elegans may suggest the existence of similar mechanisms in the nociceptive and somatosensory pathways of larger nervous systems. A complete strain list and descriptions of plasmid and strain constructions are in Supplemental Experimental Procedures. Laser ablations were carried out using a standard protocol (Bargmann and Avery, 1995). The RIHs, OLQs, and FLPs were ablated in the early L1 stage, usually check details within 3–4 hr after

hatching; the PVD cells were ablated at a slightly later stage, near the end of L1. Loss of the ablated cell was confirmed by observing loss of cameleon fluorescence in the adult animal. Optical recordings were performed essentially as described (Kerr et al., 2000 and Kerr, 2006) on a Zeiss Axioskop 2 upright compound microscope equipped with a Dual View beam splitter and a UNIBLITZ Shutter. Fluorescence images were acquired using MetaVue 6.2. Filter-dichroic pairs were excitation, 400–440; excitation dichroic 455; CFP emission, 465–495; emission dichroic 505; YFP emission, 520–550. Individual adult worms (∼24 hr past L4) were glued with Nexaband S/C cyanoacrylate glue to pads composed of 2% agarose in extracellular saline (145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 5 mM MgCl2, 20 mM D-glucose, 10 mM HEPES buffer [pH 7.2]). Serotonin was also included at a concentration of 5 mM for nose touch-imaging

experiments. Worms used for calcium imaging had similar levels of cameleon expression in sensory neurons as inferred from initial fluorescence intensity. Acquisitions were taken at 28 Hz (35 ms exposure time)

with VEGFR inhibitor 4 × 4 or 2 × 2 binning, using a 63× Zeiss Achroplan water-immersion objective. Thermal stimulation was applied as described (Chatzigeorgiou et al., 2010b). The nose touch stimulator was a needle with a 50 μm diameter made of a drawn glass Oxalosuccinic acid capillary with the tip rounded to ∼10 μm on a flame. We positioned the stimulator using a motorized stage (Polytec/PI M-111.1DG microtranslation stage with C-862 Mercury II controller). The needle was placed perpendicular to the worm’s body at a distance of 150 μm from the side of the nose. In the “on” phase, the glass tip was moved toward the worm so that it could probe ∼8 μm into the side of the worm’s nose on the cilia and held on the cilia for 1 s, and in the “off “ phase the needle was returned to its original position. To visualize the harsh head touch response in FLP, the same nose touch setup was used, but the probe was aligned in a more posterior position between the two bulbs of the pharynx. The probe was displaced ∼24 μm at a raised speed of 2.8 mm/s. The stimulus was a buzz (i.e., the probe was displaced 2.5 μm in and out for the duration of the stimulus) lasting ∼1 s. To obtain single images we used a Zeis LSM 510 Meta confocal microscope with a 40× objective. Images were exported as single TIFF files. To measure the intensity of the fluorescence, we imported the TIFF image in ImageJ.