Worm Breeder's Gazette 14(4): 74 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Molecular and Cellular Biology Program and Department of Genetics University of Washington, Seattle, WA 98195
Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and tricyclics such as imipramine have been shown to block serotonin (5-HT) reuptake in vitro and to be efficacious antidepressants in vivo. However, little direct evidence exists that demonstrates a causal relationship between these two effects. In spite of this, fluoxetine has been prescribed for over 20 million people worldwide and generates over $2 billion in sales each year. Our lab has screened a wide variety of drugs for neuromuscular effects on C. elegans. We typically assayed 8-10 adult worms in drug dissolved in 25-50 ul of M9 in microtiter wells. Previous work in our lab found that SSRIs and tricyclics rapidly induce nose and body muscle contraction1. Two pieces of evidence suggest these effects are not mediated by block of 5-HT reuptake: 1) cat-4(e1141) animals, which lack immunocytochemically detectable 5-HT, are still fully sensitive to fluoxetine-induced nose and body muscle contraction, and 2) exogenous 5-HT does not mimic the effects of fluoxetine on nose and body muscles. Previous work in our lab also suggests that similar doses of SSRIs are capable of blocking 5-HT reuptake in the egg-laying system of C. elegans1. In order to investigate the biochemical actions of fluoxetine in worms, we screened for mutants that were nose resistant to fluoxetine (Nrf). After incubating adult F2 progeny of EMS mutagenized N2 in 1 mg/ml fluoxetine for 20 minutes, we picked animals with noses that failed to contract. From 11,000 mutagenized genomes, we isolated 16 alleles that define eight complementation groups. All Nrf mutants tested so far are cross-resistant to nose contraction by a second SSRI, (paroxetine) as well as the tricyclic clomipramine. This finding suggests that SSRIs and tricyclics share a target other than 5-HT reuptake. Since we screened for mutants defective in muscle contraction, one concern was whether the Nrfs were specifically defective in fluoxetine response or their nose muscles had a non-specific excitability defect. We found that all Nrfs are sensitive to nose contraction by the cholinergic agonist levamisole, even at low doses, demonstrating that their nose muscles are still competent to contract. In addition, levamisole-resistant unc-29(e1072) animals are still sensitive to fluoxetine induced muscle contraction. Three Nrf alleles fail to complement ndg-4(lb108). This mutant was isolated in a screen for resistance to lethal exposure to the lipoxygenase inhibitor nordihydroguaiaretic acid (NDG)2. NDG has been shown to block an arachidonic acid metabolite signaling pathway that regulates S-type K+ channels in Aplysia. In worms, NDG acutely induces nose and muscle contraction in a manner similar to SSRIs and tricyclics. ndg-4 mutants have pale eggs (Peg) and we found that Nrf mutants in three other complementation groups, including one with four alleles, also have pale eggs. These Peg mutants produce roughly 50-80% dead eggs, including some that are abnormally shaped or arrest in development. Adult Peg animals accumulate large globules of what appears to be yolk in the pseudocoelomic space (W. Shreffler, pers. comm. and our observations) and the pale eggs contain less yolk than wild-type embryos. Yolk proteins are synthesized in the intestine and then transported through the pseudocoelomic space into the gonad where they are absorbed by oocytes. Peg mutants may be defective in some stage of this transport process. One speculation is that the Peg mutations disrupt the establishment of an ionic gradient that is used to transport yolk proteins across cell membranes and that a similar ionic gradient regulates the excitability of motorneurons or muscle cells. We are currently mapping the Nrf mutations and further pharmacologically analyzing the mutants. Many of the mutations have been localized to regions already sequenced by the genome project. We plan to identify molecularly several of the genes involved in this fluoxetine response. References: 1. Weinshenker et al. J. Neuroscience 15:6975 (1995) 2. Shreffler et al. Genetics 139:1261 (1995)