Fig 1.
Flow chart comparing traditional SSR with Fluorescence-guided SSR.
Traditional Single Sensillum Recordings (SSR) often requires screening many sensilla for odorant activities before identifying a correct target. Fluorescence-guided SSR (FgSSR) allows for GFP-labeled sensilla to be targeted directly for recordings. This increases the success rate for recording from a specific sensillum (e.g., ab4), and decreases the effort needed to record from the desired sensillum. FgSSR is also useful for targeting sparse sensilla (e.g. ab4 to ab10), sensilla with similar sizes and locations (ab6-10 with ai1-3; pb1-3), sensilla that house neurons with unknown odor-responses (e.g, ai1), or for targeting specific Odorant Receptors in unknown sensillar types. Thick “Yes/No” arrows indicate higher probabilities and thin decision arrows indicate lower probabilities.
Fig 2.
Expression of mCD8GFP in the olfactory neuron does not alter odor responses.
(A) ab2 sensilla were labeled using Or59b-Gal4 to drive 15xUAS-IVS-mCD8GFP expression. Antennae were visualized on the recording rig by differential interference contrast (DIC, top), and for GFP expression (middle), and the merged image is shown in the bottom row. (B) ab3 sensilla were labeled using Or22a-Gal4 to drive 15xUAS-IVS-mCD8GFP expression. (C) ab4 sensilla were labeled using Or56a-Gal4 to drive 15xUAS-mCD8GFP expression. In (A-C), arrowheads point to example cell body labeling, and arrows point to example sensillum labeling. (D) Comparing the SSR odor response profiles of wild-type (WT) and GFP-expressing neurons in ab2 and ab3 sensilla. The odor response profiles to 10 standard odorants plus mineral oil were examined for unlabeled WT and FgSSR-targeted ab2A, ab2B, ab3A, and ab3B neurons. Responses to all odorants were similar, with the exception of ab2A response to pentyl acetate, which was decreased in the FgSSR experiment (n = 4–6 for each recording). (E) The spontaneous activities of WT and GFP-labeled ab2A (Or59b), ab2B (Or85a), ab3A (Or22a), ab3B (Or85b), and at1 (Or67d) showed no significant differences (n = 9 for each recording). (F) Comparing the SSR odor response profiles of wild-type (WT) and GFP-expressing at1 Or67d+ neurons (Or67d-Gal4/15xUAS-mCD8GFP) to the pheromone ligand (cVA) at different pheromone concentrations (n = 5). Error bars indicate ± s.e.m. throughout.
Fig 3.
Suggested mounting positions and target zones.
Shown are images of mounted flies (top) and cartoons of prepared antenna (below). (A) A medial mounting position exposes ab1-3, ab7 and ab10 basiconic sensilla. (B) A posterior mounting position exposes ab4-6 and ab8-9 basiconic sensilla, as well as at1 and at4 trichoid sensilla types. (C) A lateral position exposes ai1-3 intermediate sensilla. (D) An anterior mounting position exposes at1, at4, ai2 and ai3 sensilla.
Fig 4.
Simultaneous labeling and visualization of intermediate and trichoid sensilla by FgSSR.
GFP-labeling of sensilla allows for accurate classification of sensillar types. (A-E) OrX-Gal4 with 15xUAS-IVS-mCD8GFP clearly labels a specific olfactory neuron and sensilla. Middle panels show confocal DIC images of the antenna overlaid with the GFP signal, and right panels shows a higher magnification of the GFP-labelled sensilla. Arrowheads point to example cell body labeling, and arrows point to example sensillum labeling. (A) Ai1 sensilla identified by Or13a-Gal4. (B) Ai2 sensilla identified by Or23a-Gal4 and Or83c-Gal4. (C). Ai3 sensilla identified by Or19a-Gal4 and Or43a-Gal4. (D) At1 sensilla identified by Or67d-Gal4. (E) At4 sensilla identified by Or47b-Gal4, Or65a-Gal4, and Or88a-Gal4. (F) Cartoon showing how measurement of sensillar length is performed. (G) Characteristic lengths of intermediate and trichoid sensilla. p-values are as shown. t-test, n = 15 per genotype. Error bars indicate ± 2.5 s.e.m. throughout. Data points not within this range are plotted as circles. (H) FgSSR combined with 20xUAS-IVS-CsChrimson allows light-guided stimulation of olfactory neurons. This confirms at4C neurons express Or88a.
Table 1.
Validated OrX-Gal4 reagents for FgSSR.
Fig 5.
Re-classification of Drosophila melanogaster intermediate and trichoid sensilla.
(A) Schematic for the location of trichoid and intermediate sensilla on the antennae. (B) FgSSR analyses re-classified two trichoid sensilla (names bolded) as intermediate sensilla. Intermediate sensilla identification numbers are correlated to the number of housed neurons: ai1 contains one neuron, ai2 contains two neurons, and ai3 contains three neurons.