A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans.

Standard techniques were used for nematode culture and molecular biology. A complete strain list, description of gcy-28 cloning, detailed molecular biology, and sequence analysis methods are in Supplementary Material .

Behavioral assays

Population chemotaxis assays were performed on assay agar (1.6 % agar, 1 mM MgSO 4 , 1 mM MgCl 2 , 5 mM phosphate buffer, pH 6.0) in 10 cm square plates, which are better for detecting avoidance behavior than round assay plates ( Troemel et al., 1997 ). Unless otherwise indicated, odor dilutions in ethanol were 2-butanone 1:1,000; 2,3-pentanedione 1:10,000; benzaldehyde 1:200; diacetyl 1:1,000; 2-methylpyrazine 1:1,000; 2-nonanone 1:10. 2 μl of diluted odor was placed on one side of the plate, and 2 μl of ethanol at the other side, with azide to anaesthetize animals that reached odor or ethanol sources. Washed adult animals were placed in the center of the plate, and the distribution of animals counted after 1-2 hours. A score of 1.0 represents perfect attraction, -1.0 perfect repulsion, and 0 random behavior. All data points are averages of ≥ 4 assays, with ≥ 50 animals each, repeated on at least two different days. For tracking assays ( Fig. 2 , 3 ), about 50 animals were picked onto an NGM plate without bacteria, washed with S-basal and assay buffer, and placed on round 10 cm plates with assay agar. Excess liquid was wicked off, and recording was started after a spot of odor and diluent were placed on the agar surface. Unlike standard chemotaxis assays, no sodium azide was added. Tracking assays were performed under a dissecting scope (Stemi 2000; Zeiss, Thornwood, NY) with a custom-modified base that illuminates a wide field (Serco Technical Services, Livermore, CA). The field was captured by a digital camera (MacroFire; Optronics, Goleta, CA) with 1240×1240 pixel resolution at 2 frames per second. Captured movies were analyzed using MATLAB (MathWorks, Natick, MA) scripts ( Ramot et al., 2008 ; http://wormsense.stanford.edu/ ). Tracks were segmented into turns and runs, essentially as described ( Pierce-Shimomura et al., 1999 ). Runs were analyzed in 5-second bins, and an average bearing relative to the odor was obtained for each bin. Absolute angles were binned in 30° intervals. PMA treatment was performed as described ( Okochi et al., 2005 ). Briefly, adult animals were washed onto culture plates with 1 μg/ml PMA (PMA+) or DMSO solvent (PMA-) in the agar, incubated at room temperature for two hours, and then tested for chemotaxis in drug-free assay plates. Heat shock treatment was performed as described ( L’Etoile et al., 2002 ). Animals were incubated at 33°C for two hours, and then incubated for another two hours at 20°C before testing for chemotaxis. Butanone sensitization assays were performed as described, with some modifications ( Torayama et al., 2007 ). Animals on their NGM growth plate were exposed to butanone vapor by spotting 12 μL of butanone on agar plugs on the plate lid and sealing the plate with parafilm. After 90 minutes animals were washed and tested for chemotaxis on square plates. The butanone dilution used for chemotaxis was 1:1000. Adaptation assays were performed essentially as described ( Colbert and Bargmann, 1995 ). Animals were washed and placed on 3% assay agar plates. 20 μL butanone was placed on agar plugs on the plate lid and the plates were sealed with parafilm. After two hours animals were washed off and tested for chemotaxis on square plates. Controls were treated identically except that butanone was omitted from the conditioning plate. Laser ablations were performed on L1 animals as described ( Bargmann and Avery, 1995 ). The AWC ON cell was identified by expression of str-2∷GFP in the integrated strain CX6343. About 20 animals were ablated for each tracking assay, and some animals were tested twice on two different days.

Calcium imaging

Calcium imaging was performed as described ( Chalasani et al., 2007 ; Chronis et al., 2007 ). For AWC ON imaging, the strain CX10281 expresses the calcium indicator G-CaMP2.0 ( Tallini et al., 2006 ) in AWC ON under the str-2 promoter. gcy-28(tm2411) and pkc-1(nj1) were crossed into CX10281 to generate the strains CX10223 and CX10784, respectively. For AIB imaging, the strain CX7469 expressing G-CaMP1.0 in AIB neurons ( Chalasani et al., 2007 ) was crossed with gcy-28(tm2411) to generate the strain CX8994. Animals were washed in buffer without food for ~20 minutes prior to imaging, a protocol designed to mimic the washes before chemotaxis assays. This brief washing step enhances the reliability of chemotaxis and of AWC calcium imaging. Imaging was conducted in a polydimethylsiloxane (PDMS) chamber in which an animal’s nose was exposed to a stream of buffer that could be switched between odor-containing and odor-free solutions using an electronically gated valve. The standard stimulus protocol consisted of a 5-minute step pulse of the indicated dilution of odor in S-basal (without cholesterol) followed by odor removal. G-CaMP fluorescence intensity was measured for 10 seconds before and 50 seconds after the onset or offset of the odor stimulus; the same animals were imaged for odor onset and offset. All G-CaMP strains had the appropriate olfactory behaviors for their respective genetic backgrounds.

Supplementary Material 01