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....

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