Am J Physiol Cell Physiol
280: C1616–C1622, 2001.
special communication
Identified neurons in C. elegans coexpress vesicular
transporters for acetylcholine and monoamines
Received 2 November 2000; accepted in final form 16 January 2001
Duerr, Janet S., Jennifer Gaskin, and James B.
Rand. Identified neurons in C. elegans coexpress vesicular
transporters for acetylcholine and monoamines. Am J
Physiol Cell Physiol 280: C1616–C1622, 2001.—We have
identified four neurons (VC4, VC5, HSNL, HSNR) in Caenorhabditis elegans adult hermaphrodites that express both the
vesicular acetylcholine transporter and the vesicular monoamine transporter. All four of these cells are motor neurons
that innervate the egg-laying muscles of the vulva. In addition, they all express choline acetyltransferase, the synthetic
enzyme for acetylcholine. The distributions of the vesicular
acetylcholine transporter and the vesicular monoamine
transporter are not identical within the individual cells. In
mutants deficient for either of these transporters, there is no
apparent compensatory change in the expression of the remaining transporter. This is the first report of neurons that
express two different vesicular neurotransmitter transporters in vivo.
vesicular acetylcholine transporter; vesicular monoamine
transporter; Caenorhabditis elegans; cotransmission; nematode
single VMAT gene (7), two related VMAT proteins (and
genes) have been identified in mammals: VMAT1 is
often found in neuroendocrine cells, and VMAT2 is
primarily neuronal (10, 12, 24, 44). Recombinant
VMATs have been shown to mediate the transport in
vitro of dopamine, norepinephrine, epinephrine, serotonin, and histamine (VMAT2 only) (7, 11, 12). Antibodies to the vesicular transporters have been used to
study vesicular localization and maturation (25, 30, 44,
45) as well as cell identity (28, 36, 37). The particular
neurotransmitter used by VMAT-positive cells must be
determined using additional specific markers.
We now report that several identified neurons of the
nematode C. elegans contain both VAChT and VMAT;
some of these cells contain serotonin, suggesting that
they may be both cholinergic and serotonergic. This
represents the first reported example of two different
vesicular transporters being expressed in the same
neurons in vivo.
MATERIALS AND METHODS
studies of vertebrate
neuronal and neuroendocrine tissues have shown four
distinct vesicular transmitter transport activities: a
monoamine transporter (biogenic amines), an acetylcholine transporter, a ␥-aminobutyric acid (GABA)/
glycine transporter, and a glutamate transporter (reviewed in Ref. 9). The vesicular acetylcholine
transporters (VAChTs) and the vesicular monoamine
transporters (VMATs) have been shown to be members
of the same subfamily of 12-transmembrane domain
transport proteins (2, 10, 13, 24, 42). VAChT is expressed in the same cells as choline acetyltransferase
(ChAT, the enzyme for acetylcholine synthesis) and
has been shown to transport acetylcholine in vitro (13).
Although Caenorhabditis elegans appears to have a
Nematode strains. Wild-type C. elegans (N2 Bristol) was
grown on solid medium (3, 19). The bas-1(ad446), cat1(e1111), cha-1(p1152), egl-1(n487), lin-39(n709), tph1(mg280), and unc-17(e245) mutations have been described
(3, 4, 17, 27, 33, 35, 39).
Immunocytochemistry. Goat antiserum to C. elegans
VMAT has been described (7). To stain the VAChT/UNC-17
protein, we used a mouse monoclonal antibody (1403), which
stained similarly to the previously described rabbit serum
(R383) (2). Purchased antibodies included mouse anti-dopamine (Biodesign International, Kennebunk, ME), rabbit
anti-histamine, rabbit anti-tyramine (both from Sigma, St.
Louis), rabbit anti-octopamine (Biogenesis, Sandown, NH),
and rabbit anti-serotonin (H. Steinbusch, Maastricht University, The Netherlands).
For staining with antibodies to the vesicular transporters
or choline acetyltransferase, mixed populations of nematodes
Address for reprint requests and other correspondence: J. S. Duerr, Program in Molecular and Cell Biology, Oklahoma Medical
Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104
(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
BIOCHEMICAL AND PHARMACOLOGICAL
C1616
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajpcell.org
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JANET S. DUERR, JENNIFER GASKIN, AND JAMES B. RAND
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation,
Oklahoma City, Oklahoma 73104
COEXPRESSION OF VACHT AND VMAT IN C. ELEGANS
RESULTS
Colocalization of VAChT and VMAT in specific neurons. We cloned the VAChT gene (unc-17) and the
VMAT gene (cat-1) in C. elegans and prepared specific
antibodies (2, 7). VAChT and VMAT are associated
with synaptic vesicles; generally, there is little immunoreactivity in cell somas. As expected, the cellular
expression patterns of the two transporters are quite
different. However, we have found six neurons that are
immunopositive for both proteins: a bilaterally symmetrical pair of cells in the head and four cells in the
body (Fig. 1). We have identified the four neurons in
the body as VC4, VC5, HSNL, and HSNR.
The class of VC neurons consists of six motor neurons with cell bodies in the ventral nerve cord (47).
Although the six VC cells are derived from parallel
postembryonic lineages (40), the mature cells show
considerable morphological diversity (47, 48). The VC4
and VC5 neurons flank the developing vulva and send
highly arborized processes to innervate the vulval muscles; the other VCs have less extensive output to these
muscles. In addition, all of the VC cells have sparse
output to the ventral body muscles and to other motor
neurons (47) and contain Phe-Met-Arg-Phe-NH2
(FMRF-amide) immunoreactivity (38). VC4/5 contain
weak and variable serotonin immunoreactivity and
induced fluorescence (7), but do not contain tryptophan
hydroxylase, the synthetic enzyme for serotonin (41).
We found that VC4/5 contain significant punctate
VAChT immunoreactivity and VMAT immunoreactivity (Fig. 1).
The HSN neurons are bilaterally symmetrical with
posterior lateral cell bodies. They receive input from
several interneurons and have extensive output to the
vulval muscles. In addition, the HSNs have an anterior
process that travels up the ventral nerve cord and into
the nerve ring, with minor synapses onto motor neurons and interneurons (47, 48). The HSNs are necessary for normal egg laying; ablation leads to significant
defects in egg laying (6, 17). The HSNs contain strong
serotonin immunoreactivity (6) and FMRF-amide immunoreactivity (38).
The HSN somas occasionally stained faintly for both
of the vesicular transporters, whereas the major region
of innervation by the HSNs, the vulva, stained
brightly. However, this output region is shared with
the output regions of VC4/5. Therefore, we examined
staining in unc-104 mutants, which are deficient for a
kinesin-related protein required for the transport of
synaptic vesicles from neuronal cell bodies to synapses
(16, 32). In unc-104 mutants, synaptic vesicles are
found in large clusters in cell bodies (16), and a number
of synaptic vesicle-associated antigens, such as synaptotagmin, VAChT, and VMAT, are also mislocalized to
neuronal cell bodies (2, 7, 31). In unc-104 mutants,
somas of the HSNs (as well as VC4/5) stained for both
VMAT and VAChT (Fig. 1).
We used lineage mutants to eliminate specific cells.
In egl-1 mutants, the HSNs undergo programmed cell
death (17). In lin-39 mutants, there are variable defects in the lineages that give rise to the VC cells so
that some individuals are missing VC4 and VC5 (4). In
both mutants, the output fields of the remaining cells
are positive for both VMAT and VAChT (n ⬎ 20, Fig.
2). Thus in all four of these neurons, both types of
vesicular transporters are expressed and are present in
the synaptic output zones.
Differences between VC4/5 and HSNs. VMAT immunoreactivity is more intense in the HSNs than in VC4
and VC5, whereas VAChT (and ChAT) immunoreactivity is more intense in VC4 and VC5 than in the HSNs.
Overall, the HSNs appear to be more aminergic and
less cholinergic than VC4 and VC5.
The HSNs contain the synthetic enzyme for serotonin, tryptophan hydroxylase (41), as well as high levels
of serotonin immunoreactivity and serotonin-like induced fluorescence. Therefore, they are likely to use
serotonin as their aminergic neurotransmitter. In contrast, VC4/5 do not express detectable levels of tryptophan hydroxylase (41), and they contain very low and
variable levels of serotonin immunoreactivity and serotonin-like induced fluorescence (Fig. 3). Antibodies to
dopamine, octopamine, tyramine, and histamine (all of
which work only poorly in our preparation) did not give
a detectable signal in VC4/5. VC4/5 may contain a low
level of one of these amines or may use some other
aminergic neurotransmitter.
The serotonin detected in VC4/5 could arise from
uptake of serotonin synthesized by other cells. For
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on June 16, 2017
were prepared by freeze cracking (1, 7). Fixation consisted of
2 min in methanol followed by 4 min in acetone. For staining
with anti-neurotransmitter antibodies, nematodes were prepared with two different methods. In one method, the freezecrack protocol was followed except that fixation consisted of
12–24 h in 4% formaldehyde in 0.1 M phosphate buffer. A
second method was based on the protocol of Garriga et al.
(15). Mixed populations of nematodes were collected and
placed in tubes containing 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2. Tubes were placed in a dry-ice ethanol
bath for 1 min, then thawed, refrozen, thawed, and placed on
a shaker at 4°C for 24 h. After rinsing in phosphate-buffered
saline (PBS), fixed nematodes were incubated overnight with
shaking at 37°C in a solution containing 2-mercaptoethanol
(5% 2-mercaptoethanol, 1% Triton X-100, and 120 mM Tris,
pH 7.0). Nematodes were rinsed in PBST (PBS with 0.5%
Triton X-100), and then 10–50 ␮l of nematodes were incubated in 100 ␮l of collagenase solution (100 units of Sigma
type VII collagenase, 1 mM CaCl2, 0.1% Triton X-100, and
100 mM Tris, pH 7.4) on a shaker at 37°C for 2–24 h. Finally,
fixed nematodes were rinsed with PBST.
After fixation, nematodes were rinsed in PBS and then
blocked with 10% donkey serum in antibody buffer (0.5%
Triton X-100, 1 mM EDTA, and 0.1% BSA in PBS with 0.05%
sodium azide) for 1 h. Primary antibody incubations (1:50–
1:200) were done overnight. After thorough rinsing with
antibody buffer, nematodes were incubated in secondary antibodies for 4 h. Unlabeled and Cy3-labeled secondary antibodies were obtained from Jackson ImmunoResearch (West
Grove, PA). Oregon green 488 was coupled to secondary
antibodies using a kit from Molecular Probes (Eugene, OR).
After rinsing, nematodes were mounted in antibleaching
medium (14).
Stained nematodes were visualized with a four-laser Leica
TCS NT confocal microscope.
C1617
C1618
COEXPRESSION OF VACHT AND VMAT IN C. ELEGANS
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Fig. 1. Distribution of immunoreactivity of vesicular monoamine transporter (VMAT; red), vesicular acetylcholine
transporter (VAChT; green), or both in wild-type and mutant Caenorhabditis elegans. Top row: VMAT and VAChT
immunoreactivities imaged (left) or diagramed (right) in the midbody region of a wild-type adult. Immunoreactivity
is seen in the ventral nerve cord (VNC), dorsal nerve cord (DNC), and the dorsal and ventral sublateral nerve cords
(sub). VMAT and VAChT immunoreactivities are present around the vulva (indicated on the diagram by a blue line
and seen in the fluorescent image as a thin line of autofluorescent cuticle). Although most neuronal cell bodies (gray
ovals in diagram) are not immunoreactive, the cell bodies of the VC4 and VC5 are visible. Yellow regions in the
ventral nerve cord are generally due to red (VMAT) and green (VAChT) positive regions that are very close, but are
not overlapping, whereas the yellow region around the vulva is due to colocalization (see higher magnification
views below). Second row: close-up of the region around the vulva of a wild-type C. elegans. The cell bodies of VC4
and VC5 are indicated; these cells contain both VAChT and VMAT immunoreactivity. The VNC outside of the
region of the vulva contains generally nonoverlapping spots of VAChT or VMAT immunoreactivity. A ventral
sublateral nerve cord (sub) that contains VAChT immunoreactivity is indicated. Third row: higher magnification
of a portion of the vulva innervated by the VC4/5 and HSNL. Bottom row: the region around the vulva in a unc-104
mutant, in which synaptic vesicles and VAChT and VMAT immunoreactivity are abnormally concentrated in cell
bodies. The cell bodies of VC4/5 and HSNL/R are indicated; all 4 neurons contain both VAChT and VMAT
immunoreactivity. Several motor neurons that contain only VAChT immunoreactivity are seen in the VNC. An
embryo (emb) inside the adult hermaphrodite shows VMAT immunoreactivity in a few developing neurons. All
images are maximum projections of confocal series with anterior to the left and dorsal on top. Scale bars are 5 ␮m.
COEXPRESSION OF VACHT AND VMAT IN C. ELEGANS
C1619
example, the HSNs should release serotonin near the
VC4/5 terminals and cell bodies; this serotonin might
be taken up by VC4/5. To test this hypothesis, we
examined serotonin immunoreactivity in VC4/5 in
mutants that lack HSNs. We found that the faint
serotonin immunoreactivity present in VC4/5 in
some unc-104 animals (16/46 or 35% cells positive)
was not detected in unc-104;egl-1 mutants (1/62 or
2% cells positive). Therefore, the low level of serotonin sometimes present in VC4/5 may normally originate in the HSN cells.
The different cell types show slight differences in the
development of expression of these different transporters. The six VCs are born at the end of the first larval
stages (40). VAChT (and ChAT) immunoreactivity is
generally undetectable until the fourth larval stage. At
this time, the vulva develops and the VC4/5s begin to
arborize and innervate the vulval muscle (23). VAChT
immunoreactivity increases slightly before VMAT immunoreactivity increases. The highest levels of both
VAChT and VMAT immunoreactivity are reached as
the vulva becomes completely differentiated in the
early adult and are maintained throughout adulthood.
The HSNs are born and migrate into the middle of
the body during embryogenesis. They begin to extend
axons during the second or third larval stage (15). The
axons continue to grow and differentiate through the
late fourth larval stage; final differentiation is not
complete until early adulthood. The HSNs do not show
detectable VMAT or VAChT immunoreactivity (nor
serotonin immunoreactivity) (15) before the late fourth
larval stage. Both vesicular neurotransmitter transporters are detectable in the early adult at the same
time and increase in intensity in concert. This coordi-
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Fig. 2. Distribution of VMAT (red) and VAChT (green) or both in lin-39 and egl-1 mutants. Top row: the region
around the vulva in a lin-39 mutant. Due to the (variable) lineage defects of lin-39, VC4 and VC5 are absent in this
individual; the remaining immunoreactivity at the vulva is due to HSNR and HSNL (with, perhaps, minor
contributions from VC1, VC2, VC3, and VC6). The VNC and a ventral sublateral (sub) are indicated. Middle row:
the region around the vulva in an egl-1 mutant. HSNL/R are absent in egl-1 mutants; the remaining immunoreactivity at the vulva is due to innervation by VC4 and VC5, with minor contributions from VC1, VC2, VC3, and
VC6. Bottom row: higher power view of the same egl-1 mutant shows immunoreactivity at the vulva arising from
the VCs. Top and middle rows are maximum projections of confocal series; the bottom row is a single confocal
section. Anterior is to the left and dorsal is on top. Scale bars are 5 ␮m.
C1620
COEXPRESSION OF VACHT AND VMAT IN C. ELEGANS
nate expression contrasts with the more complex timing of expression seen in VC4/5.
Subcellular localization of VAChT and VMAT. The
subcellular patterns of immunoreactivity for VAChT
and VMAT are not identical. The slightly different
distributions within somas suggest different patterns
of synthesis or trafficking. In addition, different regions of the terminal fields sometimes have different
relative amounts of the two transporters (Fig. 2). This
is true not only in wild-type animals (in which the
terminal fields of the VC4/5 and the HSNs overlap) but
also in egl-1 mutants in which all of the innervation is
derived from the VCs. This suggests that VAChT and
VMAT are present in different vesicles and/or different
vesicle types, although there may also be some membrane compartments containing both transporters.
Interactions between neurotransmitter systems. We
have been unable to demonstrate any compensatory
interactions between the two transmitter systems.
Thus the expression of ChAT and VAChT in the HSNs
and in VC4/5 (measured by immunostaining) is unchanged in cat-1 null mutants, which lack VMAT. Also,
the expression of VMAT in these four cells is unchanged in cha-1 or unc-17 mutants, which have decreased cholinergic neurotransmission. The expression
of both VAChT and VMAT was unchanged in tph-1,
which lacks serotonin (41), and bas-1 mutants, which
have reduced levels of serotonin and dopamine (27, 35).
Furthermore, neither the HSNs nor the VCs are required to induce the expression of the multitransmitter
phenotype in the other cells.
DISCUSSION
Evidence for multiple transmitter function. Previous
functional and pharmacological studies have suggested
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Fig. 3. Serotonin immunoreactivity in mutant C. elegans. Serotonin
immunoreactivity (green) and nuclei (blue) near the vulva in an
adult unc-104 mutant. A maximal projection of a confocal series
through the right half of an adult shows strong serotonin immunoreactivity in the cell body and process of HSNR. (The out-of-focus
HSNL soma is not seen in this partial projection.) Serotonin immunoreactivity is only weakly present in the cell bodies of VC4 and VC5
(arrows) and is undetectable in the processes of these two neurons.
The opening of the vulva can be seen because of the autofluorescence
of the cuticle in that region. Scale bar is 10 ␮m.
that the HSNs use both acetylcholine and serotonin
(46). In addition, serotonin and acetylcholine affect
egg-laying behavior in different ways (43). Our immunocytochemical results support and extend these conclusions. In addition, although direct physiological
analysis of C. elegans vulval muscles is not yet reported, in the nematode Ascaris suum, the vulval muscles respond directly to both acetylcholine and serotonin (34).
If both cell types (HSN and VC) release acetylcholine
and possibly serotonin on the same set of muscles, why
do decreases in either of the transmitters have only a
small effect on egg laying, while loss of the HSNs (but
not the VCs) has a very strong effect on egg laying (17,
43)? We hypothesize that there is some element of HSN
structure or function not present in VC4/5. This may
involve the synaptic output from the HSNs, such as yet
another neurotransmitter specifically released by the
HSNs. For example, the HSNs and VC4/5 all contain
FMRF-amide immunoreactivity, but they may release
different neuroactive peptides (29).
Subcellular localization of vesicular transporters. In
mammals, it has been shown that in cell somas and
axons, VAChT is localized to the precursors of small
synaptic vesicles (45), whereas VMAT2 is preferentially associated with the precursors of large dense core
vesicles (25). At synapses, rat VMAT2 can be found in
both small clear vesicles and large dense core vesicles
(30). In culture, the subcellular localization of these
two transporters can be similar or different depending
on cell type (26). It thus appears that VAChT and
VMAT2 can be present in different vesicles and/or
vesicle types, although there may also be vesicles or
pools of vesicles containing both transporters. Both
large and small vesicles are present in C. elegans
neurons (48); it is quite possible that C. elegans VAChT
and VMAT are also in different vesicle types.
Dale’s principle and multiple neurotransmitters. As
originally formulated, Dale’s principle stated that a
mature neuron makes use of the same transmitter
substance at all of its synapses (5, 8). Although Dale
did not specifically address the question of multiple
transmitters within a single neuron, over the years
Dale’s principle acquired a widely accepted corollary
that an individual neuron secretes a single neurotransmitter. After the discovery and characterization of neuropeptides, this principle was further limited to apply
only to classical neurotransmitters.
In the last fifteen years, there have been numerous
reports of more than one classical neurotransmitter
localized to the same mature neuron by immunocytochemical means (reviewed in Ref. 22). Reports include
colocalization of serotonin and ChAT immunoreactivities (21). Robust physiological and pharmacological
evidence for the release of multiple transmitters
(GABA and glycine or GABA and ATP) from single
vertebrate neurons has recently been reported (18, 20).
We have now presented evidence for the simultaneous presence of two vesicular transporters in identified neurons. Although this result was not unexpected
for the HSNs (based on previous physiological and
COEXPRESSION OF VACHT AND VMAT IN C. ELEGANS
immunocytochemical studies), it provides further evidence for synaptic vesicular release of different classes
of classical neurotransmitters from single identified
neurons. Because the ratio of the VAChT and VMAT
proteins varies in different parts of the terminal fields
of each of these cells, it is likely that VAChT and
VMAT are present in different vesicles and/or different
vesicle types, and perhaps even at different terminal
types. We are now examining the ultrastructure of the
VC and HSN terminals by electron microscopy to further study this question.
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We thank Lee Eiden, Ken Miller, Dennis Frisby, and Becky
Eustance Kohn for advice and suggestions. Some nematode strains
used in this work were provided by the Caenorhabditis Genetics
Center, which is funded by the National Institutes of Health National Center for Research Resources. tph-1(mg280) was a gift of Ji
Ying Sze.
This research was supported by National Institute of General
Medical Sciences Grant GM-38679 and by Oklahoma Center for the
Advancement of Science and Technology Grant HN3-023.
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