebrate system.