Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe

Abstract

A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain [1-7]. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants [8-11]. OSNs also exhibit odor-evoked responses with complex temporal dynamics [11], but the contribution of this activity to behavioral odor discrimination has received little attention [12]. Here, we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.

Figure 1. Or83b² flies with functional Or46a, Or67a or Or98a-expressing neurons learn to discriminate between odorants that activate these receptors

(A) Or83b² mutant flies cannot learn to discriminate between odors. Wild-type flies can learn to discriminate between six pairs of odorants whereas Or83b² mutant flies cannot. Asterisks indicate no significant difference to zero (all P>0.1, Mann Whitney U-test). (B) Upper panel, volume rendering of the fly antennal lobes highlighting the relative position of the VA7l (orange), DM6 (green) and VM5 (yellow) glomeruli innervated by Or46a, Or67a and Or98a expressing OSNs. Lower panels show corresponding confocal stack projections through the antennal lobes of flies expressing uas-n-syb::GFP driven by Or46a-GAL4, Or67a-GAL4 or Or98a-GAL4. N-syb::GFP is stained with anti-GFP (green) and neuropil is visualized with nc82 antibody (magenta) staining. Scale bar is 20µm and refers to all micrographs. (C) Flies with only functional OR46a neurons can learn to discriminate between 4-methyl phenol and methyl salicylate but flies with only OR67a neurons cannot. Asterisks indicate significant difference (all P<0.04, ANOVA) between the marked groups and all others. (D) Flies with only functional OR67a or OR98a neurons can learn to discriminate between methyl benzoate and isoamyl acetate. Asterisks indicate significant difference (all P<0.005, ANOVA) between the marked groups and all others. (E) Flies with only functional OR67a neurons can learn to discriminate between pentyl acetate and 6-methyl-5-hepten-2-one. Asterisks indicate significant difference (all P<0.005, ANOVA) between the marked groups and all others. Data are mean ± s.e.m.

Fig. 2. Limitations in learned behavior in Or83b² flies with functional Or67a-expressing neurons

(A) Flies with only functional OR67a neurons learn one component of a binary blend. Flies were trained with 6-methyl-5-hepten-2-one + isoamyl acetate versus methyl benzoate + pentyl acetate mixtures. Wild-type flies show learning when tested with all components alone whereas flies with only functional OR67a neurons exclusively show learned discrimination for the 6-methyl-5-hepten-2-one and pentyl acetate components. (B) Wild-type flies learn both components of a different binary blend but OR67a restored flies still only learn one. Flies were trained with 6-methyl-5-hepten-2-one + methyl benzoate versus pentyl acetate + isoamyl acetate mixtures. Wild-type flies learn all components whereas flies with restored OR67a neurons again only show learned discrimination for the 6-methyl-5-hepten-2-one and pentyl acetate components. (C) Flies with restored OR67a neurons do not show learned discrimination of isoamyl acetate and methyl benzoate when 6-methyl-5-hepten-2-one and pentyl acetate are also present during test. Wild-type flies trained with either set of single components show learned discrimination when tested with the additional complexity of binary mixtures, but flies with OR67a neurons only show robust performance if trained with 6-methyl-5-hepten-2-one versus pentyl acetate. (D) Flies with functional OR67a neurons do not show learned discrimination of isoamyl acetate and methyl benzoate when tested with a different composition of odorant mixtures. Wild-type flies trained with either set of single components, 6-methyl-5-hepten-2-one versus pentyl acetate or isoamyl acetate versus methyl benzoate, show learned discrimination when tested with binary mixtures but flies with OR67a neurons only show robust performance if trained with 6-methyl-5-hepten-2-one versus pentyl acetate. Individual odor concentrations in the blends were the same as those used separately in Fig.2 C and 2D and when tested for component learning (Fig.3A and 3B). Asterisks denote no significant difference to zero (all P>0.5, Mann Whitney U-test).

Figure 3. OR67a restored flies cross-adapt to odorants that activate OR67a neurons

(A) Adaptation of innate odor avoidance behavior in wild-type and OR67a restored flies. Pre-exposing wild-type flies and those with restored OR67a neurons to methyl benzoate adapts methyl benzoate avoidance behavior. Flies with OR67a restored neurons, but not wild-type flies, cross-adapt to methyl benzoate, pentyl acetate and isoamyl acetate. Asterisk indicates significant difference (P<0.002, ANOVA) (B) Pre-exposure to pentyl acetate significantly adapts pentyl acetate avoidance behavior of flies with restored OR67a neurons (P<0.002, ANOVA) but does not significantly adapt wild-type flies (P>0.1, ANOVA). Pre-exposure to pentyl acetate cross-adapts methyl benzoate and 6-methyl-5-hepten-2-one avoidance in flies with OR67a restored neurons (both P<0.001, ANOVA) but not in wild-type flies (P>0.2, ANOVA). (C) Pre-exposure to isoamyl acetate cross-adapts the methyl benzoate avoidance behavior of flies with restored OR67a neurons (P<0.002, ANOVA) but does not significantly adapt wild-type flies (P>0.1, ANOVA). (D) Pre-exposure to 6-methyl-5-hepten-2-one cross-adapts pentyl acetate avoidance behavior of flies with restored OR67a neurons (P<0.002, ANOVA) but does not significantly adapt wild-type flies (P>0.1, ANOVA). Data are mean ± s.e.m.

Figure 4. Or83b² flies with functional OR67a neurons discriminate odorants across changing concentration

(A) Wild-type flies and OR67a restored flies were trained with 6-methyl-5-hepten-2-one concentrations that were 10X less, the same or 10X more than they were tested with, while pentyl acetate concentrations were kept constant. (B) Wild-type flies and those with restored OR67a neurons were trained with pentyl acetate concentrations that were 10X less, the same or 10X more than they were tested with, while 6-methyl-5-hepten-2-one concentrations were kept constant. Asterisks indicate significant difference (all P<0.01, ANOVA). Data are mean ± s.e.m.