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Published as doi: 10.1096/fj.07-9259com.
 (The FASEB Journal. 2008;22:713-720.)
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(The FASEB Journal. 2008;22:713-720.)
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Modulatory effect of ionizing radiation on food-NaCl associative learning: the role of {gamma} subunit of G protein in Caenorhabditis elegans

Tetsuya Sakashita*,1, Nobuyuki Hamada*,{dagger}, Daisuke D. Ikeda{ddagger}, Sumino Yanase§, Michiyo Suzuki*, Naoaki Ishii|| and Yasuhiko Kobayashi*,{dagger}

* Microbeam Radiation Biology Group, Japan Atomic Energy Agency, Takasaki, Gunma, Japan;

{dagger} Department of Quantum Biology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan;

{ddagger} Molecular Genetics Research Laboratory and Graduate Program in Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan;

§ School of Sports and Health Science, Daito Bunka University, Higashi-Matsuyama, Saitama, Japan; and

|| Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan

1Correspondence: Microbeam Radiation Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki-machi, Takasaki, Gunma 370-1292, Japan. E-mail: sakashita.tetsuya{at}jaea.go.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ionizing radiation (IR) is known to impair learning by suppressing adult neurogenesis in the hippocampus. However, in a mature nervous system, IR-induced functional alterations that are independent of neurogenesis remain largely unknown. In the present study, we analyzed the effects of IR on a food-NaCl associative learning paradigm of adult Caenorhabditis elegans that does not undergo neurogenesis. We observed that a decrease in chemotaxis toward NaCl occurs only after combined starvation and exposure to NaCl. Exposure to IR induced an additional decrease in chemotaxis immediately after an acute dose in the transition stage of the food-NaCl associative learning. Strikingly, chronic irradiation induced negative chemotaxis in the exposed animals, i.e., the primary avoidance response. IR-induced additional decreases in chemotaxis after acute and chronic irradiation were significantly suppressed in the gpc-1 mutant, which was defective in GPC-1 (one of the two {gamma} subunits of the heterotrimeric G-protein). Chemotaxis to cAMP, but not to lysine and benzaldehyde, was influenced by IR during the food-NaCl associative learning. Our novel findings suggest that IR behaves as a modulator in the food-NaCl associative learning via C. elegans GPC-1 and a specific neuronal network and may shed light on the modulatory effect of IR on learning.—Sakashita, T., Hamada, N., Ikeda, D. D., Yanase, S., Suzuki, M., Ishii, N., Kobayashi, Y. Modulatory effect of ionizing radiation on food-NaCl associative learning: the role of {gamma} subunit of G protein in Caenorhabditis elegans.


Key Words: learning behavior • irradiation • nematode • GPC-1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IONIZING RADIATION (IR)-INDUCED learning impairment and abnormalities in the nervous system are important potential risks associated with interplanetary space missions (1) and radiation therapy of brain tumors (2) . An increasing body of data indicates that the response of the central nervous system to IR is an interactive, continuous, and dynamic process (2) . Experimental animal studies (2 3 4 5) have indicated the importance of IR-induced suppression of adult neurogenesis in the hippocampus, where neurons are continuously born throughout life and form an integral site for learning and memory. Although Meshi et al. (6) recently reported on a change in neurogenesis-independent learning behavior induced by environmental enrichment without IR-induced impairment in the exposed rodents, these results are highly debatable (7) . Neurogenesis-dependent and -independent mechanisms that underlie learning behavior remain unclear. To our knowledge, no study has reported on the changes in neurogenesis-independent IR-induced learning behavior in a mature nervous system. Therefore, we aimed to test the hypothesis that IR administered at nonlethal doses does not affect learning behavior in a mature nervous system.

We used Caenorhabditis elegans as a model organism to elucidate the functional response of a mature nervous system. This organism is an attractive option for analyzing learning behavior becaue of its simple neurogenesis-independent mature nervous system (in adults), its established learning paradigm, and its well-characterized genetic information (8) . C. elegans learns by using chemosensory, thermosensory, and mechanosensory inputs. Associative learning can be defined as the modification of the behavior of an animal in relation to a previous experience comprising multiple distinct sensory inputs, such as feeding experience-dependent thermotaxis behavior (9) , chemo- (10) and odor-sensory (11) associative conditioning, and experience-dependent modulation of locomotory rate (12) . Although associative learning is an interesting paradigm for studying the effect of IR on learning behavior comprised of distinct integrated inputs, the precise integral sites involved in this learning paradigm remain largely unknown. In this study, we investigated the learning behavior in a mature nervous system that is independent of neurogenesis by using the food-NaCl associative learning paradigm of C. elegans (10) in which a decrease in chemotaxis toward NaCl is observed only after a combined experience of starvation and exposure to NaCl. This study is the first to address the IR-induced responses of learning behavior in a mature nervous system that are independent of neurogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and culture
The C. elegans strains wild-type N2 and the loss-of-function mutant NL792 gpc-1 (pk298) and the Escherichia coli OP50 strain were obtained from the Caenorhabditis Genetics Center (CGC). Well-fed adult animals were grown at 20°C on a nematode growth medium (NGM) agar plate (13) spread with the E. coli.

Chemotaxis assay (CA)
To examine chemotaxis toward NaCl, a population assay was performed in triplicate using 6 cm petri dishes containing 10 ml assay agar (5 mM KPO4 pH 6.0, 1 mM CaCl2, 1 mM MgSO4, and 2% agar) and according to a previously described method with modifications (10) . To create a NaCl gradient, a cylindrical agar plug (height, 0.7 cm and diameter, 1 cm; identical composition as the assay agar except for the inclusion of 100 mM NaCl) was set at the test spot (Fig. 1 A) located 2 cm from the center of the assay plate, and the plates were incubated overnight. To examine the effects of other gustatory cues, a 6 mm diameter agar plug containing 100 mM of cAMP-NH4 (referred to hereinafter as cAMP) or 500 mM of lysine acetate (lysine) was placed on the test spot of the assay plate, as described previously (14) . Before the CA, the plug was removed and 1 µl 0.5 M NaN3 was spotted at the test spot and at a control spot located 4 cm from the test spot (Fig. 1A ). Conditioned or exposed animals (~100), rinsed with a wash buffer and collected from the conditioning plates, were placed between the test and control spots. The animals that reached the test and control spots were paralyzed using NaN3. To fix the positions of all the animals on the assay plates, the plates were chilled to 4°C at 15 min after chemotaxis was allowed at 20°C. Subsequently, the number of animals within 1.5 cm of each spot area (test or control spot) and those in other areas was counted. The chemotaxis index (CI) was calculated as the number of animals at the test spot area minus the number of animals at the control spot area divided by the total number of animals on the assay plate (Fig. 1A ).


Figure 1
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  		Figure 1. Experimental procedures. A) Conditioning, irradiation, and the CA for the food-NaCl associative learning paradigm. Animals were placed on food–/NaCl– agar petri dishes for mock conditioning, food+/NaCl+ dishes for food/NaCl conditioning, and food–/NaCl+ petri dishes for NaCl conditioning. After {gamma}-irradiation, CAs were performed on the assay plates (see Materials and Methods). T and C indicate the number of animals in the test and control spot areas, respectively. The distance between the test and control spots is 4 cm for NaCl and 6 cm for benzaldehyde, respectively. B) Timeline of learning, irradiation (hatched squares), and CA. Top panel: change in chemotaxis after NaCl conditioning for food-NaCl associative learning. The gray areas represent the food-NaCl associative learning period. The narrow lines indicate the conditioning time. We used 6 procedures: 1) chemotaxis, 2) food/NaCl irradiation, 3) prelearning irradiation, 4) during-learning irradiation, 5) postlearning irradiation, and 6) chronic irradiation (see Materials and Methods). The irradiation periods (hatched squares) are included in the conditioning time.

For the benzaldehyde CAs (15) , 10 cm plates containing 10 ml assay agar were used. The animals were rinsed with a wash buffer (10) and collected from the conditioning plates. Immediately before assays, 1 µl 0.5 M NaN3 was spotted at the center of the test and control spots that were 6 cm apart. Immediately after 1 µl ethanol or 0.5% v/v benzaldehyde in ethanol was spotted at the control or test spot, respectively, the animals were placed at the center of the assay plate, and the CAs were then performed. After 1 h of chemotaxis at 20°C, the assay plates were chilled to 4°C. The animals within 2 cm of either spot (test and control spot) were then counted and the CI calculated.

{gamma}-Ray irradiation and conditioning procedures
The animals were acutely or chronically exposed to {gamma}-rays from 60Co sources at room temperature, followed by incubation at 20°C. We used a high dose rate (32 Gy/min) for acute irradiation and a low dose rate (0.42 Gy/min) for chronic irradiation wherein 4 h were required for 100-Gy irradiation. The irradiated plates were placed horizontally, and polymer-alanine dosimeters (Hitachi Cable, Tokyo, Japan) were used for dose estimation (16) . The SD for the irradiated dose was evaluated by assuming uniformly distributed animals on the plate. For all the experiments, the control animals were sham manipulated and handled in parallel with the test animals, except that they were not exposed to irradiation.

The conditioning procedures for food-NaCl associative learning were performed using the method of Saeki et al. (10) with some modifications. For the experiments, the animals were rinsed and collected from the NGM plates (13) and washed four times with a wash buffer to reduce the effects of the E. coli (10) . To study the effects of IR on the food-NaCl associative learning paradigm, the animals were placed on the following three types of plates: 1) 6 cm petri dishes containing 10 ml agar (5 mM KPO4 pH 6.0, 1 mM CaCl2, 1 mM MgSO4, and 1.7% agar) used as mock-conditioning plates (food–/NaCl–); 2) food/NaCl-conditioning plates (food+/NaCl+) with 50 mM NaCl and E. coli lawn to test IR-induced taste aversion, which were incubated overnight at 37°C and allowed to cool to 20°C before use; and 3) NaCl-conditioning plates (food–/NaCl+) containing 50 mM NaCl used for conditioning for food-NaCl associative learning (Fig. 1A ). After the animals were transferred to the conditioning plates, excess fluids were absorbed using Kimwipes (CRECIA, Tokyo, Japan) and the plates were sealed using Parafilm (Pechiney Plastic Packaging, Chicago, IL, USA).

We used combined procedures for conditioning, irradiation, and the CA depending on the experimental purposes (Fig. 1B ). CI for the naive animals directly collected from the NGM plates was 0.81 ± 0.08, indicating that ~90% animals reached the test spot. With regard to the NaCl conditioning plate, the animals experienced starvation and simultaneous exposure to NaCl, and chemotaxis gradually decreased to 0 after 4 h and reached a plateau (Fig. 1B , top). We divided the learning period into the following 3 periods: the "During learning" 4 h period before a plateau chemotaxis and the pre- and postlearning periods designated "Prelearning" and "Postlearning" (Fig. 1B ). Based on the purpose of the experiments, six procedures were performed, i.e., chemotaxis, food/NaCl irradiation, prelearning irradiation, during-learning irradiation, postlearning irradiation, and chronic irradiation (Fig. 1B ). In the "Prelearning" periods of the chemotaxis and prelearning irradiation procedures (Fig. 1B1 , 3, respectively), we used the mock-conditioning plates. No change in the chemotaxis occurred in the animals conditioned using the mock-conditioning plate (10) . To test IR-induced taste aversion, animals were placed on the food/NaCl-conditioning plates (Fig. 1B2 ). In the "During learning" and "Postlearning" periods, the animals were placed on the NaCl-conditioning plates. Acute {gamma}-irradiation was performed during five procedures (Fig. 1B1-5 ) excluding chronic irradiation.

Statistical analyses
All values in the figures and text are means ± SE. The variation between the data sets was tested by ANOVA and significance by unpaired t tests together with a Bonferroni modification for multicomparison of data. A value of P < 0.05 was considered significant. SYSTAT software (Systat Software, San Jose, CA, USA) was used for data analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose response of chemotaxis after {gamma}-irradiation
Although exposure of C. elegans wild-type L4 larvae to IR doses of up to 800 Gy did not result in significant lethality (17) , the effects of IR on the function of the nervous system remain largely unknown. We first examined the effects of IR on chemotaxis toward NaCl before testing those on the food-NaCl associative learning paradigm because this paradigm depends on the changes in chemotaxis. To determine the limit of IR dose for normal chemotaxis, we measured the chemotaxis of animals exposed to graded doses of {gamma}-rays under mock conditions (Fig. 1A , B1). Mock conditioning did not decrease chemotaxis (10) , and the animals exhibited chemotaxis profiles almost identical to those of naive animals grown on NGM agar plates spread with the E. coli. The CIs of the exposed animals were 0.90 ± 0.03 at 0 Gy, 0.90 ± 0.02 at 114 ± 5 Gy (P=1.0 as compared with the CI at 0 Gy), 0.87 ± 0.03 at 503 ± 20 Gy (P=1.0), and 0.69 ± 0.03 at 980 ± 40 Gy (P<0.05). Less than 500 Gy {gamma}-irradiation did not significantly decrease chemotaxis toward NaCl. Thus, animals exposed to an acute dose of <500 Gy showed normal chemotaxis toward NaCl. We used doses of up to ~500 Gy in our following experiments, i.e., within a dose range that is known not to affect the survival of wild-type C. elegans larvae (17) .

IR is not an aversive unconditioned stimulus for gustatory plasticity in C. elegans
IR induces taste aversion in rodents in which IR functions as an aversive unconditioned stimulus in gustatory sense (18) . To test a taste aversion to NaCl after exposure to IR, C. elegans were placed on the food/NaCl conditioning plates and were {gamma}-irradiated. On the plates, animals sensed NaCl but did not show food-NaCl associative learning behavior (10) . We observed that despite a water-soluble attractant of NaCl, acute IR did not significantly change animal chemosensation (Fig. 2 ). Thus, simultaneous presentation of NaCl and IR did not reduce the chemotaxis toward NaCl, suggesting that IR itself dose not induce a taste aversion to NaCl and that IR would not be an aversive unconditioned stimulus for gustatory plasticity.


Figure 2
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  		Figure 2. Dose response of chemotaxis in animals exposed to acute IR on the food/NaCl conditioning plates. The error bars represent the SE for 4 experiments.

Effects of IR on chemotaxis in various stages of associative learning
To test the effects of IR on the associative learning paradigm of C. elegans, animals exposed to an acute dose of IR at various stages were analyzed during the postirradiation period. First, we performed the prelearning irradiation experiment (Fig. 1B3 ). CIs for the naive and mock-conditioned animals were 0.81 ± 0.08 and 0.90 ± 0.03, respectively. The CI of NaCl-conditioned animals gradually decreased to 0 after 4 h and reached a plateau (Fig. 1B , top). Here we present the actual CI after NaCl-conditioning and exposure to IR. Exposure to IR in the prelearning period is known to suppress learning behavior in mammals (19) . To examine whether {gamma}-irradiation suppresses the ability of C. elegans for associative learning, mock-conditioned animals irradiated with IR during the prelearning period were subjected to 4 h of NaCl conditioning (Fig. 1B3 ). The exposed animals exhibited a decrease in CI to ~0, as well as unexposed animals (Fig. 1B ). No significant difference was observed in the CI compared with that of the control animals (Fig. 3 A1). The result indicates that IR exposure did not significantly inhibit the ability for associative learning, although the CI of the animals exposed to the highest dose differed slightly from those of the nonirradiated controls. With regard to the during-learning irradiation experiment (Fig. 1B4 ), we observed a significant additional dose-dependent decrease in chemotaxis immediately after irradiation (Fig. 3A2 ). This decrease occurred temporally, and at 3 h after irradiation, the CI was higher than that of the nonirradiated control (Fig. 3B ). After 5 h of irradiation, the CIs of the exposed animals reached ~0, except at the highest dose. When the animals were irradiated during the postlearning stage (Fig. 1B5 ), we observed no significant change in CI after 1 h of irradiation (Fig. 3A3 ). These results indicate that IR exposure induces a marked change in chemotaxis in exposed animals only when associative learning is in transition. Thus, the effect of IR on chemotaxis may depend on the specific stage of associative learning.


Figure 3
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  		Figure 3. CIs of animals acutely irradiated with IR at various conditioning time points for associative learning. CIs for naive (0.81±0.08) and mock-conditioned (0.90±0.03) animals were gradually decreased to 0 after 4 h NaCl-conditioning and reached a plateau (Fig. 1B , top panel). Here we present the actual CI. A) Dose-response relationship of CIs immediately after irradiation for the following 3 experiments: 1) prelearning (shaded and dashed bars), 2) during-learning (solid bars), and 3) postlearning (shaded bars) irradiation. B) Time course of CIs in the during-learning irradiation experiment. The data for the first time point are equal to the dose response of the during-learning irradiation experiment shown in Fig. 3A2 . The error bars represent the SE for 5 or more experiments. Means ± SD for the exposed doses are indicated. *Significant differences in CI between the exposed and nonirradiated animals (P<0.05).

Additional IR-induced decrease in chemotaxis is not observed in the gpc-1 mutant
Defective mutants with gustatory plasticity have been previously reported (20 , 21) ; the gpc-1 mutant is defective in GPC-1 (one of the two {gamma} subunits of the heterotrimeric G-protein) that is predominantly expressed in some sensory neurons. In the present conditioning procedure, this mutant could learn by combined stimulation with starvation and exposure to NaCl, although the learning was slightly inhibited (P=0.296 vs. wild-type animals, Fig. 4 A). Mock-conditioned gpc-1 mutants did not exhibit an IR-induced decrease in CI [CI: 0.76±0.06 at 0 Gy, 0.72±0.06 at 115±5 Gy (P=1.0 as compared with the CI at 0 Gy), and 0.73 ± 0.06 at 509 ± 21 Gy (P=1.0)] as well as wild-type animals. Intriguingly, we observed an additional decrease in CI immediately after irradiation in the wild-type animals irradiated during the learning period (Fig. 3A2 ) was significantly suppressed in the gpc-1 mutants (Fig. 4B ). This result indicates that the gpc-1 mutant is defective in its ability to exhibit the IR-induced additional decrease in chemotaxis after during-learning irradiation but not in its ability to exhibit food-NaCl associative learning. Together, these results suggest that the effect of IR on food-NaCl associative learning in C. elegans is mediated by GPC-1, a subunit of the heterotrimeric G-protein.


Figure 4
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  		Figure 4. Comparison of the radiation response between wild-type and gpc-1 mutant animals. A) Decrease in CI in NaCl conditioning for food-NaCl associative learning of nonirradiated wild-type and gpc-1 mutant animals. B) CIs immediately after irradiation under the same condition as that of the during-learning irradiation experiment (Fig. 1B4 ). The error bars represent the SE of 4 or more experiments. *Significant differences between the wild-type and gpc-1 mutant animals (P<0.05).

Chronic IR exposure during associative learning induces negative chemotaxis
To confirm whether chronic exposure to IR induces any further decrease in chemotaxis, we examined the effect of chronic exposure to IR during the learning period (4 h, Fig. 1B6 ). After low-dose-rate chronic exposure, the exposed wild-type animals exhibited a negative CI (Fig. 5 ). This result indicates that chronic exposure to IR induces a further decrease in chemotaxis, and a population of animals with avoidance of NaCl is predominant compared with that of attractive one, i.e., avoidance response. On the other hand, CI after chronic exposure was not significantly decreased in gpc-1 mutants; however, there was a slight trend toward decreased CI (P=0.37; Fig. 5 ). Importantly, the chronic IR exposure at a low dose rate induces the primary avoidance response to NaCl which GPC-1 mediates.


Figure 5
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  		Figure 5. Chemotaxis of the wild-type and gpc-1 mutant animals after chronic irradiation (Fig. 1B6 ). Chronic irradiation experiments were performed at a low dose rate (0.42 Gy/min), and the total dose was 105 ± 5 Gy. The error bars represent the SE of 6 experiments. *Significant differences between irradiated and nonirradiated animals (P<0.05).

Chemosensation to other water-soluble attractants, cAMP and lysine
We observed a change in the chemotaxis profile of animals irradiated during the transition stage of the food-NaCl associative learning (Fig. 3A2, B ); however, it was unclear whether the IR-induced response was specific to chemotaxis toward NaCl. To examine the specificity of the effects of IR exposure, we tested chemosensation to other chemoattractants, cAMP and lysine (10 , 14) . NaCl-conditioned animals are known to show the decrease of chemotaxis to cAMP and lysine (10) . IR exposure of NaCl-conditioned animals induced an additional significant decrease in CI for cAMP during the transition stage of associative learning (Fig. 6 A). However, chemosensation to lysine was not affected by IR exposure to the contrary (Fig. 6B ). These results indicate that despite water-soluble attractants sensed at the same ASE neurons (14) , a chemosensory response of NaCl-conditioned animals after acute IR exposure differs with the kinds of chemoattractants.


Figure 6
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  		Figure 6. Chemotaxis to cAMP (A) and lysine (B) after the exposure to IR. Open and gray bars represent the results for the mock-conditioned animals and the NaCl-conditioned ones, respectively. Animals irradiated with IR under the same condition as that used in the during-learning irradiation experiment (Fig. 1B3 ), and the doses of the cAMP and lysine experiments are 506 ± 21 and 484 ± 20 Gy, respectively. The error bars represent the SE of 4 experiments. *Significant differences between irradiated and nonirradiated animals (P<0.05); ns = not significant.

Normal chemotaxis toward benzaldehyde after irradiation in the learning experiment
We are concerned with the effects of IR on olfactory sensation of NaCl-conditioned animals as well as other water-soluble chemoattrantants. Thus, we examined the effects of IR on chemotaxis toward benzaldehyde. Chemotaxis toward benzaldehyde was analyzed after irradiation of the animals that were conditioned similarly as in the during-learning irradiation experiment (Fig. 1B4 ). Here, chemotaxis toward benzaldehyde was not significantly affected immediately after irradiation (0 h) and 5 h after irradiation (P=0.515 at 0 h and P=0.778 at 5 h; Fig. 7 ); this indicates that exposure to IR does not affect chemotaxis toward benzaldehyde, even in the transition stage of food-NaCl associative learning. This further supports that IR exposure specifically affects chemotaxis toward NaCl via GPC-1.


Figure 7
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  		Figure 7. Chemotaxis toward benzaldehyde of animals irradiated with IR at a dose of 546 ± 22 Gy under the same condition as that used in the during-learning irradiation experiment (Fig. 1B4 ). Chemotaxis toward NaCl of these animals after irradiation is shown in Fig. 3B . The time after irradiation is indicated by 0 h and 5 h. Open bars represent the results for the nonirradiated animals, and the hatched bars represent results for the irradiated animals. The error bars represent the SE of 6 experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we evaluated the hypothesis that exposure to a nonlethal dose of IR does not affect learning behavior in a mature nervous system. Using the food-NaCl associative learning paradigm of C. elegans (10) , we found that IR exposure influences chemotaxis toward NaCl only during the transition stage of associative learning (Fig. 3A2, B ); however, animals under simultaneous presentation of IR and NaCl and in the pre- or postlearning stages were not affected (Figs. 2 and 3A1 , 3). Our hypothesis was not supported by the present results. Our findings may offer some novel insight into the mechanism by which the learning behavior of a mature nervous system responds to IR exposure. Chronic irradiation induced the primary avoidance response to NaCl in addition to the learning behavior (Fig. 5) . We found that the gpc-1 mutant is unable to exhibit additional decreases in chemotaxis after acute and chronic irradiation (Figs. 4B and 5) . Further, in the learning experiment, we observed an IR-induced additional decrease in CI for cAMP but not for lysine (Fig. 6A , B). Moreover, we also showed normal chemotaxis toward benzaldehyde after irradiation (Fig. 7) .

The radiation doses used in the present study (100–550 Gy) were ~2 log higher than those typically used in behavioral studies on mammals (0.5–10 Gy is a sublethal dose for mammals). The dose that results in a radiobiological effect in eukaryotic cells is inversely proportional to the size of the genome (22) . The need for higher doses in C. elegans may be consistent with this observation. The mean lethal dose (37% survival dose) of X-rays used for wild-type C. elegans adults was >800 Gy (17) . The present doses were sublethal for C. elegans and might be biologically equivalent to those required for mammals. The low dose rate (0.42 Gy/min) used for chronic exposure is equal to or less than that used in mammalian behavioral studies. This may support the idea that IR significantly influences the learning behavior of C. elegans that are independent of cell inactivation. In fact, we observed successfully hatched eggs on the NGM agar plates with E. coli after exposure at the low dose rate, although we did not evaluate the exact hatchability. Importantly, the chronic IR exposure at a low dose rate induced behavioral change in C. elegans. If one would estimate the effects of IR at the same-time exposure on the decrease of CI, one could predict a little or no decrease in CI from the linearity of the radiation dose-response relationship at the high-dose rate (Fig. 3A2 ). However, we observed a significant and large decrease in CI after a low dose-rate and chronic exposure (Fig. 5) . Thus, IR-induced response would not be a single-time event but a constitutive activity. This could be a radioadaptive response, which has been under debate for radiation protection guidelines (23 , 24) . This study is the first to demonstrate the radioadaptive response for learning behavior.

A previous work (25) reported incapacitation of the mammalian shock-avoidance response after exposure to an acute lethal dose was performed; the response began as early transient incapacitation, followed by a temporary recovery and, finally, death. In the present study, the ability of the exposed animals for associative learning was maintained after irradiation (Fig. 3A1 ). Approximately 80% of the animals irradiated at the highest dose exhibited chemotaxis in the present assays (94.2% at 0 Gy, 86.7% at 118 Gy, 88.8% at 332 Gy, and 80.0% at 546 Gy; the proportion of animals in the test and control spot areas in Fig. 3A2 ). Thus, our findings would differ from those of previous works due to the sustained capability of chemotaxis. Another similar finding is IR-induced taste-aversion learning (18) in which rats exposed to IR subsequently avoid ingestion of a tasting solution. Our findings would also differ from those regarding IR-induced taste aversion; IR is an aversive unconditioned stimulus for the taste-aversion learning paradigm (18) but not in the food-NaCl associative learning paradigm in C. elegans because simultaneous presentation of NaCl and IR dose not reduce the chemosensation to NaCl (Fig. 2) . However, a clarification of how IR-induced cue is evolutionarily conserved encourages further studies for analogy to taste aversion. To our knowledge, no other work has been preformed on the IR-induced behavioral response, except those related to neurogenesis. Taken together, our findings may establish a novel behavioral response to IR-induced learning in mature nervous systems.

Our findings offer a novel hypothesis regarding IR as a modulatory cue rather than a learning cue for food-NaCl associative learning in C. elegans. This hypothesis is supported by the following three lines of evidence: 1) we observed no decrease in CI in the mock-conditioned and food/NaCl-conditioned animals after irradiation, suggesting that IR itself played no role in the learning cue nor an unconditioned stimulus in the present learning paradigm; 2) the IR-induced additional decrease in CI occurred only during the transition stage of associative learning (Fig. 3A2 , B); and 3) wild-type animals displayed negative CI after exposure to chronic irradiation (Fig. 4) , indicating that IR exposure induces a further decrease in chemotaxis beyond the limits of our conditioning (Fig. 1B ). In addition, our findings raise a question regarding whether exposure to an acute dose of IR suppresses attraction to NaCl or enhances NaCl avoidance. After exposure to chronic IR, the primary avoidance response of wild-type animals (Fig. 5) and the lack in additional decreases in the CI of the gpc-1 mutants (Figs. 4B and 5) suggest the presence of an IR-induced primary avoidance response of NaCl in addition to a lesser degree of suppressed attraction to NaCl.

We found that the wild-type animals, but not the gpc-1 mutants, exhibit the additional decrease in CI after acute and chronic irradiation (Figs. 4B and 5) . This finding suggests the possibility of a gene responsible for the IR-induced additional decrease in CI (Fig. 3A2 ). Suppression of the primary avoidance response in the gpc-1 mutants after irradiation might suggest the presence of a modulatory pathway. C. elegans possesses two genes that encode the G-protein {gamma} subunits: gpc-1 and gpc-2. It is known that while gpc-2 is ubiquitously expressed in all neurons and muscle cells (26) , gpc-1 is predominantly expressed in specific sensory neurons, i.e., ADL, ASH, ASJ, AFD, ASI, AWB, and PHB, and is responsible for the chemosensory avoidance of NaCl (20) . Introduction of the wild-type gpc-1 gene in each neuron of gpc-1 mutants revealed that the avoidance of high NaCl concentrations after preexposure to NaCl involved cues from ASI, ASH, and ADL neurons (21) . GPC-1 also plays a central role in the sensory adaptation induced by repellent stimuli in ASH neurons (27) . Together with the findings of these studies, our findings imply that IR-induced cues for ASI, ASH, or ADL neurons mediated by GPC-1 facilitate avoidance of (or adaptation to) NaCl in the food-NaCl associative learning paradigm. Thus, we propose a novel pathway that functions via the {gamma} subunit of the G-protein (GPC-1) for the IR-induced modulatory effect in the food-NaCl associative learning paradigm of C. elegans (Fig. 8 ).


Figure 8
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  		Figure 8. Proposed schematic model of the IR-induced modulatory effects on the food-NaCl associative learning paradigm of C. elegans. The regulation pathway for attraction to NaCl is based on the findings of a previous report (28) . The "sensory neurons" may include ASE and ADF neurons in this model. The present model for the IR-induced response is represented by bold unbroken lines. The broken lines indicate the speculated pathways.

It would be noteworthy that specific neurons may play a pivotal role in the effect of IR on chemosensation of NaCl-conditioned animals. It is known that while the chemotaxis to cAMP depends on four pairs of chemosensroy neurons (ASE, ADF, ASG, and ASI), the chemotaxis to lysine involves ASE, ASG, ASI, and ASK (14) . Our results (Fig. 6A, B ) suggest the importance of ADF neurons to IR-induced decrease of chemotaxis. The signaling pathway of the odr-3 gene at ADF neurons in gustatory plasticity (21) may support our findings. On the other hand, C. elegans senses benzaldehyde via AWC neurons, which synaptically connect to ASE neurons. No change in chemosensation to benzaldehyde (Fig. 7) indicates that AWC neurons are dispensable for the IR-induced response. An identification of responsible neurons or neuron network underlying our findings awaits further investigation.

The following events in IR-induced behavioral responses remain unexplained: 1) the reason behind the activation of the primary avoidance response after IR exposure only during learning, 2) the initial target for this response, 3) the neurons responsible for this phenomenon, 4) the mechanism by which the CI of the exposed animals at the highest dose can be shifted to the stage of attraction to NaCl (Fig. 3B ), and 5 ) the relationship between the avoidance and attraction responses. Further studies are required to elucidate these mechanisms.

In conclusion, the present study revealed that IR exposure induced an additional decrease in chemotaxis in the food-NaCl associative learning paradigm in C. elegans adults that do not undergo neurogenesis. Our results suggest that IR exposure may affect the function of the mature nervous system. In addition, IR-induced responses may be localized in the nervous system, i.e., ADF and several neurons related to GPC-1. Taken together with the results of the acute and chronic irradiation experiments in the wild-type and gpc-1 mutant analyses, our findings reveal a novel modulatory effect for IR on associative learning, namely, a primary avoidance response in addition to a lesser degree of suppressed attraction to NaCl. These IR-induced responses appear to be an additional response to enhance the escape behavior for avoiding the risks associated with radiation in addition to the loss of chemotaxis induced by associative learning. Further identification of genes involved in the novel IR-induced modulatory effects would be potentially valuable since a human homologue of GPC-1 exists. In addition, further experiments in organisms exposed to particulate radiation (e.g., protons and energetic heavy ions) will be needed for protecting astronauts during interplanetary space missions, as these are the primary energetic particles that may affect cognitive function of astronauts during long-term flights.


   ACKNOWLEDGMENTS
 
We thank Dr. A. Higashitani for commenting on the manuscript; Dr. G. Jansen for helpful suggestions; and Drs. T. Funayama, S. Wada, T. Tsuji, I. Narumi, and Y. Furusawa for valuable discussions. This work was supported in part by a grant-in-aid for scientific research (17710052 to T.S.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and the REIMEI Research Resources of the Japan Atomic Energy Agency. N.H. was supported by a grant-in-aid for the 21st Century COE Program for Biomedical Research Using Accelerator Technology from MEXT. M.S. was supported by the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

Received for publication June 20, 2007. Accepted for publication September 20, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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