Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Summary

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

Abstract

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Introduction

The electrical properties of cells such as neurons are observed by measuring ion flow across cell membranes as a change in voltage or current. A variety of electrophysiological techniques have been developed to measure bioelectric events in cells. Neurons found in the eyes of animals are accessible and their circuitry is often less complex than in the brain, making these cells good candidates for electrophysiological study. Common applications of electrophysiology in the eye include electroretinography (ERG)^1,2 and microelectrode intracellular recording. ERG involves placing an electrode in or on the eye of an animal, applying a light stimulus, and measurin....

Protocol

All animals were treated as humanely as possible. Insects were shipped as pupae from Costa Rica Entomological Supply, Costa Rica.

1. Heliconius Pupae Care

1. Hang all pupae spaced 2-3 cm apart in a humidified chamber using insect pins.
2. After eclosion, allow wings to dry then keep butterflies alive for at least 1 day in a humidified chamber and feed a dilute honey solution daily before recording.
   1. Dilute honey with water to about a 20% honey solution by volume, and pour into a shallow Petri dish.
   2. Bring individual butterflies to the Petri dish, one by one. Upon touching the solution with their front tarsi, the but....

Results

For many elements of the recording setup, a written description does not provide enough detail. Figure 1 is a schematic of the components involved in the complete recording setup. In Figure 2, spectra are plotted for white light and each interference filter to give a sense of why a correction factor is needed and what is needed to calculate this correction. Figure 3 shows photos and a diagram of the Cardan arm that is used for these experiments. Figure 4.......

Discussion

Intracellular recording can be a difficult technique to master due to the many technical steps involved. For successful experiments several important points must be considered. First, it is important to have a properly vibrationally-isolated table on which the experiment is performed. Many researchers use air tables, which completely separate the tabletop from the base, giving superior vibration isolation. Our setup involves a thick marble table with a sandbox on top, into which is placed the micromanipulator/electrode h.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Butterfly pupae			Several local species available, need USDA permits for shipping. Carolina Bio Supply has several insect species that may be ordered within the U.S. without the need for additional permits
Large plastic cylinder			Any chamber that remains humidified will work
Insect pins, size 2	BioQuip	1208B2
100% Desert Mesquite Honey	Trader Joe's		Any honey or sucrose solution will work
Xenon Arc Lamp	Oriel Instruments	66003	Oriel is now a part of Newport Corporation
Universal Power Supply	Oriel Instruments	68805	Oriel is now a part of Newport Corporation
Optical Track	Oriel Instruments	11190	Oriel is now a part of Newport Corporation
Rail Carrier, Large (2x)	Oriel Instruments	11641	Oriel is now a part of Newport Corporation
Rail Carrier, Small (4x)	Oriel Instruments	11647	Oriel is now a part of Newport Corporation
Thread Adaptor, 8-32 Male to 1/4-20 Male, pack of 10	Newport Corporation	TA-8Q20-10
Optical Mounting Post, 1.0 in., 0.5 in. Dia. Stainless, 8-32 & 1/4-20 (5x)	Newport Corporation	SP-1
No Slip Optical Post Holder, 2 in., 0.5 in. Diameter Posts, 1/4-20 (5x)	Newport Corporation	VPH-2
Fixed lens mount, 50.8 mm	Newport Corporation	LH-2
Fixed lens mount, 25.4 mm	Newport Corporation	LH-1
Condenser lens assembly	Newport Corporation	60006
Convex silica lens, 50.8 mm	Newport Corporation	SPX055
Six Position Filter Wheel, x2	Newport Corporation	FW1X6
Filter Wheel Mount Hub	Newport Corporation	FWM
Concave silica lens, 25.4 mm	Newport Corporation	SPC034
Collimator holder	Newport Corporation	77612
Collimating beam probe	Newport Corporation	77644
Ferrule Converter, SMA Termination to 11 mm Standard Ferrule	Newport Corporation	77670	This adapter allows the fiber optic to fit into the collimator holder
600 μm diameter UV-vis fiber obtic cable	Oriel Instruments	78367	Oriel is now a part of Newport Corporation
Shutter with drive unit	Uniblitz	100-2B
UV Fused Silica Metallic ND Filter, 0.1 OD	Newport	FRQ-ND01
UV Fused Silica Metallic ND Filter, 0.3 OD	Newport	FRQ-ND03
UV Fused Silica Metallic ND Filter, 0.5 OD	Newport	FRQ-ND05
UV Fused Silica Metallic ND Filter, 1.0 OD	Newport	FRQ-ND10
UV Fused Silica Metallic ND Filter, 2.0 OD	Newport	FRQ-ND30
UV Fused Silica Metallic ND Filter, 3.0 OD	Newport	FRQ-ND50
LS-1-Cal lamp	Ocean Optics	LS-1-Cal
Spectrometer	Ocean Optics	USB-2000
SpectraSuite Software	Ocean Optics
Interference bandpass filter, 300 nm	Edmund Optics	67749
Interference bandpass filter, 310 nm	Edmund Optics	67752
Interference bandpass filter, 320 nm	Edmund Optics	67754
Interference bandpass filter, 330 nm	Edmund Optics	67756
Interference bandpass filter, 340 nm	Edmund Optics	65614
Interference bandpass filter, 350 nm	Edmund Optics	67757
Interference bandpass filter, 360 nm	Edmund Optics	67760
Interference bandpass filter, 370 nm	Edmund Optics	67761
Interference bandpass filter, 380 nm	Edmund Optics	67762
Interference bandpass filter, 390 nm	Edmund Optics	67763
Interference bandpass filter, 400 nm	Edmund Optics	65732
Interference bandpass filter, 410 nm	Edmund Optics	65619
Interference bandpass filter, 420 nm	Edmund Optics	65621
Interference bandpass filter, 430 nm	Edmund Optics	65622
Interference bandpass filter, 440 nm	Edmund Optics	67764
Interference bandpass filter, 450 nm	Edmund Optics	65625
Interference bandpass filter, 460 nm	Edmund Optics	67765
Interference bandpass filter, 470 nm	Edmund Optics	65629
Interference bandpass filter, 480 nm	Edmund Optics	65630
Interference bandpass filter, 492 nm	Edmund Optics	65633
Interference bandpass filter, 500 nm	Edmund Optics	65634
Interference bandpass filter, 510 nm	Edmund Optics	65637
Interference bandpass filter, 520 nm	Edmund Optics	65639
Interference bandpass filter, 532 nm	Edmund Optics	65640
Interference bandpass filter, 540 nm	Edmund Optics	65642
Interference bandpass filter, 550 nm	Edmund Optics	65644
Interference bandpass filter, 560 nm	Edmund Optics	67766
Interference bandpass filter, 570 nm	Edmund Optics	67767
Interference bandpass filter, 580 nm	Edmund Optics	65646
Interference bandpass filter, 589 nm	Edmund Optics	65647
Interference bandpass filter, 600 nm	Edmund Optics	65648
Interference bandpass filter, 610 nm	Edmund Optics	65649
Interference bandpass filter, 620 nm	Edmund Optics	65650
Interference bandpass filter, 632 nm	Edmund Optics	65651
Interference bandpass filter, 640 nm	Edmund Optics	65653
Interference bandpass filter, 650 nm	Edmund Optics	65655
Interference bandpass filter, 660 nm	Edmund Optics	67769
Interference bandpass filter, 671 nm	Edmund Optics	65657
Interference bandpass filter, 680 nm	Edmund Optics	67770
Interference bandpass filter, 690 nm	Edmund Optics	65659
Interference bandpass filter, 700 nm	Edmund Optics	67771
Faraday cage			Any metal structure will work that can be grounded and that fits the experimental setup.
Stereomicroscope, 6X, 12X, 25X, 50X magnification	Wild Heerbrugg	Wild M5	Any Stereomicroscope will do
Microscope stand with swinging arm and heavy base	McBain Instruments		Any heavy base with arm will do
Cardan arm			Custom built, See Figure 4
Fiber-lite high intensity illuminator	Dolan-Jenner	MI-150	For lighting specimen
Fiber-lite goose-neck light guide	Dolan-Jenner	EEG 2823	Any goose-neck light guide will do
Marble table
Raised wooden table			Hole should be cut through this table so that the sandbox can rest on the marble table underneath
Wooden box filled with sand			custom built, any box with sand
Manipulator	Carl Zeiss - Jena
Electrode holder
Specimen stage
Alligator clip wires for grounding
Insulated copper wire
Silver wire, 0.125 mm diameter	World Precision Instruments	AGW0510
BNC cables
Preamplifier with headstage	Dagan Corporation	IX2-700
Humbug Noise reducer	Quest Scientific	Humbug
Oscilloscope, 30 MHz, 2 CH, Dual Trace, Alt-triggering, without probe	EZ Digital	os-5030
BNC T-adapter
Powerlab hardware 2/20	ADI instruments	ML820
Labchart software	ADI instruments	Chart 5
10 MHz Pulse Generator	BK Precision	4030
Glass pipette puller	Sutter Instruments	P-87
Borosillicate glass capillaries with filament	World Precision Instruments	1B120F-4
Potassium chloride, 3 M
Slotted plastic tube
Low melting temperature wax
Soldering Iron	Weller
Platform with ball-and-socket magnetic base	Hama photo and video
Double edge carbon steel, breakable razor blade	Electron Microscopy Sciences	72004
Vaseline
Microsoft Excel	Microsoft