Extremely sparse olfactory inputs are sufficient to mediate innate aversion in Drosophila

Abstract

Innate attraction and aversion to odorants are observed throughout the animal kingdom, but how olfactory circuits encode such valences is not well understood, despite extensive anatomical and functional knowledge. In Drosophila melanogaster, ~50 types of olfactory receptor neurons (ORNs) each express a unique receptor gene, and relay information to a cognate type of projection neurons (PNs). To examine the extent to which the population activity of ORNs is required for olfactory behavior, we developed a genetic strategy to block all ORN outputs, and then to restore output in specific types. Unlike attraction, aversion was unaffected by simultaneous silencing of many ORNs, and even single ORN types previously shown to convey neutral valence sufficed to mediate aversion. Thus, aversion may rely on specific activity patterns in individual ORNs rather than the number or identity of activated ORNs. ORN activity is relayed into the brain by downstream circuits, with excitatory PNs (ePN) representing a major output. We found that silencing the majority of ePNs did not affect aversion, even when ePNs directly downstream of single restored ORN types were silenced. Our data demonstrate the robustness of olfactory aversion, and suggest that its circuit mechanism is qualitatively different from attraction.

Fig 1. Aversion and attraction after broad ORN inactivation.

(A) Definition of the Preference Index (PI). 10% acetic acid is delivered to the bottom right quadrant, and air to the other three quadrants. Each number (#1-#4) represents the positions visited by flies in a particular quadrant counted over all the frames in a defined period of time. (B) Orco > shi ^ts1 and Ir8a > shi ^ts1 impair attraction to vinegar at the restrictive (B₁, n ≥ 4) but not the permissive (B₂, n ≥ 4) temperature for shi ^ts1. (C, D) The same manipulations as in B₁ do not affect aversion to E2-hexenal (C, n ≥ 3) or acetic acid (D, n ≥ 3). Throughout this paper, n refers to the number of trials, each bar represents the mean performance index (PI), and error bars represent s.e.m.; comparisons are t-test with Holm-Bonferroni post hoc correction. For the same genotype on different plots, the same data were used. * p<0.05, ** p<0.01, *** p < 0.001, **** p < 0.0001.

Fig 2. Aversion after pan-ORN inactivation and restoration in OR+ ORNs.

(A) Venn diagrams and schematic circuits for the genetic intersections. In the Venn diagrams, green circles represent expression pattern of Pebbled-GAL4, red circles represent expression pattern of ey-FLP, blue circles represent expression pattern of Orco-GAL80, and dark green fill represent expression pattern of effector. In the circuit diagrams, the ellipses represent neuronal cell bodies, the straight lines represent their axon, the small triangles represent presynaptic terminals, and the grey circles in dashed lines represent glomeruli; each neuron represents one type of ORNs, black fill and solid lines indicate functional neurons, and dark green fill and dashed lines indicate blocking by the effector. Top right: AND intersection between Pebbled-GAL4 and ey-FLP blocks outputs from all ORNs. Bottom left: NOT intersection by adding Orco-GAL80 restores synaptic transmission in OR+ ORNs. (B ₁, B ₂ ) Visualizing the intersection between Pebbled-GAL4 and ey-FLP with UAS-FRT-stop-FRT-CD8::GFP. Single image slices are shown in (B₁), and the projection of the whole brain in (B₂). (C) Aversion to E2-hexenal is abolished by Pebbled AND ey > shi ^ts1 at the restrictive (C₁, n ≥ 3) but not the permissive (C₂, n ≥ 4) temperature for shi ^ts1, and rescued by Orco-GAL80 (C₁). (D) Aversion to acetic acid is also abolished by the same manipulation as in C₁, and rescued by Orco-GAL80 (n ≥ 4). (E, F) Orco-GAL80 suppresses Pebbled-GAL4 expression in a large subset of glomeruli, visualized with UAS-nsyb::GFP.

Fig 3. Aversion after ORN-specific restoration of synaptic transmission.

(A-H) Pebbled-GAL4 expression in specific glomeruli is suppressed by Ir64a-GAL80 (A, B), Or22a-GAL80 (C, D), Or85a-GAL80 (E, F), and Or42b-GAL80 (G, H). White texts label the glomeruli predicted from published work; yellow texts label the additionally suppressed glomeruli. Glomeruli with reduced expression of ORN outputs are outlined. Each image is representative of 5 brains of the same genotype. (I) A representative scheme of using Or22a-GAL80 to restore inputs to DM2 glomerulus in the pan-ORN inactivation background, same legend as Fig 2A. (J, K) Aversion in Pebbled AND ey > shi ^ts1 flies to E2-hexenal (J, n ≥ 3) and acetic acid (K, n ≥ 3) is recued by GAL80s, and Or42b-GAL80 causes less aversion to E2-hexenal than Or22a-GAL80 (J). The stars right below each bar indicate statistical significance as compared to the non-GAL80 control.

Fig 4. Testing the role of ePNs in aversion and attraction.

(A-C) GH146 > shi ^ts1 affects attraction to vinegar (A, n ≥ 4) but not aversion to E2-hexenal (B, n ≥ 3) or acetic acid (C, n ≥ 3). (D ₁, D ₂ ) Visualizing the intersection between 853-GAL4 and GH146-FLP with UAS-FRT-stop-FRT-CD8::GFP. The projection of the whole brain is shown in (D₁), and single image slices in (D₂). (E ₁, E ₂ ) Schemes of combining Or22a-GAL80-mediated restoration with the inactivation of its cognate ePNs (E₁) or the majority of ePNs (E₂), same legend as Fig 2A with the additional layer of ePNs connecting to cognate ORNs in the glomeruli; the color of inactivated ePNs is the intermediate between the black of intact neurons and the dark green of the inactivated ORNs, indicating possibly incomplete inactivation resulting from GH146-GAL4 not expressing in all ePNs. (F, G) Aversion to E2-hexenal (F, n ≥ 4) or acetic acid (G, n ≥ 4) in Pebbled AND ey NOT Or22a > shi ^ts1 flies (“ctrl”) is not affected by either adding 853-GAL4 and GH146-FLP to block the cognate DM2 ePNs, or adding GH146-QF and QUAS-shi ^ts1 to block the majority of ePNs including those innervating DM2. (H) Or42b-GAL80 rescue still causes less aversion than Or22a-GAL80, after blocking most of the ePNs with GH146-QF and QUAS-shi ^ts1 (n ≥ 5).

Fig 5. Summary of the manipulations and results.

Same legend as Fig 4E. “✓” indicates the ability to perform the behavior, and “✗” indicates a behavioral defect.