The light stimulus

Flashes are controlled in my lab by a Uniblitz shutter with its appropriate controller.

A 150 Watt Xenon arc and the associated power supply (Opti-Quip) is useful in providing a broad spectrum of ultraviolet (UV) and visible light.

A look inside my monochromator (Bausch and Lomb 500 mm) shows light entering, the grating, and a spectrum. A slit selects the wavelength being sent further through the optics.

Neutral densityy filters regulate the intensity of the light stimulus.

A carefully calibrated photodiode operated by batteries would be placed at the place where the fly's eye is stimulated and fed into the recording equipment for intensity calibrations.

Making electrodes

I use a Narishige PD-5 (Tokyo) horizontal puller with controls for an early magnet, a heater, and a late (stronger magnet).

The heater glows red while the first magnet pulls gently.

A microswitch with a shim detects the melt and the early pull to kick in the harder pull.

After the second pull, two electrodes are made.

Over the history of micropipettes, many tricks have been developed to get the very narrow tip to fill. Currently, a capillary tube with an inner filament has magic filling properties.

First you back fill the butt end a little with a spinal tap needle.

The electrolyte (I use saturated NaCl for ERGs) is carried to the tip. Then, you can finish back filling the elecrode with the syringe.

Sticking the fly down

The fly is stuck onto the edge of a cover slip. It is important that the head be really fused tightly, and I use l'Oreal top coat nail polish. If it gets too thick, I thin it with butyl acetate. I immobilize the legs and body with protemp (dental wax) being careful not to buile a high mound.

A nicer photograph of this is available on the research section of my home page since the same technique of sticking flies down is useful for pseudopupil analysis.

A sliver of agar (mixed in physiological saline) serves as a blanket to conduct the indifferent connection from the body to an agar block connected to the indifferent electrode.


The equipment

If equipment is dumping current into ground in various locations, then there is a circuit with voltage differences despite the infinitesimal resistance through ground. The result is ground loop noise. Thus it is wise to hook all grounds to one central ground tree. I hook this to water pipe ground with a big braided wire and bypass all the equipment grounds, connecting to the tree instead.

In the set-up, a dissecting microscope can be swung into position. I shine the stimulus down through some microscope parts. I can move the fly eye to under the focused light spot under the objective with stage controls. The probe from the amplifier is in the Faraday cage (painted flat black) near the fly. A micromanipulator allows the electrode to be advanced toward the eye. The cage should not be cluttered by electrically noisy stuff, but a microscope illuminator is necessary.

Poking the electrode into the eye

I suppose most people would put the indifferent electrode into the fly somewhere away from the eye. I hook a platinum wire in a dish full of agar and make an external bolt that the alligator clip from the probe can be hooked to.

Looking through the microscope, it is important to check the alignment insofar as the electrode and your field of view can reach the eye when the stimulus is focussed onto the eye.

A hydraulic microdrive (Kopf) [stepping motor driving water syringe on left and controller on rignt] driving a slave syringe helps to get the electrode into the eye.

A sharp micropipette will dimple in the eye surface just a little bit before penetrating the cornea.

Carefully backing off, the dent is made smooth while the electrode does not slide out of the retina.

Amplification and display

An electrometer serves as the differential preamplifier

In the old days, this could feed into a polygraph, a penwriter that graphs voltage as a function of time, limited for speed by the momentum of the pen

Also somewhat outdated is the oscilloscope

A permanent record can be made with a camera, and the most famous is the Grass camera

Nowadays, the computer is used for an oscilloscope. Here is a PowerBase 180 from Power Computing (Mac work-alike) feeding into an Optiquest monitor using the PowerLab 410 from AD Instruments as the interface

Diet

Here is our recipe: 2625 ml water, 28 g agar, 350 g yellow corn meal, 140 g brewers yeast, 17.5 g Carolina mold inhibitor, 21 ml propionic acid, .44 g beta-catotene in a few ml of ethanol

Yellow corn meal by itself probably gives enough carotenoid (Stark, Ivanyshyn and Greenberg, J. Comp. Physiol. A 121, 289-305, 1976), now known to provide precursor for the chromophore, 3-hydroxy retinal

We also supplement our food with beta carotene at a dose which is the minimum amount to maximixe sensitivity in otherwse retinoid deprived flies (Stark, Ivanyshyn and Greenberg, J. Comp. Physiol. A 121, 289-305, 1976). Also carrot juice can rapidly provide vitamin A replacement therapy in adults

Vitamin A deprivation is with Sang's medium (WWDoane, Drosophila, in Methods in developmental biology, ed. FHWilt & NKWessels, NY, Thomas Y CrowellCo. 1967, p. 234): 100 ml water, amounts in mg: 3000 agar, low vitamin casein 5500, fructose 750, cholesterol 30, lecithin (e.g. from soy) 400, yeast nucleic acid (yeast RNA) 400, thiamin HCl 0.2, riboflavin 1, nicotinic acid 1.2, calcium pantothenate 1.6, pyridoxine HCl 0.25, Biotin 0.016, folic acid 0.3, NaHCO3 140, KH2PO4 183, Na2HPO4 189, Carolina mold inhibitor 320


Some properties of the ERG

Note, these ERG's (from Stark and Wasserman, 1972, Vision Research 12, 1771-1775) are plotted up-side-down (negative up) to the usual convention (negative down). There is an "on-transient" which follows stimulus onset (mark) and an "off-transient" after the stimulus ends (also marked). These arise from R1-6 connections in the first optic neuropil, the lamina ganglionaris. There is a slow steady wave maintained for the stimulus duration (the "receptor wave") which comes from the retina. The shape of the ERG, especially the relative size of the on- and off-transients, depends on wavelength in red-eyed flies but not in white-eyed flies.


Blue light induces a prolonged depolarizing afterpotential (PDA) which yellow light repolarizes in R1-6. During that PDA, R1-6 are inactivated. Vitamin A deprivation eliminates the PDA. An expanded description of this can be found at the Vitamin A deprivation in Drosophila site.

In white-eyed otherwise wild-type flies (left), the waveform has on- and off-transients in the dark adapted fly. In the blue adapted fly (on top of a PDA) a smaller ERG (from R7/8) has no transients. In rdgB (right), a normal ERG can be obtained from a dark reared fly. Only the R7/8 ERG survives in light reared flies. (Harris and Stark, 1977, J. Gen. Physiol. 69, 261-291)

Another version of this waveform experiment is shown, with dark-adapted on top, blue-adapted in the middle and UV adapted at thebottom. (Harris, Stark and Walker, J. Physiol. 256, 415-439, 1976)

When R1-6 are inactivated with a PDA or eliminated by mutations like redB or ora, UV light induces an R7 PDA which is repolarized by blue light. A response calibration marker precedes the responses on that trace while stimuli are shown on the other trace. (Stark, J. Comp. Physiol. 115, 47-59, 1977)

The R1-6 PDA fizzles away in rdgB relative to its maintenance in white-eyed wild-type controls. (Harris, Stark and Walker, J. Physiol. 256, 415-439, 1976)

In response to a strobe (from aphoto-flash), a very fast potential called the M-potential (by Pak and co-workers) is elicited after blue light has converted most of the visual pigment into the metarhodopsin-570nm form (left, note fast time scale and stimulus monitor of fast flash). By contrast, when yellow light has converted most of the visual pigment into the rhodopsin-480nm form, a slightly slower on-transient is elicited (right). Later, it was shown (by Pak and co-workers and by Minke and co-workers) that the M-potential is an early receptor potential and the on-transient which that ERP elicits. (Stark, Ivanyshyn and Greenberg, J. Comp. Physiol. A 121, 289-305, 1976)

ERGs from ocelli are different in that they have no transients, but, when portrayed on this slow time scale, report changes in stimulus intensity. (Hu, Reichert and Stark, J. Comp. Physiol. A 126, 15-24, 1978)

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This page was created June, 1999
It was revised in July, 2000 based on work by Jon Wagnon, MS, Biology, SLU, 2000