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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
1Molecular Neuropathobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn
Fields, London WC2A 3PX, UK
2Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA
3Center for C. elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA
4Nutrition and Metabolism Research Group, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada
5EleGene AG, Am Klopferspitz 19, 82152 Martinsried, Germany
*Present address: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK
‡Author for correspondence: (e-mail:
Accepted 2 October 2003Journal of Cell Science 116, 4965-4975 2003 The Company of Biologists Ltddoi:10.1242/jcs.00918 The complex lipid constituents of the eukaryotic plasma
Expression of functional fat-3 in neurons, or application
membrane are precisely controlled in a cell-type-specific
exogenous LC-PUFAs to adult animals rescues
manner, suggesting an important, but as yet, unknown
defects. Pharmacological, ultrastructural and
cellular function. Neuronal membranes are enriched in
electrophysiological analyses demonstrate that fat-3
long-chain polyunsaturated fatty acids (LC-PUFAs) and
mutant animals are depleted of synaptic vesicles and
alterations in LC-PUFA metabolism cause debilitating
release abnormally low levels of neurotransmitter at
neuronal pathologies. However, the physiological role of
cholinergic and serotonergic neuromuscular junctions.
LC-PUFAs in neurons is unknown. We have characterized
These data indicate that LC-PUFAs are essential for
the neuronal phenotype of C. elegans mutants depleted of
efficient neurotransmission in C. elegans and may account
for the clinical conditions associated with mis-regulation of
The C. elegans genome encodes a single 6-desaturase
LC-PUFAs in humans.
gene (fat-3), an essential enzyme for LC-PUFA
biosynthesis. Animals lacking fat-3
function do not
synthesize LC-PUFAs and show movement and egg-laying

Key words: C. elegans, Neuromuscular junction, Neurotransmitter abnormalities associated with neuronal impairment.
release, Polyunsaturated fatty acids, Synapse functions of these molecules we generated Caenorhabditis One of the central challenges in biology is to understand the elegans mutants depleted of LC-PUFAs and analyzed their cellular functions of the wide variety of complex lipids present in animal cells. Long-chain polyunsaturated fatty acids (LC- The nematode C. elegans synthesizes many of the LC- PUFAs), fatty acids with multiple double bonds, are PUFAs found in humans (Wallis et al., 2002) and represents a synthesized from dietary precursors and are localized to cell good model organism to systematically study the neuronal membranes as phospholipid esters. Both the absolute LC- roles of these molecules. First, the location, structure and PUFA levels and their relative concentrations are strictly function of virtually all C. elegans neurons are known. Second, controlled in mammalian neurons (Lauritzen et al., 2001) pharmacological assays can be used to test synaptic function implying that LC-PUFAs have critical neuronal functions.
of certain neurons such as cholinergic motor neurons and Indeed, mutations in enzymes involved in LC-PUFA serotonergic neurons (see Jorgensen et al., 1995; Lackner et al., metabolism cause a form of X-linked mental retardation 1999; Miller et al., 1999). Third, it is possible to study the role (Meloni et al., 2002) and two forms of macular dystrophy of LC-PUFAs independently of the known eicosanoid- (Zhang et al., 2001) and diets deficient in essential LC-PUFAs mediated signaling pathways, since the C. elegans genome are associated with deficits in infant brain function (Anderson does not apparently encode orthologs of cyclooxygenase, et al., 1999; Helland et al., 2003; Lauritzen et al., 2001; Willatts lipooxygenase, thromboxane synthase or any orthologs of et al., 1998). In addition, LC-PUFAs induce oligomerization of prostaglandin, leukotriene or thromboxane receptors. Finally, α-synuclein, a protein found as insoluble aggregates in α- C. elegans mutants depleted of LC-PUFAs have been isolated synucleinopathies including Parkinson's disease (Sharon et al., by inactivating the gene fat-3, which encodes ∆6-desaturase, 2003). Although these and other studies suggest an important an enzyme essential for LC-PUFA biosynthesis (Fig. 1; this role of LC-PUFAs in nervous system function, their precise study and Watts and Browse (Watts and Browse, 2002)). Initial role in neurons remains unclear. To address the neuronal analysis revealed that some of the defects displayed by fat-3 Journal of Cell Science 116 (24) mutants are suggestive of neuronal impairment (Watts et al., Materials and Methods
2003), yet the molecular basis of these effects are unknown.
Strains and isolation of fat-3 deletions Here we characterize in detail the neuronal phenotypes C. elegans strains were cultured at 20°C as described (Brenner, 1974).
displayed by fat-3 mutants and demonstrate that LC-PUFAs are The strain GR1333 (IsPtph-1::gfp) was provided by I. Y. Sze and G.
essential for normal neurotransmitter release at cholinergic and Ruvkun (University of California Irvine, CA and Harvard University, serotonergic neuromuscular junctions (NMJs). These defects Cambridge, MA) (Sze et al., 2000). are not developmental but are functional, since exogenous LC- To isolate deletion of the fat-3 gene, we constructed DNA libraries PUFAs can rescue mutant adults. Consistent with these deficits of approximately 5,400,000 haploid genomes from wild-type (N2) we provide pharmacological and electrophysiological evidence animals mutagenized with ethylmethane sulfonate or with trimethylpsoralen. Using the polymerase chain reaction (PCR) with fat-3 that animals lacking LC-PUFAs release abnormally low levels primers (Jansen et al., 1997) we isolated two deletions: fat-3(lg8101) of neurotransmitter. In addition, ultrastructural analysis reveals and fat-3(qa1811). Both deletions confer a fully recessive phenotype.
that synapses in these animals are severely depleted of synaptic We used PCR-based genotyping to verify that the deleted strains do vesicles. We conclude that LC-PUFAs are required for efficient not contain duplications of the intact fat-3 gene. Analyses were carried out in fat-3(lg8101) homozygous, in fat-3(lg8101)dpy-20(e1282)/unc-24(e138)fat-3(qa1811)dpy-20(e1282) heterozygous or in fat-3(wa22)homozygous animals. fat-3 mutants were outcrossed to wild-type animals at least six times before analysis. Total RNA for RT-PCR was extracted from mixed C. elegans populations (Chomczynski and Sacchi, 1987). 1 µg of total RNA was reverse transcribed and PCR amplified using Ready-to-go RT-PCR beads (Amersham Biosciences, Chalfont St. Giles, UK). RT-PCR for unc-22 mRNA was used as positive control (data not shown). Primers used (Fig. 2A): a, 5′-CTCGAATTTTAAACAACTTCGCCGC-3′; b,5′-GGCAGCTTTAGCTTGAATGTGCTC-3′; c, 5′-CAGAAGCTTC- Rescue experiments A region comprising 1 kb 5′ of the fat-3 start, the entire fat-3 codingsequence (Napier et al., 1998) and 0.9 kb 3′ of the fat-3 end rescuedthe defects associated with fat-3 mutants. Primers containing KpnI andSacI restriction sites were used to PCR amplify the entire fat-3 coding sequence. This KpnI-SacI fragment and a HindIII-KpnI fragment from Punc-119::gfp (Maduro and Pilgrim, 1995) were cloned intopPD49.26 (provided by A. Fire, Carnegie Institution of Washington, Baltimore, MD) to generate Punc-119::fat-3. To generate Pmyo- 3::fat-3, a HindIII-BamHI fragment including the myo-3(+) promoter and the KpnI-SacI PCR fragment including the entire fat-3 coding sequence were cloned into pPD49.26. This KpnI-SacI fat-3 fragment and a PstI-SmaI fragment containing the elt-2 promoter were cloned into pPD49.26 to generate Pelt-2::fat-3. Constructs were injected (Mello et al., 1991) at 1-100 ng/µl in dpy-20(e1282) backgrounds with dpy-20(+) pMH86 (20 ng/µl). The presence of fat-3 mRNA was verified in all transgenic lines by RT-PCR (data not shown).
Fig. 1. fat-3 encodes a ∆6-desaturase. (A) The LC-PUFA synthetic
pathways (Lauritzen et al., 2001; Spychalla et al., 1997). ALA, α- linolenic acid; LIN, linoleic acid; DGLA, dihomo-γ-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 mutationthat 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.
LC-PUFAs and neurotransmission For fatty acid rescue experiments, arachidonic acid (AA), between assays, wild-type controls were included in each assay. The docosahexaenoic acid (DHA) or linoleic acid (LIN; Sigma, Poole, acetylcholine (ACh) sensitivity was tested by spreading 1 M ACh on UK) were prepared as 100 mg/ml solution in 95% ethanol and 100 µl plates (final concentration 10 mM). To minimize ACh hydrolysis, were spread over nematode growth medium plates. E. coli OP-50 were the assay was started within 10 minutes. 35-40 animals per experiment then added as a food source and 3-20 L4-adult fat-3(lg8101)dpy- were scored for complete paralysis (Lackner et al., 1999) and 3-6 independent experiments per dose and per drug were carried out.
were grown on each plate. These hermaphrodites or their progeny Statistical analysis was performed using the Mann-Whitney test with (thus exposed to LC-PUFAs from hatching), were analyzed.
InStat software (GraphPad Software, San Diego, CA).
Motility, egg-laying and paralysis assays Visualization of neurons and synapses These assays were carried out in fat-3(lg8101)dpy-20(e1282)/unc- To visualize serotonergic neurons in fat-3 mutants, we constructed the strain fat-3(lg8101);Is(Ptph-1::gfp,rol-6D). tph-1 encodes a fat-3(lg8101) mutants transgenic for the indicated constructs, or N2 tryptophan hydroxylase and is expressed in serotonergic neurons (Sze animals. Motility was quantified by placing adult worms in the centre et al., 2000). Animals were immobilized with 10 mM levamisole, of a bacterial lawn on a Petri dish and allowing them to move. After mounted on a 2% agarose pad and observed under a 40× or a 63× 30 seconds worms were immobilized with heat. Pictures of the tracks objective on a Zeiss LSM510 confocal microscope (Carl Zeiss, left on the bacterial lawn were taken using a Leica MZ 125 Oberkoken, Germany). microscope (Leica Microsystems, Milton Keynes, UK) equipped with For electron microscopy, young adult nematodes were fixed by a Photometrics CoolSnap digital camera (Roper Scientific, Tucson, immersion in buffered aldehydes and stained in osmium tetroxide AZ) and Openlab software (Improvision, Coventry, UK). Single (Hall, 1995) or fast frozen under high pressure followed by freeze tracks were highlighted with Adobe Illustrator (Adobe Systems, substitution into osmium tetroxide in acetone (McDonald, 1999).
Uxbridge, UK) and their pixels counted using NIH Image (National Samples were embedded into plastic resin, thin sectioned using a Institutes of Health, Bethesda, MD). 20-47 tracks from at least two diamond knife, counterstained with uranyl acetate and lead citrate, independent transgenic stable lines per genotype were analyzed. and examined on a Philips CM10 electron microscope (Philips Egg-laying-defective animals were determined by cloning L4 Electron Optics, Eindhoven, The Netherlands). We used 2 fixation hermaphrodites to single plates and by scoring them every day for 4 conditions because fat-3 mutants are, for some unknown reason, days for embryos that hatched inside the mother. difficult to fix well and the quality of the images obtained was not The egg-laying assay in the presence of drugs was performed in optimal. However, we found similar results with either fixation microtiter wells as described previously (Trent et al., 1983) using method. Both, fat-3(lg8101) and dpy-20(e1282)fat-3(lg8101)/unc- serotonin or fluoxetine (Sigma) dissolved at the indicated doses in M9 24(e138)fat-3(qa1811)dpy-20(e1282) mutants displayed similarly buffer. 12-36 animals for each dose were analyzed. depleted synapses. Morphometric analysis was carried out in animals For the paralysis assay, plates containing 1 mM aldicarb stained with osmium tetroxide. Vesicles of approximately 30 nm (Greyhound Chromatography, Birkenhead, UK) or 0.2 mM diameter were counted. Statistical analysis was performed using levamisole (Sigma) were prepared fresh for each set of assays as SAS/STAT software (SAS Institute Inc., Cary, NC). Significance described previously (Miller et al., 1999). To allow comparisons values were calculated using Student's t-test.
ACh and fatty acid quantificationACh was quantified in fat-3(lg8101)dpy-20(e1282)/unc-24(e138)fat-3(qa1811)dpy-20(e1282) or dpy-20(e1282) animals as describedpreviously (Nonet et al., 1993).
The genotypes of animals used for fatty acid quantification were as follows: fat-3(lg8101), N2, or fat-3(lg8101)dpy-20(e1282);Ex(fat-3(+)dpy-20(+)). Lipids were extracted and partitioned (Hargreavesand Clandinin, 1988). Phospholipid-derived fatty acid methyl esterswere separated by capillary gas liquid chromatography using a fullyautomated Varian 6000 GLC (Varian Instruments, Mississauga,Ontario). Data were expressed as a percentage of the area count foreach individual fatty acid relative to all fatty acids combined. ElectrophysiologyElectrophysiological methods were performed as previouslydescribed (Richmond et al., 1999; Richmond and Jorgensen, 1999)with minor adjustments. Briefly, the animals were immobilized in Fig. 2. fat-3 deleted mutants do not synthesize fat-3 mRNA.
cyanoacrylic glue and a lateral incision was made to expose the ventral (A) Schematic of the fat-3(lg8101) and fat-3(qa1811) deletions and medial body wall muscles. The preparation was then treated with the primers used for fat-3 mRNA analysis. (B,C) No wild-type fat-3 collagenase (type IV; Sigma) for 15 seconds at a concentration of 0.5 mRNA is detected in fat-3(lg8101) or fat-3(lg8101)/fat-3(qa1811) mg/ml. The muscle was then voltage clamped using the whole cell mutant animals. (B) RT-PCR of total mRNA from fat-3(lg8101) or configuration at a holding potential of –60 mV. All recording were wild-type animals with primers a and b, in the presence (+) or in the made at room temperature (21°C) using an EPC-9 patch-clamp absence (–) of reverse transcriptase. Predicted PCR products: cDNA, amplifier (HEKA, Southboro, MA) run on an ITC-16 interface 281 bp; genomic DNA, 326 bp. (C) RT-PCR with primers b and c of (Instrutech, Port Washington, NY). Data were acquired using Pulse total mRNA from fat-3(lg8101), wild-type or fat-3(lg8101)/fat- software (HEKA).
3(qa1811) animals. Predicted PCR products: cDNA, 999 bp; The extracellular solution contained: 150 mM NaCl, 5 mM KCl, genomic DNA, 1,451 bp. 0.5 mM CaCl2, 4 mM MgCl2, 10 mM glucose, 15 mM Hepes, pH Journal of Cell Science 116 (24) 7.35, and sucrose to 340 mOsm. The pipette solution contained: 120 Using a highly sensitive and quantitative chromatographic mM KCl, 20 mM KOH, 4 mM MgCl2, 5 mM N-tris (hydroxymethyl) method, we demonstrated that fat-3 deletion mutants are methyl-2-aminoethane-sulphonic acid, 0.25 mM CaCl2, 4 mM defective in LC-PUFA production and display a fatty acid NaATP, 36 mM sucrose, 5 mM EGTA, pH 7.2, sucrose to 335 mOsm.
composition similar to that reported for fat-3(wa22) mutants All data analysis and graph preparation was performed using Pulsefit (Watts and Browse, 2002). Wild-type worms produce (HEKA), Mini Analysis (Synaptosoft, Decatur, GA), and Igor Pro significant levels of dihomo-γ-linolenic acid (DGLA), AA and (Wavemetrics, Lake Oswego, OR).
eicosapentaenoic acid (EPA; Table 1), three LC-PUFAssynthesized only in the presence of active ∆6-desaturase.
Consistent with loss of ∆6-desaturase activity, fat-3(lg8101) mutant animals have drastically reduced levels of these three Generation of mutants depleted of LC-PUFAs LC-PUFAs and accumulate two ∆6-desaturase substrates, ALA LC-PUFAs are synthesized from dietary precursors by and LIN (Table 1). The residual levels of DGLA, AA and EPA sequential double bond insertion (desaturation) and elongation.
detected in these animals may reflect the activity of another ∆6-desaturase catalyses desaturation at the ∆6 position of desaturase or a dietary contribution to the animal. These α-linolenic acid (ALA) and LIN (Los and Murata, 1998; observations suggest that the defects observed in fat-3 animals Nakamura et al., 2001) (Fig. 1A). The C. elegans gene fat-3, are caused by depletion of LC-PUFAs.
also called W08D2.4 (Fig. 1B), is a ∆6-desaturase. This proteincontains three histidine clusters distinctive of desaturases(Los and Murata, 1998), harbors a cytochrome-b The behavioral defects observed in fat-3 mutants are observed in other ∆6-desaturases (Napier et al., 1999), is caused by LC-PUFA depletion homologous to human and plant ∆6-desaturases (Fig. 1C), and fat-3 mutants display a variety of phenotypes that include both has ∆6-desaturase enzymatic activity on C18 fatty acids behavioral and non-behavioral defects (Watts et al., 2003).
(Napier et al., 1998). Since we were interested in clarifying the role of LC-PUFAs fat-3(wa22) results in a serine to phenylalanine substitution in the nervous system, we focused our analysis on the two most at position 186 (Fig. 1B,C) (Watts and Browse, 2002); this prominent fat-3 behavioral phenotypes, namely, the deficits in serine is not conserved in human or plant desaturases and it is movement and egg laying. fat-3 mutants show deficiencies in not clear that this mutation is a null allele. To determine the both forward and backward movements, and are particularly null phenotype we generated two fat-3 deletion mutations unable to respond to head-touch, that is, when touched gently using PCR to screen chemically mutagenized C. elegans near the head, wild-type animals respond by reversing direction libraries with fat-3 specific primers (Jansen et al., 1997). Both and proceeding rapidly away from the stimulus. However, of these mutations are likely to strongly reduce or completely when fat-3 mutants are stimulated in the same manner, they eliminate fat-3 activity: fat-3(qa1811) lacks 1,324 bp that stop or proceed backwards only very slowly. In addition, while include the three histidine clusters necessary for desaturase fat-3 mutants do lay eggs, they frequently retain eggs in the activity (Los and Murata, 1998). fat-3(lg8101) lacks 2,076 bp uterus abnormally as they age. Some of these eggs hatch before that include the start codon, an invariant heme-binding site being laid, causing the mother to eventually be consumed by indispensable for enzymatic activity (Sayanova et al., 1999) hatched embryos. This phenotype is both qualitatively and and a large portion of the predicted promoter (Fig. 1B) and quantitatively similar to the egg-laying defects caused by results in no detectable fat-3 mRNA transcripts (Fig. 2B,C).
mutations in the egl-1 gene, which cause a specific fat-3(lg8101) homozygous animals develop very slowly and developmental disruption of the hermaphrodite specific their phenotype is more severe than that of fat-3(lg8101)/fat- neurons (HSNs) (Desai and Horvitz, 1989; Trent et al., 1983), a pair of serotonergic neurons innervating the egg-laying homozygotes (data not shown). Therefore, it is likely that fat- muscles. In particular, 29% of fat-3(lg8101)/fat-3(qa1811) 3(lg8101) is a molecular null while fat-3(qa1811) and fat- (n=24), 39% of fat-3(lg8101) (n=23) and 47% of egl-1(n487) 3(wa22) are severe loss-of-function mutations. The phenotype (n=34) animals were consumed by hatched embryos late in of fat-3(qa1811) homozygous animals could not be directly adult life. Based on these observations and on the fact that these assessed because the qa1811 allele is tightly associated with behavioral defects were rescued by selective expression of fat- an independent lethal mutation. 3 in the nervous system (see below) we hypothesized that fat- Table 1. LC-PUFA composition
Percentage (w/w) of total fatty acids LC-PUFA composition of total phospholipids isolated from wild-type and fat-3(lg8101) C. elegans grown in normal growth medium or medium containing DHA. Transgenic fat-3(lg8101)Exfat-3(+) worms carry cosmid C24G5, which contains coding region and regulatory sequences of fat-3. Data are themean±s.e.m. of 3-4 independent measurements. ND, not detected (<0.2%).
LC-PUFAs and neurotransmission Fig. 3. Rescue of the behavioral
defects associated with loss of fat-3 activity. (A) ExogenousAA and DHA, but not LIN, rescue the movement defects of mutants. Animals were exposed to fatty acids from egg to adult.
*P<0.0001 versus wild-type Exfat-3(+) Exfat-3(+) Exfat-3(+) animals. (B) fat-3 expressedunder the control of theneuronal promoter unc-119 (Exfat-3(+) neuron) but not under the control of the muscularpromoter myo-3 (Exfat-3(+) muscle), completely rescues the egg-laying defect of fat-3(lg8101)/fat-3(qa1811) animals. Egl+ indicateshermaphrodites that were not consumed byembryos by the fourth day after reachingadulthood. (C) fat-3 expressed under the control of the neuronal promoter but not under the control of the muscular promoter or theintestinal 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 Exfat-3(+) Exfat-3(+) in A and C are plotted as mean ± s.e.m.
3 activity might be required for normal neuronal development involved in a variety of biological functions. However, or function.
eicosanoids do not appear to be produced or used in C. To determine whether the behavioral defects observed in fat- elegans. Thus, AA exerts its function via a distinct as yet 3 mutants are caused by deficits in LC-PUFA levels, we asked uncharacterized lipid pathway. Conversion of AA into DHA whether exogenous LC-PUFAs rescue the movement defects precursors by a C. elegans enzyme has been previously associated with loss of fat-3 function. We grew fat- observed (Spychalla et al., 1997). Therefore, the active LC- 3(lg8101)/fat-3(qa1811) hermaphrodites from egg to adult in PUFA species are DHA or its related metabolic products and the presence of AA, whose synthesis is dependent on ∆6- precursors, which could be generated catabolically. desaturase (Fig. 1A). AA fully rescued the lack of coordinationof fat-3 mutants (Fig. 3A). Similarly, exogenous application ofDHA, another LC-PUFA product of ∆6-desaturase activity, fat-3 expressed in neurons rescues the behavioral was also sufficient to rescue the locomotion defects associated defects of fat-3 mutants with fat-3 mutant animals. As a negative control we used LIN The FAT-3 protein is expressed in the intestine, body-wall (Fig. 1A). Since fat-3 mutants have inactive ∆6-desaturase and muscles, pharynx and several neurons (Watts et al., 2003). To can convert LIN to LC-PUFAs only very poorly, if at all (Table determine where fat-3 expression is required, we generated 1), LIN administration is not expected to rescue the fat-3 constructs that drive gene expression in specific tissues and phenotype. Indeed, exogenous LIN did not have any effect on tested their ability to rescue the behavioral defects associated the motility of fat-3 mutants (Fig. 3A).
with loss of fat-3 activity. As expected, a construct comprisingthe endogenous promoter, the coding sequence and the 3′untranslated region of fat-3 (Fig. 1B) restored normal LC- DHA or its metabolic products mediate ∆6-desaturase PUFA levels (Table 1) and rescued the egg-laying impairment and the reduced motility of fat-3(lg8101) homozygous animals To test whether ∆6-desaturase function is mediated by DHA or (Fig. 3B,C). Since the uncoordinated and egg-laying AA, we measured the levels of each lipid in fat-3 mutant phenotypes of fat-3 mutants are suggestive of neuronal defects, animals rescued with DHA. We observed that exogenous we investigated whether fat-3 selectively expressed in the application of DHA did not increase AA levels in fat-3 mutant nervous system could rescue the impaired motility and the egg- animals (Table 1), suggesting that AA by itself is not laying defect of fat-3 mutant animals. We made a chimeric responsible for the observed phenotypic rescue. In other construct, Punc-119::fat-3, in which the fat-3 coding sequence systems, AA is a precursor of eicosanoids, bioactive lipids is placed under the control of a promoter driving gene Journal of Cell Science 116 (24) expression in the entire nervous system (Maduro and Pilgrim, serotonergic neurons (Fig. 4B). Moreover, when we visualized 1995). Transgenic fat-3 mutants bearing this construct the entire nervous system using a pan-neuronal GFP marker recovered normal egg-laying capability (Fig. 3B) and almost (Punc-119::gfp) we were also unable to detect any normal motility (Fig. 3C). Conversely, expression of the fat-3 morphological defect in fat-3 mutant animals (data not shown).
coding sequence under the control of the muscle-specific myo- Finally, when we analyzed the ultrastructure of the nervous 3 promoter did not rescue the egg-laying defect (Fig. 3B) and system, we found that the general arrangement, structure and only minimally rescued the motility defect (Fig. 3C) associated positioning of neurons as well as synaptic morphology are with the fat-3 mutation. We also tested whether intestine- normal in fat-3 mutants (data not shown). These results suggest specific expression was sufficient to rescue the fat-3 mutant that the neuronal impairments associated with loss of fat-3 phenotype. We placed fat-3 under the control of the intestine- activity are likely to be functional rather than developmental.
specific promoter elt-2 (Fukushige et al., 1998), which is If the behavioral defects observed in fat-3 mutants are indeed expressed in the intestine and its precursors cells from early functional it should be possible to rescue these phenotypes by embryonic stages. This chimeric construct could not rescue the providing adult animals, in which the nervous system is fully uncoordinated phenotype of fat-3 mutants (Fig. 3C). Since we developed, with the metabolic products of fat-3 activity. We restored normal egg-laying and near-normal motility only therefore exposed adult fat-3 mutants to exogenous LC-PUFAs when we expressed fat-3 in the nervous system, it is likely that and analyzed movement. Adult homozygotes exposed to AA fat-3 activity is required in neurons for their normal function.
for 24 hours recovered almost normal motility and were However, we cannot exclude the possibility that fat-3 may have completely rescued after 48 hours (Fig. 4C). These results are additional normal functions in other cells.
consistent with the notion that LC-PUFAs are required forneuronal function rather than development. Loss of fat-3 activity causes functional, notdevelopmental defects fat-3 mutants display defects in neurotransmitter release The behavioral deficits observed in fat-3 mutants could reflect To better assess these defects in neuronal function, we developmental defects in the nervous system. As a first measured the transmission efficiency of both serotonergic approach to this issue, we analyzed the neuronal morphology NMJs involved in egg-laying and cholinergic NMJs involved of fat-3 mutants at the light microscope level. We generated in body wall muscle contraction and movement. Egg-laying is animals in which specific subsets of neurons were fluorescently mainly controlled by the serotonergic HSN motor neurons labeled with green fluorescent protein (GFP) and could not (Trent et al., 1983). Both exogenous serotonin and fluoxetine detect any morphological defect in HSN structure or in the induce wild-type animals to lay eggs (Desai and Horvitz, 1989; attachments of the HSN to its egg-laying muscle target (Fig.
Trent et al., 1983; Weinshenker et al., 1995). Fluoxetine 4A). We also could not detect morphological alterations in potentiates the effect of endogenous serotonin by selectivelyinhibiting its presynaptic re-uptake (Hyttel, 1994). Animalsmissing the HSN neurons are sensitive to serotonin andinsensitive to fluoxetine (Trent et al., 1983; Weinshenker et al.,1995). Animals with defective egg-laying muscles areinsensitive to both drugs. The egg-laying response of fat-3 mutant animals exposed to serotonin wasnormal (Fig. 5A). For example, at 5mg/ml, the dose eliciting the highestresponse, wild-type animals laid16.42±1.17 eggs and fat-3(lg8101)/fat-3(qa1811) mutants laid 13.00±1.04 eggs.
This demonstrates that muscle functionin these animals is not disrupted.
However, fat-3 animals responded very poorly to fluoxetine (Fig. 5B). Wild-typeanimals laid an increasing number ofeggs when exposed to higher doses offluoxetine. They reached a peak of11.88±0.97 eggs laid at 0.5 mg/mlfluoxetine. However, fat-3(lg8101)/fat-3(qa1811) animals laid an almost constant number of eggs whatever the Fig. 4. fat-3 mutant animals display functional and not morphological neuronal defects.
dose of fluoxetine (from 3.29±0.64 eggs (A,B) Serotonergic neurons visualized with GFP under the control of a tryptophane at 0.1 mg/ml fluoxetine to 5.00±0.94 hydoxylase promoter (tph-1), which is expressed in serotonergic neurons (Sze et al., 2000).
eggs at 1 mg/ml). Similarly, fat-3 (A) An HSN motor neuron in proximity of the vulva. Bars, 5 µm. (B) Serotonergic neurons mutants were only inefficiently in the head. Images shown are projections of confocal xy sections. Bars, 10 µm.
stimulated by imipramine, another (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 potentiator of endogenous serotonin animals. Data is plotted as mean ± s.e.m. (Fig. 5C). These observations suggest LC-PUFAs and neurotransmission Levamisole after AA fat-3Exfat-3(+) neuron fat-3Exfat-3(+) muscle 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. (G-
H) 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.
that fat-3 mutant animals release abnormally low levels of paralysis. However, mutants with decreased ACh release are serotonin into the synaptic cleft.
resistant to aldicarb (Jorgensen et al., 1995; Nonet et al., 1993; Cholinergic NMJ function was probed using the agonist Nonet et al., 1998). Consistent with reduced ACh release into levamisole, which binds to muscular ACh receptors, and the the synaptic cleft, fat-3 mutant animals were significantly less ACh esterase inhibitor aldicarb, which enhances the effect of responsive to aldicarb than wild-type animals. For example, endogenously released ACh. Treatment of wild-type animals exposure to 1 mM aldicarb for 60 minutes resulted in with either drug results in muscle hypercontraction and 90.5±3.3% of wild-type animals and only 54.5±7.4% of fat- Journal of Cell Science 116 (24) 3(lg8101)/fat-3(qa1811) mutant animals being paralyzed (Fig.
defects in ACh biosynthesis are unlikely to account for the 5D). Both fat-3(lg8101)/fat-3(qa1811) and fat-3(wa22) movement defects displayed by fat-3 mutants. Taken together, animals responded to aldicarb in similar fashion (Fig. 5D,E).
these results suggest that the fat-3 lesion causes inefficient ACh This reduced response to aldicarb is comparable in severity to release from cholinergic neurons. that observed in the "weak" synaptic transmission mutant rab- To test directly whether fat-3 mutants have decreases in ACh 3 (Fig. 5D) (Nonet et al., 1997). To verify that this response is release, we measured both evoked excitatory postsynaptic associated with loss of fat-3 activity, we exposed fat-3 mutants, currents and endogenous miniature excitatory postsynaptic expressing fat-3 in neurons, to aldicarb. These, but not animals currents from voltage-clamped muscles at the C. elegans NMJ.
expressing fat-3 in muscles, recovered almost normal fat-3(wa22) mutants displayed a decrease in evoked amplitude sensitivity to aldicarb (Fig. 5F). This decreased response is not (417±41 pA) compared to the wild type (830±144 pA; Fig.
due to ACh receptor impairment because fat-3 mutants were 6A,B). In addition, the frequency of miniature postsynaptic hypersensitive to both levamisole (Fig. 5G,H) and ACh (data currents, caused by the fusion of one or a few synaptic vesicles, not shown) as compared to wild-type animals. For example, was reduced in fat-3(wa22) mutants (10±2 fusions/second) exposure to levamisole for 40 minutes resulted in paralysis of compared to the wild type (15±1 fusions/second; Fig. 6C,D).
47.5±7.1% of wild-type and 94.7±1.5% of fat-3(lg8101)/fat- The amplitude of the miniature currents was not significantly 3(qa1811) mutant animals (Fig. 5G). Normal response to different in fat-3(wa22) (41±3 pA) compared to wild type levamisole was restored in fat-3 mutants grown in the presence (37±2 pA; Fig. 6E). These data suggest that in fat-3 mutants of AA (Fig. 5I), confirming that the hypersensitivity to synaptic vesicles are correctly filled with neurotransmitter and levamisole is dependent upon LC-PUFA levels. Such the postsynaptic receptor field is normal. However, synaptic hypersensitivity may reflect an adaptive response to decreased vesicles are either reduced in number or in release probability ACh availability and is observed in other mutant backgrounds at fat-3 mutant synapses. defective in cholinergic transmission (Nonet et al., 1993).
Lower levels of ACh at the synaptic cleft of fat-3 mutants couldarise from either inefficient release of ACh or decreased ACh Presynaptic sites are depleted of synaptic vesicles in fat- biosynthesis. To discriminate between these possibilities we quantitatively assessed the level of ACh produced in fat-3 Decrease in neurotransmitter release in fat-3 mutants could be mutants. Since total ACh levels do not diminish in fat-3 caused by a number of possible mechanisms. For example, mutants [65.1±10.8 fmoles per µg of protein in fat-3 mutants synaptic vesicles could be assembled and localized normally to (n=20) versus 48.7±4.0 in wild-type (n=20), P=0.3040], NMJs but undergo exocytosis only inefficiently. In this view, the number of vesicles at NMJs is expected toincrease. Alternatively, fat-3 mutants couldlocalize fewer synaptic vesicles at the nerveterminal or could be defective in endocytosis.
In this view, the number of synaptic vesicles isexpected to decrease (Harris et al., 2000;Jorgensen et al., 1995). To distinguish betweenthese possibilities, we examined the synapticultrastructure of fat-3 mutants and determinedthe distribution of synaptic vesicles at NMJs.
We found that while synapses in wild-typeanimals had clusters of vesicles in theproximity of the active zone (Fig. 7A),synapses in fat-3 mutants were depleted ofvesicles (Fig. 7B). The number of synapticvesicles per synaptic terminal (within 300 nmof the active zone) was 2.7-fold smaller infat-3 mutants than in wild-type animals(10.32±0.96 versus 28.07±3.22, P<0.0001;Fig. 7C). In addition, both the number ofsynaptic vesicles docked to the presynapticmembrane (0.72±0.15 versus 2.00±0.36,P<0.001) and the number of those in itsvicinity (within 100 nm; 3.40±0.30 versus6.88±0.87, P=0.0001) were significantly Fig. 6. fat-3 mutants have reduced evoked amplitude and reduced rates of spontaneous
decreased in fat-3 mutant animals (Fig. 7C).
fusion but normal quantal size. (A) Representative evoked responses from wild-type Moreover, our ultrastructural analysis did not and fat-3 animals. (B) The mean amplitude of the evoked responses is reduced in fat-3 reveal accumulation of vesicles in neuronal (n=6) compared to wild-type (n=7) animals (P<0.03). (C) Representative traces of cell bodies in fat-3 mutants (data not shown).
spontaneous fusion events in wild-type and fat-3 mutants. (D-E) The mean frequency of These results indicate that LC-PUFAs are 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 required to maintain a normal pool of synaptic as mean ± s.e.m.
vesicles at NMJs.

LC-PUFAs and neurotransmission PUFAs causes functional rather than developmental defects inthe nervous system. These defects can be rescued by selectiveFAT-3 expression in the nervous system or by dietarysupplementation of LC-PUFAs to adult worms. Usingpharmacological techniques, we identify neurotransmitterrelease defects in both cholinergic and serotonergic neuronsof fat-3 mutants. Electrophysiological studies suggest thata decrease in neurotransmitter release rather than neurotransmitter loading is responsible for the neuronal defectsof fat-3 mutants. Finally, ultrastructural analysis of synapticterminals demonstrates that synapses are depleted of synapticvesicles. We conclude that LC-PUFA depletion results ininsufficient neurotransmitter release.
LC-PUFAs and neurotransmitter releaseThe locomotion and egg-laying defects observed in fat-3mutants are neuronal in nature and could be rescued byexpressing fat-3 in the nervous system but not by expressing itin muscles or intestine. This suggests that LC-PUFAs areproduced and act in neurons. The defects associated with lossof fat-3 activity were also rescued by providing exogenous LC-PUFAs. Free fatty acids are known to diffuse across biologicalmembranes from intercellular spaces (Frohnert and Bernlohr,2000). The observation that fat-3 expressed in intestine andmuscles did not result in rescue, suggests that fat-3 expressionin these tissues does not provide enough LC-PUFAs inintercellular spaces to support normal neuronal function.
Therefore it is likely that fat-3 activity is required in neuronsfor their normal function.
Although we cannot rule out that fat-3 mutants might have subtle neuronal developmental defects, several lines ofevidence suggest that the neuronal developmental defects donot contribute significantly to the behavioral phenotypesobserved in C. elegans depleted of LC-PUFAs. First, in fat-3mutants we could not observe gross morphological defectsin neurons visualized with GFP markers. Second, at theultrastructural level we found that both the general organizationof the nervous system and neuronal specializations such as theNMJs appeared normal in animals without fat-3 activity. Third,the locomotion defects of adult fat-3 mutants were rescuedacutely by providing exogenous LC-PUFAs. Therefore, our Fig. 7. Synapses are partially depleted of synaptic vesicles in fat-3
results indicate that LC-PUFAs are important for neuronal mutant animals. Electron micrographs of wild-type (A) and fat- function rather than neuronal development. 3(lg8101) (B) synapses fixed by fast-freezing. (A) Clusters of Two lines of evidence indicate the defects in neuronal vesicles (ves and arrowheads) in ventral nerve cord neurons arelocalized close or docked to the active zone (white arrows).
function observed in fat-3 mutants are due to decreases in (B) Ventral ganglion synapses are depleted of most vesicles close to neurotransmitter release. First, fat-3 mutant animals displayed the active zone (black arrows). A few vesicles lie at a distance (ves presynaptic defects in neuronal function at both cholinergic and arrowheads). (C) Quantification of synaptic vesicles in fat- and serotonergic neurons in pharmacological assays. Second, 3(lg8101)/fat-3(qa1811) mutant animals. The average fraction of electrophysiological recordings demonstrate that fat-3 mutants synaptic vesicles per section at given distances in fat-3 mutants release abnormally low levels of neurotransmitter. This compared to wild-type animals (100%). Vesicles were considered impaired neurotransmission is probably caused by decreases in docked when they were touching the active zone. Total number of synaptic vesicle number rather than neurotransmitter loading synapses scored: 50, fat-3; 42, wild-type. *P<0.001; **P<0.0001.
or release probability. In fat-3 mutants an abnormally low Scale bar: 0.5 µm.
quantity of neurotransmitter is released upon stimulation. Thisreduction in neurotransmitter release does not result from decreases in loading of neurotransmitter into synaptic vesicles The complex lipids LC-PUFAs are highly enriched and as fat-3 mutants have similar amplitudes of mini currents as precisely regulated in neurons. By inactivating the gene fat-3, wild-type animals. Thus, a decrease in the number of synaptic we have generated animals depleted of LC-PUFAs and vesicles undergoing exocytosis is probably responsible for the analyzed their neuronal deficits. We show that depletion of LC- impaired neurotransmission. This reduced number of vesicles Journal of Cell Science 116 (24) undergoing exocytosis is probably due to a decrease in 1988). Conversely, Weisinger and collegues reported a available vesicles rather than defects in release since our complete functional recovery (Weisinger et al., 1999). Here we ultrastructural analysis of fat-3 mutant synapses showed have demonstrated that LC-PUFA depletion leads to functional reductions in both total and morphologically docked synaptic but not developmental defects in C. elegans neurons. Our vesicles. The decrease in available vesicles being responsible findings are consistent with recent reports showing that for fat-3 mutant defects is also supported by the correlation supplementation of certain LC-PUFAs can improve between the extent of synaptic vesicle depletion and the neurological conditions associated with LC-PUFA mis- decrease in synaptic vesicle fusion. fat-3 mutants carry regulation (Martinez et al., 2000).
approximately 40% of the synaptic vesicles of wild-type These experiments provide a foundation for a genetic animals. Similarly, the evoked responses in fat-3 mutants is analysis of LC-PUFA function. We anticipate that genetic approximately 50% that of wild-type animals. We conclude screens aimed at identifying proteins regulated by LC-PUFAs that the abnormally low number of synaptic vesicles present at in C. elegans will uncover the cellular targets and define the NMJs of fat-3 mutants is insufficient to support normal molecular mechanisms underlying the roles of LC-PUFAs in neurotransmission. The decrease in synaptic vesicle number at synaptic terminals could be due to defects in transport, endocytosis, or This work was supported by the Wellcome Trust (G.M.L.), by synaptic vesicle biogenesis. fat-3 mutants are unlikely to have Cancer Research UK (G.M.L. and G.S.), by The Natural Sciences and significant defects in synaptic vesicle transport, since in Engineering Research Council of Canada (M.T.C.) and by the NIH,grant RR12596 (D.H.H.). We thank Tom R. Clandinin, Neil Hopper, our ultrastructural analysis we did not observe vesicle Enzo Lalli, Raffi Aroian, Cori Bargmann, Amanda Kahn-Kirby, Julian accumulation in the proximity of the Golgi apparatus or in the Lewis, Caetano Reis e Sousa, Nirao Shah, Jennifer Watts, Axel rest of the neuronal cell body (data not shown). Therefore it is Behrens and Ralf Adams for critically reading the manuscript, Tom likely that fat-3 mutants are defective in synaptic vesicle R. Clandinin for advice and discussions, Yeow Goh for fatty acid biogenesis and/or synaptic vesicle recycling. Further work is analysis, Ji Ying Sze and Gary Ruvkun for strains, John Sulston for needed to clarify in detail the mechanism(s) responsible for the cosmids, Andy Fire for plasmids, Giovanna Lalli for help with synaptic vesicle depletion observed in fat-3 mutant animals.
confocal microscopy, Graham Clark for help with PCR screening, andKen Nguyen, Toni Long and Nancy Hall for help with EM analysis.
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