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: [email protected]
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
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
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
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-
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
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
. 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
mRNA was used as positive control (data not shown). Primers
used (Fig. 2A): a, 5′-CTCGAATTTTAAACAACTTCGCCGC-3′; b,5′-GGCAGCTTTAGCTTGAATGTGCTC-3′; c, 5′-CAGAAGCTTC-
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 Kpn
I restriction sites were used to PCR amplify the entire fat-3
sequence. This Kpn
I fragment and a Hin
I fragment from
(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-
, a Hin
HI fragment including the myo-3(+)
and the Kpn
I PCR fragment including the entire fat-3
sequence were cloned into pPD49.26. This Kpn
and a Pst
I 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)
pMH86 (20 ng/µl). The presence of fat-3
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
. A 4.7 kb genomic
fragment including 977 bp 5′ and 862 bp 3′ of the 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)
deletions and their breakpoints are shown. T
indicates an A to T substitution. Dp indicates a 17 bp duplication
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)
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
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)
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
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)
serotonin or fluoxetine (Sigma) dissolved at the indicated doses in M9
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
ACh and fatty acid quantificationACh was quantified in fat-3(lg8101)dpy-20(e1282)/unc-24(e138)fat-3(qa1811)dpy-20(e1282)
animals as describedpreviously (Nonet et al., 1993).
The genotypes of animals used for fatty acid quantification were as
, 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
cyanoacrylic glue and a lateral incision was made to expose the ventral
(A) Schematic of the fat-3(lg8101)
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)
mg/ml. The muscle was then voltage clamped using the whole cell
mutant animals. (B) RT-PCR of total mRNA from fat-3(lg8101)
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-
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
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)
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
Nakamura et al., 2001) (Fig. 1A). The C. elegans
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
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
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
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
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
mutants are stimulated in the same manner, they
lacks 1,324 bp that
stop or proceed backwards only very slowly. In addition, while
include the three histidine clusters necessary for desaturase
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
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)
is a molecular null while fat-3(qa1811)
=24), 39% of fat-3(lg8101)
=23) and 47% of egl-1(n487)
are severe loss-of-function mutations. The phenotype
=34) animals were consumed by hatched embryos late in
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.
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
Rescue of the behavioral
defects associated with loss of
activity. (A) ExogenousAA and DHA, but not LIN,
rescue the movement defects of
mutants. Animals were exposed
to fatty acids from egg to adult.
<0.0001 versus wild-type
Exfat-3(+) Exfat-3(+) Exfat-3(+)
animals. (B) fat-3
expressedunder the control of theneuronal promoter unc-119
but not under the control of the muscularpromoter myo-3
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
intestine),almost completely rescues the movement
defects of fat-3 (lg8101)
<0.0001 versus fat-3Exfat-3(+)
in A and C are plotted as mean ± s.e.m.
activity might be required for normal neuronal development
involved in a variety of biological functions. However,
eicosanoids do not appear to be produced or used in C.
To determine whether the behavioral defects observed in fat-
. Thus, AA exerts its function via a distinct as yet
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-
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
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)
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
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
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
, in which the fat-3
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
) 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
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
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
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
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
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
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.
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
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
<0.005. (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)
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
<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.
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)
mutant animals being paralyzed (Fig.
defects in ACh biosynthesis are unlikely to account for the
5D). Both fat-3(lg8101)/fat-3(qa1811)
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
(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
currents and endogenous miniature excitatory postsynaptic
in neurons, to aldicarb. These, but not animals
currents from voltage-clamped muscles at the C. elegans
in muscles, recovered almost normal
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
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
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
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
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
synaptic vesicles could be assembled and localized normally to
=20) versus 48.7±4.0 in wild-type (n
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
animals. (B) The mean amplitude of the evoked responses is reduced in fat-3
reveal accumulation of vesicles in neuronal
=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
=13) compared to wild-type (n
<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
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.
Some nematode strains were supplied by the Caenorhabditis Genetics
Link with human brain function
Center, which is supported by the NIH and the University ofMinnesota.
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ORIGINAL INVESTIGATION HEALTH CARE REFORM An Empirical Model to Estimate the Potential Impactof Medication Safety Alerts on Patient Safety, HealthCare Utilization, and Cost in Ambulatory Care Saul N. Weingart, MD, PhD; Brett Simchowitz, BA; Harper Padolsky, MD; Thomas Isaac, MD, MBA, MPH;Andrew C. Seger, PharmD; Michael Massagli, PhD; Roger B. Davis, ScD; Joel S. Weissman, PhD
Gabinete de Alcaldía Alkatetzako Kabinetea REGLAMENTO ORGÁNICO DEL AYUNTAMIENTO DE PAMPLONA. Pza Consistorial s/n, 3º 31001 Pamplona www.pamplona.es Udaletxe Plaza z/g, 3º 31001 Iruña Gabinete de Alcaldía Alkatetzako Kabinetea El Pleno del excelentísimo Ayuntamiento de Pamplona con fecha 27 de febrero de 1998, aprobó el Reglamento Orgánico del Ayuntamiento de Pamplona.