Article Figures & Data
Figures
- Figure 1.
TAX-6 is required for associative learning between temperature and feeding state. A , Thermotaxis assay system. The thermograph shows the stable radial temperature gradient from 17 to 25°C. Single animals were placed on agar at ∼22°C, as indicated by a cross. B , The thermotaxis behavior of animals cultivated at 17, 20, and 23°C. The colored boxes indicate the phenotypic categories as described in Materials and Methods. Each bar represents the average of five independent thermotaxis assays using 20 animals per assay. Three independent transgenic lines were tested for tax-6(sensory+, inter−). For all strains tested, n = 100. C , Responses to AWA and AWC olfactory neuron-sensed odorants, as well as ASE gustatory neuron-sensed NaCl. DIA, 1:100 diacetyl; PYR, 100 mg/ml pyrazine; TRI, 1:100 2,4,5-trimethylthiazole; IAA, 1:1000 isoamyl alcohol; BEZ, 1:100 benzaldehyde. Each bar represents the average of nine independent chemotaxis assays using 10 animals per assay. Two independent transgenic lines were tested for tax-6(sensory+, inter−). For all strains tested, n = 90. D , Avoidance behavior from Cu2+. Each bar represents the average of eight independent chemotaxis assays using ∼100 animals per assay. Three independent transgenic lines were tested for tax-6(sensory+, inter−). For wild-type, n = 834. For tax-6(db60), n = 875. For each tax-6(sensory+, inter−) transgenic line, n = ∼800. E , Tracks of individual 17°C- and 23°C-cultivated animals on a radial temperature gradient. F , Results of the thermotaxis assay for well fed or starved animals cultivated at 17°C. Each bar represents the average of at least 16 independent thermotaxis assays using 20 animals per assay. Three and two independent transgenic lines were tested for tax-6(sensory+, inter−) and tax-6(sensory+, inter+), respectively. For wild-type and for each tax-6 transgenic line, n = ∼320. n = 318 for wild-type. For each tax-6 transgenic line, n = ∼320. G , Results of the thermotaxis assay for 20°C-cultivated animals. Each bar represents the average of at least four independent thermotaxis assays using 20 animals per assay. For wild-type, n = 80. For tax-6(sensory+, inter+), n = 78. H , Results of the thermotaxis assay for 23°C-cultivated animals. Each bar represents the average of at least six independent thermotaxis assays using 20 animals per assay. Three and two independent transgenic lines were tested for tax-6(sensory+, inter−) and tax-6(sensory+, inter+), respectively. For each genotype, n ≥ 120. I , J , Modulation of the locomotory rate. Well fed animals (F) were transferred to assay plates without food (gray bars; F > S) or with food (black bars; F > F) and starved animals (S) were transferred to assay plates without food (hatched bars; S > S) or with food (dotted bars; S > F), and the number of body bends in 20 s was counted. Each bar represents the average of three independent locomotory rate assays using 10 animals per assay. Three independent transgenic lines were tested for tax-6(sensory+, inter−). K , Attraction to E. coli (OP50). Each bar represents the average of 10 independent attraction assay using 10 animals per assay.
- Figure 2.
TAX-6 calcineurin regulates neural function in the mature nervous system for feeding state-dependent learning behavior. A , Schematic diagram for the assay for salt gustatory plasticity for NaCl (Saeki et al., 2001). B , Chemotaxis toward NaCl of animals of each genotype conditioned without food and with NaCl or mock conditioned without food and without NaCl. Each bar represents the average of at least four independent chemotactic plasticity assays using ∼150 animals per assay. Three and two independent transgenic lines were tested for tax-6(sensory+, inter−) and tax-6(sensory+, inter+), respectively. For wild-type, n = 649. For hen-1(tm501), n = 621. For each tax-6 transgenic line, n = ∼670. C , Schematic diagram for the interaction assay (Ishihara et al., 2002). D , Results of the interaction assay. Each bar represents the average of three independent interaction assays using ∼60 animals per assay. Three independent transgenic lines were tested for tax-6(sensory+, inter−). For wild-type, n = 182. For hen-1(tm501), n = 191. For each tax-6(sensory+, inter−) transgenic line, n = ∼180. E , tax-6(db60) animals carrying hsp::tax-6 were heat shocked at various developmental stages and analyzed by the chemotactic plasticity assay. Each bar represents the average of at least three independent chemotactic plasticity assays using ∼50 animals per assay. Four and three independent transgenic lines were tested for tax-6(sensory+, hsp::tax-6cDNAwt) and tax-6(sensory+, hsp::tax-6(H176Q), respectively. For wild-type and tax-6(sensory+, inter−), n=∼150. For each tax-6(sensory+, hsp::tax-6cDNAwt) and tax-6(sensory+, hsp::tax-6(H176Q) transgenic line, n = ∼140.
- Figure 3.
Essential interneurons for TAX-6/calcineurin-mediated thermotactic associative learning. A , Schematic diagram of the neural circuit underlying thermotaxis (Mori and Ohshima, 1995). A temperature signal is sensed by the AFD and unidentified X thermosensory neurons; it is then transmitted to AIY and AIZ interneurons, and then finally integrated in the RIA interneuron. The AIY thermophilic (T) and AIZ cryophilic (C) arms of the circuit mediate movement to higher (red) and lower (blue) temperatures, respectively. B , Rescue experiments of defective temperature-feeding state associative learning in the tax-6 mutant. Each bar represents the average of at least three independent thermotaxis assays using 20 animals per assay. Two or three independent transgenic lines were tested for tax-6(sensory+) animals expressing tax-6cDNAwt driven by various promoters. For wild-type and tax-6(sensory+, inter−), n = 120. For each tax-6(sensory+, promoter::tax-6cDNAwt) transgenic line, n ≥ 100. C , Summary for rescue experiments of defective associative learning between temperature and feeding state of tax-6 mutant. D , The effect of the TAX-6 dominant-negative form on thermotaxis. Each bar represents the average of at least three independent thermotaxis assays using 20 animals per assay. Three independent transgenic lines were tested for tax-6(sensory+) animals expressing the tax-6 dominant-negative form driven by various promoters. n ≥ 120 animals for each genotype. E , F , Results of the thermotaxis assay conducted at 17 ( E ) or 23°C ( F ) on 17°C-cultivated fed wild-type animals carrying various types of tax-6 cDNA in AIZ and RIA interneurons. H176Q indicates the TAX-6 phosphatase negative form. Each bar represents the average of at least three independent thermotaxis assays using 20 animals per assay. Three independent transgenic lines were tested wild-type animals expressing various tax-6 cDNAs. n ≥ 80 animals for each genotype.
- Figure 4.
In vivo calcium ratio imaging of the neural circuit for feeding state-dependent thermotactic plasticity. Calcium ratio imaging of AIZ ( A–D , F , G ) and RIA interneurons ( E ) in 17°C-grown animals cultivated under the fed or starved condition. Each graph represents the average response of AIZ or RIA interneurons to thermal stimuli. Each data were aligned at stimulus onset. Relative increases or decreases in the intracellular calcium concentration were measured as increases or decreases in the YFP/CFP fluorescence ratio of the cameleon protein (ratio change), respectively. Temperature (temperature) is shown as a thin black line at the bottom of the graph. A , The AIZ interneuron in wild type responds to warming. The average ratio change from baseline ± SEM is 0.143 ± 0.008; n = 12. B , The AIZ interneuron in wild type responds to cooling. The average ratio change from baseline ± SEM is −0.155 ± 0.013; n = 11. C , A schematic diagram of an AIZ interneuron (green, top) in the C. elegans head, and corresponding pseudocolor images (bottom) depicting the fluorescence ratio before (left) and during (right) temperature changes. Blue and red pixels represent lower and higher fluorescence ratios, respectively. D , The AIZ interneurons respond to step-like temperature changes; n = 10. E , Calcium ratio imaging of RIA interneurons in wild-type animals cultivated under the fed or starved condition. The average ratio changes from baseline ± SEM are 0.04 ± 0.004 (fed) and 0.038 ± 0.007 (starved). Fed, n = 10; starved, n = 10. F , Calcium ratio imaging of AIZ interneurons in wild-type animals cultivated under the fed or starved condition. Fed, n = 20; starved, n = 21. The average ratio changes from baseline ± SEM are 0.145 ± 0.009 (fed) and 0.073 ± 0.010 (starved). G , Calcium ratio imaging of AIZ interneurons in tax-6(sensory+, inter−) mutant animals cultivated under the fed or starved condition. The average ratio changes from baseline ± SEM are 0.122 ± 0.010 (fed) and 0.140 ± 0.013 (starved). Fed, n = 21; starved, n = 27.
- Figure 5.
A neural circuit model for associative learning between temperature and feeding state. In fed wild-type animals cultivated at 17°C, the AIZ–RIA neural pathway is activated, thereby causing fed animals to be attracted to the cultivation temperature (top left and middle). In starved wild-type animals, a starvation signal negatively regulates the AIZ-mediated cryophilic driving pathway through TAX-6 calcineurin (right), leading to a relatively more active state of the thermophilic driving pathways such as the AIY-mediated pathway (bottom middle or bottom left). As a result, starved animals avoid the cultivation temperature (bottom left).
