Olfactory processing and behavior downstream from highly selective receptor neurons

Abstract

In both the vertebrate nose and the insect antenna, most olfactory receptor neurons (ORNs) respond to multiple odors. However, some ORNs respond to just a single odor, or at most to a few highly related odors. It has been hypothesized that narrowly tuned ORNs project to narrowly tuned neurons in the brain, and that these dedicated circuits mediate innate behavioral responses to a particular ligand. Here we have investigated neural activity and behavior downstream from two narrowly tuned ORN types in Drosophila melanogaster. We found that genetically ablating either of these ORN types impairs innate behavioral attraction to their cognate ligand. Neurons in the antennal lobe postsynaptic to one of these ORN types are, like their presynaptic ORNs, narrowly tuned to a pheromone. However, neurons postsynaptic to the second ORN type are broadly tuned. These results demonstrate that some narrowly tuned ORNs project to dedicated central circuits, ensuring a tight connection between stimulus and behavior, whereas others project to central neurons that participate in the ensemble representations of many odors.

Figure 1. Two narrowly tuned ORN types

(a) Single-sensillum recording from a t1-type trichoid sensillum showing spikes from the DA1 ORNs. (b) A tuning curve for DA1 ORNs. Odors are arranged on the x-axis so that the strongest responses are in the center. (See Supplementary Table 1 for odor order.) Negative values mean that the odor suppressed firing rates below spontaneous levels. The specificity (measured as lifetime sparseness, S) of this cell type is 1.00. Error bars in all panels are s.e.m. (c) A cis-vaccenyl acetate dose-response curve for DA1 ORNs. All other data in this study are collected at the 0.1 dilution (see Methods). (d) Single-sensillum recordings from an ab5 sensillum; large spikes originate from the VA6 ORNs; a few spikes (“B” symbols, smaller spikes) originate from the other ORN in the ab5 sensillum. (e) A tuning curve for VA6 ORNs. Note that odor order is different from (a). (f) An average ORN tuning curve. Data from ref. 28 was used to construct a normalized tuning curve for each of 24 olfactory receptors in the Drosophila antennae, and these normalized tuning curves were averaged together to produce a composite picture of the ORN cohort. (g) Distribution of specificity (S) values for DA1 and VA6 ORNs, as compared to all 24 olfactory receptors in ref. 35. Gray symbol indicates the S value obtained by ref. 28 for Or82a; this slightly lower S value may reflect experimental differences between the two studies. Among the open symbols (from ref. 28), the lowest S value corresponds to Or85f and the highest S value corresponds to Or47b.

Figure 2. DA1 ORNs are required for behavioral attraction to cis-vaccenyl acetate

(a) Response index of control (w¹¹¹⁸) flies in a Y-maze (see Supplementary Methods). Flies demonstrate attractive, repulsive, and neutral responses. All odors were diluted 1:250 in water. H₂0 = water control, CVA = cis-vaccenyl acetate, PYR = pyrrolidine, BNZ = benzaldehyde, PRO = propionic acid, GER = geranyl acetate. Numbers inside each bar indicate the number of trials run for the given condition. Error bars on all bar graphs are s.e.m. Symbols indicate significance: *ANOVA, P = 10^-18, post hoc Tukey HSD, P < 0.01; ‡ t-test, P = 0.02). (b) Control (w¹¹¹⁸), Gal4-only (Or67d-Gal4), and UAS-only (UAS-DTl) flies are attracted to cis-vaccenyl acetate plus propionic acid, as compared to propionic acid alone. This synergistic attraction is absent in Or67d-Gal4;UAS-DTl flies (*ANOVA, P = 0.0007, post hoc Tukey HSD, P < 0.05). As an additional control, we tested Or82a-Gal4;UAS-DTl flies, which showed normal attraction. (c) Extracellular recordings from t1-type trichoid sensilla control flies, and in flies where Or67d-Gal4 drives expression of diphtheria toxin. (d) Projections of confocal stacks through the antennal lobe. Dual immunofluorescence uses an anti-CD8 antibody (green) to visualize ORN axons, and nc82 antibody (gray) to visualize glomeruli. One fly (left) carries the Or67d-Gal4 transgene plus a UAS-CD8:GFP transgene; the other fly (right) also carries a UAS-DTl trangene. In the latter fly, the Gal4-expressing axons are ablated. Scale bar = 20μm. (e) Flies lacking DA1 ORNs show normal attraction to propionic acid, an attractive odor that does not activate DA1 ORNs.

Figure 3. VA6 ORNs are required for behavioral attraction to geranyl acetate

(a) Control (w¹¹¹⁸), Gal4-only (O82a-Gal4), and UAS-only (UAS-DTl) flies are attracted to geranyl acetate. This attraction is absent in Or82a-Gal4;UAS-DTl flies (*ANOVA, P = 0.0003; post hoc Tukey HSD, P < 0.01). Numbers inside each bar indicate the number of trials run for the given condition. Error bars on all bar graphs are s.e.m. (b) Extracellular recordings from ab5 sensilla in control flies, and in flies where Or82a-Gal4 drives expression of diphtheria toxin. In the top trace, the larger spikes (“A” symbols) arise from the VA6 ORNs (refs. 31, 32, 41). The “A” spikes are absent in the bottom trace. (c) Projections of confocal stacks through the antennal lobe. Dual immunofluorescence uses an anti-CD8 antibody (green) to visualize ORN axons, and nc82 antibody (gray) to visualize glomeruli. One fly (left) carries the Or82a-Gal4 transgene plus a UAS-CD8:GFP transgene; the other fly (right) also carries a UAS-DTl trangene. In the latter fly, the Gal4-expressing axons are ablated. Scale bar = 20μm. (d) Flies lacking VA6 ORNs show normal attraction to propionic acid. (e) A dose-response curve for geranyl acetate. Flies lacking VA6 ORNs are not attracted to geranyl acetate at any concentration tested. Six trials were run for each condition, except as indicated in (a). Symbols indicate a significant difference between the Or82a-Gal4;UAS-DTl genotype and both controls (*ANOVA, P = 10^-4; post hoc Tukey HSD, P < 0.01; **ANOVA, P < 0.005, post hoc Tukey HSD, P < 0.05; ***ANOVA P = 10^-6, post hoc Tukey HSD, P<0.01). Error bars are s.e.m.

Figure 4. DA1 PNs are narrowly tuned to cis-vaccenyl acetate

(a) Projection of a confocal stack through the antennal lobe. A portion of a biocytin-filled PN is shown (magenta) extending a dendritic tuft into glomerulus DA1. Glomeruli are outlined with nc82 antibody (gray). Scale bar = 20μm. (b) A raw trace illustrating the response of a DA1 PN to cis-vaccenyl acetate. Magenta symbols indicate the timing of action potentials. Gray bar shows timing of odor stimulation. (c) Tuning curves for DA1 PNs and ORNs. Odors are arranged so that the strongest responses are in the center, meaning that the odor order for the ORN and PN graphs is not the same (Supplementary Table 1). Error bars are s.e.m. (d) PSTHs showing the responses of DA1 ORNs (green) and PNs (magenta) to 19 odor stimuli and three controls (paraffin oil, water, and empty vial). Error bars (in lighter colors) are s.e.m.

Figure 5. VA6 PNs are more broadly tuned to odors than their presynaptic ORNs

(a) Projection of a confocal stack through the antennal lobe. A portion of a biocytin-filled PN is shown (magenta) extending a dendritic tuft into glomerulus VA6. Glomeruli are outlined with nc82 antibody (gray). Scale bar = 20μm. (b) A raw trace illustrating the response of a VA6 PN to geranyl acetate. Magenta symbols indicate the timing of action potentials. Gray bar shows timing of odor stimulation. (c) Tuning curves for VA6 PNs and ORNs. Odors are arranged so that the strongest responses are in the center, meaning that the odor order for the ORN and PN graphs is not the same (Supplementary Table 1). Error bars are s.e.m. (d) PSTHs showing the responses of VA6 ORNs (green) and PNs (magenta) to 19 odor stimuli and three controls (paraffin oil, water, and empty vial). Error bars (in lighter colors) are s.e.m.

Figure 6. There is a significant transformation of odor tuning in glomerulus VA6, but not in DA1

Tuning curves for DA1 (a) and VA6 (b). ORN responses are in green, PN responses in magenta. Error bars are s.e.m. Odors are arranged so that the strongest ORN responses are at the right-hand side of the graph. Odor order is the same for ORN and PN graphs. All values are normalized to the maximum average response for that cell type. The P value is indicated below the odor for ORN-PN comparisons that are significant at the criterion P<0.05 (unpaired 2-tailed t-tests). Only P values in blue remain significant after a Bonferroni correction for multiple comparisons (P<0.0026). Lower panels plot the mean normalized PN response minus the mean normalized ORN response for each odor (black symbols). Violet bars indicate the confidence interval corresponding to P<0.05 for each test, showing a similar level of statistical power for the DA1 dataset as compared to the VA6 dataset.