Ichiro Torayama et al. (2004) East Asia Worm Meeting "Isolation and analysis of mutants defective in olfactory learning in C.elegans"

Isao Katsura, Takeshi Ishihara, Ichiro Torayama

[East Asia Worm Meeting, 2004]

C.elegans shows a diversity of behavioral plasticity in response to environmental stimuli. We observed apparent behavioral plasticity by pre-exposure to food and butanone, but not benzaldehyde and isoamyl alcohol. Namely, animals conditioned with butanone and food exhibited enhanced chemotaxis to butanone, while those conditioned with benzaldehyde or isoamyl alcohol and food did not change the efficiency of chemotaxis to the same odorants. On the basis of these observations, we developed an assay system and isolated mutants defective in this olfactory learning. Among these mutants, ut305 and ut306 exhibited weaker chemotaxis to butanone after conditioning with butanone and food. Interestingly, ut305 also showed defects in adaptation to benzaldehyde or isoamyl alcohol, while ut306 showed normal adaptation to these odorants. This observation may indicate that a specific pathway for butanone exists in olfactory learning. Furthermore, ut305 also showed weak defects in chemotaxis to butanone. ut305 was mapped to the region containing a single candidate gene C02C6.2, which encodes a novel protein with two or three transmembrane domains. In this meeting, we will report and discuss the result of the expression of ut305 protein, the rescue experiments of cell-specific expression and the experiment of cell ablation.

Ichiro Torayama et al. (2002) Japanese Worm Meeting "Isolation and Analysis of Mutants - Defective in Olfactory Learning Behavior in C.elegans"

Isao Katsura, Takeshi Ishihara, Ichiro Torayama

[Japanese Worm Meeting, 2002]

The nematode C.elegans shows behavioral plasticity in response to environmental stimuli. For instance, animals conditioned with NaCl and food migrate toward NaCl, while animals conditioned with NaCl and starvation do not migrate toward NaCl (Saeki S et al, 2001). This learning behavior can be regarded as associative learning, because it is probably based on modulation by the integration of paired stimuli, food and chemical stimuli. However, only limited results have been obtained on the cellular and molecular events in the sensing of food and the integration of paired signals in associative learning. Food and starvation also modulate sensory adaptation to AWC-sensed odorants. Food inhibits sensory adaptation (Ishihara T and Katsura I, personal communication), while starvation enhances sensory adaptation (Colbert HA and Bargmann CI, 1997). On the basis of these observations, I developed an assay system to isolate mutants defective in olfactory learning behavior. In the assay system, wild-type animals were exposed to AWC-sensed odorants in the presence or absence of food, and then tested for their response to the same odorants. The results showed that animals conditioned with butanone and food exhibited enhanced chemotaxis to butanone, while those conditioned with benzaldehyde (or isoamyl alcohol) and food did not change the efficiency of chemotaxis to the same odorants. Using this assay system, we screened 5,000 genomes and obtained 8 mutants defective in the olfactory learning behavior. Among these mutants, ut305 and ut306 showed defects in olfactory learning behavior induced by butanone and food. Interestingly, ut305 also showed defects in adaptation to benzaldehyde, while ut306 showed normal adaptation to benzaldehyde. This observation may indicate that a specific pathway for butanone exists in olfactory learning. We mapped ut305 to LG X and ut306 to LG V. Further mapping and rescue experiments are underway to determine the identities of these genes.

Ichiro Torayama et al. (2003) International Worm Meeting "Olfactory learning induced by paired stimuli, butanone and food, is regulated by a novel protein"

Isao Katsura, Takeshi Ishihara, Ichiro Torayama

[International Worm Meeting, 2003]

The nematode C.elegans shows behavioral plasticity in response to environmental stimuli. For instance, animals conditioned with NaCl and food migrate toward NaCl, while animals conditioned with NaCl and starvation do not migrate toward NaCl (Saeki S et al, 2001). This learning behavior is probably based on modulation by the integration of paired stimuli, food and chemical stimuli. However, only limited results have been obtained on the cellular and molecular events in the sensing of food and the integration of paired signals. Food and starvation also modulate sensory adaptation to an AWC-sensed odorant, benzaldehyde. Food inhibits sensory adaptation (Ishihara T and Katsura I, unpublished results; Bargmann CI, pers. comm.; Nuttley WM et al, 2002), while starvation enhances sensory adaptation (Colbert HA and Bargmann CI, 1997). On the basis of these observations, we developed an assay system to isolate mutants defective in olfactory learning behavior. In the assay system, wild-type animals were exposed to AWC-sensed odorants in the presence or absence of food, and then tested for their response to the same odorants. The results showed that animals conditioned with butanone and food exhibited enhanced chemotaxis to butanone, while those conditioned with benzaldehyde or isoamyl alcohol and food did not change the efficiency of chemotaxis to the same odorants. Using this assay system, we screened 5,000 genomes and obtained mutants defective in the olfactory learning behavior. Among these mutants, ut305 and ut306 showed defects in olfactory learning behavior induced by butanone and food. Namely, these mutants exhibited weaker chemotaxis to butanone after conditioning with butanone and food. Interestingly, ut305 also showed defects in adaptation to benzaldehyde or isoamyl alcohol, while ut306 showed normal adaptation to these odorants. This observation may indicate that a specific pathway for butanone exists in olfactory learning. We mapped ut305 between R03A10 and C02C6 on chromosome X and ut306 between F44C4 and VC5 on chromosome V. ut305 was mapped to the region containing a single candidate gene C02C6.2, which encodes a novel protein with two or three transmembrane domains.

Ichiro Torayama et al. (2005) International Worm Meeting "olrn-1 gene regulates the olfactory learning of butanone and food through a switch involved in the asymmetry of AWC neurons."

Ichiro Torayama, Isao Katsura, Takeshi Ishihara, Kotaro Kimura

[International Worm Meeting, 2005]

C.elegans shows a diversity of behavioral plasticity in response to environmental stimuli. We previously found a novel type of behavioral plasticity, in which pre-exposure to food and an AWC-sensed odorant (butanone) enhances animal's chemotaxis to the same odorant. We have isolated mutants defective in this olfactory learning (olrn), of which olrn-1(ut305) and olrn-2(ut306), animals exhibit reduced chemotaxis to butanone after conditioning with butanone and food. Interestingly, the olrn-1 mutant also shows defects in adaptation to benzaldehyde and isoamyl alcohol as well as defects in chemotaxis to low concentrations (but not high concentrations) of butanone. olrn-1 encodes a novel membrane protein.In this study, we report that olrn-1 shows abnormality in the asymmetry of AWC neurons. In wild-type animals, str-2::GFP is expressed randomly in either the left or the right AWC neuron but not both (Troemel et al., 1999). In contrast, olrn-1 animals (but not olrn-2 animals) expressed str-2::GFP in neither of AWC neurons. Expression of olrn-1 gene in a small subset of neurons including AWCs rescued the mutant phenotypes in the expression of str-2::GFP, olfactory learning, and chemotaxis. The lack of str-2::GFP expression and poor chemotaxis to butanone in the olrn-1 mutant is consistent with the poor chemotaxis to butanone by AWCON-ablated wild type animals, reported by Wes and Bargmann (2001). To study the relationship between the asymmetry of AWC neurons and the olfactory learning, we analyzed the phenotypes of known neuronal asymmetry mutants as well as double mutants of these mutations and the olrn-1 mutation. The nsy-1(ky397) mutant, which expresses str-2::GFP in both AWC neurons (Troemel et al., 1999), showed the phenotype of normal olfactory learning. Moreover, the nsy-1 mutation partially suppressed the phenotypes of olrn-1 in the olfactory learning and the expression of str-2::GFP. The slo-1(ky389);olrn-1(ut305) double mutant animals consisted of those expressing str-2::GFP in two, one, and neither of AWC neurons (2ON, 1ON, and 2OFF), respectively. The olfactory learning assay of the mixture of these animals revealed that 2ON and 1ON animals are normal, while 2OFF animals are abnormal in the olfactory learning. These results suggest that the olfactory learning requires AWCON neuron and that the learning abnormality of the olrn-1 mutant originates from a decision leading to the 2OFF state of AWC neurons.

Hiroshi Ichijo et al. (2006) East Asia C. elegans Meeting "Analysis of mutants defective in olfactory learning induced by AWC-sensed odorants."

Isao Katsura, Hiroshi Ichijo, Ichiro Torayama, Kotaro Kimura

[East Asia C. elegans Meeting, 2006]

C.elegans changes its behavior based on its experience, which can be regarded as learning. For example, animals decrease the efficiency of taxis to a stimulus after they have received the same stimulus under starving conditions: Animals conditioned with starvation and NaCl no longer migrate toward NaCl, and the animals that had been starved at certain temperature avoid the starvation-temperature upon temperature gradient (Saeki et al., 2001; Mohri et al., 2005). In contrast, we discovered that conditioning with food and the AWC-sensed odorant butanone (Bu) enhances chemotaxis to this odorant, which we call butanone enhancement (Torayama et al., submitted).Recently we found that such olfactory enhancement was not specific to butanone: animals showed olfactory enhancement after conditioning with food and AWC-sensed odorant Isoamyl alcohol (Ia) or 2,3-pentanedione (Pd), although animals that conditioned with food and the AWC-sensed odorant benzaldehyde or the AWA-sensed odorant diacetyl exhibited almost no change or decrease (i.e., adaptation) rather than increase in the response to the same odorants.To elucidate the molecular mechanism of olfactory enhancement, we used a forward genetic approach. We screened 7000 genomes and isolated seven candidate mutants for the Ia olfactory enhancement (ut321 - ut327). We are analyzing the phenotypes of these mutants and other mutants that were previously isolated for the Bu olfactory enhancement defective (ut301-ut315). One of the Ia olfactory enhancement mutants (ut324) and some of the Bu olfactory enhancement mutants were also defective in the Pd olfactory enhancement. In this meeting, we will also report on the progress in the genetic mapping of ut324 and some other mutants.

Hiroshi Ichijo et al. (2007) International Worm Meeting "Genetic mapping of a novel butanone enhancement mutant."

Isao Katsura, Hiroshi Ichijo, Kotaro Kimura, Ichiro Torayama

[International Worm Meeting, 2007]

We have previously reported that C. elegans enhances its chemotaxis to butanone (bu) after conditioning with food and the odorant, which we call butanone enhancement. This behavioral plasticity is independent to serotonin, which is requires for the modulation of many other food related behavioral plasticity. Analysis of olrn-1(ut305) and olrn-2/bbs-8(ut306) (olrn: olfactory leaning defective) revealed that butanone enhancement is probably conducted in the STR-2-expressing AWC (AWCON) neuron and required the function of Bardet-Biedl syndrome genes, suggesting the importance of sensory cilia in this plasticity (Torayama et al., 2007). Such olfactory enhancement was not specific to butanone: animals also showed olfactory enhancement after conditioning with food and AWC-sensed odorants Isoamyl alcohol (Ia) or 2,3-pentanedione (Pd) (Ichijo et al., EAWM 2006). To elucidate the molecular mechanism of olfactory enhancement, we used a forward genetic approach. We screened 7000 genomes and isolated seven candidate mutants for the Ia olfactory enhancement (ut321 - ut327). We analyzed the phenotypes of these mutants and other mutants that were previously isolated as Bu olfactory enhancement defective mutants (ut301-ut315). Among these mutants, ut311 showed nearly normal chemotaxis to AWC-sensed odorants (Bu, Ia and Pd), and defects in Bu- and Pd-enhancement. To assess whether ut311 has defects in asymmetrical differentiation of AWC neurons and sensory cilia formation, we checked the expression pattern of str-2::gfp and the dye filling of sensory neurons with the fluorescence dye DiI in ut311. ut311 showed neither the 2 AWCOFF phenotype nor dye-filling defects. This suggests that the Bu enhancement defects in ut311 is due to a reason independent of the olrn-1 and olrn-2. To clone the responsible gene for ut311, we mapped ut311 to the 2.5 map unit interval on the chromosome V by snip-SNPs. In this meeting, we will report on the progress of detailed mapping and cloning of ut311.

Hiroshi Ichijo et al. (2005) International Worm Meeting "Behavioral plasticity induced by conditioning with isoamyl alcohol and food"

Kotaro Kimura, Hiroshi Ichijo, Ichiro Torayama, Isao Katsura

[International Worm Meeting, 2005]

It is well known that C. elegans decreases the efficiency of taxis to an attractive stimulus or even changes its behavior to avoidance, if it has received the same stimulus under starving conditions. For instance, animals conditioned with starvation and NaCl no longer migrate toward NaCl, while animals conditioned with starvation and the cultivation temperature change their behavior to avoid the temperature (Saeki et al., 2001; Mohri et al., 2005). On the other hand, we investigated the effect of food rather than starvation and found a different type of behavioral plasticity, which we call olfactory learning. Namely, conditioning with food and butanone enhances chemotaxis to butanone (Torayama et al., 2003 IWM). In this study, we asked whether animals show olfactory learning with odorants other than butanone. We used an AWA-sensed odorant, diacetyl (Di), and three AWC-sensed odorants, butanone (Bu), benzaldehyde (Bz), and isoamyl alcohol (Iaa), for the experiments. Wild-type animals were exposed to one of these odorants in the presence of food for 90 min, and then tested for a selection assay between the same odorant (test odorant) and another odorant (counter odorant). The selection assay was used instead of chemotaxis assay to detect small changes in the efficiency of chemotaxis. We changed three parameters: (1) the concentrations of odorants for conditioning, (2) the species of counter odorants, and (3) the concentrations of test odorants in the selection assay, while the concentrations of counter odorants were determined so that unconditioned animals were attracted to counter odorants as strongly as test odorants in the selection assay. As expected, Bu-conditioned animals exhibited olfactory learning over wide concentration ranges of the conditioning odorant and the test odorant (both Bu), and with different species of counter odorants. In contrast, Bz- or Di-conditioned animals exhibited almost no change or decrease (i.e., adaptation) rather than increase in the response to the test odorants. Furthermore, although Iaa-conditioned animals showed adaptation at high concentrations of the conditioning odorant, they exhibited olfactory learning at low concentrations of the conditioning odorant and with Bu or Di as the counter odorant. We confirmed that the Iaa-conditioned animals enhanced the efficiency of chemotaxis toward Iaa itself, by carrying out chemotaxis assay after the conditioning. Since the behavioral change induced by food and Iaa was large enough. we screened 4000 genomes and obtained some candidates for mutants abnormal in this olfactory learning.