Optogenetically Induced Olfactory Stimulation in Drosophila Larvae Reveals the Neuronal Basis of Odor-Aversion behavior

Abstract

Olfactory stimulation induces an odor-guided crawling behavior of Drosophila melanogaster larvae characterized by either an attractive or a repellent reaction. In order to understand the underlying processes leading to these orientations we stimulated single olfactory receptor neurons (ORNs) through photo-activation within an intact neuronal network. Using the Gal4-UAS system two light inducible proteins, the light-sensitive cation channel channelrhodopsin-2 (ChR-2) or the light-sensitive adenylyl cyclase (Pacalpha) were expressed in all or in individual ORNs of the larval olfactory system. Blue light stimulation caused an activation of these neurons, ultimately producing the illusion of an odor stimulus. Larvae were tested in a phototaxis assay for their orientation toward or away from the light source. Here we show that activation of Pacalpha expressing ORNs bearing the receptors Or33b or Or45a in blind norpA mutant larvae induces a repellent behavior away from the light. Conversely, photo-activation of the majority of ORNs induces attraction towards the light. Interestingly, in wild type larvae two ligands of Or33b and Or45a, octyl acetate and propionic ethylester, respectively, have been found to cause an escape reaction. Therefore, we combined light and odor stimulation to analyze the function of Or33b and Or45a expressing ORNs. We show that the larval olfactory system contains a designated neuronal pathway for repellent odorants and that activation of a specific class of ORNs already determines olfactory avoidance behavior.

Keywords: Drosophila; channelrhodopsin-2; electrophysiology; olfaction; olfactory behavior; optogenetics; photo-activated adenylyl cyclase; photo-activation.

Figure 1

Behavior test assay for larvae. (A) Gray quadrants show dark areas while white sections represent illuminated areas of the Petri dish. Numbers indicate the light intensity at 480 nm. (B) Individual wild type larval crawling traces show negative phototactic reaction to a blue light stimulus (n = 12). (C) Traces individual transgenic larvae that express ChR-2 under control of Or83b-Gal4 in all ORNs (n = 12). (D) Traces of wild type larvae (n = 12) exposed to benzaldehyde on a filter paper indicated as black squares. (E,F) Response indices (RI) of wild type larvae, transgenic larvae and controls. (E) Wild type and transgenic larvae that express either ChR-2 or Pacα in all ORNs under control of the driver line Or83b-Gal4. *RI values are statistically different from those of wild type larvae (t-test, P < 0.05); **RI values are statistically different from those of transformed larvae stimulated with white light (t-test, P < 0.05). Negative phototaxis is strongly reduced in the transgenic strains expressing the light-sensitive proteins in all ORNs. (F) As control strains wild type larvae fed with retinal (wild type + retinal) or parental lines (Or83b-Gal4 and UAS lines) were used (n = 15 each). Here, a strong light avoidance can be observed.

Figure 2

Individual elecrophysiological recording from the dorsal organ of the third instar larvae (EDG) with (A) application of pure ethyl acetate and (B) application of light at 480 nm wavelength for 1 s each. (C) Application of light for 2 min with simultaneous application of ethyl acetate (Ea) for 1 s. (D) The mean of EDG response (n = 10) in mV in response to the application of either Ea or 480 nm or Ea together with 480 nm. The upper bar indicates the duration of stimuli with 480 nm, whereas the lower short bar represents the stimulation with Ea.

Figure 3

(A) Behavior of wild type larvae exposed to either white or blue light and benzaldehyde in the illuminated quadrants (n = 15). The anosmic mutant Or83b⁻ shows only a negative phototactic response, while wild type larvae are more attracted by increasing concentrations of benzaldehyde, as indicated by the decreasing RI values. (B) Wild type larvae exposed to different concentrations of octyl acetate. With undiluted concentration of octyl acetate applied in the dark quadrants larvae escaped into illuminated sections of the Petri dish. Bars represent means ± SEM (n = 15).

Figure 4

Orientation behavior of transgenic larvae in the four quadrant assay with two quadrants illuminated either with blue or white light, showing the photo-activation of individual ORNs (n = 20). Expression of (A) ChR-2 or (B) Pacα in ORNs that express different Ors as indicated. *RI values are statistically different from RI values of wild type larvae (t-test, P < 0.05) Bars represent mean ± SEM.

Figure 5

Behavior in the presence of octyl acetate and blue light (n = 10). (A) Reaction of wild type, anosmic (Or83b⁻) and transgenic larvae that express Pacα under the control of the indicated driver lines. The repellent octyl acetate was presented on filter papers in the dark areas of the Petri dish. Stimulation was either with octyl acetate (white bars), with blue light (dark bars) or both together over a period of 30 s. (B) The larvae treated with odor and light simultaneously were tested for an additional 2 min in the presence of octyl acetate in the dark quadrants, but in complete darkness. Bars represent mean ± SEM.

Figure 6

Expression of either ChR-2 or Pacα in the mutant norpA background. Larvae expressing ChR-2 or Pacα in all ORNs (Or83b) are attracted by the blue light. On the contrary, larvae expressing these proteins in Or33b or Or45a neurons are repelled by blue light. The UAS-ChR-2 or UAS-Pacα lines in the mutant norp A background (norp A) show no phototaxis. Bars represent mean ± SEM (n = 10 each).