Electrophysiological Recordings from Drosophila Sensilla in Response to Volatile Odorants

Protocol

1. Preparation of the Hardware for at4 Recording

1. Use a pipette puller instrument to prepare electrodes with aluminosilicate glass capillaries (O.D 1.0 mm, I.D. 0.64 mm). Blunt the tip of the reference electrode slightly with a pair of fine forceps to facilitate insertion into the clypeus of the fly (i.e. a rounded plate at the front of the fly head, above the mouthparts).
   NOTE: 7-day-old WT(Wild type) males (Berlin) were used in this study. Use AHL saline solution as the electrolyte for both electrodes.
2. Prepare 1 L of AHL(Adult Hemolymph-Like) by mixing 900 mL of distilled water with 6.312 g of NaCl, 0.373 g of KCl, 0.337 g of NaHCO₃, 0.1120 g of NaH₂PO₄, 1.892 g of Trehalose ּ2H₂O, 3.423 g of sucrose, 1.192 g of HEPES(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), and 8.2 mL of 1M MgCl₂. Using distilled water, bring the total volume up to 1 L. Bring the pH to 7.4 using 1 N NaOH and sterilize the solution with a vacuum-driven filter system. For long-term storage, keep the AHL aliquots at 4 °C.
   NOTE: Consistent deliveries of palmitoleic acid are contingent upon uniformity between cartridges. Each cartridge must be assembled in a reproducible manner.
3. Using a razor blade, remove 0.9 cm from the tip of a 200 µL pipette tip to create the first cartridge section, measuring 4.1 cm; refer to the dimensions detailed in Figure 1A. Use another 200 µL pipette tip and remove 1.7 cm and 1.5 cm from the tip and base to create the second cartridge section, measuring 1.8 cm (Figure 1A). Use a ruler to ensure reproducibility.
4. Use a ⅛" hole puncher to cut discs from filter paper.
5. Use forceps to place a filter paper disc at the tip of the second cartridge section. Visually confirm an opening in the cartridge tip through which air can pass.
6. Attach the first and second cartridge sections, as shown in Figure 1A. Angle the second cartridge section downward to facilitate square aiming at the prep (Figure 1B).
7. Connect the cartridge with the odor-delivery tube mounted on a micromanipulator.
   NOTE: This design allows the cartridge to be swiveled outward to facilitate exchange (Figure 1C).
8. Set the constant humidified airflow to 2 L/min in one mass controller and the odorant flow to 500 mL/min in another mass controller.
9. Using the software (see the Table of Materials), program the procedure to administer a 500 ms odor puff.

2. Preparation of Palmitoleic Acid Odorant Solutions for Delivery

NOTE: Or47b ORNs respond to both cis- and trans-palmitoleic acid. As palmitoleic acid is unstable at RT, stocks are stored at -20 °C and used within a month upon opening. Ethanol is the solvent of choice for palmitoleic acid.

1. Use a vortex mixer to thoroughly mix 10 µL of cis- or trans-palmitoleic acid stocks or dilutions with 90 µL of 100% ethanol for ten-fold serial dilutions in 1.7 mL microtubes. Prepare fresh palmitoleic acid dilutions daily before experiments and use within a day.
   NOTE: A glass vial is recommended for odorants not soluble in ethanol to prepare odor dilutions with other types of organic solvents.
2. Using a P10 micropipette, apply 5 µL of cis-palmitoleic acid solutions of the desired dilutions to the filter paper in each corresponding cartridge.
   NOTE: The highest dosage (10^-1) contains 450 µg of the compound. For trans-palmitoleic acid solutions, apply 4.5 µL instead so that the highest dosage (10^-1 ) contains 450 µg of the compound.
3. Place the palmitoleic acid cartridges in a vacuum desiccator for 1 h at RT and 7.59 mmHg of pressure to completely evaporate the solvent.
   NOTE: The cartridges can be used for up to 4 h at RT.

3. Preparation of Drosophila for Ready Access to the at4 Sensilla for In Vivo Electrophysiological Recordings

NOTE: WT flies (Berlin) are reared in standard cornmeal medium at 25°C in a 12:12 light-dark cycle. Upon eclosion, flies are separated by sex into groups of ten, whereby they are group-housed until 7 d of age. Or47b ORNs (Olfactory Receptor Neurons) in both male and female flies respond to palmitoleic acid. For simplicity, only male flies are examined in the current study.

1. Assemble a fly-prep slide: on a glass slide, place a glass coverslip (18 x 18 mm2) on a small amount of modeling clay, forming a ~3° angle with the glass slide. Place double-sided tape on the inner edge of the coverslip and the area of the slide immediately below. Replace with fresh tape for every day of recording (Figure 2A).
2. Use a fly aspirator15 to collect the fly of interest in the tubing and then fit a 200-µL pipette tip over the end of the tubing. Simultaneously, flick the tube forward while blowing air into it to push the fly to the end of the pipette tip. Use a razor blade to cut just below the body of the fly and 2 heads' lengths above the fly.
3. Tamp the bottom of the pipette tip with modeling clay, pushing the fly upward until both the antennae and the clypeus are exposed (Figure 2B). To avoid killing the fly, add only enough clay to expose the antennae and aristae, as this prevents the fly's abdomen from being crushed. Furthermore, add clay slowly and gently to prevent any sudden constriction. Confirm that the fly is alive by checking for antennal or proboscis movement.
4. Use forceps to maneuver the pipette tip that houses the fly. Orient the head so that the clypeus faces the observer's right. Adjust the prep along the coverslip using fine forceps until the lateral side of the antenna lies against the taped coverslip surface (Figure 2B).
5. Place a holding rod on the arista to secure the antenna to the double-sided tape to prevent movement (Figure 2B).
   NOTE: The holding rod is pulled from a borosilicate glass capillary with a pipette puller and held in position with modeling clay (Figure 2A).
6. Place the prep on the rig's stage (Figure 2C). Using the microscope, confirm that the trichoid is visible along the distal-lateral edge of the third segment of the antenna.
   NOTE: The sensilla should be silhouetted against the background, simplifying their identification and facilitating recording (Figure 3). In this preparation, the most accessible trichoid sensilla are of the at4 type.
7. As described previously, 2, 15, keep the prep under constant humidified airflow (2 L/min) delivered via a separate air delivery tube about 2 cm from the prep (Figure 4).

4. Recording of at4 Sensillum Activity from Or47b ORNs in the at4 Trichoids in Response to Palmitoleic Acid

1. Insert the reference electrode into the clypeus (Figure 3A). With a swift and smooth motion, ensure that the electrode is inserted just below the surface, where it can contact the hemolymph under the cuticle to avoid tissue damage.
2. Lower the recording electrode slowly until it enters the same view plane as the target sensillum (Figure 3B). Record under a 50X objective lens.
   NOTE: The tough trichoidal cuticle necessitates inserting the recording electrode into the sensillar base, whose wider area provides a larger target that reduces the likelihood that the electrode is deflected away (Figure 3B, inset).
3. Before applying odor stimuli to a sensillum, observe the following selection criteria: any trichoid failing to meet these standards should be rejected, and another sensillum should be chosen instead.
   1. Observe a high signal-to-noise ratio (see Figure 3C for an example).
   2. Observe identifiable spikes from at4A and at4C neurons (Figure 3C).
      NOTE: The at4B spike amplitude appears very similar to at4A10 and cannot be identified easily without odor stimulation.
   3. Observe that the basal firing rate of the at4A neurons is around or under 20 Hz.
      NOTE: This criterion is specific for at4A because the basal firing rate for the neuron is higher than that of the basiconic ORNs. A much higher basal firing indicates that the neurons may have been damaged during electrode insertion.
4. Connect the cartridge to the odorant-delivery tube. Start with the solvent control and then the odorants, from low to high concentrations. Use the micromanipulator to maneuver the cartridge towards the prep while aiming the cartridge squarely at the head of the prep. Visually confirm that the cartridge is pointed directly at the antenna (Figure 4) from a few millimeters away.
   NOTE: The goal is to orient the opening of the cartridge directly at the antenna and position it near the target tissue.
5. Ensure that the odorant cartridge is separated from the recording electrode on its right by 1 - 2 mm and from the fly prep slide below by approximately 1 mm.
   NOTE: In the setup described here, the odorant cartridge is closely bordered by the recording electrode, the reference electrode, and the fly-prep slide (Figure 4).
   NOTE: Consider the distance between the cartridge and the recording/reference electrodes. A distance of around 4 mm is recommended. Unintentional contact may terminate the signal and break the tip of the recording electrode, damaging the current neuron and complicating further recordings.
   NOTE: Consider the distance separating the cartridge and the fly-prep slide. Touching the coverslip may also dislodge the recording electrode, disrupting the recording.
6. Press "Record" in the data acquisition software to begin the recording.
   NOTE: For each 10 s recording, a single 500 ms odor pulse is delivered directly to the antenna, as described in step 1.9.
7. After the odorant is applied, carefully retract the cartridge before replacing it with a cartridge of the next-highest concentration. Continue until the entire dosage range is obtained.
   NOTE: Only one Or47b ORN is recommended to be recorded from each fly to avoid any possible effects of adaptation.
8. Thoroughly rinse the recording electrode with distilled water after finishing recording for the day.
9. Analyze and plot the data using commercially available offline analysis software.

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النتائج

Figure 1: Cartridge and Olfactometer Setup. (A) Preparation of odor cartridges. From left to right: a standard 200-µL pipette tip, the first and second cartridge sections, and a completed odorant cartridge. (B) The cartridge connected to the olfactometer, showing the downward angling of the second section. (C) Olfactometer setup depicting the odor delivery tube mounted on the micromanipulator, with an attached odorant cartridge.

Figure 2: Drosophila Preparation. (A) A complete preparation, showing the relative positions of the fly, coverslip, and holding rod. (B) Close-up view of the prep, showing the positioning of the fly, its antennal orientation, and its clypeus. The holding rod is placed over the arista, securing the third antennal segment to the double-sided tape. (C) Rig setup. All major components are annotated.

Figure 3: Identification of the at4 Sensillum for SSR. (A) 4X view of the prep, showing the reference electrode inserted in the clypeus, the holding rod atop the arista, and the recording electrode positioned near the third antennal segment. (B) 50X view of the electrode, poised for insertion into the at4 trichoid. Inset: Illustration of the position of the recording electrode. (C) Representative SSR traces of baseline spike activity, demonstrating good (top) or poor (bottom) signal-to-noise ratio. Good signal-to-noise ratio permits the reliable identification of at4A and at4C spikes.

Figure 4: Cartridge Placement. (A) The odorant cartridge is aimed squarely at the head of the fly from a distance of a few mm. (B) Another view of the prep and olfactometer from a different angle. (C) A close-up view of the prep and olfactometer, showing the position of the odorant cartridge above the fly-prep slide.

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Materials

Name	Company	Catalog Number	Comments
Prep Setup & Miscellaneous Materials
Pipette Puller Instrument	Sutter Instruments ,Novato CA USA	P97	Pipette Puller
Borosilicate Glass Capillaries	World Precision Instruments, Sarasota FL USA	1B100F-4	to make holding rods
Aluminosilicate Glass Capillaries	Sutter Instruments, Novato CA USA	AF100-64-10	to make electrodes
Superfrost Microscope Slides	Fisher Scientific, Pittsburgh PA USA	12-550-143	for fly-prep station
Permanent Double Sided Tape	Scotch St. Paul MN USA	NA	for fly-prep station
Upright microscope	Olympus, Shinjuku Tokyo Japan	BX51	for recording rig
Plastalina modeling clay	Van Aken, North Charleston SC USA	B0019QZMQQ	for prep station and to stablize the holding rod
Rapid-Flow Sterile Disposable Filter Unit with SFCA Membrane, 0.45 mm	Nalgene, Rochester NY USA	#156-4045	to sterilize AHL solution
200 µL pipette tip	VWR Radnor, PA USA	53508-810	to make odor cartridges and fly prep
Filter Paper	Whatman, Maidstone Kent UK	740-E	to make odor cartridges
Vacuum Desiccator	Cole-Parmer, Vernon Hills IL USA	VX-06514-30	to vaporize ethanol solvent
cis-palmitoleic acid	Cayman Chemical, Ann Arbor MI USA	#10009871 (CAS # 373-49-9)	Or47b odorant
trans-palmitoleic acid	Cayman Chemical, Ann Arbor MI USA	#9001798 (CAS # 10030-73-6)	Or47b odorant
Ethanol	Spectrum Chemical MFG.  New Brunswick NJ USA	E1028-500MLGL	to dilute palmitoleic acid
Odorant Cartridge Micromanipulator	Siskiyou, Grants Pass OR USA	MX130R	to position the olfactometer
Flow Vision software	Alicat, Tuscon AZ USA	FLOWVISIONSC	software to control flow rate
Mass Controller	Alicat, Tuscon AZ USA	MC-2SLPM-D	to control the flow rate for humidified air
Mass Controller	Alicat, Tuscon AZ USA	MC-500SCCM-D	to control the flow rate for odor stimulation
Clampex	Molecular Devices, Sunnyvale CA USA	Ver. 10.4	Data acquisition software
Air delivery tube	Ace Glass, Vineland NJ USA	8802-936	to deliver humidified air
50X objective lens	Olympus, Shinjuku Tokyo Japan	LMPLFL50X	recording rig
Clampfit 10	Molecular Devices, Sunnyvale CA USA	Ver. 10.4	software for spike analysis
Igor Pro 6	WaveMetrics, Lake Oswego OR USA	Ver. 6.37	software for data analysis
Audio Monitor	ALA Scientific Instruments, Farmingdale NY USA	NPIEXB-AUDIS-08B	Aurally reports individual spikes
Extracellular Amplifier	ALA Scientific Instruments, Farmingdale NY USA	NPIEXT-02F	to increase the amplitude of electrical signals
Valve Controller	Warner Instruments	VC-8	to control the opening of the valve for odor stimulation
Recording Electrode Micromanipulator	Sutter Instruments, Novato CA USA	MP-285	to position recording electrode
Headstage Amplifier	ALA Scientific Instruments, Farmingdale NY USA	EQ-16.0008	to increase the amplitude of electrical signals
Oscilloscope	Tektronix, Beaverton OR USA	TDS2000C	Visual report of individual spikes