Left-right olfactory asymmetry results from antagonistic functions of voltage-activated calcium channels and the Raw repeat protein OLRN-1 in C. elegans

<p>Abstract</p> <p>Background</p> <p>The left and right AWC olfactory neurons in <it>Caenorhabditis elegans </it>differ in their functions and in their expression of chemosensory receptor genes; in each animal, one AWC randomly takes on one identity, designated AWC^OFF, and the contralateral AWC becomes AWC^ON. Signaling between AWC neurons induces left-right asymmetry through a gap junction network and a claudin-related protein, which inhibit a calcium-regulated MAP kinase pathway in the neuron that becomes AWC^ON.</p> <p>Results</p> <p>We show here that the asymmetry gene <it>olrn-1 </it>acts downstream of the gap junction and claudin genes to inhibit the calcium-MAP kinase pathway in AWC^ON. OLRN-1, a protein with potential membrane-association domains, is related to the <it>Drosophila </it>Raw protein, a negative regulator of JNK mitogen-activated protein (MAP) kinase signaling. <it>olrn-1 </it>opposes the action of two voltage-activated calcium channel homologs, <it>unc-2 </it>(CaV2) and <it>egl-19 </it>(CaV1), which act together to stimulate the calcium/calmodulin-dependent kinase CaMKII and the MAP kinase pathway. Calcium channel activity is essential in AWC^OFF, and the two AWC neurons coordinate left-right asymmetry using signals from the calcium channels and signals from <it>olrn-1</it>.</p> <p>Conclusion</p> <p><it>olrn-1 </it>and voltage-activated calcium channels are mediators and targets of AWC signaling that act at the transition between a multicellular signaling network and cell-autonomous execution of the decision. We suggest that the asymmetry decision in AWC results from the intercellular coupling of voltage-regulated channels, whose cross-regulation generates distinct calcium signals in the left and right AWC neurons. The interpretation of these signals by the kinase cascade initiates the sustained difference between the two cells.</p

Isolation and analysis of mutants defective in olfactory learning in C.elegans

　Animals, including humans, sense environmental cues and select appropriate behaviors depending upon the environment in which they live. This plasticity, called learning, is based on changes in their neural functions at cellular and molecular levels. Until now, many investigators have studied the molecular mechanism of neural plasticity using various species of animals. Because of the complexity of most nervous systems controlling this plasticity, its studies have often focused on relatively simple nervous systems and on reduced preparations. 　The nematode Caenorhabditis elegans (C.elegans ) is a good model organism for studying behavioral and neural science because of its simple multicellular structure consisting of only 959 somatic cells including 302 neurons (Brenner S, 1974, Sulston JE, Horvitz HR, 1977, White JG et al, 1986). Despite its simple structure, C.elegans shows the diversity of 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 odorants. 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 Cl, 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. This phenomenon cannot be explained by the inhibition of olfactory adaptation by food, but can be explained by learning through the association of butanone and food. 　Using this assay system, we screened 5,000 genomes and obtained mutants defective in the olfactory learning behavior induced by butanone and food. Among these mutants, ut305 and ut306 showed defects in olfactory learning behavior induced by butanone and food. Namelv these mutants exhibited weaker chemotaxis to butanone after conditioning with butanone and food. Interestingly, ut306 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, ut306 also showed weak defects in chemotaxis to butanone. Serotonin (5-HT) mediates some of the effects of food on many behaviors: it stimulates pharyngeal pumping (Avery L and Horvitz HR, 1990) and egg laying (Trent C et al, 1983), but inhibits locomotion (Horvitz HR et al, 1982). Exogenous 5-HT also inhibits the effect of starvation in sensory adaptation (Colbert HA, Bargmann CI, 1997, Nuttley WM et al, 2002). Analysis of mutants defective in 5-HT and catecholamine synthesis indicated that neither 5-HT nor catecholamine is required for the olfactory learning behavior induced by butanone and food. Furthermore, the experiments of exogenous 5-HT on ut305 olfactory learning behavior revealed that ut305 gene and 5-HT control distinct pathways: one for the olfactory learning (increase of chemotaxis index induced by butanone and food) and the other for the inhibition of olfactory adaptation. 　I mapped ut305 between R03Al0 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. This protein was predicted to have 3 transmembrane domains and showed partial homology to the fruitfly raw protein. Raw protein is required for restriction of JNK signaling in embryogenesis. ut305::-GFP fusion gene, Which rescued the behavioral defects, was expressed in some neurons in pharynx and a pair of head neurons (AIA interneurons). Furthermore, killing of AIA neurons resulted in abnormality in the olfactory learning. AIA neurons are interneurons and receive synaptic inputs from many amphid sensory neurons including AWC. On the other hand, the experiment of extopic expression was shown that the expression of the wild type ut305 gene in AWC and AWB sensory neurons (using gcy-10 promoter) was sufficient for normal olfactorv learning induced by butanone and food. Here, I would like to propose a hypothesis for the mechanism of action of ut305 protein. Wild type ut305 gene is thought to be involved in the sensitization of the olfaction of butanone because ut305 animals show weak defects in response to butanone. If this sensitization system is regulated by the signals of butanone, wild type animals may show olfactory leaning behavior. Therefore, the connection between AWC sensory neurons and AIA interneurons would be important for this sensitization and the olfactory leaning behavior by wild type ut305 gene, when the gene is expressed in AIA neurons