The overall goal of the following cut loading experiment is to examine the extent of gap junction tracer coupling between neurons in the intact retina under controlled illumination conditions. The first step is achieved by cutting retinas with a sharp blade dipped in a solution of neuro biotin, a tracer molecule that can diffuse through open gap junction channels, but not cell membranes. In the second step, the retinas are incubated in ringer's solution, which allows tracer diffusion through open gap junction channels.
If gap junction coupling is weak, neuro biotin fluorescence will be restricted to retinal neurons that are near the cut. In contrast, if gap junction coupling is strong, the fluorescence will be detected in retinal neurons far from the cut. The main advantage of cut loading over existing techniques is that it is easier to perform on neurons with small cell bodies in intact neural retinal tissue.
It is also easier to control the illumination conditions during cut loading than during an electrophysiological study of single neurons in intact retinal tissue. Because the light stimuli used during electrical recording can themselves lead to changes in gap junction coupling demonstrating the technique will be Dr.Chu Choi, a postdoc from my laboratory. In this procedure, a goldfish is kept in constant darkness in the day for at least one hour before surgery, and all of the following steps until the retinal tissue is fixed, are also performed during the day in constant darkness, deeply anesthetize the goldfish by adding 150 milligrams per liter methane sulfonate to the aquarium water.
Then sacrifice the goldfish according to federal and university guidelines and remove its eyeballs carefully. Next, remove the anterior portion of each eyeball. Then place a piece of filter paper on top of the posterior portion of the eye and invert the filter paper and eye so that the filter paper is underneath the posterior portion of the eye.
Now, gently remove the back of the eye, including the sclera and pigment epithelium from the dark adapted goldfish. Retina cut the optic nerve with a pair of fine scissors. The neural goldfish, retina oriented photoreceptor side up should now be attached to the filter paper before enucleation, while the fish is undergoing anesthesia at five milliliters oxygenated by carbonate based ringer solution with or without test drug to each well of a six well plate following dissection of the intact neural retina from the posterior portion of the eye.
Transfer the retinas to the wells during constant darkness. Then seal the wells with paraform and let the tissue incubate for 30 minutes. Prepare 100 microliters of tracer solution by dissolving 0.5%neuro biotin in ringer solution immediately before cutting through the retina.
Next, add the neuro biotin solution to a glass petri dish. Then gently tap the filter paper to which the retina is attached on a paper towel. To remove excess ringers, dip a razor blade in the neuro biotin solution and to make a perpendicular radial cut through the retina and the filter paper.
After that, dip the razor blade in the neuro biotin solution again and cut the retina a second time. Repeat this up to four cuts per retina. At this step, transfer the retina to fresh ringers, medium with or without test drug for 15 minutes to allow loading and diffusion.
Wash it three times with fresh ringers, medium with or without test drug for five minutes each. Then fix the retina with 4%para formaldehyde in 0.1 molar phosphate buffer solution at room temperature for one hour. Subsequently, wash the retina with 0.1 molar PBS overnight at four degrees Celsius by placing it in a refrigerator on the following day.
React the retina with 2%streptavidin Alexa 4 88 in 0.1 molar PBS and 0.3%Triton X 100 to a final concentration of 10 micrograms per milliliter and incubated overnight at four degrees Celsius. The next day. Wash the retina three times with 0.1 molar PBS at room temperature each time for 10 minutes.
Detach the retina from the filter paper in PBS. Then using a fine brush, mount the retina photoreceptor side up onto a slide with vector shield mounting medium. Transfer the slide to a laser scanning confocal microscope and take pictures at the same magnification resolution and settings for every experimental condition so that comparisons of the effects of different experimental conditions on the extent of gap functional tracer coupling can be made shown.
Here is the extent of photoreceptor cell tracer coupling during the day in constant darkness compared to during the day in constant darkness in the presence ofone a selective dopamine D two receptor antagonist. Note that the razor cut in both images is on the left. Although a single image may contain all of the labeled cells, it is recommended that one collect a Z stack of the area of interest and collapse it into a single image shown.
Here is the extent of photoreceptor cell tracer coupling as one moves sequentially through such a Zack. Note that the razor cut in this image is on the right. To quantify the extent of tracer coupling in the LSM image browser, open the image, then click export and save the picture as a TIF 16 bit non compressed file scale bar should be included in this TIFF image using NIH image J software.
Measure the fluorescence intensity of Alexa 4 88 labeled neuro biotin from low magnification images of hole mount retinas. Open the TIFF file using Image J software and then draw a straight line corresponding to the scale bar. Next, go to analyze, set scale, and enter the known distance and unit of length.
Then click okay. Now the fluorescence measurements can be presented in calibrated units such as millimeters. Following this, select the area of interest using the rectangular selection tool.
The peak of fluorescent should be positioned at the left border of your selection. Make sure there is no fluorescent signal near the right border. Click analyze and plot profile for the daytime control data obtained in constant darkness.
A curve with an exponential decay is displayed from the left to the right. Then click copy in the pop-up window and paste it in Excel. Now there are two columns, one showing the distance from the cut and the other showing fluorescence intensity as a function of the distance from the cut.
As a comparison, the above steps are repeated with data obtained in constant darkness in the day. In the presence of spit bro, divide each raw fluorescence value by the maximum fluorescence value to obtain the relative fluorescence intensity. Copy two columns corresponding to the distance from the cut and the relative fluorescence intensity, and then paste them into origin software.
These steps are shown for both the daytime control and the daytime spit. Run data at this point fit the results with the exponential decay. Number one function, compare the space constant values at different experimental conditions using a T-test or Inova.
Exponential functions and statistical analyses of daytime control, daytime pip rone, and nighttime control data are shown for comparison.Shown. Here are examples of the extent of photoreceptor cell tracer coupling in the dark adapted goldfish retina in the day A, the night B, and in the day in the presence ofone a selective dopamine D two receptor antagonist as revealed by the cut loading technique. All of the confocal images were taken using the same settings for comparison.
In this graph, the fluorescence intensity for three experimental conditions is plotted as a function of the distance from the cut and fitted by the exponential function. The cut loading technique is used to quantify the extent of gap junctional tracer coupling by calculating space constant values as shown here for three experimental conditions. The cut loading technique can also be used to investigate the electrical synapses between other cell types in the retina and in other species.
For example, these two figures illustrate that rabbit, A type and B type horizontal cells exhibit homologous tracer coupling following, cut loading, and diffusion of neuro biotin under dark adapted conditions. We have used the cut loading technique to study the extent of tracer diffusion through the gap junctions between rods and cones in the day and night. In addition, this technique can be used to determine whether tracer coupling between other retinal neurons is controlled by the retinal clock and or by specific light dark conditions.
It can also be used to study tracer coupling between neurons in other brain regions.
