Name: determination of photoreceptor cell spectral sensitivity in an insect model from in vivo intracellular recordings
Uploaded: 2016-02-26T14:00:00
Description: Watch this Scientific Journal Video about Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings at JoVE.com

We thank the late&#160;Rudy Limburg for fabricating the cardan arm perimeter, Kimberly Jamison, Matthew McHenry, and Raju Metherate for lending us equipment, and Almut Kelber and Kentaro Arikawa, for encouragement. This work was supported by a National Science Foundation (NSF) Graduate Research Fellowship to KJM and NSF grant IOS-1257627 to A.D.B.

All animals were treated as humanely as possible. Insects were shipped as pupae from Costa Rica Entomological Supply, Costa Rica.
1. Heliconius Pupae Care
<ol>
	<li>Hang all pupae spaced 2-3 cm apart in a humidified chamber using insect pins.</li>
	<li>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.
	<ol>
		<li>Dilute honey with water to about a 20% honey solution by volume, and pour into a...

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...

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 measuring the change in voltage as a sum of the responses of all nearby cells3-6. If one is specifically interested in characterizing spectral sensitivities of individual photoreceptor cells, often multiple cell types simultaneously respond at different strengths&#160;to a given stimulus; thus it can be difficult to determine the sensitivities of specific cell types from ERG data especially if there are several different kinds of spectrally-similar photoreceptor cells in the eye. One potential solution is to create transgenic Drosophila with the photoreceptor (opsin) gene of interest expressed in the majority R1-6 cells in the eye and then perform ERGs7. Potential drawbacks of this method include no to low-expression of the photoreceptor protein8, and the long time frame for the generation and screening of transgenic animals. For eyes with fewer kinds of spectrally distinct photoreceptors, adaptation of the eye with colored filters can help with lowering the contribution of some cell types to the ERG, thereby permitting estimation of spectral sensitivity maxima9.
Intracellular recording is another technique where a fine electrode impales a cell and a stimulus is applied. The electrode records only that individual cell&#39;s response so that recording from and analyzing multiple individual cells can yield specific sensitivities of physiologically different cell types10-14. Although our protocol focuses on analysis of spectral sensitivity, the basic principles of intracellular recording with sharp electrodes are modifiable for other applications. Using a different preparation of a specimen, for instance, and using sharp quartz electrodes, one may record from deeper in the optic lobe or other regions in the brain, depending on the question being asked. For example, response times of individual photoreceptor cells15, cell activity in the optic lobes16 (lamina, medulla or lobula17), brain18 or other ganglia19 can also be recorded with similar techniques, or color stimuli could be replaced with polarization20-22 or motion stimuli23,24.
Phototransduction, the process by which light energy is absorbed and converted into an electrochemical signal, is an ancient trait common to nearly all present day animal phyla25. The visual pigment found in photoreceptor cells and responsible for initiating visual phototransduction is rhodopsin. Rhodopsins in all animals are made up of an opsin protein, a member of the 7 transmembrane G protein-coupled receptor family, and an associated chromophore which is derived from retinal or a similar molecule26,27. Opsin amino acid sequence and chromophore structure affect the absorbance of rhodopsin to different wavelengths of light. When a photon is absorbed by the chromophore the rhodopsin becomes activated, initiating a G-protein cascade in the cell that ultimately leads to the opening of membrane-bound ion channels28. Unlike most neurons, photoreceptor cells undergo graded potential changes that can be measured as a relative change in response amplitude with changing light stimulus. Typically a given photoreceptor type expresses only one opsin gene (though exceptions exist8,10,29-31). Sophisticated color vision, of the kind found in many vertebrates and arthropods, is achieved with a complex eye of hundreds or thousands of photoreceptor cells each expressing one or occasionally more rhodopsin types. Visual information is captured by comparing responses over the photoreceptor mosaic via complex downstream neural signaling in the eye and brain, resulting in the perception of an image complete with color and motion.
After measuring the raw responses of a photoreceptor cell to different wavelengths of light via intracellular recording, it is possible to calculate its spectral sensitivity. This calculation is based on the Principle of Univariance, which states that a photoreceptor cell&#39;s response is dependent on the number of photons it absorbs, but not on the particular properties of the photons it absorbs32. Any photon that is absorbed by rhodopsin will induce the same kind of response. In practice, this means that a cell&#39;s raw response amplitude will increase due to either an increase in light intensity (more photons to absorb), or to a shift in wavelength toward its peak sensitivity (higher probability of rhodopsin absorbing that wavelength). We make use of this principle in relating cellular responses at known intensity and the same wavelength to responses at different wavelengths and the same intensity but unknown relative sensitivity. Cell types are often identified by the wavelength at which their sensitivity peaks.
Here we show one method for intracellular recording and analysis of spectral sensitivity of photoreceptors in the eye of a butterfly, with a focus on making this method more accessible to the wider research community. Although intracellular recording remains common in the literature, particularly with respect to color vision in insects, we have found that descriptions of materials and methods are usually too brief to allow for reproduction of the technique. We present this method in video format with the aim of permitting its easier replication. We also describe the technique using easily obtainable and affordable equipment. We address common caveats that often are not reported, which slow down research when optimizing a new and complex technique.

determination of photoreceptor cell spectral sensitivity in an insect model from in vivo intracellular recordings

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.

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...

Watch this Scientific Journal Video about Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings at JoVE.com

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

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.

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

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

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