Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Abstract

Source: Katz, B., et al., Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors. J. Vis. Exp. (2017)

The video demonstrates recording electrical activity in the photoreceptor neurons of Drosophila's ommatidium by establishing a whole-cell configuration. It highlights the activation of transient receptor potential channels by light pulses, resulting in measurable quantum bumps.

Protocol

NOTE: During all of the following steps, use only dim red light illumination and ensure that the ommatidia's exposure to light is minimal (i.e., work rapidly and turn off the dissecting light source and chamber red illumination used for viewing the ommatidia when performing tasks that do not demand viewing the ommatidium). In addition, perform all of the following steps according to standard electrophysiological protocol.

1. Under an inverted microscope (40X objective), carefully inspect all of the ommatidia in the bath and choose a suitable ommatidium for the experiment.​
   1. Ensure that the outer membrane of the ommatidium is smooth and intact, that the long axis is approximately at a right angle relative to the electrode approach direction (as seen in Figure 1A), and that the distal section of the ommatidium is not surrounded by any excess tissue. Place the chosen ommatidium at the optical axis of the objective lens (in the center of the field of vision) to ensure uniform illumination.
2. Fill a patch pipette with intracellular solution (IS1 or IS2).
   NOTE: To measure the intensity response and bump analysis, use IS1. To measure the reversal potential of the light-sensitive channels, use IS2.
3. Mount the patch pipette onto the electrode holder.
4. Blow into the pipette by mouth through the tube connected to the electrode holder, causing it to fill with positive pressure. Close the tube valve to maintain the pressure.
5. Using the micromanipulator, insert the electrode into the bath chamber.
6. Guide the electrode close to the distal section of the ommatidium, such that there is no contact between the electrode and the ommatidium, until a small dimple (due to the positive pressure in the patch pipette) can be observed in the ommatidium.
7. Open the recording software (see the Table of Materials). Open the "membrane test module" to apply continuous square voltage pulses of 2 mV at a rate of 100 Hz.
8. Set the junction potential to "zero," by adjusting the appropriate knob in the patch clamp amplifier to set the base of the square pulse to "zero" current
   NOTE: The electrophysiological setup includes a head stage (i.e. first-stage amplification) connected to an amplifier (i.e. second-stage amplification). The amplified analog signal is converted to a digital signal using the A/D converter, which is controlled by software installed on a PC computer.
9. Release the positive pressure in the pipette by opening the valve of the tube connected to the electrode holder. Gently create negative pressure in the pipette by sucking out of the tube, leading to the association of the pipette to the cell membrane. Close the tube valve to maintain the pressure.
10. Ensure that the electrode resistance viewed on the computer screen is elevated to 100 - 150 MΩ. Release the negative pressure in the pipette by manually opening the valve of the tube connected to the electrode holder.
11. Ensure that the electrode resistance is elevated to at least 1-2 GΩ.
    NOTE: At this point, a seal has been formed between the electrode and the photoreceptor.
12. Offset the capacitive currents of the pipette by adjusting the appropriate knob in the patch clamp amplifier.
13. Create rapid, short, and forceful bouts of negative pressure in the electrode, sucking by mouth out of the tube connected to the electrode holder to "break" into the photoreceptor membrane and create a whole-cell configuration. Alternatively, use the "Zap button" to apply short, rectangular electrical pulses, starting with a duration of "0.1 ms," or apply a combination of both methods.
    NOTE: The generation of the whole-cell configuration is revealed by a sudden increase in pipette capacitance (typically ~60 pF for a wildtype R1-6 photoreceptor; a capacitance of only ~20 pF indicates a recording from an R7 photoreceptor; a capacitance above ~90 pF indicates a recording from two photoreceptors).
14. Set the holding potential of the photoreceptor to the required voltage (usually -70 mV) manually using the appropriate knob in the patch clamp amplifier.
    NOTE: This step can be performed after a seal has been obtained and before the whole-cell configuration has been achieved.
15. Offset the capacitive currents and series resistance (a measured series resistance value greater than 25 MΩ indicates that the electrode pipette has been clogged) and, if required (i.e. for larger currents), apply series resistance compensation using the appropriate knobs in the patch clamp amplifier.
16. Close the black front curtain of the Faraday cage to obtain maximal darkness and electrical isolation.
17. Begin the recording process using the software and administer light stimuli and/or pharmacological substances according to the desired experimental procedure.

Results

Figure 1: The inaC^P209 and inaD^P215 Mutants Reveal Slow Response Termination of the Macroscopic Response to Light and of the Single Quantum Bumps. (A)The isolated ommatidium preparation with a patch pipette filled with fluorescent Lucifer ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polyethylene Tubing	Becton Dickinson		1.57 x 1.14 mm (35 cm)
Cover slips			22 x 22 mm No. 0
Paraplast Plus	Sigma	Paraffin – polyisobutylene mixture	To glue the coverslip onto the bottom of the bath chamber
Ground	Warner Instruments	64-1288	Hybrid Assembly Ag-AgCl Wire Assembly
Stereoscopic zoom Microscope	Nikon	SMZ-2B
Cold light source	Schott	KL1500 LCD
Filter (Color) for cold light source	Schott	RG620
Electrode holder	Warner Instruments	QSW-T10P	Q series Holders compatible with Axon amplifiers, straight body style
Silver Wire	Warner Instruments		0.25 mm diameter, needs to be chloridized
Micromanipulator	Sutter Instruments	MP 85	Huxley-Wall Style Micromanipulator
Faraday cage	home made		Electromagnetic noise shielding and black front curtain
Anti-vibration Table	Newport	VW-3036-OPT-01
Perfusion system	Warner Instruments	VC-8P	Pinch valve control system
Perfusion valve controller	Scientific instruments	BPS-8
Suction system
Amplifier	Molecular Device	Axopatch-1D
Head-stage	Molecular Device	CV - 4	Gain: x 1/100
A/D converter	Molecular Device	Digidata 1440A
Clampex	Molecular Device	10	software
pCLAMP	Molecular Device	10	software
Light source (Xenon Arc lamp)	Sutter Instruments	Lambda LS
Light detector	home made		phototransistor
Filter wheel and shutter controller	Sutter Instruments	Lambda 10-2 with a Uniblitz shutter
Filters (Natural density filter)	Chroma	6,5,4,3,2,1,0.5,0.3
Filter (Color)	Schott	OG590, Edge filter
Xenon Flash Lamp system	Dr. Rapp OptoElecftronic	JML-C2
Light guide			Quartz
Pulse generator	AMPI	Master 8
Microscope	Olympus	IX71, Inverted
Red illumination filter (Microscope)			RG630 / RG645 ø45mm
Microscope objective	Olympus	X60/0.9 UplanFL N air or X60/1.25 UplanFI oil
CCD Camera	Andor	iXon DU885K
NIS Element	Nikon	AR	software
Calcium indicator	Invitrogen	Calcium green 5N
Excitation & emission filters and dichroic mirror	Chroma	19002 - AT - GFP/FITC Longpass set
Vertical pipette puller	Narishige	Model PP83	Use either vertical or horizontal puller, as preferred.
Horizontal pipette puller	Sutter Instrument	Model P-1000 Flaming/Brown Micropipette Puller
Filament	Sutter Instrument		3 mm trough or square box