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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.


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.
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pone.0139675.g003: 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.

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)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592000&req=5

pone.0139675.g003: 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.
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.