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Re-Classification of Drosophila melanogaster Trichoid and Intermediate Sensilla Using Fluorescence-Guided Single Sensillum Recording.

Lin CC, Potter CJ - PLoS ONE (2015)

Bottom Line: Drosophila olfactory receptor neurons are found within specialized sensory hairs on antenna and maxillary palps.Fluorescence-guided SSR further revealed that two antennal trichoid sensilla types should be re-classified as intermediate sensilla.This approach provides a simple and practical addition to a proven method for investigating olfactory neurons, and can be extended by the addition of UAS-geneX effectors for gain-of-function or loss-of-function studies.

View Article: PubMed Central - PubMed

Affiliation: The Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Drosophila olfactory receptor neurons are found within specialized sensory hairs on antenna and maxillary palps. The linking of odorant-induced responses to olfactory neuron activities is often accomplished via Single Sensillum Recordings (SSR), in which an electrode inserted into a single sensory hair records the neuronal activities of all the neurons housed in that sensillum. The identification of the recorded sensillum requires matching the neuronal responses with known odor-response profiles. To record from specific sensilla, or to systematically screen all sensillar types, requires repetitive and semi-random SSR experiments. Here, we validate an approach in which the GAL4/UAS binary expression system is used for targeting specific sensilla for recordings. We take advantage of available OrX-Gal4 lines, in combination with recently generated strong membrane targeted GFP reporters, to guide electrophysiological recordings to GFP-labeled sensilla. We validate a full set of reagents that can be used to rapidly screen the odor-response profiles of all basiconic, intermediate, and trichoid sensilla. Fluorescence-guided SSR further revealed that two antennal trichoid sensilla types should be re-classified as intermediate sensilla. This approach provides a simple and practical addition to a proven method for investigating olfactory neurons, and can be extended by the addition of UAS-geneX effectors for gain-of-function or loss-of-function studies.

No MeSH data available.


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.
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pone.0139675.g002: 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.

Mentions: Sensilla of targeted ORNs were identified using 10x and 50x objectives with an optovar 1.6x attachment (Zeiss, EC Epiplan-Neofluar 10x, LC EC Epiplan-Neofluar 50x and Optovar Module 1.6x P&C ACR) on a Zeiss AxioExaminer D1 compound microscope, using a light source and eGFP filter cube (FL Filter Set 38 HE GFP shift free). Green fluorescence signals in flies were visualized from OrX-Gal4 and 10xUAS-IVS-mCD8GFP (Bloomington Stock #32186; for the OrX-Gal4 on Chr. II) or 15xUAS-IVS-mCD8GFP (Bloomington Stock #32193; for the OrX-Gal4 on Chr. III). The representative images shown in Fig 2A–2C and S1 Fig were taken on the recording rig. The suggested mounting positions of antenna are shown in Fig 3. The electrode was filled with Beadle-Ephrussi ringers solution (7.5g of NaCl+0.35g of KCl+0.279g of CaCl2-2H2O in 1L of H2O). Extracellular activity was recorded by inserting a glass electrode into the base of the sensillum of 4–8 day-old flies. Signals were amplified 100X (USB-IDAC System; Syntech, Hilversum, The Netherlands), inputted into a computer via a 16-bit analog-digital converter and analyzed off-line with AUTOSPIKE software (USB-IDAC System; Syntech). The low cutoff filter setting was 50Hz, and the high cutoff was 5kHz. Stimuli consisted of 1000 ms air pulses passed over odorant sources. The Δspikes/second was obtained by counting the spikes in a 1000ms window from 500 ms after odor stimuli were triggered, subtracting the spikes in a 1000ms window prior to stimulation. 10 standard odors for identification of sensillar types are acquired at highest purity and listed as follows: Ethyl acetate (Sigma #270989), Pentyl acetate (Sigma #109584), Ethyl butyrate (Sigma #W242705), Methyl salicylate (Sigma #76631), Hexanol (Sigma #471402), 1-octen-3-ol (Sigma #68225), E2-hexenal (Sigma #W256005), 2,3-butanedione (Sigma #B85307), Geranyl acetate (Sigma #45896), 2-heptanone (Sigma #537683), 11-cis vaccenyl acetate (50mg in 1ml ethanol, Cayman Chemical Company #0424297–6). The odors except cVA were diluted in mineral oil (Sigma #330779) at 1:100 and 30 μl was used for stimulation. Odors were delivered to the antenna as previously described [4, 6, 10]. Stimuli were delivered by placing the tip of an odor Pasteur pipette through a hole in a pipette (Denville Scientific Inc, 10ml pipette) that carried a purified continuous air stream (8.3 ml/s) directed at the antenna. A solenoid valve (Syntech) diverted delivery of a 1 s pulse of charcoal-filtered air (5 ml/s) to a Pasteur pipette containing odorant dissolved onto filter paper. Fresh odorant pipettes were used after no more than 3 odor presentations.


Re-Classification of Drosophila melanogaster Trichoid and Intermediate Sensilla Using Fluorescence-Guided Single Sensillum Recording.

Lin CC, Potter CJ - PLoS ONE (2015)

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.
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pone.0139675.g002: 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.
Mentions: Sensilla of targeted ORNs were identified using 10x and 50x objectives with an optovar 1.6x attachment (Zeiss, EC Epiplan-Neofluar 10x, LC EC Epiplan-Neofluar 50x and Optovar Module 1.6x P&C ACR) on a Zeiss AxioExaminer D1 compound microscope, using a light source and eGFP filter cube (FL Filter Set 38 HE GFP shift free). Green fluorescence signals in flies were visualized from OrX-Gal4 and 10xUAS-IVS-mCD8GFP (Bloomington Stock #32186; for the OrX-Gal4 on Chr. II) or 15xUAS-IVS-mCD8GFP (Bloomington Stock #32193; for the OrX-Gal4 on Chr. III). The representative images shown in Fig 2A–2C and S1 Fig were taken on the recording rig. The suggested mounting positions of antenna are shown in Fig 3. The electrode was filled with Beadle-Ephrussi ringers solution (7.5g of NaCl+0.35g of KCl+0.279g of CaCl2-2H2O in 1L of H2O). Extracellular activity was recorded by inserting a glass electrode into the base of the sensillum of 4–8 day-old flies. Signals were amplified 100X (USB-IDAC System; Syntech, Hilversum, The Netherlands), inputted into a computer via a 16-bit analog-digital converter and analyzed off-line with AUTOSPIKE software (USB-IDAC System; Syntech). The low cutoff filter setting was 50Hz, and the high cutoff was 5kHz. Stimuli consisted of 1000 ms air pulses passed over odorant sources. The Δspikes/second was obtained by counting the spikes in a 1000ms window from 500 ms after odor stimuli were triggered, subtracting the spikes in a 1000ms window prior to stimulation. 10 standard odors for identification of sensillar types are acquired at highest purity and listed as follows: Ethyl acetate (Sigma #270989), Pentyl acetate (Sigma #109584), Ethyl butyrate (Sigma #W242705), Methyl salicylate (Sigma #76631), Hexanol (Sigma #471402), 1-octen-3-ol (Sigma #68225), E2-hexenal (Sigma #W256005), 2,3-butanedione (Sigma #B85307), Geranyl acetate (Sigma #45896), 2-heptanone (Sigma #537683), 11-cis vaccenyl acetate (50mg in 1ml ethanol, Cayman Chemical Company #0424297–6). The odors except cVA were diluted in mineral oil (Sigma #330779) at 1:100 and 30 μl was used for stimulation. Odors were delivered to the antenna as previously described [4, 6, 10]. Stimuli were delivered by placing the tip of an odor Pasteur pipette through a hole in a pipette (Denville Scientific Inc, 10ml pipette) that carried a purified continuous air stream (8.3 ml/s) directed at the antenna. A solenoid valve (Syntech) diverted delivery of a 1 s pulse of charcoal-filtered air (5 ml/s) to a Pasteur pipette containing odorant dissolved onto filter paper. Fresh odorant pipettes were used after no more than 3 odor presentations.

Bottom Line: Drosophila olfactory receptor neurons are found within specialized sensory hairs on antenna and maxillary palps.Fluorescence-guided SSR further revealed that two antennal trichoid sensilla types should be re-classified as intermediate sensilla.This approach provides a simple and practical addition to a proven method for investigating olfactory neurons, and can be extended by the addition of UAS-geneX effectors for gain-of-function or loss-of-function studies.

View Article: PubMed Central - PubMed

Affiliation: The Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Drosophila olfactory receptor neurons are found within specialized sensory hairs on antenna and maxillary palps. The linking of odorant-induced responses to olfactory neuron activities is often accomplished via Single Sensillum Recordings (SSR), in which an electrode inserted into a single sensory hair records the neuronal activities of all the neurons housed in that sensillum. The identification of the recorded sensillum requires matching the neuronal responses with known odor-response profiles. To record from specific sensilla, or to systematically screen all sensillar types, requires repetitive and semi-random SSR experiments. Here, we validate an approach in which the GAL4/UAS binary expression system is used for targeting specific sensilla for recordings. We take advantage of available OrX-Gal4 lines, in combination with recently generated strong membrane targeted GFP reporters, to guide electrophysiological recordings to GFP-labeled sensilla. We validate a full set of reagents that can be used to rapidly screen the odor-response profiles of all basiconic, intermediate, and trichoid sensilla. Fluorescence-guided SSR further revealed that two antennal trichoid sensilla types should be re-classified as intermediate sensilla. This approach provides a simple and practical addition to a proven method for investigating olfactory neurons, and can be extended by the addition of UAS-geneX effectors for gain-of-function or loss-of-function studies.

No MeSH data available.