First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00918
Long chain polyunsaturated fatty acids are required for efficient neurotransmission in C. elegans
Giovanni M. Lesa1,*,
,
Mark Palfreyman2,
David H. Hall3,
M. Thomas Clandinin4,
Claudia Rudolph5,
Erik M. Jorgensen2 and
Giampietro Schiavo1
1 Molecular Neuropathobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
2 Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA
3 Center for C. elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA
4 Nutrition and Metabolism Research Group, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada
5 EleGene AG, Am Klopferspitz 19, 82152 Martinsried, Germany
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Fig. 1. fat-3 encodes a  6-desaturase. (A) The LC-PUFA synthetic pathways (Lauritzen et al., 2001 ; Spychalla et al., 1997). ALA,  -linolenic acid; LIN, linoleic acid; DGLA, dihomo- -linolenic acid; EPA, eicosapentaenoic acid; AA, arachidonic acid; DHA, docosahexaenoic acid. (B) The fat-3 gene (W08D2.4) is located on chromosome IV, between unc-24 and dpy-20. A 4.7 kb genomic fragment including 977 bp 5' and 862 bp 3' of the fat-3 coding region rescues fat-3 mutants. The coding regions are in boxes and the non-coding regions are shown as lines. The cytochrome b5-like domain is in gray. Asterisks indicate histidine-rich regions. The fat-3(lg8101) and fat-3(qa1811) deletions and their breakpoints are shown. T indicates an A to T substitution. Dp indicates a 17 bp duplication (GAAAATGGTTGAATCAT). fat-3(wa22) is a C to T point mutation that changes S186 to F. (C) ClustalX alignment of FAT-3 protein with human and plant (Borago officinalis) 6-desaturases. The triangle indicates the fat-3(wa22) mutation. Single-letter abbreviations for amino acid residues are used. Identical and similar amino acids are identified by gray and light gray shading, respectively. The putative cytochrome b5-like domain is indicated with a line. Histidines important for catalytic activity are marked by asterisks.
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Fig. 2. fat-3 deleted mutants do not synthesize fat-3 mRNA. (A) Schematic of the fat-3(lg8101) and fat-3(qa1811) deletions and the primers used for fat-3 mRNA analysis. (B,C) No wild-type fat-3 mRNA is detected in fat-3(lg8101) or fat-3(lg8101)/fat-3(qa1811) mutant animals. (B) RT-PCR of total mRNA from fat-3(lg8101) or wild-type animals with primers a and b, in the presence (+) or in the absence (-) of reverse transcriptase. Predicted PCR products: cDNA, 281 bp; genomic DNA, 326 bp. (C) RT-PCR with primers b and c of total mRNA from fat-3(lg8101), wild-type or fat-3(lg8101)/fat-3(qa1811) animals. Predicted PCR products: cDNA, 999 bp; genomic DNA, 1,451 bp.
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Fig. 3. Rescue of the behavioral defects associated with loss of fat-3 activity. (A) Exogenous AA and DHA, but not LIN, rescue the movement defects of fat-3(lg8101)/fat-3(qa1811) mutants. Animals were exposed to fatty acids from egg to adult. *P<0.0001 versus wild-type animals. (B) fat-3 expressed under the control of the neuronal promoter unc-119 (Exfat-3(+) neuron) but not under the control of the muscular promoter myo-3 (Exfat-3(+) muscle), completely rescues the egg-laying defect of fat-3(lg8101)/fat-3(qa1811) animals. Egl+ indicates hermaphrodites that were not consumed by embryos by the fourth day after reaching adulthood. (C) fat-3 expressed under the control of the neuronal promoter but not under the control of the muscular promoter or the intestinal promoter elt-2 (Exfat-3(+) intestine), almost completely rescues the movement defects of fat-3 (lg8101) homozygous animals. *P<0.0001 versus fat-3Exfat-3(+) animals. Data in A and C are plotted as mean ± s.e.m.
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Fig. 4. fat-3 mutant animals display functional and not morphological neuronal defects. (A,B) Serotonergic neurons visualized with GFP under the control of a tryptophane hydoxylase promoter (tph-1), which is expressed in serotonergic neurons (Sze et al., 2000). (A) An HSN motor neuron in proximity of the vulva. Bars, 5 µm. (B) Serotonergic neurons in the head. Images shown are projections of confocal xy sections. Bars, 10 µm. (C) Exogenous arachidonic acid (AA) restores wild-type movement in adult fat-3(lg8101)/fat-3(qa1811) mutant animals within 48 hours. *P=0.0017 versus wild-type animals. Data is plotted as mean ± s.e.m.
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Fig. 5. Cholinergic and serotonergic synapses are functionally disrupted in fat-3 mutant animals. (A-C) Synaptic release of endogenous serotonin was measured by determining the egg-laying response of fat-3(lg8101)/fat-3(qa1811) mutants to serotonin, fluoxetine and imipramine. Muscles are functional in fat-3 mutants because they respond well to serotonin (A,C). However, they are defective in serotonin release, because they respond only inefficiently to the endogenous serotonin potentiators fluoxetine (B) and imipramine (C). In C, a single dose of serotonin (5 mg/ml) and imipramine (0.75 mg/ml) were used. (D,E) Synaptic release of endogenous ACh was measured by determining the response to the endogenous ACh potentiator aldicarb. fat-3 mutants are defective in ACh release because aldicarb induces spastic paralysis faster in wild-type animals than in fat-3(lg8101)/fat-3(qa1811) (D) or in fat-3(wa22) (E) mutants. The defect of fat-3 mutants is weaker but comparable to that of rab-3(y250), a synaptic vesicle transmission mutant. *P<0.01; **P<0.005. (F) The altered aldicarb sensitivity of fat-3 mutants is of neuronal origin. fat-3 expressed in neurons, but not in muscles, restores normal aldicarb sensitivity to fat-3(lg8101) mutants. (GH) The ACh mimetic levamisole induces paralysis faster in fat-3(lg8101)/fat-3(qa1811) (G) and in fat-3(wa22) (H) mutants than in wild-type animals. *P<0.003. (I) Normal sensitivity to levamisole is restored in fat-3(lg8101)/fat-3(qa1811) mutant animals exposed to AA from egg to adult. In all panels, data points show the mean ± s.e.m.
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Fig. 6. fat-3 mutants have reduced evoked amplitude and reduced rates of spontaneous fusion but normal quantal size. (A) Representative evoked responses from wild-type and fat-3 animals. (B) The mean amplitude of the evoked responses is reduced in fat-3 (n=6) compared to wild-type (n=7) animals (P<0.03). (C) Representative traces of spontaneous fusion events in wild-type and fat-3 mutants. (D-E) The mean frequency of spontaneous fusion is reduced in fat-3 mutants (n=13) compared to wild-type (n=14; *P<0.02), while the mean amplitude of the individual events is normal. Data is plotted as mean ± s.e.m.
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Fig. 7. Synapses are partially depleted of synaptic vesicles in fat-3 mutant animals. Electron micrographs of wild-type (A) and fat-3(lg8101) (B) synapses fixed by fast-freezing. (A) Clusters of vesicles (ves and arrowheads) in ventral nerve cord neurons are localized close or docked to the active zone (white arrows). (B) Ventral ganglion synapses are depleted of most vesicles close to the active zone (black arrows). A few vesicles lie at a distance (ves and arrowheads). (C) Quantification of synaptic vesicles in fat-3(lg8101)/fat-3(qa1811) mutant animals. The average fraction of synaptic vesicles per section at given distances in fat-3 mutants compared to wild-type animals (100%). Vesicles were considered docked when they were touching the active zone. Total number of synapses scored: 50, fat-3; 42, wild-type. *P<0.001; **P<0.0001. Scale bar: 0.5 µm.
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© The Company of Biologists Ltd 2003
