To test this, we built a temperature-controlled stage (see Supplemental Experimental Procedures). In animals grown at 22°C, AFD responded to CO2 both at 15°C and at 22°C (Figures S1E and S1F). The shape of the response was similar at the two temperatures but smaller at 15°C than at 22°C.

These data support the idea that AFD CO2 and temperature-sensing pathways are at least partly distinct. Recent work has shown that the BAG neurons are transiently activated when O2 levels see more drop below 10% (Zimmer et al., 2009). Hallem and Sternberg (2008) showed that feeding animals lacking the BAG neurons have reduced avoidance of a 10% CO2/10% O2 mixture. We have previously shown that O2 responses can modulate CO2 avoidance (Bretscher et al., 2008). These data suggest that either BAG responds exclusively to O2 but modulates neural circuits mediating CO2 responses or that BAG is a primary sensor of both O2 and CO2. To test BAG neuron CO2 sensitivity, we created animals expressing cameleon YC3.60 in BAG from a pflp-17::YC3.60 Palbociclib transgene and imaged Ca2+ levels. The BAGL and BAGR neurons were exquisitely sensitive to a rise in CO2 ( Figures 3A–3C). Cameleon reported a rise in Ca2+ that peaked after

∼30 s and then decayed ( Figures 3A and 3B). The excitability threshold of BAG was below 0.25% CO2. A plot of mean fluorescence ratio change against percent (%) CO2 suggests that BAG reaches half-maximal activity at ∼2.9% CO2 through ( Figure 3D). Thus, BAG neurons respond to both O2 and CO2. Elevated CO2 persistently stimulates locomotory activity in feeding C. elegans, suggesting that some CO2-sensing circuits can signal tonically in high CO2 ( Bretscher et al., 2008). During prolonged high CO2 the BAG Ca2+ spike decayed to a plateau that persisted until CO2 removal, at which point Ca2+ returned to resting levels ( Figure 3E). Thus, BAG exhibits both a transient peak and a perduring Ca2+ plateau in response to elevated CO2. As with AFD, we asked whether BAG neurons habituate. During

five stimulus cycles of 3% CO2, BAG showed a decrement in response amplitude after the first CO2 stimulus, but no habituation thereafter ( Figures 3F–3H). To test if the BAG neurons are primary CO2 sensors, we disrupted synaptic input to BAG using the unc-13 and unc-31 mutations. unc-31 mutants are defective in dense-core vesicle release, but not synaptic vesicle release ( Speese et al., 2007). Neither the unc-13 nor the unc-31 mutations disrupted BAG Ca2+ responses, suggesting that BAG neurons are intrinsically CO2 sensitive ( Figures 3I–3K). However, the magnitude of Ca2+ responses in these mutants was significantly enhanced, particularly in unc-31 animals, suggesting that BAG activity is normally inhibited by neuromodulators.