(The FASEB Journal. 2000;14:2414-2422.)
© 2000 FASEB
Neurogenetics of vesicular transporters in C. elegans
JAMES B. RAND1,
JANET S. DUERR and
DENNIS L. FRISBY
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA
1Correspondence: Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104, USA. E-mail: James-Rand{at}omrf.ouhsc.edu
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ABSTRACT
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The nematode Caenorhabditis elegans
has a number of advantages for the analysis of synaptic molecules.
These include a simple nervous system in which all cells are identified
and synaptic connectivity is known and reproducible, a large collection
of mutants and powerful methods of genetic analysis, simple methods for
the generation and analysis of transgenic animals, and a number of
relatively simple quantifiable behaviors. Studies in C.
elegans have made major contributions to our understanding of
vesicular transmitter transporters. Two of the four classes of
vesicular transporters so far identified (VAChT and VGAT) were first
described and cloned in C. elegans; in both cases, the
genes were first identified and cloned by means of mutations causing a
suggestive phenotype (1
, 2)
. The phenotypes of
eat-4 mutants and the cell biology of the EAT-4 protein
were critical in the identification of this protein as the vesicular
glutamate transporter (3
, 4)
. In addition, the unusual
gene structure associated with the cholinergic locus was first
described in C. elegans (5)
. The
biochemical properties of the nematode transporters are surprisingly
similar to their vertebrate counterparts, and they can be assayed under
similar conditions using the same types of mammalian cells (6
, 7)
. In addition, mild and severe mutants (including knockouts)
are available for each of the four C. elegans vesicular
transporters, which has permitted a careful evaluation of the role(s)
of vesicular transport in transmitter-specific behaviors. Accordingly,
it seems appropriate at this time to present the current status of the
field. In this review, we will first discuss the properties of
C. elegans vesicular transporters and transporter
mutants, and then explore some of the lessons and insights C.
elegans research has provided to the field of vesicular
transport.—Rand, J. B., Duerr, J. S., Frisby, D. L.
Neurogenetics of vesicular transporters in C. elegans.
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CAENORHABDITIS ELEGANS AS A MODEL ORGANISM
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These studies rely on the strengths of Caenorhabditis
elegans as a research organism: ease of manipulation, a simple
nervous system, and powerful tools for the analysis of mutants and
genes. Genetically, C. elegans is advantageous because of
its short generation time (3 days), its prolific progeny yield (280 per
parent), its small size (1.5 mm long), and its ease of laboratory
culture (on Escherichia coli lawns on agar in Petri dishes).
There are now thousands of mutant strains of C. elegans,
exhibiting a wide variety of behavioral, morphological, and
developmental phenotypes, and hundreds of genes have now been mapped on
the animals’ six chromosomes.
C. elegans is particularly suited for studying neural
function. Its nervous system has many morphological and biochemical
similarities to those of mammals. Most proteins identified as important
for neurotransmitter release have homologues in both C.
elegans and mammals (8
, 9)
. In addition, because
C. elegans hermaphrodites can self-fertilize and produce
progeny without mating, it is possible to maintain strains carrying
severe neural defects that disrupt movement or other behaviors.
Cellularly, C. elegans is remarkably simple. At hatching,
there are 550 somatic cells (10)
; over the course of the
next 48 h, this number increases to produce 959 adult somatic
cells (11)
. The adult contains 302 neurons, and
reproducibility of neuron structure and connectivity has been
demonstrated by serial section electron microscopy for most portions of
the nervous system (12
13
14
15)
.
Molecular biology using this organism is simplified by its relatively
small genome size of 108 bp, which has now been
fully sequenced (16
, 17)
. C. elegans can
maintain injected DNA as extrachromosomal arrays, and it is therefore
relatively straightforward to express cloned genes by transformation
(18
, 19)
.
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NEUROTRANSMITTER STUDIES IN C. ELEGANS
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Although the C. elegans nervous system has only 302
neurons, the diversity of classical neurotransmitters and neuropeptides
seems comparable to that in vertebrate nervous systems
(20)
. Analysis of specific neurotransmitters and their
functions has relied on a combination of pharmacology, biochemistry,
mutant analysis, and cell ablation studies. In particular, the
relatively simple neural circuitry of C. elegans has made it
possible to assess the involvement of specific neurotransmitters in
particular behaviors.
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CHOLINERGIC FUNCTION IN C. ELEGANS
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Acetylcholine is the major excitatory neurotransmitter controlling
motor functions in nematodes (21
, 22)
. C.
elegans contains the enzymes of acetylcholine synthesis and
degradation (23
24
25)
, and acetylcholine is present in
extracts (26
27
28)
. In addition, C. elegans are
paralyzed by cholinesterase inhibitors (organophosphates and
carbamates) and by nicotinic agonists (29)
.
The transmitter is synthesized by choline acetyltransferase (ChAT)
(25)
, encoded by the cha-1 gene
(30)
. Partially ChAT-deficient mutants grow slowly and
display deficits in a number of neuromuscular behaviors, such as
locomotion, feeding, and defecation (30
31
32
33)
.
ChAT-deficient mutants are also resistant to AChE inhibitors (30
, 34)
, presumably because decreased synthesis of acetylcholine
counteracts the toxin-induced accumulation of excess synaptic
transmitter. Mutations that eliminate all gene function (null
mutations) are lethal; mutant homozygotes are able to complete
embryogenesis and hatch, but the young larvae are unable to move or
feed normally, and they shrink and die within a few days
(31)
.
Based on immunostaining with anti-ChAT antibodies, 
115 C.
elegans neurons appear to be cholinergic; almost all of these
cells are motor neurons ( J. S. Duerr et al., unpublished
results).
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VAChT AND unc-17 MUTANTS
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Mutations in the unc-17 gene (so named because
mutations conferred uncoordinated locomotion) were first
described by Brenner (29)
. In addition to the locomotion
deficit, unc-17 mutants with a slight amount of residual
gene function are slow-growing, small, resistant to inhibitors of
cholinesterase, and contain excess acetylcholine (26
, 28
, 29)
. Null mutations for unc-17 are lethal, with the
same phenotype displayed by null cha-1 mutants (1
, 31)
.
The unc-17 gene was cloned and sequenced by Alfonso et al.
(1)
; when it was first cloned, the unc-17 gene
product (UNC-17) had no homologs in the databases. The predicted UNC-17
protein contained 532 amino acid residues and 12 putative transmembrane
domains. Immunocytochemical studies demonstrated that ChAT and UNC-17
were colocalized at the cellular and the subcellular level.
Immunoreactivity to both proteins is present in synaptic regions of
115 neurons, and additional experiments showed that UNC-17 was
associated with synaptic vesicles (Fig. 1
) (1)
. Almost all of the ChAT- and UNC-17-positive cells
are (apparently excitatory) motor neurons. When the cloning of the rat
VMATs was published (35
, 36)
, it became clear that UNC-17
was related to the VMATs, and it was therefore likely to be the
C. elegans VAChT (Fig. 2
) (1)
. The C. elegans unc-17 gene was then used
to isolate vertebrate homologs, and the identity of the corresponding
gene products as VAChTs was confirmed by binding and uptake studies
(6
, 37)
.
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Figure 1. Distribution of immunoreactivity of VAChT (green) and VMAT (red) in
C. elegans. A) VAChT and VMAT
immunoreactivity in the head of a wild-type nematode. Immunoreactivity
is seen in the nerve ring (NR), ventral nerve cord (VNC), dorsal nerve
cord (DNC), and the dorsal and ventral sublateral nerve cords of the
head and body (subs). In the pharynx (ph), numerous immunoreactive
processes are also seen. Immunoreactivity is predominantly in synaptic
regions along neuronal processes and is sparse in neuronal cell bodies
(compare with diagram in panel B). The yellow regions in
the ventral nerve cord are generally due to green (VAChT) and red
(VMAT) positive regions that are very close, but are not overlapping.
B) Diagram of the nervous system of the nematode
illustrated in panel A. Neuronal processes are shown as
black lines; neuronal somas are shown as white circles with gray
outlines. The pharynx is indicated with medium gray; the neurons within
the pharynx are not diagramed for the sake of clarity. The beginning of
the intestine is shown as a darker gray. C) The region
around the nerve ring in an unc-104 mutant at slightly
higher magnification. In unc-104 mutants, synaptic
vesicles as well as VAChT and VMAT immunoreactivity are abnormally
concentrated in cell bodies. The cell bodies of numerous neurons in the
lateral ganglia and the ventral ganglion are apparent. Many neurons are
immunoreactive for VAChT or VMAT, but very few are immunoreactive for
both.
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Figure 2. Dendrogram of published VMAT and VAChT sequences. The PILEUP program
(Genetics Computer Group Wisconsin Package, Version 8) was used.
(Modified slightly from 7). Prefix abbreviations: Bos, bovine; Cel,
C. elegans; Dro, Drosophila; Hum, human;
Tor, Torpedo. The individual Genbank Accession numbers
are: HumVMAT2, #L23205 (79)
; BosVMAT2, #U02876
(80)
; RatVMAT2, #L00603 (35)
; HumVMAT1,
#U39905 (81)
; RatVMAT1, #M97380 (36)
;
CelVMAT, (7)
; HumVAChT, #U10554 (6)
;
RatVAChT, #U09211 (6)
; TorVAChT #U05591 (37)
;
DroVAChT, #AF030197 (39)
; CelVAChT, #L19621
(1)
.
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An unusual aspect of unc-17 is its genomic organization: it
is part of a complex transcription unit that also includes the
cha-1 gene (5)
. The cha-1 and
unc-17 transcripts appear to be derived by alternative
splicing of a common precursor. The two genes use a common
5'-untranslated exon; the remainder of the unc-17 gene is
nested within the long first intron of cha-1 (Fig. 3
). A similar genomic organization (i.e., the VAChT gene nested within
the first intron of the ChAT gene) was subsequently identified in
mammals and in Drosophila (6
, 38
, 39)
. Thus, in
mammals, insects, and nematodes, the synthesis and the vesicular
transport of acetylcholine are coupled at the genomic level (Fig. 3)
.
This suggests that the organization of this cholinergic locus is
somehow important for its function. It is noteworthy that a similar
organization is not found for VMAT or for VGAT.
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CATECHOLAMINES IN C. ELEGANS
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Three amine neurotransmitters have been identified thus far in
C. elegans. Dopamine was originally identified in eight
sensory neurons using the technique of formaldehyde-induced
fluorescence (40)
. Exogenous dopamine inhibits locomotion
and egg laying (41)
. Dopamine is synthesized in a two-step
enzymatic process, using the enzymes tyrosine hydroxylase and aromatic
amino acid decarboxylase (AAAD; often called DOPA decarboxylase). In
C. elegans, these enzymes are encoded by the
cat-2 and bas-1 genes, respectively (42
, 43)
. Using mutations in these genes, it has been possible to
analyze the involvement of dopamine in specific behaviors. The deficits
in such mutants include difficulties sensing and responding to the
presence of food (42
, 44)
. Males contain three additional
pairs of dopamine-containing sensory neurons in the tail, and mutants
lacking dopamine are deficient in male mating behaviors (42
, 45)
.
Serotonin (5HT) has been identified in C. elegans neurons by
formaldehyde-induced fluorescence (41)
and by anti-5HT
immunostaining (46
, 47)
. In C. elegans,
exogenous 5HT stimulates egg laying and pharyngeal pumping and inhibits
locomotion and defecation (41
, 48)
. There are 11 neurons
in C. elegans with anti-5HT immunoreactivity (7
, 42
, 46)
; these include sensory neurons, interneurons, motor neurons,
and secretory cells. Serotonin is synthesized by the sequential action
of tryptophan hydroxylase (encoded by the tph-1 gene) and
AAAD (the same enzyme used for dopamine synthesis, encoded by
bas-1) (42
, 49)
. Mutant analysis and drug
studies have shown that, in addition to regulating egg laying,
serotonin mediates the response to starvation and is required for male
mating behaviors (42
, 44
, 50
, 51)
.
Epinephrine and norepinephrine were not detected in C.
elegans; however, octopamine
(p-hydroxyphenylethanolamine) was present in C.
elegans homogenates, and exogenous octopamine inhibited egg laying
and stimulated locomotion (41)
. In addition, the presence
of N-acetyl serotonin has been reported in several parasitic nematodes,
and tissue extracts of Ascaris and C. elegans
contain an enzymatic activity (arylalkyl amine N-acetyl transferase)
capable of acetylating serotonin, dopamine, and octopamine (52
, 53)
. It is not known whether the N-acetylated derivatives are
merely catabolic intermediates or if they might represent additional or
alternative neurotransmitters.
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VMAT AND cat-1 MUTANTS
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The C. elegans Genome Sequencing Consortium identified
sequence on genomic cosmid W01C8 that encoded a VMAT-like protein; this
appears to be the only VMAT homologue in the C. elegans
genome. Analysis of cDNAs indicated an open reading frame corresponding
to a 553 amino acid protein; the predicted C. elegans
protein is 47% identical to rat VMAT1 and 49% identical to rat VMAT2
(Fig. 2)
(7)
. When expressed in mammalian cells, the
C. elegans protein is functional and mediates time-dependent
transport of serotonin and dopamine; transport is reserpine sensitive
and tetrabenazine sensitive, and is competitively inhibited by
dopamine, octopamine, tyramine, norepinephrine, and histamine
(7)
.
Immunolocalization studies indicate that the VMAT protein is primarily
localized to synaptic regions of a subset of neurons and is associated
with synaptic vesicles (Fig. 1)
(7)
. There are 25
VMAT-positive neurons; these include the 8 dopamine-containing cells
and the 11 serotonin-containing neurons, as well as a few still
unidentified cells. These neurons are (with at least two clear
exceptions) distinct from those that are immunopositive for VAChT.
The previously identified cat-1 gene corresponds to the
C. elegans VMAT homologue. In cat-1 mutants,
levels of dopamine (visualized by formaldehyde-induced fluorescence;
40
) and 5HT (visualized by immunocytochemistry;
42
) are decreased in neuronal processes and increased in
cell bodies. Furthermore, the apparent abundance of dopamine is
decreased by 60%, and VMAT immunoreactivity is eliminated in
cat-1 mutants (7
, 40)
. It is noteworthy that
these are the same phenotypes obtained by treating wild-type animals
with reserpine (40)
. cat-1 mutants are also
deficient in dopamine- and 5HT-mediated behaviors, such as slowing
their rate of locomotion in the presence of food, egg laying, and male
mating (7
, 42)
.
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GABA FUNCTION IN C. ELEGANS
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The evidence for GABA function in C. elegans includes
pharmacological studies using GABA-related compounds,
immunohistochemical demonstration of the presence of GABA in specific
cells, and analysis of mutants defective in GABA synthesis and function (54
, 55)
. Twenty-six neurons contain GABA
immunoreactivity; most of these are inhibitory motor neurons, but some
are apparently excitatory GABAergic motor neurons (55)
. In
each of these cells, the GABA immunoreactivity is uniformly distributed
throughout the cytoplasm and is not restricted to synaptic regions
(55)
.
Mutants with GABAergic transmission defects are viable, although they
have several motor defects. The most obvious phenotype is a tendency to
contract dorsal and ventral body wall muscle simultaneously in response
to touch (‘shrinker’ phenotype); this appears to result from lack of
function of the GABA-containing DD and VD inhibitory motor neurons
(54
, 55)
.
GABA is synthesized from glutamic acid by the enzyme glutamic acid
decarboxylase (GAD). In C. elegans, unc-25
encodes a protein that is 45% identical to mammalian GAD
(56)
. unc-25 mutants have no apparent GABA
(54)
and extracts prepared from these animals lack GAD
activity (C. Johnson and A. Stretton, personal communication). The
unc-25 GAD gene is expressed in 26 neurons
(56)
, which correspond to the 26 GABA-containing neurons.
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VGAT AND unc-47 MUTANTS
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Mutants of the unc-47 gene were first reported by
Brenner (29)
and extensively characterized by McIntire et
al. (54)
. These mutants were deficient in GABA-mediated
behaviors and accumulated excess GABA in presynaptic neurons, but
displayed normal responses to GABA agonists. This led to the suggestion
that unc-47 might encode a VGAT (54)
.
Subsequent cloning of the locus revealed a 486 amino acid protein
containing 10 putative transmembrane domains, bearing a weak similarity
to some plant amino acid permeases (2)
. The UNC-47 protein
was associated with synaptic vesicles and its expression was limited to
the 26 previously identified GABA-containing neurons (2)
.
A rat homologue of the UNC-47 protein (38% identical) conferred GABA
transport activity to PC12 cells, with an apparent
Km of 5 mM (2)
. Additional studies
have suggested that the mammalian transporter is also capable of
vesicular transport of glycine (57)
; it would therefore
seem more appropriate to consider the protein to be a vesicular
inhibitory amino acid transporter (VIAAT) (58
, 59)
.
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GLUTAMATE FUNCTION IN C. ELEGANS
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Molecular studies have identified and characterized several of the
components of glutamatergic transmission in C. elegans, and
mutant analysis has suggested a role for glutamate in specific neural
circuits and the behaviors they mediate.
A C. elegans plasma membrane glutamate transporter
(glt-1) similar to the vertebrate excitatory amino acid
transporters has been cloned and shown to exhibit glutamate transport
activity when expressed in Xenopus oocytes
(60
61
62)
. In addition, several genes encoding glutamate
receptor subunits have been cloned and studied; these include
glr-1, which encodes an AMPA-type receptor (63
, 64)
, and three genes—avr-14, avr-15, and
glc-1—that encode subunits of glutamate-gated chloride
channels (65
66
67
68)
. These glutamate-gated chloride channels
have been shown to be the sites of action of the antiparasitic drug
ivermectin (69)
.
In general, the identification of glutamate releasing neurons and
glutamate-dependent behaviors has been inferred from analysis of the
phenotypes of glr-1 mutants (and eat-4 mutants,
described below) and from the expression patterns of the receptor
proteins. These studies suggest that glutamate is released by many
sensory neurons and interneurons, and a few of the behavioral circuits
have been identified. These include 1) the response to light
mechanical stimulation of the head mediated by the ASH sensory neurons
through the GLR-1 postsynaptic receptor (63
, 64)
;
2) the response to strong mechanical stimulation in the
anterior body mediated by the ALM and AVM sensory neurons through the
AVR-15 postsynaptic receptor (3)
; and 3) the
effects of the M3 motor neuron on the pharyngeal muscle, mediated
through the AVR-15 postsynaptic receptor (described below).
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VGluT AND eat-4 MUTANTS
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Mutations in the eat-4 gene were originally identified
because of altered feeding behavior (33)
. The M3 cells are
inhibitory motor neurons that trigger repolarization of the pharyngeal
muscle, and many of the pharyngeal pumping defects present in
eat-4 mutants are phenocopied by laser ablation of the M3
neurons (70)
. M3 neurotransmission is mediated by the
postsynaptic avr-15 glutamate receptor (67)
;
after M3 ablation, the function(s) of the M3 neurons could be restored
by application of glutamate but not by other putative neurotransmitters
(71)
. This strongly suggested that glutamate was the
neurotransmitter released by the M3 cells. In addition, because the
pharyngeal muscles of eat-4 mutants have normal postsynaptic
responses to glutamate, the eat-4 gene product EAT-4
apparently functions in the presynaptic (M3) neurons (67)
.
In addition to the pharyngeal effects, eat-4 mutants exhibit
many of the sensory defects associated with glr-1 mutants
(3)
. There are also alterations in the habituation
behavior of eat-4 animals: they habituate more rapidly than
wild-type, recover from habituation more slowly, and show no
dishabituation (72)
.
Cloning and sequencing of the eat-4 gene showed that the
EAT-4 protein was quite similar to the previously identified mammalian
brain-specific, sodium-dependent inorganic phosphate transporter
BNPI (3
, 73)
. The EAT-4 protein is expressed in at least
38 neurons; these include the M3 cells as well as a number of sensory
neurons known to be presynaptic to GLR-1-expressing interneurons
(3)
.
Although the mammalian BNPI protein was originally characterized as a
plasma membrane phosphate transporter, reexamination of its properties
showed that it was associated preferentially with small synaptic
vesicles, rather than with the plasma membrane (74)
, and
that the expressed protein could transport glutamate (4)
.
These data, together with the EAT-4 expression pattern and the
eat-4 mutant phenotypes, suggested strongly that BNPI and
EAT-4 were in fact vesicular glutamate transporters.
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USE OF REPORTERS AND STUDIES OF GENE REGULATION
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One of the advantages of simple model organisms (e.g., C.
elegans and Drosophila) is the relative ease with which
genes and altered gene constructs may be introduced and expressed. This
has permitted the use of reporter proteins to monitor the patterns of
gene expression. A further advantage of C. elegans is its
transparency; in combination with the new generation of fluorescent
reporter proteins (e.g., GFP), it is now possible to monitor cellular
expression patterns directly, in live, developing nematodes (75
, 76)
.
For example, in a transgenic C. elegans strain containing a
GFP reporter gene under the control of the
unc-17–cha-1 promoter region, strong GFP
expression is seen in cholinergic neurons and not in other cells
(Fig. 4
). Similarly, GFP constructs have been used to demonstrate that both
unc-25 and unc-47 are positively regulated by the
UNC-30 homeodomain-containing transcription factor (77)
.
It is noteworthy that even though unc-25 and
unc-47 are both expressed in all 26 of the GABA-positive
cells, their regulation by UNC-30 occurs in only 19 of these cells.
These are the 19 so-called type D Inhibitory motor neurons, which are
responsible for the reciprocal (ventral-dorsal) inhibition of body
muscle contraction involved in sinusoidal locomotion (55)
.
Because of the relative ease with which reporter genes can be
engineered and expressed, it is possible to perform relatively
sophisticated promoter analysis of C. elegans genes.
However, there are significant experimental differences between
promoter analysis in C. elegans and mammals. In both cases,
a common experimental design involves ‘promoter bashing’ using
ordered deletions of regulatory regions, followed by analysis of
reporter expression. In C. elegans (or
Drosophila), the expression of the reporter gene is observed
in living animals, with very little control over the humoral
environment of each differentiating neuron. Mammalian regulation
experiments generally involve transfected cell lines and often measure
the response of the reporter to a particular signal, e.g., NGF. In this
case, there is great experimental control over the humoral environment
of the cells, but little or no positional information available to let
a cell know where it is in the organism. As a result, promoter analysis
in worms or flies will tell you that a particular element is required
for gene expression in a specific set of cells, whereas promoter
analysis in mammals will tell you that a particular element is involved
in the response to NGF. This is not to say that the genes are regulated
differently in the two taxa (although they may be), but rather that the
type of information we can readily obtain about the regulation is
different.
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LESSONS AND INSIGHTS FROM C. ELEGANS
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In addition to the initial identification of VAChT and VGAT (and a
major role in identification of VGluT), C. elegans research
has made important contributions to our understanding of the biology of
vesicular neurotransmitter transport. The biological information
derives in part from the simplicity of the nematode body plan,
particularly its nervous system. Often a neurotransmitter is used by
only a limited number of neurons and is required for a (relatively)
limited number of nonvital functions. For many transmitters, the
biosynthetic enzymes are known and cloned, and mutants are available
that are deficient in the synthesis of each. It is therefore possible
to compare specific behaviors in synthesis-deficient mutants and
transport-deficient mutants or to compare either type of genetic lesion
with laser ablation of the transmitter-containing cells.
Evolution of VMAT isoforms: C. elegans
Although clearly a close structural relative of the mammalian
VMATs, C. elegans VMAT is neither a VMAT1 nor a VMAT2 (Fig. 2)
. Since there is only a single VMAT gene in the C. elegans
genome, it appears that the two mammalian VMAT genes diverged from each
other subsequent to the divergence of nematode and mammalian ancestors.
However, the transport properties of C. elegans VMAT (in
particular, its affinity for histamine) appear more like those of the
mammalian VMAT2 (‘neuronal’) isoform than the VMAT1
(‘neuroendocrine’) isoform (7)
. The ability of C.
elegans VMAT to recognize histamine suggests that the ability to
transport histamine was not a function acquired by VMAT2, but rather a
preexisting trait of VMAT that was retained by VMAT2 and lost by VMAT1.
How essential is vesicular transport?
As described above, cha-1 (ACh synthesis) and
unc-17 (ACh vesicular transport) mutants have the same
phenotypes, and unc-25 (GABA synthesis) and
unc-47 (GABA vesicular transport) mutants have the same
phenotypes. It therefore appears that for these two neurotransmitters,
loss of vesicular transport seems to be functionally equivalent to loss
of transmitter synthesis and that vesicular transport is therefore
essential for neural function. This is not meant to exclude the
possibility of nonvesicular release mechanisms for these transmitters
(e.g., GABA efflux through plasma membrane GABA transporters); however,
under standard laboratory conditions, nonvesicular release makes little
or no contribution to the development of the nervous system or the
behavior of the animals.
For some neurotransmitters and for some cells, loss of vesicular
transport is equivalent to loss of cell function. Thus, for example,
the dopamine-related phenotypes associated with loss of VMAT
(cat-1 mutants) are quite similar to those associated with
ablation of the 8 dopaminergic sensory neurons, and the phenotypes
associated with loss of VGAT (unc-47 mutants) are quite
similar to those associated with ablation of the GABAergic motor
neurons (7
, 44
, 54
, 55)
.
In other cases, however, loss of vesicular transport is associated with
only a partial loss of cell function. For example, although the HSN
cells (which regulate egg laying) contain both serotonin and VMAT,
mutational elimination of either has a much milder effect than laser
ablation of the HSN cells (7
, 42
, 46)
. Discrepancies such
as these point to the involvement of other neurotransmitters and/or
neuropeptides in the functioning of these cells (51)
.
Another example is the difference between the loss of VGluT (EAT-4)
expression in the ALM and AVM sensory neurons and ablations of these
cells; this is presumably because the ALM and AVM cells make electrical
as well as chemical synapses onto their postsynaptic partners, and in
eat-4 mutants only the chemical transmission is compromised
(3)
.
Regulation of cytoplasmic transmitter levels
In vesicular transport mutants, it is possible to unmask some of
the homeostatic mechanisms involved in the regulation of transmitter
levels. A striking difference between the acetylcholine and GABA
vesicular transporters on the one hand, and the vesicular monoamine
transporter on the other is that for both VAChT and VGAT mutants, there
is a net accumulation of the transmitter (presumably in the presynaptic
cytoplasm) (26
, 28
, 55)
, whereas VMAT mutants are
associated with a decrease in presynaptic transmitter (7
, 40)
. Presumably, sequestering the dopamine or serotonin into
synaptic vesicles leads to a net increase in the total levels of
dopamine and serotonin, but it also seems that the biogenic amines in
the cytoplasmic compartment are maintained at a low level whether or
not there is sequestration. There are several plausible explanations
for this. Sequestration might improve the stability of the amines,
either because of the low pH or by protection from catabolic enzyme
activities. In addition, it is likely that C. elegans
tyrosine hydroxylase is similar to its vertebrate cousins and its
activity is feedback-inhibited by dopamine.
Regulation of the cholinergic gene locus
Although it has long seemed likely that the expression of
transmitter-specific genes would be coregulated, the genomic
organization of the ChAT and VAChT genes provided a plausible mechanism
for such regulation. In theory, if the mRNAs for both ChAT and VAChT
were derived from the same primary transcript, one would not expect
transcriptional initiation factors to have any differential effects on
ChAT and VAChT expression. In mammals, however, there are several
documented ChAT-specific and VAChT-specific promoters, and transcripts
derived from these promoters appear to be more abundant than
transcripts derived by alternative splicing from the common (R-exon)
promoter (reviewed in 78). It is therefore likely that ChAT and VAChT
would be differentially expressed in some cells and/or at some
developmental stages.
Immunostaining studies in C. elegans indicate that
ChAT and VAChT proteins are present in the same cells at the same times
(1
; Duerr et al., unpublished results), although it would
be difficult to detect subtle changes in the relative expression of the
two proteins or differential expression limited to a few cells and/or
brief developmental stages. Thus far, we have been unable to
demonstrate the existence of additional (ChAT-specific or
VAChT-specific) promoters in C. elegans (unpublished
observations); it therefore seems likely that there is only a single
type of primary transcript, which is then processed to yield either a
cha-1 mRNA or an unc-17 mRNA. Even under these
conditions, it is possible that regulation of the alternative splicing
(or other post-transcriptional mechanisms) or translation control
mechanisms could lead to differential ChAT and VAChT expression.
Note added in proof
In addition to Bellocchio et al. cited in the text (4), another recent paper identifies BNPI as the vesicular glutamate transporter [Takamori, S., Rhee, J. S., Rosenmund, C., and Jahn, R. (2000) Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature (London) 407, 189-194]
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ACKNOWLEDGMENTS
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|---|
We are grateful to many members of our laboratory, past and
present, who have helped with the analysis of C. elegans
vesicular transporter genes, proteins, and mutants. These include Aixa
Alfonso, Tony Crowell, Angie Duke, Kiely Grundahl, He-Ping Han, and
John McManus. Ken Miller helped greatly in the development of
behavioral assay strategies and Erik Jorgensen provided us with data
about VGAT before publication. We also wish to acknowledge our
collaborators Lee Eiden and Jeff Erickson at the National Institute of
Mental Health, who have helped with the biochemical and kinetic studies
of C. elegans VAChT and VMAT. Our transport
investigations have been supported by research grants from the National
Institutes of Health (GM38679) to J.B.R. and the Oklahoma Center for
the Advancement of Science and Technology to J.S.D., and National
Research Service Award NS10031 to D.L.F.
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ABSTRACT
CAENORHABDITIS ELEGANS AS A...
