Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila

Podsumowanie

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

Streszczenie

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential food source, compounds from the food source enter through the pore and activate neurons within. For over 50 years, these responses have been recorded using a technique called tip recording. However, this method has major limitations, including the inability to measure neural activity before or after stimulus contact and the requirement for tastants to be soluble in aqueous solutions. We describe here a technique that we call base recording, which overcomes these limitations. Base recording allows the measurement of taste neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It can be used to study hydrophobic compounds such as long-chain pheromones that have very low solubility in water. In summary, base recording offers the advantages of single-sensillum electrophysiology as a means of measuring neuronal activity - high spatial and temporal resolution, without the need for genetic tools - and overcomes key limitations of the traditional tip recording technique.

Wprowadzenie

Insects, including drosophilid flies, are endowed with a sophisticated taste system that enables them to extract complex chemical information from their surroundings. This system allows them to discern the chemical composition of various substances, distinguishing between those that are nutritious and those that are harmful^1,2.

At the core of this system are specialized structures known as taste hairs or sensilla, strategically located on various body parts. In drosophilid flies, these sensilla are located on the labellum, which is the major taste organ of the fly head^1,2,3,4, as well as on the legs and wings^1,2,5,6. The labellum is located at the tip of the proboscis and contains two lobes^4,7,8. Each lobe is covered with 31 taste sensilla categorized as short, long, and intermediate^4,7,8. These sensilla each house 2-4 taste neurons^1,2,9,10. These taste neurons express members of at least four different gene families, namely the Gustatory receptor (Gr), Ionotropic receptor (Ir), Pickpocket (Ppk), and Transient receptor potential (Trp) genes^1,2,11,12,13. This diversity of receptors and channels equips insects with the ability to recognize a wide array of chemical compounds, including both nonvolatile and volatile cues^1,2,14.

For over 50 years, scientists have quantified the response of taste neurons and their receptors using a technique called tip recording^{3,4,6,8,13,15,16,17,18,19,20,21,22,23,24,25,26,27,28,
29,30,31,32,33,34,35}. However, this method has major limitations. First, neural activity can be measured only during contact with the stimulus, and not before or after contact. This limitation precludes the measurement of spontaneous spiking activity and prevents the measurement of OFF responses. Second, only tastants that are soluble in aqueous solutions can be tested.

These limitations can be overcome by a rarely used alternative electrophysiological technique called "base recording." Here we describe this technique, which we have adapted from a method used by Marion-Poll and colleagues²⁴, and show the crucial taste coding features that it can now conveniently measure¹⁴.

Protokół

The following protocol complies with all the animal care guidelines of Yale University.

1. Flies

1. Place 10-15 newly emerged flies in fresh standard culture vials at 25 °C and 60% relative humidity in a 12:12 h light-dark cycle.
2. Use flies when 3-7 days old.

2. Chemosensory stimuli

1. Obtain chemosensory stimuli of the highest available purity. Store them as recommended by the vendor until use.
2. Dissolve chemosensory stimuli and dilute to the desired concentrations in water or another desired, non-toxic solvent such as paraffin oil. Stir the prepared solutions for a minimum of 1 h in the case of dissolved solid compounds.

3. Glass stimulus capillary

1. Pull a glass capillary to hold the stimulus from a borosilicate glass capillary (100 mm length, 1 mm outer diameter, 0.58 mm inner diameter) using a pipette puller instrument. Aim to achieve a tip diameter between 3 µm and 10 µm.
2. Fill the glass capillary with the preferred stimulus solution using a microloader pipette tip. Take care to avoid bubbles, which can be removed by gentle tapping.
3. If the stimulus crystallizes at the tip, clean or replace the glass stimulus capillary.

4. Reference and recording electrodes

1. Use tungsten rods (127 µm diameter and 76.2 mm length) for both reference and recording electrodes. Sharpen the reference and recording electrodes to approximately 1 µm diameter at the tip (the shapes of these electrodes are depicted in Delventhal et al.³⁶) by dipping them repeatedly for several seconds in either a 10% KNO₃ (~1 M) solution or a 10% KOH (1.8 M) solution.
   NOTE: This solution requires current (0.3-3 mA) to facilitate this process.

5. Preparation of the fly for base recording

1. Draw a single fly from the vial into an aspirator. Withdraw the aspirator and trap the animal by placing a finger over the end.
2. Expel the fly into a 200 µL plastic pipette tip. Keeping the end of the aspirator in the pipette tip, use the end to push the fly forward, headfirst, toward the narrow end of the pipette tip.
3. Trim the pipette tip at each end (i.e., anterior and posterior to the animal) using a razor blade.
4. Use either clay or a small piece of cotton to push the fly forward further, until half of the head protrudes from the end of the trimmed pipette tip. Use forceps to gently push until the labellum at the front of the head is exposed.
5. Fix the trimmed pipette tip onto a glass microscope slide using clay (Figure 1).
6. Under the stereomicroscope, position the labellum laterally on a cover slip so that one lobe, along with its 31 taste sensilla, is exposed (Figure 1). The cover slip keeps the labellum in place.

6. Electrophysiology rig

1. Select a room for the rig setup that has stable temperature and relative humidity (<70%) and is isolated from sources of electrical and mechanical noise, such as refrigerators and centrifuges.
2. Place the microscope on the center of an antivibration table.
3. Secure a manual micromanipulator to the antivibration table (Figure 2).
4. Attach a stainless-steel shaft that holds the tungsten reference electrode to the manual micromanipulator (Figure 2).
5. Connect motorized manipulators-one with a holder for the recording electrode probe and a second one with a holder connected to a stainless-steel shaft for the glass stimulus capillary-to the same table using stands (Figure 2).
6. Connect the recording electrode probe to an Intelligent Data Acquisition Controller (IDAC) system or another amplifier/digitizer system.
7. Link this IDAC system to the computer at the workstation.
8. Ground the manual and motorized manipulators to the same location within the rig.
9. Install appropriate acquisition software for the IDAC system on the computer. Ensure that the digital acquisition drivers are compatible with the operating system (e.g., Windows XP-7, -8, or -10) on the computer.

7. Recording from taste sensilla

1. Place the preparation slide on the microscope stage with a low magnification (e.g., 10x) objective in position. Move the stage until the labellum is in focus at the center of the field of view at both low-magnification and high-magnification (e.g., 50x) objectives.
2. Insert the reference electrode into the eye using the low magnification objective. To insert the reference electrode, target the eye on the side of the fly opposite the side with the recording electrode, for example, if the recording electrode is approaching from the right, place the reference electrode in the left eye. Utilize a manual micromanipulator for precise insertion.
3. Bring the tip of the glass stimulus capillary into focus at the center of the field of view of both low-magnification and high-magnification objectives using a motorized micromanipulator (Figure 3).
4. Under low magnification, bring the recording electrode close to the labellum using a second motorized micromanipulator.
5. Under high magnification, insert the recording electrode into the base of a taste sensillum using the motorized micromanipulator until the sound of neuronal firing activity from the audio output of the IDAC system is heard.
6. Once a stable signal has been established, start recording the signal using the software that comes with the IDAC system (Figure 4A-D). To start recording, press the Start recording button.
7. Bring the tip of the stimulus glass capillary to cover the tip of the taste sensillum using the motorized manipulator.
8. To end the stimulus, remove the glass stimulus capillary from the sensillum using the motorized manipulator.
9. Mark the start and end of the stimulation manually using a pedal. The pedal is connected to the IDAC, and its communication with the software is facilitated through the IDAC to mark the start/end of the stimulus.

8. Analysis

1. Use the different functions of the software that comes with the IDAC system to sort spike populations by amplitude (when possible) and analyze response dynamics.
   1. To count spikes, left click on the recording of interest, bringing up a window to select from. Choose To Spikes, initiating another window named Convert waves to spikes. Enter a name in the New field and press the OK button.
   2. Entering the name in the New field in step 8.1.1 leads to the Amplitude Histogram view. Choose the amplitude to count, then close this view. Left click to add a counter.
   3. Examine the spikes manually to confirm conclusions based on analysis with software.
      NOTE: The software also allows the exportation of data in different formats for further analysis.

Wyniki

Figure 4A shows spontaneous spikes that arise from a sensillum. They fall into two classes based on amplitude, with the larger spikes deriving from the neuron that is sensitive to bitter compounds and the smaller spikes from the neuron that responds to sugars. The relationship between spike amplitude and functional specificity has been corroborated by genetic experiments^4,14,37,

Dyskusje

In recordings from some types of sensilla, it can be challenging to differentiate the spikes of different neurons. For example, the sugar neurons and mechanosensory neurons of S and I sensilla produce spikes of similar amplitudes, making it difficult to distinguish them^4,14. We find that the use of a very sharp tungsten recording electrode reduces the firing of the mechanosensory neuron, as does the judicious placement of the recording electrode. Insertion of the...

Materiały

Name	Company	Catalog Number	Comments
Microscope	Olympus	BX51WI	equipped with a 50X objective (LMPLFLN 50X, Olympus) and 10X eyepieces.
Antivibration Table	TMC	63-7590E
motorized Micromanipulators	Harvard Apparatus and Märzhäuser Micromanipulators	Micromanipulator PM 10 Piezo Micromanipulator
manual Micromanipulators	Märzhäuser Micromanipulators	MM33 Micromanipulator
Magnetic stands	ENCO	Model #625-0930
Reference  and recording Electrode Holder	Ockenfels Syntech GmbH
Stimulus glass capillary Holder	Ockenfels Syntech GmbH
Universal Single Ended Probe	Ockenfels Syntech GmbH
4-CHANNEL USB ACQUISITION CONTROLLER , IDAC-4	Ockenfels Syntech GmbH
Stimulus Controllers	Ockenfels Syntech GmbH	Stimulus Controller CS 55
Personal Computer	Dell	Vostro	Check for compatibility with digital acquisition system and software
Tungsten Rod	A-M Systems	Cat#716000
Aluminum Foil and/or Faraday Cage			Electromagnetic noise shielding
Borosilicate Glass Capillaries	World Precision Instruments	1B100F-4
Pipette Puller	Sutter Instrument Company	Model P-97 Flaming/Brown Micropipette Puller
Stereomicroscope	Olympus	VMZ 1x-4x	For fly preparation
p200 Pipette Tips			Generic
Microloader tips	Eppendorf	E5242956003
1 ml Syringe			Generic
Crocodile clips
Power Transformers	STACO ENERGY PRODUCTS	STACO 3PN221B	Assembled from P1000 pipette tips, flexible plastic tubing, and mesh
Modeling Clay			Generic
Forceps			Generic
Plastic Tubing	Saint Gobain	Tygon S3™ E-3603
Standard culture vials	Archon Scientific		Narrow 1-oz polystyrene vails, each with 10 mL of glucose medium, preloaded with cellulose acetate plugs
Berberine chloride (BER)	Sigma-Aldrich	Cat# Y0001149
Denatonium benzoate (DEN)	Sigma-Aldrich	Cat# D5765
N,N-Diethyl-m- toluamide (DEET)	Sigma-Aldrich	Cat# 36542