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.

Serotonin-Independent Neuromuscular Targets of Fluoxetine (Prozac)

Robert K.M. Choy, James H. Thomas

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.
        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.

1. Weinshenker et al. J. Neuroscience 15:6975 (1995)
2. Shreffler et al. Genetics 139:1261 (1995)