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The Journal of Neuroscience, March 1, 2001, 21(5):1510-1522
Differential Expression of Glutamate Receptor Subunits in the Nervous System of Caenorhabditis elegans and Their Regulation by the Homeodomain Protein UNC-42
Penelope J. Brockie, David M. Madsen, Yi Zheng, Jerry Mellem, and Andres V. Maricq Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
In almost all nervous systems, rapid excitatory synaptic communication is mediated by a diversity of ionotropic glutamate receptors. In Caenorhabditis elegans, 10 putative ionotropic glutamate receptor subunits have been identified, a surprising number for an organism with only 302 neurons. Sequence analysis of the predicted proteins identified two NMDA and eight non-NMDA receptor subunits. Here we describe the complete distribution of these subunits in the nervous system of C. elegans. Receptor subunits were found almost exclusively in interneurons and motor neurons, but no expression was detected in muscle cells. Interestingly, some neurons expressed only a single subunit, suggesting that these may form functional homomeric channels. Conversely, interneurons of the locomotory control circuit (AVA, AVB, AVD, AVE, and PVC) coexpressed up to six subunits, suggesting that these subunits interact to generate a diversity of heteromeric glutamate receptor channels that regulate various aspects of worm movement. We also show that expression of these subunits in this circuit is differentially regulated by the homeodomain protein UNC-42 and that UNC-42 is also required for axonal pathfinding of neurons in the circuit. In wild-type worms, the axons of AVA, AVD, and AVE lie in the ventral cord, whereas in unc-42 mutants, the axons are anteriorly, laterally, or dorsally displaced, and the mutant worms have sensory and locomotory defects.
Key words: glutamate receptor; neuron; neural circuit; development; mechanosensation; homeodomain transcription factor; Caenorhabditis elegans; locomotion; glr-1; unc-42
INTRODUCTION
Glutamate is a neurotransmitter that is required for synaptic communication in vertebrate and invertebrate nervous systems. Signaling by glutamate is mediated by a large and diverse number of receptors that include ionotropic receptors that mediate rapid excitatory neurotransmission. Ionotropic glutamate receptors belong to either the NMDA family, which contains receptors that are selectively gated by the agonist NMDA, or the non-NMDA family, which contains receptors that are gated by the agonists AMPA and kainate (Dingledine et al., 1999
; Hollmann, 1999
Invertebrate nervous systems also appear to use multiple subtypes of glutamate receptor subunits. Analysis of the genomes of both Drosophila melanogaster and Caenorhabditis elegans has revealed large families of genes that encode potential ionotropic glutamate receptor subunits (Littleton and Ganetzky, 2000
In C. elegans, the command interneurons form part of the locomotory control circuit and are required for the escape response to tactile stimuli (Chalfie et al., 1985
In a subset of the command interneurons, GLR-1 expression is dependent on the homeodomain protein UNC-42 (Baran et al., 1999
Here we describe 10 ionotropic glutamate receptors that are expressed in the nervous system of C. elegans. The receptor subunits can be categorized into NMDA and non-NMDA subtypes and are expressed in a variety of interneurons and motor neurons that suggest their involvement in the control of movement, thermotaxis, and pharyngeal function. We have also characterized the regulation of receptor expression and shown that the expression of GLR-4 and GLR-5 is also dependent on the homeodomain protein UNC-42. Furthermore, we show that connections made by the "backward" command interneurons are disrupted in unc-42 mutants, suggesting that defects in synaptic wiring of these interneurons are the probable cause of the unc-42 phenotype.
MATERIALS AND METHODS
Strains. Nematodes were grown at 20°C under standard conditions that included uncrowded conditions and the presence of ample food (the Escherichia coli strain OP50). Wild-type nematodes were C. elegans strain N2. Mutant strains were obtained from the Caenorhabditis Genetic Center.
cDNA clones and expression constructs. Using degenerate oligonucleotide primers designed to amplify conserved regions of ionotropic glutamate receptors, we amplified DNA fragments from first-strand mixed-stage C. elegans cDNA that encoded portions of glr-3 and glr-5. These partial gene products were used to screen cDNA libraries at high stringency. After screening ~106 clones, we obtained several partial cDNAs for each gene, indicating that mRNA encoding these glutamate receptor subunits is present at relatively low levels. Hence, we were unable to isolate full-length cDNAs using this approach. We also searched the published C. elegans genome for genes related in sequence to glr-1, glr-3, and glr-5 and identified seven additional genes that appeared to encode glutamate receptor subunits (see below). Partial cDNA clones were amplified from first-strand cDNA generated from mixed-stage N2 worms. Oligonucleotide primers were designed to amplify sequence that encoded a highly conserved region found in ionotropic glutamate receptor subunits. The PCR products were then subcloned into pCR2.1 (Invitrogen, San Diego, CA) and sequenced.
Green fluorescent protein (GFP) fusions with glr-3, glr-4, glr-6, glr-7, glr-8, and nmr-2 were made by PCR amplification of a genomic DNA fragment that included ~5-7 kb of upstream regulatory sequence followed by coding sequence terminating immediately upstream of that encoding TMIII (see Figs. 1, 4). The PCR products were subcloned into one of four GFP expression vectors: pPD95.75, pPD95.77, pPD95.79, or pPD95.81 (gifts from A. Fire). Partial, rather than full-length, GFP fusions were made to achieve maximum GFP fluorescence intensity while still including introns that might contain intragenic regulatory regions. For those subunits in which both a full-length fusion and a partial fusion have been made (glr-1 and nmr-1) there was no difference in expression pattern between the two transgenes. glr-1:: GFP and glr-5:: GFP were full-length GFP fusions. nmr-1:: GFP (see Fig. 5) was a partial fusion that included 5 kb of upstream regulatory sequence followed by the first five codes fused in-frame with GFP. The following plasmids were used: pPB45 (glr-1:: GFP), GFP excised from pPD119.45 (A. Fire) and inserted into a HindIII site of pV1 (glr-1 genomic clone), inserting GFP 16 amino acids upstream of the GLR-1 C terminal; pMJ3 (glr-2:: GFP), the XhoI-XmaI fragment from pV29 (glr-2 genomic clone) subcloned into pPD95.79, truncating the last 13 codons of the Genefinder-predicted glr-2-coding sequence; pDM46 (glr-7:: GFP), a 6.7 kb PCR product subcloned into the PstI and XmaI sites of pPD95.75, fusing GFP after S191 (for amino acid numbers, see those in Fig. 1); pDM38 (glr-4:: GFP), a 15.9 kb PCR product subcloned into the SalI and BamHI sites of pPD95.75, fusing GFP after A196; pDM4 (glr-3:: GFP), an 11.9 kb PCR product subcloned into the SphI and XbaI sites of pPD95.77, fusing GFP after S190; pYZ4 (glr-5:: GFP), a 12 kb PCR product amplified from cosmid ZC196 and subcloned into SphI and BamHI sites of pPD95.77, fusing GFP immediately upstream of the Genefinder-predicted stop codon; pDM42 (glr-6:: GFP), a 15 kb PCR product subcloned into the SphI and XbaI sites of pPD95.75, fusing GFP after T184; pDM41 (glr-8:: GFP), a 12.5 kb PCR product subcloned into the SalI and BamHI sites of pPD95.77, fusing GFP after S186; pPB1 (nmr-1:: GFP), a 5 kb PCR product subcloned into the SphI and SalI sites of pPD95.81, fusing GFP after the first five codons predicted by Genefinder; pPB17 (nmr-2:: GFP), an 8 kb product subcloned into the SphI and XmaI sites of pPD95.75, fusing GFP after S221; and pPB38, a 4.6 kb genomic fragment that encodes UNC-42 subcloned into the KpnI and NotI sites in MCSII of pV145.
Transgenic strains. Transgenic strains were generated by microinjection of lin-15(n765ts) mutants to achieve germ line transformation (Mello et al., 1991
Sequence analysis and alignments. To identify genes encoding potential ionotropic glutamate receptors, we used the tblastn BLAST algorithm (Altschul et al., 1997
Analysis of the amino acid sequences of glutamate receptor subunits was performed using the CLUSTALW program (Thompson et al., 1994
Behavioral assays. Nose touch assays were performed as described previously (Kaplan and Horvitz, 1993
Microscopy. Epifluorescence images were acquired using a Zeiss Axioskop microscope and a Princeton Instruments Micromax charge-coupled device camera. Confocal images were acquired using an Optiphot-2 microscope (Nikon) and a Bio-Rad Confocal Imaging System.
RESULTS
Both NMDA and non-NMDA classes of ionotropic glutamate receptors are expressed in C. elegans
We have identified by sequence similarity searches (BLAST) 10 genes encoding putative glutamate receptor subunits in C. elegans (see Materials and Methods). glr-1 has been described and encodes a subunit most similar to the non-NMDA receptor subtypes (Hart et al., 1995
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Figure 1. Partial sequence alignments of predicted C. elegans ionotropic glutamate receptors. Sequence alignment of a highly conserved region of ~400 amino acids of GLR-1 and the additional nine putative C. elegans glutamate receptor subunits is shown. This region includes the four hydrophobic domains (TMI-TMIV, solid underline) and two ligand-binding domains (S1, S2, dashed underline with arrows) that are similar in sequence to bacterial amino acid-binding proteins (Nakanishi et al., 1990
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Table 1. Percentage (%) of sequence identity between selected C. elegans and rat glutamate receptor subunits The topological arrangement of each protein is predicted to be similar to that of other NMDA and non-NMDA receptors. We identified four hydrophobic regions, three of which are predicted to be transmembrane domains (Fig. 1, TMI, TMIII, and TMIV). TMII, the pore-forming region, is a reentrant loop that does not traverse the membrane (Hollmann et al., 1994
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Figure 2. Comparison of C. elegans, Drosophila, and rat ionotropic glutamate receptor subunits. Sequence alignment of C. elegans, Drosophila, and rat ionotropic glutamate receptor subunits beginning immediately at TMI and terminating at TMIII is shown. C. elegans subunits have signature features found in both vertebrate and invertebrate subunits. These include predicted hydrophobic domains (solid underline), a conserved glutamine residue in TMII (filled circle; a target of RNA editing in a subset of vertebrate non-NMDA subunits), and a sequence of nine amino acids in TMIII (dashed line) found in almost all known ionotropic glutamate receptor subunits. This region also contains a conserved alanine residue (filled triangle) that when mutated to threonine causes the ion channel to be constitutively open (Zuo et al., 1997
The relationship between the putative C. elegans and known vertebrate glutamate receptor subunits was analyzed by generating a neighbor-joining tree of receptor subunits (Fig. 3). Clear groupings of the subunits can be distinguished. Thus, GLR-1 and GLR-2 group together and are most similar to rat AMPA receptors. GLR-3 and GLR-4, and more weakly GLR-5 and GLR-6, also group together. GLR-3-GLR-7 are clearly non-NMDA receptor subunits but cannot be obviously classified into pharmacological subtypes. GLR-8 is the farthest outlier and is even more divergent than glutamate receptors identified in Arabidopsis. NMR-1 groups with rat NR1 and Drosophila NR1, and NMR-2 groups with rat NR2 receptors.
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Figure 3. Phylogenetic tree of the amino acid sequences for glutamate receptor subunits. Unrooted phylogenetic tree of the amino acid sequences for the 10 identified C. elegans putative glutamate receptor subunits and selected rat, Drosophila, and Arabidopsis subunits (see Materials and Methods) is shown. The predicted C. elegans polypeptides are based on the cloned partial cDNAs (Fig. 1). The schematic shows clusters of relationships between the amino acid alignments of the glutamate receptor subtypes. The total length of the horizontal lines between different receptors is proportional to the difference between sequences; distance along the vertical axis has no significance. Five hundred bootstrap replicates were performed, and the bootstrap values are indicated by dots on the supporting branches. No dot indicates a value <50%. At, A. thaliana.
Many neurons in C. elegans express multiple glutamate receptor subunits
To determine which cells in C. elegans expressed a particular glutamate receptor subunit, we examined transgenic worms that expressed chimeric fusion proteins to the reporter molecule GFP (Chalfie et al., 1994
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Figure 4. Glutamate receptor-GFP fusion proteins. Schematic representation of the genomic fusions to GFP for each of the glutamate receptor subunits. Boxes represent exons, and horizontal lines between boxes represent introns. Black and gray boxes represent genomic sequence that was either included or omitted from the GFP fusion construct, respectively. The underlined regions represent intron and exon boundaries that have been confirmed by cDNA sequence that was used to predict the amino acid sequence shown in Figures 1 and 2. The amount of upstream regulatory sequence included in each fusion is indicated as is the site of GFP fusion (inverted triangle).
Ionotropic glutamate receptor subunits were expressed in a locomotory control circuit and in a thermosensory circuit
Many of the glutamate receptor subunits were found to be expressed in the command interneurons that control worm locomotion (Fig. 5). For example, the neuron AVA expresses both the non-NMDA subtypes GLR-1, GLR-2, GLR-4, and GLR-5 and the NMDA subtypes NMR-1 and NMR-2. Other command interneurons expressed various subsets of these subunits (Table 2). Several patterns emerged from the expression data. First, the NMDA-type receptor subunits NMR-1 and NMR-2 were always coexpressed and were found in a relatively small number of neurons that included four of the five command interneurons. Second, many neurons expressed multiple non-NMDA receptor subunits in various combinations. For example, AVB expressed GLR-1 and GLR-5; AVD expressed GLR-1, GLR-2, and GLR-5; RIM expressed GLR-1, GLR-4, and GLR-5; and RIF expressed GLR-4 and GLR-5. Presumably, these neurons could create a variety of mature heteromeric receptors from these subunits. Third, some neurons expressed only one subunit. For example, RIB expressed only GLR-4, LUA expressed GLR-5, and AIA expressed GLR-2. Whether these subunits can form functional homomeric receptors or whether other subunits were expressed at levels too low to detect remains to be determined.
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Figure 5. Four non-NMDA and two NMDA ionotropic glutamate receptor subunits are expressed in the command interneurons of the locomotory control circuit. Confocal micrographs of transgenic worms expressing GFP fusions to glr-1, glr-2, glr-4, glr-5, nmr-1, and nmr-2 are shown. Only the anterior head and posterior tail regions of the worm are shown (two left columns). A schematic reconstruction of the neuronal cell bodies in the head and tail of the worm are also shown [two right columns; adapted from White et al. (1986)
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Table 2. Neuronal distribution of glutamate receptor subunits Although most glutamate receptor subtypes are expressed in multiple neurons, two non-NMDA subunits, GLR-3 and GLR-6, were exclusively expressed in the thermosensory interneuron RIA (Fig. 6). This neuron also expressed the GLR-2 receptor subunit (Fig. 5). RIA is considered the major integrative thermosensory interneuron because it receives input from the interneurons AIY and AIZ. In addition, as revealed by laser ablation studies, it is required for worms to thermotax in defined temperature gradients (Mori and Ohshima, 1995
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Figure 6. Two ionotropic glutamate receptor subunits are exclusively coexpressed in the thermoregulatory interneuron RIA (arrows). A, B, Confocal micrographs (anterior region only) of transgenic worms expressing glr-3:: GFP (A) and glr-6:: GFP (B) are shown. C, Expression of both fusion proteins is detected in a single neuron, the thermoregulatory interneuron RIA (arrow). Scale bars, 5 µm.
Two genes, glr-7 and glr-8, were primarily expressed in the pharyngeal nervous system
The pharynx of C. elegans functions to ingest and process food. It contains 58 cells, including 20 neurons that comprise the pharyngeal nervous system. This collection of neurons is almost entirely separate and independent from the rest of the C. elegans nervous system (Albertson and Thomson, 1976
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Figure 7. Two ionotropic glutamate receptors are coexpressed in the pharyngeal nervous system. A, Confocal image of a transgenic worm expressing the glr-7:: GFP transgene in the pharyngeal nervous system (head region only). B, Diagram of the pharynx and the neuronal cell bodies that express glr-7:: GFP. C, D, Confocal images of a transgenic worm expressing glr-8:: GFP in the pharyngeal nervous system (C, head region only) and in a single neuron in the tail (arrow; D). E, F, Neuronal cell bodies that express glr-8:: GFP. Neurons filled in black coexpress glr-7:: GFP and glr-8:: GFP. Neurons filled in gray only express glr-8:: GFP in either the pharyngeal nervous system (E; URB is the only neuron of this subset that is not considered part of the pharyngeal nervous system) or the tail (F). Scale bars, 5 µm.
Expression of all glutamate receptor subunits began during embryogenesis
Expression of the GFP reporter constructs could be observed at all larval stages and in the adult. The onset of expression was typically noted in late embryogenesis (Fig. 8). glr-5 had the earliest onset of expression in which GFP fluorescence could be observed at the twofold stage (~450 min after fertilization). Within another 100 min of development (at the threefold stage), expression of glr-1, glr-2, glr-4, glr-7, glr-8, nmr-1, and nmr-2 was first observed. We could not detect glr-3 or glr-6 expression until near hatching. However, the GFP signal from the transgenic strains that expressed these constructs was rather weak, and the late onset of expression may simply reflect low expression levels.
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Figure 8. Expression of ionotropic glutamate receptor subunits can be detected during embryogenesis. A-C, Image of a twofold stage embryo that expressed glr-5:: GFP (arrowheads). Expression of this fusion protein was detected the earliest of all of the GFP fusion transgenes. D-F, Image of a threefold stage embryo that expressed glr-1:: GFP. Most of the GFP fusions were first detected at this stage. G, Time course of embryogenesis (minutes) indicating the approximate time of the earliest detected expression of each of the receptor subunits. glr-3:: GFP and glr-6:: GFP expression was not detected during embryogenesis. A, D, Nomarski. B, E, FITC. C, F, Overlay. Scale bars, 5 µm.
Expression of GLR-4 and GLR-5 was dependent on the homeodomain protein UNC-42
The genetic control of glutamate receptor expression is not well understood. In a recent study, the homeodomain gene unc-42 was found to be required for the development of neuronal identity and specifically required for the expression of GLR-1 in the AVA, AVD, and AVE interneurons and the RMD motor neurons (Baran et al., 1999
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Figure 9. UNC-42 is required for expression of GLR-1, GLR-4, and GLR-5. A-C, Confocal images of unc-42(e270) transgenic worms that expressed glr-1:: GFP (A), glr-4:: GFP (B), and glr-5:: GFP (C). D-F, Diagram of neuronal cell bodies that express glr-1 (D), glr-4 (E), and glr-5 (F). Cell bodies filled in gray and black represent those that expressed the respective subunits in wild-type worms. Cell bodies filled in gray represent those that no longer express the subunits in unc-42 mutants. unc-42 is required for glr-1 expression in the command interneurons AVA, AVD, and AVE and in the RMD motor neurons (Baran et al., 1999
unc-42 regulates axon outgrowth in a subset of the command interneurons
The command interneurons have been divided into two interconnected subclasses, those that primarily regulate backward movement (AVA, AVD, and AVE) and those that primarily regulate forward movement (AVB and PVC) (Chalfie et al., 1985
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Figure 10. unc-42 disrupts the axon outgrowth of AVA, AVD, and AVE. A-D, Confocal images of transgenic unc-42(e419) mutants (A, B) and unc-42(e270) mutants (C, D) that expressed the nmr-1:: GFP transgene. Abnormal processes can be observed extending anterior to the nerve ring (arrows) or along dorsal or lateral paths (arrowheads). E, Confocal image of a transgenic unc-42(e270) mutant expressing both nmr-1:: GFP and a wild-type unc-42 genomic clone. Axon outgrowth is indistinguishable from that of wild-type worms. F, Wild-type worm that expressed nmr-1:: GFP. All images show the head region only, with anterior on the left and more posterior regions on the right of each panel. See Figure 5 for the identity of neurons that express nmr-1:: GFP. G, Processes of the ventral cord in an nmr-1:: GFP; unc-42(e270) mutant. Only four processes were visible in the ventral cord (three of these indicated by arrows), whereas nine are expected in a wild-type worm. Migration of a subset of the processes terminated prematurely in the ventral cord (arrowhead). H, I, Schematic diagram of the locomotory control circuit in wild-type (H) and unc-42 mutants (I). The circuits have been divided into forward (black) and backward (gray) components. Gap junctions and chemical synapses between sensory neurons (triangles), command interneurons (hexagons), and motor neurons (circles) are represented by lines and arrows, respectively. In wild-type worms (H) the DA motor neurons that direct backward movement receive synaptic input from the backward command interneurons (AVA, AVD, and AVE). In unc-42 mutants (I) these connections are disrupted, and the worms have difficulty moving backward. Note that both the forward and backward command interneurons receive synaptic input from sensory neurons (AVM and ASH) that normally drive backward movement in response to anterior tactile stimulation. Scale bars, 5 µm.
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Table 3. unc-42 behavioral defects
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Table 4. unc-42 axon outgrowth defects Because nmr-1:: GFP expression was not dependent on unc-42, we were able to use this transgene to study the morphology of the command interneurons in unc-42 mutants. Transgenic unc-42 mutants showed severe axon outgrowth defects in the backward command interneurons AVA, AVD, and AVE (Fig. 10). Normally, the processes of these neurons enter the nerve ring and then travel posteriorly in the ventral cord (Fig. 10F) (White et al., 1986
DISCUSSION
A detailed analysis of how individual receptor subunits influence synaptic transmission and contribute to neuronal processing and behavior is difficult in the vertebrate nervous system because of its complexity. We have begun to address these questions by undertaking a comprehensive analysis of the distribution of glutamate receptors in the relatively simple nervous system of C. elegans. This analysis is a first step toward identifying the various combinations of subunits that interact to form ion channels and determining the specific contribution of these channels to worm behavior. In the C. elegans nervous system, individual neurons can be identified, receptor expression can be modified, and neuronal function can be studied in parallel with behavioral analysis.
Neuronal expression of glutamate receptors in C. elegans
In the vertebrate nervous system, 18 different receptor subunits, as well as alternatively spliced or RNA-edited variants, are differentially expressed throughout the CNS (Petralia and Wenthold, 1996
In C. elegans, our cloning and expression studies revealed a surprising number of putative glutamate receptor subunits for an organism with only 302 neurons. By comparing the amino acid sequence of these subunits with known vertebrate receptor subunits, we have assigned subunits to NMDA and non-NMDA classes. However, whether receptors formed from these subunits are characteristically activated by the selective pharmacological agents AMPA, kainate, and NMDA remains to be determined. The receptor subunits were predominantly expressed in interneurons, and most neurons expressed a number of receptor subunits. Subunits closely related by sequence were likely to be coexpressed in the same neurons, e.g., GLR-1 and GLR-2 or NMR-1 and NMR-2. However, some neurons expressed only one subunit, which might therefore form functional homomeric receptors. Alternatively, we have not identified all of the ionotropic glutamate receptor subunits in C. elegans (see Materials and Methods for additional candidate glutamate receptors). In contrast, other neurons, such as AVA, expressed six receptor subunits. Interestingly, unlike vertebrate neurons, most C. elegans neurons do not express NMDA receptors. Consequently, the majority of neurons that express non-NMDA receptor subtypes do not express either of the NMDA subtypes NMR-1 or NMR-2. Unlike the case for vertebrate glutamate receptor subunits, we found no evidence of alternative splicing or RNA editing of any subunits. In Drosophila, neuromuscular transmission is glutamatergic; however, in C. elegans, glutamate receptor expression in muscle cells was not observed.
The subunit composition of vertebrate glutamatergic synapses changes with activity. Whether the initial formation of glutamatergic synapses is also dependent on activity is unclear. One model suggests that glutamatergic synapses initially contain only NMDA receptors and that non-NMDA receptors are later recruited to the synapse in an NMDA receptor and activity-dependent process. Thus, synaptic plasticity is achieved by modulating the relative level of non-NMDA receptor expression in response to patterns of synaptic activity. In this view, the expression of glutamate receptors at synapses is a dynamic process and is partly dependent on the activation of NMDA receptors. Thus, in hippocampal CA1 pyramidal neurons, certain synapses, termed "silent synapses," contain only NMDA receptors (Liao et al., 1995
What are the functions of C. elegans glutamate receptor subunits? How might they contribute to neuronal processing in the worm? Hints are provided by their respective neuronal expression patterns. Thus, GLR-3 and GLR-6 were expressed in an interneuron required for thermotaxis and may be required for normal thermotaxis behavior; GLR-7 and GLR-8 were expressed in the pharyngeal nervous system and may participate in pharyngeal function; and GLR-1, GLR-2, GLR-4, GLR-5, NMR-1, and NMR-2 were expressed in command interneurons and may contribute to locomotory control. The large number of subunits in some neurons suggests that considerable receptor diversity may exist. However, which subunits form functional glutamate-gated channels remains to be determined.
How many neurons are glutamatergic?
The widespread expression of glutamate receptors in interneurons and motor neurons suggests that many sensory neurons and interneurons are glutamatergic. A recent study identified EAT-4 as a putative Na-Pi cotransporter in C. elegans that is required for vesicular uptake of glutamate (Lee et al., 1999
Expression of glutamate receptor subunits is differentially regulated by UNC-42
Mutations that affect the connections among the locomotory command interneurons should disrupt locomotion. For example, vab-8 mutants are uncoordinated and have particular difficulty moving backward. In these mutants the outgrowth of most posteriorly directed axons, including the axons of a subset of the command interneurons, is disrupted (Wightman et al., 1996
UNC-42 is required for axon outgrowth of the backward command interneurons
By using the nmr-1 promoter to express GFP in AVA, AVD, and AVE in unc-42 mutants, we have shown that the most likely cause of uncoordinated movement in unc-42 mutants was disruption of synaptic communication secondary to defective axon outgrowth of AVA, AVD, and AVE. Whether these axon outgrowth defects were secondary to defects in glutamate receptors, defects in the expression of additional gene products in interneurons, or defects in the target tissues remains to be determined. Our results suggest that the sensory defects (nose touch and body touch) may also be caused by disruption of the interneuronal circuitry. When these neurons were killed either by a laser (Chalfie et al., 1985
UNC-42 is also expressed in the ASH sensory neurons that are required for the nose touch response. However, the processes of these neurons are not disrupted in unc-42 mutants (Baran et al., 1999
The forward movement of unc-42 mutants is disrupted, although the forward command interneurons do not express UNC-42 and their processes are normal in unc-42 mutants. These results support a model in which the function of the command interneurons is, in part, distributed (Zheng et al., 1999
Summary
In a small and relatively simple nervous system, such as that found in C. elegans, single neurons perform many tasks undertaken by ensembles of neurons in larger nervous systems. For example, the amphid sensory neurons that detect olfactory cues express numerous genes encoding odorant receptors, whereas in the vertebrate olfactory system each neuron is thought to express only one specific receptor (Chess et al., 1994
Note added in proof. Several recent reviews have addressed the predicted glutamate receptors that were identified by the C. elegans genome sequencing consortium. These reviews have used different receptor nomenclatures. Littleton and Ganetsky (2000)
FOOTNOTES Received Aug. 9, 2000; revised Nov. 22, 2000; accepted Dec. 12, 2000.
This research was supported in part by the Sloan Foundation, by National Institutes of Health Grant NS35812, and by a University of Utah Graduate Research Fellowship (P.J.B.). We thank M. Vetter and members of the Maricq laboratory for comments on this manuscript. We also thank C. Bargmann for assistance with cell identifications and for helpful discussions, the C. elegans sequencing consortium for cosmid clones, Chris Rongo and Josh Kaplan for nuIs25, the Caenorhabditis Genetic Center for some nematode strains, and the HCI DNA Sequencing Core Facility.
Correspondence should be addressed to Dr. Andres Villu Maricq, Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840. E-mail: maricq{at}biology.utah.edu.
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