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Genetics. Mar 2005; 169(3): 1437–1450.
PMCID: PMC1449549

Genetic Control of Temperature Preference in the Nematode Caenorhabditis elegans

Abstract

Animals modify behavioral outputs in response to environmental changes. C. elegans exhibits thermotaxis, where well-fed animals show attraction to their cultivation temperature on a thermal gradient without food. We show here that feeding-state-dependent modulation of thermotaxis is a powerful behavioral paradigm for elucidating the mechanism underlying neural plasticity, learning, and memory in higher animals. Starved experience alone could induce aversive response to cultivation temperature. Changing both cultivation temperature and feeding state simultaneously evoked transient attraction to or aversion to the previous cultivation temperature: recultivation of starved animals with food immediately induced attraction to the temperature associated with starvation, although the animals eventually exhibited thermotaxis to the new temperature associated with food. These results suggest that the change in feeding state quickly stimulates the switch between attraction and aversion for the temperature in memory and that the acquisition of new temperature memory establishes more slowly. We isolated aho (abnormal hunger orientation) mutants that are defective in starvation-induced cultivation-temperature avoidance. Some aho mutants responded normally to changes in feeding state with respect to locomotory activity, implying that the primary thermosensation followed by temperature memory formation remains normal and the modulatory aspect of thermotaxis is specifically impaired in these mutants.

ANIMALS can modulate behavioral responses by sustained training and retain memory for a certain time. Learning and memory have been successfully investigated using model organisms such as Aplysia, Drosophila, and mice. Various learning paradigms have been developed in each model and analyzed behaviorally, physiologically, and pharmacologically (Mayford and Kandel 1999). From the standpoint of genetics, the development of conditioning assays in Drosophila made it possible to identify and manipulate genes involved in learning and memory (Waddell and Quinn 2001). On the basis of olfactory associative conditioning, Drosophila learning mutants were isolated. For example, the molecular and genetic analysis of dunce and rutabaga mutants revealed that cAMP-specific phosphodiesterase and Ca2+/calmodulin-stimulated adenylyl cyclase are essential for establishment of learning and memory (Chen et al. 1986; Qiu et al. 1991; Levin et al. 1992).

The nematode Caenorhabditis elegans is well suited to a behavioral genetic approach to dissecting the molecular mechanism of learning and memory because of its accessible genetics (Brenner 1974), its stereotyped behavioral responses, and the ease of controlling experimental conditions in the laboratory. C. elegans is also ideal for the study of the cellular basis of learning and memory because of our complete anatomical knowledge of its nervous system, consisting of 302 identifiable neurons with all neuronal connections known (White et al. 1986). C. elegans can respond to various environmental cues, such as taste, smell, touch, and temperature, to ensure its own survival and reproduction in the natural environment. In addition to these primary behavioral responses, neural plasticity, learning, and memory can be observed in C. elegans despite its simple nervous system. Recently, various studies aiming to understand these complex mechanisms have been carried out on olfactory adaptation, nonassociative learning in mechanical tap response, and locomotory rate modulated by feeding state (Rankin et al. 1990; Colbert and Bargmann 1995; Sawin et al. 2000).

Hedgecock and Russell (1975) reported and defined thermotaxis of C. elegans. Well-fed animals migrated to their cultivation temperature and then moved isothermally, temperature preference of well-fed animals changed a few hours after cultivation temperature was shifted, and animals dispersed from their cultivation temperature after cultivation under starved and overcrowded conditions. These phenomena implicate two types of neural modulation in thermotaxis. First, animals store the temperature information after exposure to a certain temperature for a few hours. Second, animals somehow associate the cultivation temperature with the environmental condition, either pleasant or unpleasant, and drastically change the response to the temperature. Neural-circuit-regulating thermotaxis under well-fed conditions was proposed by laser-killing experiments (Mori and Ohshima 1995). It is highly likely that some of the neurons in the neural circuit are also required for neural modulations of thermotaxis.

In this study, we have demonstrated that C. elegans changes temperature preference in response to cultivation temperature and feeding states and that the memory formations for temperature and for feeding state are discrete processes. We also found that, as in the case of pharyngeal pumping and egg laying, exogenous serotonin and octopamine can mimic a well-fed and a starved state, respectively, in thermotaxis. We isolated several aho mutants, some of which are probably able to recognize the feeding state normally but are defective in reversal of temperature preference. We believe that some of these aho mutants are the novel example of thermotaxis-defective mutants, in which thermotactic neural modulation by the feeding state is specifically damaged. It is thus likely that the molecular and cellular analysis of aho genes should shed light on understanding mechanisms for neural plasticity, learning, and memory.

MATERIALS AND METHODS

Strains and genetics:

The techniques used for culturing and handling C. elegans were essentially as described by Brenner (1974). The following strains were used in this work: wild-type C. elegans variety Bristol strain (N2), CB1111 cat-1(e1111)X, CB1141 cat-4(e1141)V, MT7988 bas-1(ad446)III, GR1321 tph-1(mg280)II, MT2426 goa-1(n1134)I, aho-1(nj5), aho-2(nj32), aho-3(nj15), nj22, nj23, nj24, nj26, nj27, nj28, nj30, nj31, wild-type C. elegans variety Hawaiian strain (CB4856), CB61 dpy-5(e61)I, CB51 unc-13(e51)I, CB3297 vab-9(e1744)II; him-5(e1490)V, CB189 unc-32(e189)III, CB185 lon-1(e185)III, CB49 unc-8(e49)IV, CB270 unc-42(e270)V, CB678 lon-2(e678)X, and CB151 unc-3(e151)X.

Thermotaxis assay:

A radial temperature-gradient assay was performed using a 9-cm agar plate without Escherichia coli (OP50), a food source, and a vial containing frozen acetic acid, as described by Mori and Ohshima (1995) with some modification. C. elegans was usually cultured at 20° from egg to L4 larval stage, and then L4 larvae, ~40 animals/6-cm NGM plate or ~120 animals/9-cm NGM plates, were cultured for 1 day under uncrowded conditions at designated temperature. In this study, we used a 45-ml glass vial (catalog no. 224833, Wheaton) to maintain a stable temperature gradient, and animals were allowed to move freely for 50 min on an assay plate. After the assay, animals were categorized into four groups, “17,” “20,” “25,” and “17/25,” on the basis of the tracks left on the assay plates (see legend of Figure 1, B and C, for detailed classification of thermotactic phenotype). Thermotaxis was evaluated using “fraction of 17,” which is the fraction of animals categorized as “17,” or “fraction of 25,” which is the fraction of animals categorized as “25.” The evaluation of thermotactic responses was made in a completely blind manner: an individual, who had done the experiment, and at least an additional individual with a career in thermotaxis assay for >3 years, who had not done the experiments and had not been told the experimental design, blindly evaluated the track left on each assay plate and categorized it into one of four groups. Then, the evaluations that had been individually made were put together and the final classification of each assay was determined by at least two individuals. The statistically significant differences of thermotaxis behavior were determined by using ANOVA for repeated measures.

Figure 1.
Radial temperature-gradient assay. (A) Radial temperature-gradient assay was performed using a 9-cm agar plate and a vial containing frozen acetic acid. Thermograph (TVS-610, Nippon Avionics) showed the surface temperature of the plate contacting a glass ...

Cultivation-temperature-shift assay:

Well-fed naive animals were cultured with fresh food for 1 day under uncrowded conditions at designated temperatures of 17° or 25°. For cultivation-temperature-shift assay, well-fed naive animals were washed three times with NG buffer (0. 3% NaCl, 1 mm CaCl2, 1 mm MgSO4, 25 mm, pH 6.0, potassium phosphate/liter) and transferred to conditioning plates (2% agar, 1 mm CaCl2, 1 mm MgSO4, 25 mm, pH 6.0, potassium phosphate) at designated temperatures of 17° or 25°. The number of animals placed on a conditioning plate was restricted to 20 animals/6-cm plate or 120 animals/9-cm plate to assure cultivating animals under uncrowded conditions. Conditioning plates with fresh food or without food were preincubated at designated temperature overnight until the thermotaxis assays were conducted. Thermotaxis of cultivation-temperature-shifted animals was assayed at designated times after transfer (see Figures 2, 4, and 5 for details of time-course assays). To investigate the thermotactic responses of each time point after temperature shift, thermotaxis of animals after shifting the temperature was assayed at each time point in a 1-day cultivation-temperature-shift experiment, which was repeated over more than a few days.

Figure 2.
The memory formation to a new temperature takes a few hours for well-fed animals in the radial temperature-gradient assay. (A) Experimental procedure for cultivation-temperature-shifted assay at the well-fed state. The solid ellipsoid indicates a conditioning ...

Feeding-state-shift assay:

Naive animals were prepared using the same procedure as described for well-fed naive animals in Cultivation-temperature-shift assay above. For food-deprived animals, well-fed naive animals were washed three times with NG buffer and transferred to conditioning plates without food. The number of animals placed on a conditioning plate was restricted to 120/9-cm plate to cultivate animals under uncrowded conditions. Conditioning plates were preincubated at designated temperatures overnight until the thermotaxis assays were conducted. To prevent animals from swimming off the agar, conditioning plates contained a high-osmolarity ring of 8 m glycerol at the periphery (Culotti and Russell 1978). The 8-m glycerol ring did not affect thermotaxis behavior (data not shown). Thermotaxis of food-deprived animals was assayed at designated time after transfer (see Figures 35, 7, and 8 for details of starvation time). For the experiment on recultivation with food, starved animals were transferred to conditioning plates with food as described by Colbert and Bargmann (1997). The number of animals was restricted to 20/6-cm conditioning plate to cultivate the animals under uncrowded conditions. Food drops were spotted on conditioning plates and incubated at 17° or 25° before transfer for at least a few hours. About 10 animals recultivated with food were assayed every 10 min after transfer. Thermotaxis of conditioned animals was continuously assayed in a day, and thermotaxis assays were repeated over more than a few days.

Figure 3.
Feeding states solely modulated thermotaxis behavior. (A and D) Experimental procedure for feeding-state-shifted assay at the same cultivation temperature. An ellipsoid indicates conditioning plate: a solid ellipsoid indicates cultivation with food and ...
Figure 5.
(A) Animals conditioned to be well fed at 17° were cultivated without food. (B) Animals conditioned to be well fed at 25° were cultivated without food. (A and B) About 20 animals were examined at each time point in four trials. Top arrows ...

Pharmacological treatment:

For serotonin-treated animals, well-fed animals grown at 25° were cultivated on serotonin plates (10 mm serotonin, 2% agar, 1 mm CaCl2, 1 mm MgSO4, 25 mm, pH 6.0, potassium phosphate) without food at 25° for 2 hr. For octopamine-treated animals, well-fed animals grown at 25° were cultivated on octopamine plates (20 mg/ml octopamine, 2% agar, 1 mm CaCl2, 1 mm MgSO4, 25 mm, pH 6.0, potassium phosphate) with food at 25° for 2 hr.

Mutant screen:

Wild-type (N2) hermaphrodites at L4 stage were mutagenized by ethyl methanesulfonate, and aho mutant candidates were isolated from F2 progeny (Brenner 1974). About 5700 genome-F2 progeny grown at 25° were transferred to conditioning plates without food under uncrowded conditions for ~2–4 hr. These starved animals were then placed at 22.5°–23.5° on a Sephadex gel slurry (Sephadex G-200, superfine, Sigma, St. Louis), which had a linear temperature gradient from ~20° to 25° (Hedgecock and Russell 1975). After 1 hr, animals migrating at ~25° were picked to individual plates. Thermotaxis of these aho candidates was retested at well-fed and food-deprived states at least five times after conditioning at 17° or 25° to exclude athermotactic and thermophilic mutants from aho mutant candidates, and we selected as aho mutants strains that exhibit normal thermotaxis in a well-fed state but with abnormal cultivation-temperature avoidance in a starved state.

Locomotory rate assay:

The procedure for the locomotory rate assay using a bacterial lawn was essentially according to Sawin et al. (2000) with some modifications. The 6-cm assay plates (2% agar, 1 mm CaCl2, 1 mm MgSO4, 25 mm, pH 6.0, potassium phosphate) with food were prepared before a few hours by spreading an OP50-enriched solution, a much concentrated overnight-cultured OP50, as described previously (Colbert and Bargmann 1997). OP50 was spread in a square with ~3 cm. Food-deprived animals grown at 17° or 25° were left in conditioning plates under uncrowded conditions for 2.5 hr or 30 min, respectively. Two minutes after transfer to assay plate, the number of body bends was counted for 10 animals for each strain.

Genetic mapping of aho-1(nj5), aho-2(nj32), aho-3(nj15), nj26:

All mutations were outcrossed by N2 at least two times before mapping and were mapped using the classical genetic method and the snip-SNPs method, a rapid gene mapping method using a high-density polymorphism map. aho-1(nj5) mutation was mapped to linkage groups using the following visibly marked tester strains: dpy-5(e61)I, unc-13(e51)I, vab-9(e1744)II; him-5 (e1490)V, unc-32(e189)III, lon-1(e185)III, unc-8(e49)IV, unc-42(e270)V, and lon-2(e678)X. aho-2(nj32), aho-3(nj15), and nj26 mutations were mapped to linkage groups using the snip-SNPs method, a procedure essentially as described by Wicks et al. (2001). In addition, nj26 mutation was also mapped using unc-3(e151)X.

RESULTS

Modulation of temperature preference by cultivation temperature and feeding state:

Hedgecock and Russell (1975) reported that well-fed animals migrate to their cultivation temperature and move isothermally and that this thermotaxis behavior is modified by two kinds of experience. First, behavioral acclimation to a new cultivation temperature requires a few hours, after cultivation temperature of well-fed animals was shifted. Second, starved and overcrowded conditions make animals disperse from their cultivation temperature. In this study, we mostly analyzed thermotaxis of wild-type animals individually by changing condition of cultivation temperature and/or feeding state, whereas Hedgecock and Russell (1975) analyzed thermotaxis of population of animals mainly using a liner temperature-gradient assay. In an individual assay, a stable radial temperature gradient, ranging from 17° to 25°, was established in a 9-cm agar plate without food (Figure 1A), and thermotaxis of individual animals was evaluated by categorizing animal tracks into four groups (Figure 1, B and C). Most well-fed animals grown at 17°, 20°, and 25° migrated to their cultivation temperature in an individual assay with a radial temperature gradient (Table 1 and Figure 10A) as observed in a population assay with a linear temperature gradient (Hedgecock and Russell 1975; data not shown).

Figure 10.
aho-2(nj32) mutant exhibits normal thermotaxis at the well-fed state. (A) Typical tracks of well-fed wild-type animals cultivated at 17°, 20°, and 25°. (B) Typical tracks of well-fed aho-2(nj32) mutants cultivated at 17°, ...
TABLE 1
Phenotypic distributions of well-fed wild-type animals

We shifted the cultivation temperature of animals with a well-fed state and analyzed the time course of behavioral response to a new cultivation temperature using an individual radial temperature-gradient assay (Figure 2A). After the cultivation temperature was shifted from 17° to 25° for a few hours, most well-fed animals migrated to 25°, although naive animals that had been cultivated continuously at 17° seldom migrated to 25° (Figure 2B). After the cultivation temperature was shifted from 25° to 17° for a few hours, most well-fed animals migrated to 17°, although naive animals continuously cultivated at 25° seldom migrated to 17° (Figure 2C). These results suggest that memory formation for a new cultivation temperature takes a few hours for well-fed animals and the speed of memory formation appears to be independent of cultivation temperature.

Hedgecock and Russell (1975) observed that animals from very old plates (>30% dauer larvae) as well as animals grown at high density in liquid culture dispersed from their cultivation temperature. To start illuminating more precisely the environmental parameters that influence thermotactic responses, we first investigated the effect of food deprivation on thermotaxis (Figure 3A). When well-fed animals preconditioned to migrate to 17° were cultivated without food at the same temperature (17°), they seldom migrated to the cultivation temperature after experiencing food deprivation for 3 hr (Figure 3B). When well-fed animals preconditioned to migrate to 25° were cultivated without food at the same temperature (25°), they seldom migrated to the cultivation temperature after experiencing food deprivation for 30 min (Figure 3C). The evaluation of individual thermotactic responses at various time points revealed that starvation induces avoidance and not simple dispersion from the temperature associated with a food-deprived condition (Figure 3, B and C). For example, most 17°-grown animals migrated to 25° while they hardly migrated to 17°, and most 25°-grown animals migrated to 17° while they hardly migrated to 25° (Figure 3, B and C). These results suggest that food deprivation alone can modulate thermotaxis and the speed of the modulation depends on cultivation temperature.

We next examined the effect of food supply on thermotactic response of the animals that had been conditioned to avoid cultivation temperature (Figure 3D). The starved animals cultivated at either 17° or 25° immediately recovered from the starved state and migrated to their cultivation temperature after recultivation with food for only 10 min (Figure 3, E and F). These results suggest that the transition from a starved to a well-fed state is quickly triggered by food and that, in contrast to the transition from a well-fed to a starved state, this neural modulation is independent of cultivation temperature.

Distinct memory formations for temperature and feeding state:

We showed that application of food to starved animals instantaneously triggered the switch from an aversive to an attractive response to cultivation temperature. To investigate this quick transition in more detail, we changed simultaneously two parameters, cultivation temperature and feeding state. We first shifted cultivation temperature at the time when starved animals were provided food. When starved animals preconditioned to avoid 17° were exposed to food at 25° for 10 min, most animals migrated to the former cultivation temperature of 17°, the temperature that used to act as an aversive signal for the animals (Figure 4A). Likewise, when animals that had been starved at 25° were recultivated with food at 17°, most animals initially migrated to 25°, the noxious temperature associated with food-deprived experience (Figure 4B). In similar temperature/feeding state shift experiments, we next shifted cultivation temperature at the time when well-fed animals experienced starvation prior to recultivation with food. When well-fed animals cultivated at 17° were transferred to conditioning plates without food for 3 hr at 25° and then recultivated with food at 25°, the conditioned animals seldom migrated to 17° and mostly migrated to 25° after 10 min of recultivation with food (Figure 4C). When well-fed animals cultivated at 25° were transferred to conditioning plates without food at 17° for 3 hr and then recultivated with food at 17°, the conditioned animals seldom migrated to 25° and a fraction of animals migrating to 17° started to increase immediately after recultivation with food (Figure 4D). In this latter shift experiment, a fraction of animals migrating to 17° increased even faster after recultivation with food when animals were cultivated at 17° under food-deprived conditions for 4 instead of 3 hr (data not shown). Despite the initial and transient attractive response to the previous aversive temperature, the fraction of animals that migrated to 25° or 17°, a new temperature associated with food signal, gradually increased (Figure 4, A and B). These results suggest that food intake quickly causes the transition of thermotactic response from aversion to attraction to the temperature in memory. Interestingly, starved animals started to prefer the new attractive temperature associated with food much faster than well-fed animals (Figures 2 and and4),4), which suggests that starvation accelerates the formation of new temperature memory.

Figure 4.
The memory formation processes for temperature and feeding states are biologically discrete. (A) Animals conditioned to be starved at 17° for 3 hr were recultivated with food at 25°. (B) Animals conditioned to be starved at 25° ...

We also shifted cultivation temperature when well-fed animals were cultivated under food-deprived condition (Figure 5). After well-fed animals that had been preconditioned to migrate to 17° were cultivated without food at 25°, they first behaved to avoid 17°, the former attractive temperature, and after 1 hr of starvation the fraction of animals migrating to 17° started to increase (Figure 5A). In the converse temperature/feeding state shift experiment, where well-fed animals preconditioned to migrate to 25° were transferred to plates without food and cultivated at 17°, the conditioned animals first avoided 25°, and after a few hours of starvation the fraction of animals migrating to 25° started to increase (Figure 5B). These results suggest that food deprivation stimulates the transition of thermotactic responses from attraction to aversion to direct avoidance from the temperature in the animals' memory.

On the basis of the results obtained in this study, we propose a conceptual model for thermotaxis (Figure 6). First, both temperature and feeding state, indicative of environmental condition, are sensed independently (reception). Second, a prolonged exposure to the same condition leads the animals to store thermal information and feeding state independently (learning and memory), although these two parameters, temperature memory and feeding-state memory, interact with each other. For example, the speed of inducing cultivation-temperature avoidance depends on cultivation temperature, and starvation accelerates the formation of new temperature memory. Third, on a temperature gradient, the animals assess the changes in thermal information relative to the stored thermal and feeding-state memories (assessment). Finally, the animals thus migrate to either a colder or a warmer area and move isothermally around the region of interest as a final decision (movement). We suggest that thermotaxis is a kind of associative learning, an ideal behavioral paradigm advantageous to investigating neural plasticity at molecular and cellular levels.

Figure 6.
A conceptual model for thermotaxis.

Serotonin and octopamine can mimic a well-fed or a food-deprived state, respectively, in thermotaxis:

Our results demonstrated that feeding condition is an important trigger to stimulate the transition between aversive and attractive responses to temperature in animals' memory. Feeding state is also known to modulate egg laying, pharyngeal pumping, and locomotion in C. elegans. The previous pharmacological analysis of these behaviors showed that exogenous serotonin and octopamine can mimic well-fed and food-deprived states, respectively (Horvitz et al. 1982; Trent et al. 1983; Avery and Horvitz 1990; Y. Shibata and S. Takagi, personal communication). Thus, serotonin and octopamine are likely to act as neurotransmitter or neurohormone to regulate feeding states antagonistically in C. elegans.

We addressed whether these biogenic amines are also involved in neural modulation of thermotaxis. When well-fed animals preconditioned to migrate to 25° were placed on food-deprived plates containing serotonin for 2 hr, most of these serotonin-treated animals migrated to 25° on a thermal gradient, regardless of food-deprived experience (Figure 7A). We also examined the effect of exogenous octopamine on thermotactic behavior. When well-fed animals preconditioned to migrate to 25° were placed on food plates containing octopamine for 2 hr, most octopamine-treated animals moved away from 25°, although they were physically fed with real food (Figure 7B). Consistent with the previous report on other behaviors, our results showed that exogenous serotonin and octopamine can also mimic a well-fed and a food-deprived state, respectively, in thermotaxis.

Figure 7.
Exogenous serotonin and octopamine can mimic well-fed or food-deprived states, respectively, in thermotaxis. (A and B) Well-fed animals conditioned to migrate to 25° were transferred onto plates with or without drug and with or without food for ...

In C. elegans, serotonin is thought to be transported into secreted vesicles by CAT-1, a vesicular monoamine transporter, which is required for neurotransmitter release at synaptic terminal and has serotonin transport activity in mammalian cells (Duerr et al. 1999). Serotonin is absent from neuronal processes but present in cell bodies in cat-1(e1111) mutants, which are abnormal in egg laying, pharyngeal pumping, locomotion, and male mating (Sulston et al. 1975; Duerr et al. 1999). Interestingly, we found that cat-1(e1111) mutants that had been preconditioned to avoid 25° exhibited a significant delay in the recovery from the starved state (Figure 7C). This result suggests that monoamines transported by CAT-1 act as feeding signals in thermotaxis.

Serotonin is synthesized from tryptophan via two steps. In mutants defective in the biosynthesis pathway, cat-4(e1141), bas-1(ad446), and tph-1(mg280), the endogenous serotonin level appears to be reduced (Sulston et al. 1975; Loer and Kenyon 1993; Sze et al. 2000). We analyzed these mutants and found that they are essentially normal in all aspects of thermotactic responses tested, although tph-1(mg280) mutants cultivated at 25° were too sick to test any aspects of thermotactic responses (data not shown). Two possibilities explain the normal responses of these serotonin mutants. Serotonin is not necessary but sufficient to convey feeding signal, and other neurotransmitters or neurohormones transported by CAT-1 act as additional feeding signals. Alternatively, endogenous serotonin is still synthesized at low levels in these mutants and the small amount of serotonin is sufficient to work as a feeding signal. Previous immunostaining analysis showed that the endogenous serotonin was undetectable in cat-4(e1141), bas-1(ad446), and tph-1(mg280) mutants (Desai et al. 1988; Loer and Kenyon 1993; Sze et al. 2000), although a report suggests that at least the cat-4(e1141) mutation did not lead to a complete loss of serotonin (Ranganathan et al. 2001).

Isolation of mutants defective in modulation of thermotaxis by cultivation temperature and feeding state:

We performed a genetic screen to identify genes and cells required for feeding-state-dependent modulation of thermotaxis (Figure 8A). We mutagenized a wild-type strain and sought animals that were defective in cultivation-temperature avoidance. Among those animal lines, we sought mutant strains that exhibited normal thermotaxis after cultivation under well-fed conditions. It is highly possible that the primary thermosensation followed by temperature storage remains normal in these mutants, designated aho (abnormal hunger orientation). Finally, we examined whether aho mutants were able to recognize change of feeding state normally, since in theory the failure to respond to the feeding state per se could cause the Aho phenotype, defective in cultivation-temperature avoidance.

Figure 8.
A screen for mutants defective in modulation of thermotaxis affected by feeding states. (A) An experimental strategy to obtain mutants defective in neural modulation of thermotaxis by feeding states. (B) Results of 17°-thermotaxis assay for aho ...

Fifteen aho mutants were isolated among ~5700 genomes screened. Of 11 mutants, aho-1(nj5) was a dominant mutation and the others were recessive mutations (data not shown). These 11 aho mutants were defective in cultivation-temperature avoidance compared to wild-type animals (Figure 8, B and C). Statistical significances of aberrant thermotactic responses of aho mutants at starved state were evaluated by ANOVA for repeated measures. Statistical analysis was applied independently between wild type and each aho mutant, although Figure 8, B and C, shows the averages of all thermotaxis assays for each strain. Cultivated at 17°, nj26, nj30, and aho-2(nj32) had significant differences (P < 0.0002), nj28 and nj31 had significant differences (P < 0.004), and nj27 had significant differences (P < 0.03). Cultivated at 25°, aho-1(nj5), aho-3(nj15), and nj22 had significant differences (P < 0.0005), nj23 and nj24 had significant differences (P < 0.001), nj28 had significant differences (P < 0.07), and aho-2(nj32) had significant differences (P < 0.08). Although the thermotactic abnormalities of nj28 and aho-2(nj32) mutants cultivated at 25° were statistically not as significant as other aho mutants, both mutants showed apparently impaired responses to starvation. Most of aho mutants seem to be classified into three groups. The first group exhibits Aho after cultivation at both 17° and 25° (i.e., aho-2(nj32)), the second group exhibits Aho at 17° (i.e., nj26), and the third group exhibits Aho at 25° (i.e., aho-3(nj15)).

To assess whether food recognition is normal or not in these aho mutants, we tested the feeding-state modulation of locomotory activity. Well-fed wild-type animals move more slowly in plates with food than in plates without food (basal slowing response) and starved animals move even more slowly in plates with food than well-fed animals in plates with food (enhanced slowing response; Sawin et al. 2000). Some aho mutants exhibited nearly the same responses as wild type in locomotory rate assay (Figure 9). Our results suggest that these aho mutants are normal in food recognition and defective in modulation of thermotaxis by cultivation temperature and feeding state, although we cannot rule out the possibility that a yet-to-be-known food recognition pathway, which is not reflected in change in locomotory activity, could be abnormal in these aho mutants. In addition, it is likely that aho mutants that exhibited abnormality in locomotory assay this time will come to exhibit a normal response of change of feeding state, if these are outcrossed several times with wild-type N2 strain.

Figure 9.
Locomotory rate assay of aho mutants. (A) Modulation of locomotory rate in aho mutants cultivated at 17°. Starved animals were conditioned by cultivation under food-deprived conditions for 2.5 hr. (B) Modulation of locomotory rate in aho mutants ...

Molecular dissection and behavioral analysis of aho mutants:

To understand mechanisms for thermotactic neural modulation and to gain insights into the nature of the defects, we tried to investigate aho mutants in detail and analyzed the other sensory behaviors.

The aho-2(nj32) mutant exhibited normal thermotaxis at the well-fed state (Figure 10), but was defective in reversal of temperature preference modulated by starvation at 17° and 25° (Figure 11). aho-2(nj32) was normal in chemotaxis to Na+ ion, avoidance of Cu2+ ion, and olfaction (diacetyl, isoamyl alcohol, benzaldehyde, and trimithylthiazole; data not shown). According to mapping results using snip-SNPs method, nj32 mapped to the region near the center of chromosome IV (Figure 12).

Figure 11.
aho-2(nj32) mutant defective in cultivation-temperature avoidance induced by starvation. (A) Typical tracks of wild-type animals and aho-2(nj32) mutants cultivated at 17°. Starved animals were conditioned by cultivation under food-deprived conditions ...
Figure 12.
The map position for each aho gene. Linkage groups are designated by roman numerals and X for the sex chromosome. The hen-1 mutant also exhibits Aho phenotype at 17° and 25° (Ishihara et al. 2002).

The aho-1(nj5) mutant exhibited Aho at 25°. aho-1(nj5) was normal in olfactory adaptation (diacetyl, pyrazine, isoamyl alcohol, and benzaldehyde), pharyngeal pumping, and defecation, and aho-1(nj5) had a slight defect of chemotaxis to NaCl (data not shown). According to linkage analysis using visible markers, aho-1(nj5) was linked to the region between the left and the center of chromosome I (Figure 12).

The aho-3(nj15) mutant exhibited Aho at 25°. aho-3(nj15) was normal in chemotaxis to NaCl, avoidance of Cu2+ ion, and olfaction (diacetyl, pyrazine, isoamyl alcohol, and benzaldehyde), and aho-3(nj15) was defective in male mating behavior (data not shown). Using snip-SNPs method, aho-3(nj15) was mapped near the center of chromosome I (Figure 12).

The nj26 exhibited Aho at 17°. nj26 was normal in olfaction (diacetyl and benzaldehyde) and slightly abnormal in chemotaxis to Na+ ion (data not shown). Using the classical genetic method and snip-SNPs method, nj26 was mapped to the right of chromosome X (Figure 12).

DISCUSSION

Neural modulation by cultivation temperature and feeding state in thermotactic responses:

Hedgecock and Russell (1975) reported that C. elegans cultured under very harsh conditions, such as cultivation in very old culture plates or in high density, dispersed from their cultivation temperature. Under these conditions, starvation, high density of dauer pheromone, and other unpleasant environmental factors are all likely to cause dispersion from cultivation temperature. In this study, we focused on food condition and showed that feeding state alone could be an important cue to modulation of behavioral responses to environmental temperature. These results suggest that C. elegans can associate cultivation temperature with feeding-state information and modify behavioral outputs in response to environmental changes.

After animals that had been preconditioned to migrate to cultivation temperature were cultivated under food-deprived and uncrowded conditions, they showed the complete reversal of temperature preference, from attractive to aversive response to the cultivation temperature (Figure 3, B and C). We also found that avoidance of the cultivation temperature turned into attraction to the temperature immediately after recultivation with food (Figure 3, E and F). Intriguingly, the transition from attractive to aversive thermotactic response stimulated by food deprivation occurred much more slowly at low (17°) temperature than at high (25°) temperature, although the time required for the transition from aversive to attractive response by food supply was similar at both temperatures. It is plausible to suppose that different mechanisms participate in feeding-state-dependent thermotactic modulation at low and high cultivation temperatures.

Our conditioning experiments with shifting temperature and feeding state simultaneously suggest that the memory formation processes for temperature and for feeding states occur independently but both processes are closely related and hardly separable with one another (Figures 4 and and5).5). Recultivation of starved animals with food first exhibited thermotaxis to the cultivation temperature that used to be an aversive cue (Figure 4, A and B). Further, food deprivation of well-fed animals initially evoked avoidance of the temperature that used to be an attractive cue (Figure 5, A and B). These results suggest that the switch between attraction and aversion stimulated by the change in feeding state establishes much more quickly than the formation of a new temperature memory. Our results also revealed that starvation experience accelerated the acquisition of a new temperature memory (Figures 2 and and4)4) and that the exposure to a new temperature influenced the time required for the transition from attractive to aversive response to cultivation temperature (Figure 3, B and C; Figure 5, A and B). These results implicate the close interaction between temperature memory and feeding-state memory processes (Figure 6).

Ryu and Samuel (2002) reported that C. elegans migrate to lower temperature but not to higher than cultivation temperature within several minutes after temporal temperature change. However, analyses using special (linear and radial) temperature-gradient assays indicate that animals do migrate to their cultivation temperature by moving up no less than down on a thermal gradient (Hedgecock and Russell 1975; Mori and Ohshima 1995; Komatsu et al. 1996; Kuhara et al. 2002). In addition, another study using temporal thermal gradients in different ways suggests that animals respond to up and down temporal thermal changes (Zariwala et al. 2003). At least two possibilities explain the different results between our observations and those of Ryu and Samuel (2002). The difference in duration of assay time may affect evaluation of thermotaxis if both studies observed the same phenomenon. We allowed animals to move freely for 50 min, while Ryu and Samuel observed animals for only several minutes. Alternatively, the different results may reflect the fact that it is harder to observe migration up the gradient of the animals cultivated at higher temperature, as compared to observing migration down the gradient of the animals cultivated at lower temperature. For example, animals cultivated at 25° started to avoid 25° for only 10 min of starvation (Figure 3A). Thus, it is highly possible that animals cultivated at higher temperature started to starve during experimental manipulation and did not migrate to cultivation temperature. It is important to make clear the biological link between our results and the different conclusions deduced from these behavioral studies.

Feeding-state signals in thermotaxis:

C. elegans eats bacteria as its food source. It is difficult to determine what C. elegans recognizes as a feeding signal. Bacteria are a composite stimulus that perhaps consists of taste, smell, touch, and so forth. Also, upon food intake the stimulation of an animal's metabolism likely takes place in addition to the activation of food-sensing signaling. We showed that exogenous serotonin and octopamine mimicked a well-fed and a starved state in thermotaxis, respectively, as in other behaviors such as egg laying, pharyngeal pumping, and locomotion (Figure 7, A and B). These results suggest that serotonin and octopamine act as feeding and food-deprived signals, respectively, to regulate temperature preference.

Importantly, CAT-1, a vesicular monoamine transporter with high affinity to serotonin, is required for the recovery from a starved state to a well-fed state at 25° (Figure 7C). One plausible scenario is that serotonin is released at a synaptic terminal as neurotransmitters or neurohormones as C. elegans eats food, although other components might act as feeding signals redundantly. Although CAT-1 seems to be a sole vesicular monoamine transporter (VMAT) in C. elegans, the cat-1(e1111) null mutant has no severe defects such as lethality and sterility (Duerr et al. 1999). We found that the cat-1(e1111) mutant exhibited normal reversal of thermotaxis at 17° and no severe defects other than the delay in thermotactic recovery at 25° (data not shown). It is possible that CAT-1 as well as other VMATs yet to be identified may act redundantly and that other VMATs substitute the role of CAT-1 in the cat-1(e1111) mutant.

The mechanism of modulation of thermotactic responses by feeding state:

Neural plasticity is probably concerned with sensory transduction and synaptic or hormonal modulation in sensory neurons and interneurons. HEN-1, a secretory protein with an LDL receptor motif, is known to regulate sensory processing non-cell-autonomously in the mature neuronal circuit and the hen-1 mutant exhibits Aho phenotype at both 17° and 25° (Ishihara et al. 2002). This result suggests that the neuroendocrine system may be important for modulation of thermotaxis. Ishihara et al. (2002) also reported that AIY interneurons are necessary for integration of two sensory signals and the expression of HEN-1 in other neurons is insufficient for substituting its function in AIY neurons (Ishihara et al. 2002). We anticipate that AIY is also crucial for the modulation of thermotaxis. It is conceivable that the synapses connecting with AIY act essentially in two different mechanisms, primary thermosensation followed by temperature memory formation and feeding-state-dependent neural plasticity. Consistent with this hypothesis, some, if not all, thermotaxis-defective mutants isolated to date are also defective in neural modulation by feeding states. For example, mutations in ttx-3, a LIM homeodomain-containing transcription factor expressed in AIY, lead not only to a cryophilic phenotype on a thermal gradient, but also to a defect in integration of two sensory signals (Hobert et al. 1997; Ishihara et al. 2002).

To identify genes and cells required for modulation of thermotaxis by feeding state, we sought mutants that are defective in cultivation-temperature avoidance. aho mutants exhibit normal thermotaxis at a well-fed state and some of them responded to the change of feeding states normally, namely they exhibited normal sensorimotor responses in thermosensation and food recognition. They are the first example of novel thermotaxis-defective mutants in which modulation of thermotaxis by feeding state is specifically impaired.

aho mutants are classified into three groups: the first group exhibits Aho at both 17° and 25°, the second group exhibits Aho at 17°, and the third group exhibits Aho at 25°. This result predicts two possibilities. First, there are at least two mechanisms, a lower-temperature mechanism and a higher-temperature mechanism, for starvation-induced temperature avoidance. For example, in the second group of aho mutants, only the lower-temperature mechanism is damaged and the higher-temperature mechanism remains normal. Alternatively, aho mutants classified into the second and the third groups are simply temperature-sensitive and cold-sensitive mutants, respectively. We favor the former possibility, because prominent differences in time required for the transition from well-fed to starved state were observed in a cultivation-temperature-dependent manner (Figure 3, B and C). The analysis of other behaviors such as olfaction, habituation, and other learning paradigms for aho mutants would help to distinguish these possibilities.

What do aho genes encode? If the underlying mechanism for this paradigm is shared by a mechanism of learning and memory in higher animals, it is likely that some aho genes encode molecules already known to be important for learning and memory such as cyclic-nucleotide signaling pathway, neurotransmission, and cell adhesion. There are many candidates for aho genes in the C. elegans genome, for example, protein kinase A, cAMP response element-binding protein, and neuropeptides (Bargmann 1998). It is also highly possible to identify novel molecules, leading to the discovery of new features to understand neural plasticity. We believe that the further dissection of aho genes will give us great insight and will clarify the mechanism of neural modulation in thermotaxis, which should lead to understanding a mechanism of learning and memory in mammals.

Acknowledgments

We thank Y. Shibata, S. Takagi, M. Hino, and K. Matsumoto for strains; Y. Shibata and S. Takagi for permission to use their unpublished data; Nippon Avionics for thermal video system TVS-610; Y. Ohshima for help at the initial stage of this work; N. Nishio for photographs of animal tracks; H. Inada and other members of the Mori laboratory for stimulating discussion; and O. Hobert, M. Okumura, A. Kuhara, and H. Inada for comments on the manuscript. Caenorhabditis Genetics Center provided some of the nematode strains used in this study. This work was supported by research grants from PRESTO, Japan Science and Technology Corporation, the Ministry of Education, Culture, Science, and Sports of Japan, and the Human Frontier Science Program Organization (to I.M.).

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