Valence opponency in peripheral olfactory processing

Significance Are olfactory receptor neurons (ORNs) arranged in a functionally meaningful manner to facilitate information processing? Here, we address this long-standing question by uncovering a valence map in the olfactory periphery of Drosophila. Within sensory hairs, we find that neighboring ORNs antagonistically regulate behaviors: stereotypically compartmentalized large- and small-spike ORNs, recognized by their characteristic spike amplitudes, either promote or inhibit the same type of behavior, respectively. Systematic optogenetic and thermogenetic assays—covering the majority of antennal sensilla—highlight a valence-opponent organization. Critically, odor-mixture behavioral experiments show that lateral inhibition between antagonistic ORNs mediates robust behavioral decisions in response to countervailing cues. Computational modeling predicts that the robustness of behavioral output depends on odor mixture ratios.

According to the spike responses of these ORNs to 110 odorants, the Euclidean distances between any two ORNs were determined. A shorter distance means that the two neurons have more similar response profiles. The distances between neurons housed in the same sensillum are outlined in red.
(B) Euclidean distance between any two randomly paired neurons (gray circles) or between paired ORNs in the same sensillum (red circles  Antennal lobe images of receptor-GAL4>mCD8::GFP flies from z-projections of multiple adjacent confocal sections. The ORN axons were stained with anti-GFP (green), and counterstained with nc82 to visualize the glomerular structure (magenta). All driver lines were backcrossed to w 1118 or Berlin, and were identical to the ones used for optogenetic or thermogenetic behavioral assays (see Tables S2 and Table S3). (A) Specific GAL4 drivers for grouped ORNs housed in 10 different antennal sensillum types (ab1, ab4, ab5, ab9, ab10, ai2, ai3, ac3, ac4, and at4). Only a single glomerulus in each antennal lobe was labeled by each driver. The positions of labeled glomeruli were as reported (2,3). (B) The GAL4 drivers for the ac1 ORNs labeled additional neurons. Left: apart from the reported VM1 glomerulus (4), the Ir92a-GAL4 driver also labeled neurons projecting to the subesophageal zone. Right: the Ir31a-GAL4 driver labeled two glomeruli: the reported VL2p glomerulus (4), as well as the column/VP1 glomerulus receiving input from hygrosensory neurons (5,6). The ac1 ORNs were therefore excluded in our behavioral assay. (C) The GAL4 drivers for the ab2 ORNs also labeled additional neurons. Left: apart from the reported DM4 glomerulus (2, 3), the Or59b-GAL4 driver also labeled neurons which likely project to the VC4 glomerulus (outlined). Right: the Or85a-GAL4 driver labeled two glomeruli: the reported DM5 (2, 3) as well as DM4 (outlined). Multi-glomerular labeling was also reported with the other available Or85a-GAL4 driver (7). The ab2 ORNs were therefore excluded in our behavioral assay. Scale bars, 20 µm.  A C + + + + + no effect no effect no effect no effect no effect  Negative controls were age-matched siblings without retinal feeding (gray). Irradiance was 0.3 µW/mm 2 for both ORN types. n=20 for each condition, **p<0.01, ***p<0.001, unpaired t-test.
(C) Thermogenetic courtship competition assay. Each circle denotes the normalized copulation rate of a given experiment with lines connecting data from the same experiments. The numbers of successful matches where copulation occurred were shown in the parentheses. p values are indicated; one-sample z-test. (D-F) As with (A-C) except that ab4 ORNs were tested. Irradiance was 1.2 µW/mm 2 for both ORNs in (D). (G-I) As with (D-F) except that at4 ORNs were tested. Data shown in (A) were the same as in Figure 1C-D, (E) as in Figure 2B, and (I) as in Figure 2F and 2H.
(J) Summary table.   Figure S6. Pheromonal modulation of courtship was abolished in receptor mutants.
Single-pair courtship assays with a 7-day-old male and a 3-day-old female perfumed with either solvent (acetone) or indicated pheromone(s).
(A) Cumulative copulation rates of wildtype males when the females were perfumed with either solvent (acetone) or palmitoleic acid (~0.1 ng/fly); the final copulation rates are shown on the right. Lines connect results from parallel experiments. The total numbers of copulated males are indicated in the parentheses.
(B) As with (A) except that Or47b mutant males were tested. ** p < 0.01, Wilcoxon rank-sum test. n=5-6, total 125-150 matches, experiments conducted at ~25°C. (C) Cumulative copulation rates of Or88a mutant males when the wildtype females were perfumed with either solvent or methyl palmitate (~87 ng/fly); the final copulation rates are shown on the right. Lines connect results from parallel experiments. The total numbers of copulated males are indicated in the parentheses.
(D) As with (C) except that Or67d mutant males were tested and the females were perfumed with cVA (~38 ng/fly).