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. 2022 Apr 20;11:e72599. doi: 10.7554/eLife.72599

Figure 4. Confirmation of co-receptor co-expression.

(A) snRNAseq of adult fly antennae (McLaughlin et al., 2021) confirms expanded expression of olfactory co-receptors. Top: tSNE plots show expression of each co-receptor in 20 decoded olfactory sensory neuron (OSN) clusters. Bottom: clusters were mapped to 24 glomeruli. Opacity of fill in each glomerulus indicates the proportion of cells in that cluster expressing the given co-receptor, normalized to total expression for that co-receptor gene (see Figure 4—source data 1). Right column: clusters color-coded according to original chemoreceptor gene family. Compass: D = dorsal; L = lateral. (B) Anti-Orco antibody staining in antennal cryosections (top) and whole-mount palps (bottom) confirms co-expression of Orco and Ir25a in the periphery (genotype: Ir25a-T2A-QF2>GFP). Right panels show cells pseudo-colored gray with specific single- or double-labeled cells indicated by colored cell markers (GFP+ only in blue, GFP+Orco+ in orange, Orco+ only in red). (C) Co-labeling experiments with various transgenic Gal4 lines driving mCD8::RFP (orange) and the Ir25a-T2A-QF2 knock-in driving GFP (green). Ir25a-T2A-QF2 labels glomeruli innervated by both antennal (top) and palpal (bottom) OSNs. (D) Verification of Ir25a expression in antennal ab3 sensilla using optogenetics. Single sensillum recordings (SSR) from ab3 Orco+ neurons in Ir25a-T2A-QF2>QUAS-CsChrimson flies. Representative traces from ab3 using 1.5 V of 627 nm LED light (red box) to activate CsChrimson. Bottom trace is control animal, which has the same genotype as the experimental animal but was not fed the required all-trans retinal cofactor (-ATR). Spikes from the ab3A and ab3B neurons are indicated by blue and green dots, respectively. Right: quantification of neuronal activity in response to light at various LED intensities (N = 7–12). These optogenetic experiments support Ir25a expression in both ab3A neurons (Or22a/b, top; corresponding to DM2 glomerulus) and ab3B neurons (Or85b, bottom; corresponding to VM5d glomerulus). Scale bars = 25 µm. See also Figure 4—figure supplement 1, Table 3, Figure 4—source data 1, Figure 4—source data 2, and Figure 4—source data 3.

Figure 4—source data 1. snRNAseq co-receptor expression in adult olfactory sensory neurons (OSNs).
Figure 4—source data 2. Individual glomerular analyses.
Figure 4—source data 3. Optogenetic validation of Ir25a expression.

Figure 4.

Figure 4—figure supplement 1. Optogenetic experiments to examine Ir25a expression in Orco+ neurons.

Figure 4—figure supplement 1.

(A–D) Confirmation of Ir25a expression in ab2 and ab9 sensilla using optogenetic stimulation. CsChrimson expression is driven by Ir25a-T2A-QF2, and recordings are performed in neurons previously determined to express only Orco. In all optogenetic experiments, control animals have the same genotypes as the corresponding experimental animals but have not been fed all-trans retinal. (A) Representative single sensillum recording (SSR) traces from ab2 using 1.5 V of a 627 nm LED light (red box) to activate CsChrimson. Bottom trace is control animal, which was not fed the necessary cofactor all-trans retinal (-ATR). ab2 has two neurons: blue dots indicate A neuron spikes, while green dots indicate B neuron spikes (genotype: Ir25a-T2A-QF2; QUAS-CsChrimson). (B) Quantification of neuronal activity in response to light stimulation at various intensities (N = 7–12). Optogenetic experiments confirm Ir25a expression in ab2A (expressing Or59b, top; DM4 glomerulus) but not ab2B (expressing Or85a and Or33b, bottom; DM5 glomerulus). (C) Combination of optogenetics and fluorescent-guided SSR (Lin and Potter, 2015) to examine Ir25a expression in ab9 sensilla (genotype: Ir25a-T2A-QF2; QUAS-CsChrimson/Or67b-Gal4, 15XUAS-IVS-mcd8::GFP). Representative traces from ab9 in response to 3 V of 627 nm LED light (red box). As in (A), -ATR indicates the inactive CsChrimson control; blue dots indicate A neuron spikes, green dots indicate B neuron. (D) Quantification of activity in response to light stimulation verified that Ir25a is expressed in ab9B (Or67b, bottom; VA3 glomerulus) but not in ab9A neurons (Or69aA/aB, top; D glomerulus). N = 5.