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Molecular Genetics Research Laboratory, The University of Tokyo, Tokyo 113-0033, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresSupplementary materialsReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialsReferences |
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In the olfactory systems, adaptation is also commonly observed in a broad range of animals. Olfactory adaptation is generally considered to occur at multiple levels, that are often categorized into the peripheral level (occurring within sensory neurons) and the central level (occurring at higher-order relay neurons and interconnections between them) (Dalton 2000). Odor adaptation at the peripheral level has been intensively analyzed, mostly using isolated olfactory neurons from various species, and its molecular mechanisms are well documented. In vertebrate olfactory neurons, modulation by calcium/calmodulin of cyclic nucleotide-gated channels plays a major role in odor adaptation, which occurs within seconds (Munger et al. 2001). Modulation of adenylyl cyclase by calcium/calmodulin-dependent protein kinase II is also responsible for adaptation (Leinders-Zufall et al. 1999). On the other hand, carbon monoxide (CO) and cGMP mediate a slower form of adaptation (Zufall & Leinders-Zufall 2000).
In addition to the peripheral adaptation, the involvement of central adaptation (also called habituation), which depends on neural circuits, has been recognized through various observations, and is becoming a focus of interest in the context of olfactory information processing. In humans, the cognitive perception of odor adapts more rapidly than electrophysiological responses of olfactory receptor neurons (Hummel et al. 1996). In rats, electrophysiological studies have shown adaptation to occur at the connections between olfactory receptor neurons and second-order neurons in the main olfactory bulb (MOB), as well as at the connections between MOB projection neurons and third-order neurons such as those in the piriform cortex (PCX) (Potter & Chorover 1976; Wilson 1998). Lateral neural connections may play roles for adaptation in both MOB and PCX (Yokoi et al. 1995; Wilson 2003). Plasticity in both of these relay structures is implicated not only in sensory adaptation but also in the refinement of odor receptive fields and odor discrimination (Fletcher & Wilson 2003). In Drosophila, long-term odor exposure causes long-lasting adaptation (lasting for more than a week). This form of adaptation involves the reduction of both volume and the number of synapses in the antennal robes, which are the insect olfactory centers. A mutant impaired in the cAMP phosphodiesterase, dunce, is defective in this form of adaptation (Devaud et al. 2001). Despite these observations, the functioning of the neural circuitry and the underlying molecular mechanisms for odor adaptation are poorly understood due to the complexity of the structures in these organisms.
In order to analyze neural circuit-dependent odor adaptation at the molecular level, simple and genetically tractable model organisms are beneficial. The soil nematode Caenorhabditis elegans has a compact nervous system consisting of 302 neurons, whose connections have been fully documented (White et al. 1986), including the whole circuitry of the olfactory system. Outputs of the olfactory nervous system can be detected as chemotaxis behavior toward odorants (Bargmann et al. 1993). Based on the powerful genetics available in C. elegans, the functions of olfactory neurons and the molecular mechanisms of odor perception have been well documented in this organism. In AWC olfactory neurons, the sensory transduction pathway consists of GPCR odorant receptors, G proteins, the cGMP pathway, and cyclic nucleotide-gated channels (Bargmann & Kaplan 1998). The sensory transduction pathway is similar overall to those of higher organisms, thus providing a basis for using C. elegans as a model for understanding olfactory systems. In addition, the Ras-MAPK pathway is activated by odor stimuli in olfactory neurons (Hirotsu et al. 2000). The response of MAPK is also conserved in mammalian olfactory neurons (Watt & Storm 2001).
It is known that C. elegans shows odor adaptation as well at the behavioral level. After continuous exposure to an odorant for more than 30 min, C. elegans shows declined chemotaxis to that odorant (Colbert & Bargmann 1995). Impairment of this form of adaptation was reported for mutants in the osm-9 gene, which encodes a TRPV ion channel, and overproduction of ODR-1, a guanylyl cyclase. The cyclic-GMP-dependent protein kinase EGL-4 is also important for adaptation. Phosphorylation of the beta subunit of the cyclic nucleotide gated channels TAX-2, which may be catalyzed by EGL-4, is essential for adaptation after 30 min of odor exposure, while the nuclear localization of EGL-4 is required for adaptation caused by odor exposure for one hour (L’etoile et al. 2002). On the other hand, calcineurin A subunit TAX-6 negatively regulates adaptation and acts antagonistically with OSM-9 (Kuhara et al. 2002). It has been suggested that these molecules act in the olfactory neurons, thereby altering the response of the olfactory neurons to odorants. However, neural circuit-dependent odor adaptation has not so far been reported in C. elegans.
In this study, we establish an assay system for analyzing neural circuit-dependent odor adaptation in C. elegans. This adaptation behavior requires AIY interneurons, which have synaptic inputs from olfactory neurons. The mutants of the Ras-MAPK pathway exhibit defects in early adaptation. MAP kinase is activated by odorant stimuli not only in the olfactory neurons, but also in the AIY interneurons. While activity of the Ras-MAPK pathway in the olfactory neurons is important for odor perception, its activity in the AIY interneurons is important for odor adaptation. These results reveal that the Ras-MAPK pathway regulates neural circuit-dependent adaptation and suggest a dual role of the pathway in olfactory sensory processing.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialsReferences |
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C. elegans exhibits chemotaxis to a set of odorants. Earlier studies showed that this response was decreased through adaptation after long pre-exposure to odorants, typically for an hour (Colbert & Bargmann 1995; Bernhard & Van Der Kooy 2000). We made several modifications to the assay protocols, including the procedure for odor pre-exposure and the plate layout for chemotaxis assays (see Experimental procedures). In this assay, we found that odor adaptation is observed after a very short pre-exposure to odorants: when worms were pre-exposed to odorants for 5 min, they showed greatly reduced chemotaxis to the same odorants at normally attractive concentrations (Fig. 1A, Supplementary Movie S1). This form of adaptation, early adaptation, has been observed for isoamyl alcohol, benzaldehyde (known to be sensed by AWC olfactory neurons, Bargmann et al. 1993), pyrazine and diacetyl (sensed by AWA). Pre-exposure to butanone, however, did not cause significant decrease in chemotaxis (data not shown). Extents of early adaptation depended on concentrations of both pre-exposed odorants and attractants on chemotaxis plates. We have observed that 10–4 dilution at pre-exposure is optimal for all odorants that cause early adaptation. On the other hand, optimal concentrations of attractants on chemotaxis plates varied between odorants, with adaptation to isoamyl alcohol and diacetyl being less pronounced at high attractant concentrations (Fig. 1A). Based on these observations, we fixed the assay conditions (see Experimental procedures). The decreased chemotaxis is unlikely to be due to a nonspecific effect of the pre-exposure treatment, since chemotaxis to NaCl (which is sensed by sensory neurons other than AWC and AWA) is not affected by pre-exposure to the odorants (Fig. 1B). The persistence of the adaptation is graded with the duration of the odor pre-exposure. After pre-exposure to the odorants for 5 min, 30 min are required for recovery, while the adaptation induced by a longer pre-exposure (30 min) requires a longer recovery time (Fig. 1C), suggesting that the underlying mechanisms may be different between the short and long exposures (see below).
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Cross-adaptation is observed between the two AWC-sensed odorants, isoamyl alcohol and benzaldehyde, as previously reported for the conventional adaptation assay (Fig. 2A, filled and stippled bars) (Colbert & Bargmann 1995), and observed between two AWA-sensed odorants, pyrazine and diacetyl, where pyrazine exposure causes significant decrease in chemotaxis to diacetyl (Fig. 2B, hatched and cross-hatched bars). Unexpectedly, we found that cross adaptation occurs between AWC-sensed and AWA-sensed odorants in our assay. Five minutes of pre-exposure to AWA-sensed pyrazine and diacetyl caused a reduction in the chemotaxis to the AWC-sensed odorants, isoamyl alcohol and benzaldehyde (Fig. 2C, hatched and cross-hatched bars). Pre-exposure to AWC-sensed benzaldehyde also caused decreased chemotaxis to AWA-sensed pyrazine (Fig. 2D, stippled bars). No significant cross-adaptation was observed in other combinations of odorants. Although the basis for this limited occurrence of cross adaptation is currently unknown, cross-adaptation appears to occur only for chemotaxis mediated by olfactory neurons, because pre-exposure to odorants does not affect chemotaxis to NaCl.
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The AIY interneurons are essential for early adaptation
The observation of cross-adaptation led us to test the possible involvement of AIY interneurons, which receive synaptic inputs from both the AWA and AWC olfactory neurons and are known to be important for sensory processing (White et al. 1986; Mori & Ohshima 1995; Ishihara et al. 2002) (Fig. 3A). The ttx-3 mutants have strong defects in the differentiation of AIY and weak defects in that of the AIA interneurons (Hobert et al. 1997; Altun-Gultekin et al. 2001). All the ttx-3 mutants tested (ks5, mg158, ot22, ot23) exhibited severe defects in cross-adaptation between AWA-sensed and AWC-sensed odorants (pyrazine 
isoamyl alcohol; Fig. 3B, benzaldehyde pyrazine; Fig. 3C and data not shown). However, the defects are not restricted to such types of cross-adaptation, since the mutants also exhibited deficits in cross-adaptation between odorants sensed by same olfactory neurons and in adaptation to single odorants (Fig. 3D–F and data not shown). Therefore, the interneurons appear to be important for early adaptation. Although we cannot exclude the involvement of AIA for adaptation, lack of functional AIY neurons is probably responsible for the adaptation defect observed in the mutants, because of the results of AIY-specific rescue experiments described below.
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Mutants of the Ras-MAPK pathway show defects in early adaptation
We have previously reported that mutants of the Ras-MAPK pathway exhibit weak defects in chemotaxis to low concentrations of odorants, and that exposure to an odorant causes activation of MAP kinase in the olfactory neurons (Supplementary Fig. S1 and Hirotsu et al. 2000). Because the Ras-MAPK signaling pathway is known to be versatile and seemed a good candidate for a mediator of the plasticity observed in the early adaptation assay, we tested the mutants in this pathway and found that they show large defects in the early adaptation assay (Fig. 4A and Supplementary Fig. S1). The mutants tested included the gain-of-function (gf) mutant of let-60 ras, let-60(n1046) (Beitel et al. 1990), the loss-of-function (lf) mutant of let-60 ras, let-60(n2021) (Han & Sternberg 1990), and the presumed null mutant mek-2(n2678), where mek-2 encodes a MAP kinase kinase (Kornfeld et al. 1995; Wu et al. 1995). In contrast to the small or no defects of the mutants in chemotaxis at the odorant concentrations and plate format used in the early adaptation assay, adaptation was severely impaired in the mutants in most odorant combinations (Fig. 4A and Supplementary Fig. S1).
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In contrast to the early adaptation, the let-60(gf), let-60(lf) and mek-2(0) mutants show normal adaptation after longer exposure to the odorants for 30 min or 1 h (Fig. 4D). This suggests that the Ras-MAPK pathway is mainly required for the early phase of adaptation. The ttx-3 mutants also show a normal level of adaptation after long exposure to the odorants (Fig. 4D), in accordance with the presumption that adaptation to a long exposure to odorants (including both ‘short-term’ and ‘long-term’ components (L’etoile et al. 2002)) is caused by the molecular machinery in the olfactory neurons (Colbert & Bargmann 1995; L’etoile & Bargmann 2000; L’etoile et al. 2002). The osm-9 mutant, which has defects in the conventional adaptation assay (Colbert & Bargmann 1995; Colbert et al. 1997), shows no defects in the early adaptation assay (data not shown), further implying that different types of adaptation depend on partially non-overlapping molecular mechanisms.
MAP kinase is activated by odorant stimulus in the AIY neurons
As mentioned above, MAP kinase is activated in the olfactory neurons by odor exposure, whereas the AIY interneurons appear to be important for early adaptation. A question therefore arises as to whether MAP kinase would be activated in the AIY interneurons by an olfactory stimulus as well. We therefore performed immunofluorescence with an antibody against activated (diphosphorylated) MAPK. After 5 min of application of a 10–4 dilution of isoamyl alcohol, the same treatment as that used for the adaptation assays, the activation of MAPK was detected in both AWC and AIY. The staining was observed in the cell bodies, nuclei and neural processes in both neurons (Fig. 5A,B). In contrast, phospho-MAPK staining in the salt-sensing ASE neuron did not increase above background by the same treatment, indicating that the activation of MAPK is not due to nonspecific effect of odorant treatment (Fig. 5B). Staining of activated MAPK in AIY was eliminated in the let-60(lf) mutants (Fig. 5B), indicating that this activation of MAPK in AIY depends on LET-60 Ras. Application of other odorants, benzaldehyde or pyrazine, also caused activation of MAPK in AIY (Fig. 5B). These results are consistent with the general requirement of AIY interneurons for early adaptation and suggest the possibility that the Ras-MAPK pathway may function in AIY interneurons as well as in olfactory neurons.
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To determine whether the Ras-MAPK pathway functions in AIY interneurons or olfactory neurons in early adaptation, we tested whether the expression of the wild-type form of let-60 ras in AIY interneurons or AWC olfactory neurons could rescue the defects of the let-60(lf) mutants in adaptation to AWC-sensed odorants. As reported previously (Hirotsu et al. 2000), expression of let-60(+) in AWC (and two other neurons, AWB and I1) by the gcy-10 promoter rescues the weak chemotaxis defects of the let-60(lf) mutant to AWC-sensed odorants (Fig. 6A). However, AIY-specific expression of let-60(+) by the cryptic ttx-3 promoter did not significantly affect chemotaxis (Fig. 6A). In contrast, AIY-specific expression of let-60(+) rescued the adaptation defects of the let-60(lf) mutants in isoamyl alcohol adaptation, benzaldehyde adaptation, and cross-adaptation between these odorants, while AWC-specific expression did not appear to rescue (Fig. 6B,C and Supplementary Fig. S2). AIY-specific expression also rescued defects in cross-adaptation between AWA-sensed pyrazine and AWC-sensed odorants (Fig. 6D and data not shown). Expression of let-60(+) in AIY using sra-11 promoter, which drives expression in AIY, AVB and a pharyngeal neuron (Troemel et al. 1995), also rescued the adaptation defects under all conditions (data not shown). Taken together, these results strongly suggest that the function of LET-60 in AIY interneurons is important for early adaptation. Its function in sensory neurons, on the other hand, is required for efficient odor perception.
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AIY neurons are known for several functions in the regulation of worm behaviors (Mori & Ohshima 1995; Ishihara et al. 2002; Tsalik & Hobert 2003). To see whether the Ras-MAPK pathway is required for all the functions of AIY neurons, we determined the reversal frequency of the let-60 mutants. AIY neurons are known to act to reduce reversal frequency (Tsalik & Hobert 2003). In accordance, the ttx-3 mutants showed enhanced reversal frequency. In contrast, neither the let-60(gf) nor let-60(lf) mutants showed this defect (Supplementary Fig. S3). These results suggest that the Ras-MAPK pathway is not required for all AIY functions but has some specialized roles, including those for odor adaptation.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialsReferences |
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In this study, we established an assay system for odor adaptation in C. elegans that does not occur solely in olfactory neurons but depends on the function of AIY interneurons. We modified the conventional assay protocols for odor adaptation, which allowed us to detect a form of adaptation that occurs early. Modifications in the plate layout for chemotaxis were particularly essential for the early adaptation assay (See Experimental procedures). On the other hand, it was not essential to pre-expose the animals to the odorants in solution, because similar results were obtained by exposing the animals to vaporized odorants, as described in Colbert & Bargmann (1995). We speculate that our plate layout is more sensitive to odorant avoidance behaviors, which may have a significant contribution to early adaptation (see below). In fact, the layout is similar to that used previously for the long-range odorant avoidance assay (Troemel et al. 1997).
C. elegans, as in most other animals, can sense a wide range of concentrations of volatile odorants (about six orders of magnitude). To detect changing concentrations of odorants, animals probably need to dynamically fine-tune the sensitivity of the sensory system. Sensory adaptation that occurs in a time range of seconds is generally believed to contribute to such tuning mechanisms in sensory systems. Because early adaptation occurs faster than conventional odor adaptation, it might be manifestation of such a dynamic sensitivity-modulation mechanism.
In another view, the mechanisms of early adaptation may serve as a short-term memory necessary for sustained alteration of behaviors. Large amounts of undiluted attractive odorants often induce avoidance behaviors in chemotaxis assays (our unpublished data and Nuttley et al. 2001). In such cases, worms initially move towards the undiluted odorant, then switch the direction of locomotion near the odorant, and leave the odorant until they reach the opposite side of the assay plates. This long-range avoidance requires a memory that lasts for minutes, because worms continue to show the avoidance behavior throughout the assay once they experience a high concentration of odorants. Early adaptation might be a basis for such a behavioral switch from attraction to avoidance. Because avoidance of extreme high concentrations of odorants seems to be required for keeping away from the danger of organic solvents, early adaptation may be essential for the survival of worms.
Although the ethological significance of early adaptation remains unclear, the assay system will be useful for studies on neuronal functions. Several assay systems for analyzing behavioral plasticity in C. elegans are known. However, molecular mechanisms for the plasticity have been mostly uncharacterized (Hobert 2003). As an addition to the repertoire of assay systems for behavioral plasticity, our new assay system provides an opportunity to analyze the molecular mechanisms of sensory adaptation that occur at the interneuron level.
AIY interneurons control early adaptation
Our results suggest that AIY interneurons are essential for early adaptation. These neurons are known to act to reduce reversal frequency of locomotion (Tsalik & Hobert 2003). In principle, this change in the basal locomotion behavior could affect chemotaxis. However, in the ttx-3 mutants, which lack functional AIY neurons, efficient chemotaxis is still observed to high concentration of odorants used in our assays, suggesting that unbiased increase of reversal frequency does not itself impair chemotaxis (Fig. 3B–F, open bars, and data not shown). On the other hand, the let-60(gf) and let-60(lf) mutants, which have normal reversal frequency, show defects in early adaptation. These results suggest that apart from their functions in regulation of basal reversal frequencies, AIY interneurons have other important roles in regulation of chemotaxis after pre-exposure to odorants, and this function depends, at least partly, on the Ras-MAPK pathway.
As mentioned above, large amounts of undiluted attractive odorants often induce avoidance behaviors (our unpublished results and Nuttley et al. 2001), indicating that the same odorants can give both attraction and avoidance signals, depending on the concentration. The behavioral output is probably determined by a balance between these opposing signals, and it is therefore conceivable that AIY regulates the balance of attraction and avoidance signals by inhibiting attraction signals and/or enhancing avoidance signals.
Both positive and negative regulations by AIY have been suggested in the neural circuit. There is suggestive evidence that AIZ interneurons, which are the most prominent synaptic targets of AIY (Fig. 3A and White et al. 1986), may be essential for attraction to odorants, and that modulation of AIZ functions can accelerate odor adaptation (Bargmann & Mori 1997; Sze & Ruvkun 2003). In the neural circuit for thermotaxis and regulation of reversal, removal of AIY and that of AIZ cause opposite effects, suggesting that AIY might be inhibitory to AIZ (Mori & Ohshima 1995; Tsalik & Hobert 2003; Wakabayashi et al. 2004; Gray et al. 2005).
The AIY interneurons also have other synaptic targets (White et al. 1986). These include RIA, RIB and RIM interneurons (Fig. 3A). Through analyses of locomotory behavior and the influence of odorants on that behavior, a negative relationship between AIY and AIZ and a positive relationship between AIY and RIM have been proposed (Gray et al. 2005; Tsalik & Hobert 2003). Although the roles for the interneurons RIA, RIB and RIM in chemoattraction or chemorepulsion to odorants have not been formally addressed so far, they are also good candidate components of the regulatory circuit for early adaptation, and could possibly modulate chemotaxis by regulating reversal and turning behaviors during chemotaxis.
It is also possible that neurons other than AIY have important functions at early steps of sensory processing, because cross-adaptation is not observed in all combinations of odorants (Fig. 2), and pyrazine adaptation is not completely impaired in ttx-3 mutants (Fig. 3F). These observations possibly suggest that some neurons other than AIY may also act in parallel for early adaptation.
Various observations implicate regulation by interneurons in processing of olfactory signals in mammals. These include centrifugal inputs that inhibit excitation of mitral cells in the MOB (main olfactory bulb) (Potter & Chorover 1976); granule cells that convey lateral inhibition signals between mitral/tufted cells in the MOB (Yokoi et al. 1995); interglomerular connections by short axon cells and periglomerular cells that also mediate lateral inhibition (Aungst et al. 2003); and association fiber connections within the piriform cortex that may be important for higher-order refinement of odor information (Haberly 2001). These interneuron connections may also mediate experience-dependent modulation of sensory information (Wilson 2003), similar to the functions of AIY neurons in early adaptation in C. elegans.
The Ras-MAPK pathway plays key roles in early adaptation
Rescue experiments demonstrate that the functions of Ras in AIY interneurons are important for odor adaptation, while the functions of Ras in olfactory neurons are important for odor perception. These results suggest that the Ras-MAPK pathway plays dual roles in olfactory sensory processing in the nervous system of C. elegans.
In response to continued sensory inputs, MAPK becomes activated in AIY. The signals and molecules in AIY that mediate this activation are currently unknown. However, we speculate that calcium signaling plays important roles, because EGTA treatment impairs early adaptation (data not shown) and calcium signaling activates the Ras-MAPK pathway in olfactory neurons of C. elegans (Hirotsu et al. 2000). If this assumption is true, the calcium signaling may be mediated by calcium-dependent activators of Ras (Farnsworth et al. 1995), or UNC-43 CaMKII, as in rat hippocampal neurons (Zhu et al. 2002).
The downstream targets of MAPK important for odor adaptation may not be transcription factors since odor adaptation and recovery from it occurs in a relatively short time. In other organisms, MAPK is known to regulate synapsin I-actin interactions by phosphorylation of synapsin I (Jovanovic et al. 1996), to phosphorylate the A-type potassium channel Kv4.2 (Adams et al. 2000), to stimulate translation (Kelleher et al. 2004) and to regulate the insertion of AMPA receptors into postsynaptic sites during long-term potentiation (Zhu et al. 2002). The last mechanism may be relevant to early adaptation in C. elegans, because mutants affected in GLR-1, a homolog of non-NMDA type glutamate receptors with closet sequence similarity to the AMPA subtype, exhibit defects in early adaptation (our unpublished data).
An ideal system for in vivo analysis of neuronal plasticity
Accumulating evidence shows that Ras and MAP kinase (ERK1/2) play fundamental roles in neuronal plasticity, such as long-term potentiation (LTP), in both vertebrates and invertebrates. Ras and the MAPK cascade are activated downstream of several receptors or channels, such as NMDA receptors, muscarinic acetylcholine receptors, or voltage-gated Ca2+ channels. Activated MAPK then translocates to the nucleus, resulting in phosphorylation and activation of transcription factors such as CREB (reviewed in Adams & Sweatt 2002). This transcription-dependent pathway regulates the late phase of LTP (L-LTP), which is mediated by the de novo expression of genes (Patterson et al. 2001). Apart from this scenario, the Ras-MAPK pathway is also involved in transcription-independent regulation of plasticity (Morozov et al. 2003), but few reports so far point to this function. Thus early adaptation may provide an ideal assay system for understanding the functions of the Ras-MAPK pathway in neural plasticity at the behavioral level. Based on the advantages of the simple neural circuits of C. elegans and powerful genetics, further studies of early adaptation should provide valuable insights in the general molecular mechanisms for behavioral and neural plasticity, as well as those for the processing of sensory information.
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Experimental procedures |
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Strains were maintained under standard conditions. The strains used were the following: N2(wild-type), odr-7(ky4), odr-4(n2144), osm-9(ky10), ttx-3(ks5), ttx-3(mg158), ttx-3(ot22), ttx-3(ot23), let-60(n1046), let-60(n2021), mek-2(n2678).
For the let-60(n1046gf) strain, we recently found that the standard strain, MT2124, carries a side mutation (Hirotsu et al. 2004). Therefore in this study we used the outcrossed strain JN130 for all the assays. This strain shows reduced chemotaxis to the two odorants tested, isoamyl alcohol and diacetyl, at low odorant concentrations, and shows better chemotaxis to isoamyl alcohol than MT2124 (Supplementary Fig. S4 and Hirotsu et al. 2000). We also outcrossed the MT4866 strain, the let-60(n2021lf) strain used in our previous study, and obtained the JN148 strain. This strain shows chemotaxis defects, the extents of which are comparable to the original MT4866 let-60(n2021lf) strain (Supplementary Fig. S4). Therefore, we used the MT4866 strain for all the assays.
Behavioral assays
Early adaptation assays were performed as described below. Animals grown on E. coli NA22 were collected in microfuge tubes and washed three times with basal buffer (5 mM potassium phosphate, 1 mM CaCl2, 1 mM MgSO4 and 0.5 g/L gelatin) and then 100 µL of 10–4 dilutions of odorants in water was added. For control animals, 100 µL of water was similarly added. After 5 min, 1 mL of basal buffer was added and centrifuged for 5 s at 1000 r.p.m. The animals that settled at the bottom were taken and about 50 animals were spotted at the center of 9 cm assay plates. Excess liquid was removed with Kimwipes, at the same time dispersing the animals along the midline of the plates (Supplementary Fig. S5). The number of animals spotted on a plate was critical, because when a greater number of animals were spotted, they tended to form clumps and failed to leave the origin. Residual odorants did not affect the chemotaxis, since animals which were pre-exposed to water and washed with buffer containing the odorants at the same concentration as above showed normal chemotaxis. Assay plates were made as previously described (Bargmann et al. 1993) with some modifications (Supplementary Fig. S5): 1 µL each of odorant and 1 M sodium azide were spotted on two points separated by about 2.5 cm at one end of the plates. Only sodium azide was similarly spotted on the other side. Odorant dilutions for the adaptation assays were: isoamyl alcohol, 10–4; benzaldehyde, 10–2; diacetyl, 10–3; pyrazine, 10–2, except for Fig. 2D and Supplementary Fig. S1H, where 10–4 dilutions were used. Thirty minutes after placing the animals at the center of the plates, the number of animals was counted and the chemotaxis index was calculated.
The chemotaxis index = (A – B)/(A + B)
where A was the number of animals on the odorant-spotted side of the plate and B was the number of the animals on the opposite side, while animals that remained within 0.5 cm of the midline were not counted to exclude immotile animals from consideration. These modifications in the plate layout for chemotaxis were particularly essential for the early adaptation assay. On the other hand, it was not essential to pre-expose the animals to the odorants in solution, because similar results were obtained by exposing the animals to vaporized odorants, in a manner similar to the conventional adaptation assay (Colbert & Bargmann 1995).
The chemotaxis to NaCl was assayed on a plate in which an NaCl gradient was formed for 15 h by placing an agar plug containing 100 mM NaCl on one end of a plate.
The recovery from adaptation was assessed by placing worms on blank chemotaxis plates without odorants after a pre-exposure treatment. Animals were then collected with buffer and spotted on chemotaxis assay plates.
Simple odorant chemotaxis assays (those shown in Figs 4B and 6A and Supplementary Fig. S1A-D) were performed on naive animals using the chemotaxis plates described above for adaptation assays, except for Supplementary Fig. S4. The animals were counted after 1 h. Note that this assay format is different from that used in our previous study (Hirotsu et al. 2000), causing minor discrepancies in chemotaxis indices between the two reports.
In Figs 1–4 and 6 and Supplementary Figs S1, S2 and S4, each set of data represents at least three independent assays. The error bars show the SEM.
Reversal assays
Reversal assays were performed as previously described (Tsalik & Hobert 2003). Reversal frequency was scored for 3 min at 25 °C.
Immunohistochemistry
For identification of the AWC, AIY or ASEL neurons, animals carrying the gcy-10p::GFP, ttx-3p::GFP or gcy-7p::GFP constructs, respectively, were used. After pre-exposure to odorants for 5 min, immunohistochemistry was performed as previously described (Hirotsu et al. 2000). Samples were observed by Zeiss Axioplan 2 epifluorescence microscope and Yokogawa CSU21 confocal unit.
Cell-specific expression of let-60
The construction of gcy-10p::let-60 was previously described (Hirotsu et al. 2000). To drive expression in AIY, a genomic fragment containing about 5 kb of the upstream regulatory region and the first two introns of ttx-3 was used (Wenick & Hobert 2004). let-60 ORF was placed downstream of this fragment so that the coding sequence of let-60 was out of frame relative to that of ttx-3, and a strong translation-initiation signal AAAA was added immediately upstream of the let-60 initiation codon. It has been reported that, although the ttx-3 gene is expressed in several classes of neurons, this fragment drives expression only in AIY (Altun-Gultekin et al. 2001). To test whether the downstream gene is expressed in this frame-shift construct, let-60 ORF was replaced with GFP and introduced into C. elegans. GFP fluorescence was observed in AIY neurons in this strain, indicating the expression of the downstream gene. sra-11 promoter contained about 2.7 kb of upstream region. We confirmed with GFP that this region drives expression in AIY.
These constructs were injected with myo-3p::GFP as a transformation marker at a concentration of 50 ng/µL each. Germ-line transformation was performed as previously described (Mello et al. 1991).
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Supplementary materials |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialsReferences |
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Supplementary Move S1
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Figure S4
Supplementary Figure S5
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Acknowledgements |
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Footnotes |
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aPresent address: Department of Biology, Graduate School of Science, Kyushu University, Fukuoka 812-8581, Japan.
* Correspondence: E-mail: iino{at}gen.s.u-tokyo.ac.jp |
References |
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Received: 20 January 2005
Accepted: 27 February 2005
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chemotaxis after pre-exposure to water; 
chemotaxis after pre-exposure to the odorants. Iaa, Bz, Pz and Da indicate isoamyl alcohol, benzaldehyde, pyrazine and diacetyl, respectively. The attractant dilutions are indicated at the bottom. (B) Chemotaxis to 100 mM NaCl after pre-exposure to water (open bar), isoamyl alcohol (filled bar), benzaldehyde (stippled bar), pyrazine (hatched bar) and diacetyl (cross-hatched bar). (C) Recovery from adaptation. Animals were pre-exposed to a 10–4 dilution of isoamyl alcohol for 5 min (•) or 30 min (
) or 30 min (
