A Specific Olfactory Circuit Mediates an Avoidance Behavior in Drosophila

Greg S.B. Suh¹, Allan W. Wong², Anne Hergarden^1,3, Jing Wang^2,3, Anne Simon¹, Richard Axel^2,3, Seymour Benzer¹, and David J. Anderson^1,3

California Institute of Technology, Division of Biology¹; Columbia University, School of Medicine²; Howard Hughes Medical Institute³

e-mail: gregsuh@caltech.edu

We have developed a novel behavioral paradigm for an innate avoidance response in Drosophila. This paradigm involves avoidance of a substance (called Drosophila Stress Odorant or dSO) emitted by flies subjected to mechanical stress or electrical shock. Responder flies were given a choice in a T-maze between a fresh tube and a conditioned tube in which a set of emitter flies were previously stressed. Most responders chose the fresh tube; the Performance Index (a measure of avoidance) typically falls between 95 and 80 under optimal conditions.

Gas Chromatography & Mass Spectrometry (GC/MS) analyses indicated that CO₂ is a component of dSO. Consistent with this, flies exhibited avoidance response to CO₂ in a dosage dependent manner. Surgical removal of the 3rd antennal segment which houses olfactory receptor neurons (ORNs) rendered the flies defective in responding to dSO and CO₂ indicating ORNs are likely required for avoidance to dSO and CO₂. We next sought to functionally map olfactory circuits mediating CO₂ avoidance in collaboration with Richard Axel laboratory in Columbia University. Flies in which a genetically encoded, calcium-responsive indicator (GCaMP) was expressed throughout the antenna lobe revealed that a single pair of ventral glomeruli, known as the V glomeruli, were activated by CO₂. Furthermore, none of 26 other odorants tested thus far activates the V glomeruli. Moreover, functional inactivation of GR21A+ sensory neurons projected to V, using UAS-Shibire^ts was sufficient to abolish the avoidance response to CO₂.

We have independently mapped groups of neurons essential for the avoidance response to dSO by carrying out an unbiased neuronal inactivation screen using the Gal4 x UAS-Shibire^ts system. Flies bearing a Gal4 enhancer trap and a UAS-Shibire^ts were assayed in the behavioral paradigm at a non-permissive temperature (32°C), and their performance compared to that at a permissive temperature (21°C). We have screened ~260 Gal4 enhancer trap lines and identified 14 lines defective in this assay only at 32°C. Among the 14 lines, 5 retained normal olfactory responses to various odorants and locomotor activities. In one of the 5 lines, c761, avoidance response to CO₂ was also defective at 32°C. In this line, Gal4 is expressed in the V glomeruli, consistent with the lack of CO₂-responsiveness.