Olfactory Neurons and Brain Centers Directing Oviposition Decisions in Drosophila

Abstract

The sense of smell influences many behaviors, yet how odors are represented in the brain remains unclear. A major challenge to studying olfaction is the lack of methods allowing activation of specific types of olfactory neurons in an ethologically relevant setting. To address this, we developed a genetic method in Drosophila called olfactogenetics in which a narrowly tuned odorant receptor, Or56a, is ectopically expressed in different olfactory neuron types. Stimulation with geosmin (the only known Or56a ligand) in an Or56a mutant background leads to specific activation of only target olfactory neuron types. We used this approach to identify olfactory sensory neurons (OSNs) that directly guide oviposition decisions. We identify 5 OSN-types (Or71a, Or47b, Or49a, Or67b, and Or7a) that, when activated alone, suppress oviposition. Projection neurons partnering with these OSNs share a region of innervation in the lateral horn, suggesting that oviposition site selection might be encoded in this brain region.

Keywords: genetic technique; genetics; geosmin; odorant receptors; olfaction; olfactogenetics; olfactory; oviposition; projection neurons; vinegar fly.

Figure 1.. Or56a-Geosmin Olfactogenetic Method for Investigating Odor-Guided Behaviors

(A) Schematic of the olfactogenetic approach. In wild-type (WT) conditions, the ab4 sensillum contains 2 OSNs: the “A” neuron expresses Or7a (gray), and the “B” neuron expresses Or56a (green). The ab3 sensillum also contains 2 OSNs: the ab3A neuron expresses Or22a (magenta), and the ab3B neuron expresses Or85b (gray). Geosmin (dark green circles) activates (orange star) only Or56a-positive neurons and not other neurons. Mutating the Or56a receptor results in an olfactory system that does not detect nor behaviorally respond to geosmin. To create olfactogenetic flies, we use the GAL4/UAS system to ectopically express UAS-Or56a in a specific olfactory neuron (e.g., Or22a-GAL4, magenta plus green with magenta outline) in an Or56a^−/−mutant background. This allows the odorant geosmin to activate olfactory neurons with high specificity to drive olfactory behaviors. (B) Single sensillum recordings (SSR) of ab4 sensilla. The B neuron in an Or56a^−/−mutant no longer responds to geosmin. The A neuron in an Or56a^−/−mutant responds normally to 9-tricosene. ab, antennal basiconic; PNs, projection neurons; OSNs, olfactory sensory neurons. See Figure S1 for details on generation of the Or56^−/−mutant.

Figure 2.. Ectopic Expression of Or56a in Olfactory Neurons Confers a Response to Geosmin

(A) Representative single sensillum recordings of basiconic, intermediate, and trichoid sensilla containing neurons expressing Or56a. The blue bar highlights the 1 s odor pulse. (B) Quantification of recordings. (C) Representative SSR of ab1 sensilla that contain 4 olfactory neurons. The “C” neuron expresses Gr21a/Gr63a and is sensitive to CO₂. (D) Quantification of recordings. Error bars represent SEM. See also Figure S2.

Figure 3.. Behavioral Effects in the Two-Choice Oviposition Assay Using Geosmin Rely on Olfaction

(A) Schematic of oviposition assay. (B) Olfactory mutant animals do not exhibit behavioral responses to geosmin. Control w¹¹¹⁸ animals are slightly repelled by geosmin. The oviposition index is calculated as (the number of eggs laid in odor well the average number eggs laid in the no-odor wells)/the total number of eggs. The different genotypes are not statistically significant as determined by a Dunnett’s many-to-one comparisons test. (C) Positional controls for two-choice oviposition assay. The position of the odorant well in the three-well assay does not affect behavior. Differences are not statistically significant as determined by a Dunnett’s many-to-one comparisons test.

Figure 4.. Olfactogenetic Activation of Specific OSN Types Mediates Negative Oviposition

(A) Oviposition assays using geosmin. OrX-Gal4 lines were combined with UAS-Or56a in the Or56a^−/−mutant background (gray bars). Gr63a-GAL4 combined with UAS-Or56a and UAS-Orco in the Or56a^−/−background (gray bar). Mutant Or56^−/−and wild-type (Or56a) responses denoted by white bars. (B) Oviposition assays comparing results obtained from an olfactogenetic approach to those ob-tained using the indicated odorants. Statistics are a Dunnett’s many-to-one comparisons test compared to Or56a^−/−. Asterisks indicate p values: *p < 0.1, **p < 0.05, and ***p < 0.001. See also Figure S4 and Table S1.

Figure 5.. Representation of Negative Oviposition Olfactory Cues in the Lateral Horn

(A) PN traces corresponding to the listed OSN type for the most statistically significant responses in the olfactogenetic oviposition assay (p < 0.001). LH, lateral horn; MBc, mushroom body calyx; A, anterior; P, posterior; D, dorsal; V, ventral; L, lateral; M, medial. (B) Comparison of negative oviposition PNs (purple) to all other PN types (black in top trace, blue and red in categorized bottom). See also Figure S5.