Visualizing Rhabdom Shedding in Whole Mounts of Photoreceptors from Limulus polyphemus

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1 Proceedings of The National Conference On Undergraduate Research (NCUR) 2006 The University of North Carolina at Asheville Asheville, North Carolina April 6 8, 2006 Visualizing Rhabdom Shedding in Whole Mounts of Photoreceptors from Limulus polyphemus Brian C. Oveson Department of Biology Union College 807 Union Avenue Schenectady, NY USA Faculty Advisor: Leo Fleishman, Ph.D. Abstract The purpose of this project was to determine if a new whole-mount procedure for visualizing rhabdom shedding in photoreceptors of the lateral eye of Limulus polyphemus would eliminate the need for serial sections, thus avoiding several hours of cutting and aligning frozen sections of ommatidium. The shedding of multi-vesicular bodies (MVB) in photoreceptors currently requires an arduous procedure of freezing, cutting sections, and performing immunocytochemistry on samples as small as 15 micrometers in thickness. Confocal microscopy is performed on each section, and the resulting images are then aligned so that all of the rhabdom shedding of one ommatidium can be quantified. This is done in an effort to track transient and light-driven shedding in the ommatidium. It was hypothesized that the rhabdom shedding could be visualized through the whole mount of the cornea instead of using serial sections. To test this hypothesis, the neural plexus and retinal epidermis were removed from the cornea, and immunocytochemistry was performed on the whole mount cornea. A z-series of the fluorescent images of the whole cornea was constructed using confocal microscopy techniques. The z-series was accomplished, but it was hypothesize that the rhabdom shedding could not be properly quantified because the screening pigment complicated the staining process of immunocytochemistry. Keywords: Limulus polyphemus, ommatidium, confocal microscopy. 1. Introduction The lateral compound eyes of Limulus polyphemus (American Horseshoe Crab) are used for pattern detection primarily during mating. As mating occurs during the night, the sensitivity to light of the lateral eyes are increased while visual resolution is decreased. This process is modulated by circadian rhythms of the animal. The lateral compound eye contains about 800 lenses called ommatidia. Each ommatidium contains about photoreceptors called retinular cells which are arranged in a star-like pattern surrounding the eccentric cell. The center of the ommatidium contains specialized light-sensitive membrane called rhabdom. The rhabdom surrounds the eccentric cell and screening pigment cell columns are located at the ends of the rhabdom (Fig. 1). When a light stimulus is presented to the compound eye, it enters the ommatidium through the cuticular cone. The cuticular cone refracts the light rays towards the center of the aperture. The amount of light and that enters the retinular cells is modulated by the shape of the aperture. Once the light is absorbed by the visual pigment in the rhabdom, electrical events occur in the retinular cells which are coupled to the eccentric cell. The summation of the electrical signals from each retinular cell is expressed in the spike initiation zone of the eccentric cell. The combination of all eccentric cell firings in the compound eye creates a visual image of the environment in which the objects have enhanced edges 1.

2 Figure 1. A single ommatidium. Figure 1 Cutaway view of a single ommatidium showing the location of the cuticular cone (CC), the aperture (Ap), the rhabdom (Rh), the screening pigment cells (PP), the pigment granules (RP), the eccentric cell (ED), the retinular cells (R), and the axon (Ax). There is an initial shedding of about 10% of the total rhabdom at the onset of dawn (Fig 2). This transient shedding requires input from circadian efferent fibers to the retina prior to the initial light stimulus. The rhabdom is removed by coated vesicles that pack together to form multivesicular bodies (MVB). The MVBs are immunoreactive for opsin. Throughout the day light-driven shedding occurs. This is described as smaller amounts of rhabdom shedding proportionately to the amount of light intensity from the sun. These MVBs are immunoreactive for opsin, and arrestin 2. (A) (B) 25 µm Figure 2. Transient rhabdom shedding.

3 Figure 2 TEM of transient rhabdom shedding in an ommatidium. (A) was taken from an animal before dawn and it shows almost no shedding. (B) was taken from the same animal 15 minutes after the initial onset of dawn and shows transient shedding occurring. Immunocytochemistry combined with confocal microscopy yield images of ommatidia with fluorescently labeled MVBs from which, the amount of rhabdom shedding can be quantified from reconstructed images of the whole ommatidia 3. The current method requires serial sections of the ommatidia to be cut, and immunocytochemistry performed on each section in order to accurately count the rhabdom shedding. This process is extremely time consuming and tedious. I hypothesize that the need for cutting sections and making reconstructions can be eliminated by performing immunocytochemistry on whole ommatidium, and then constructing a z-series of the sample with confocal microscopy. 2. Methodology 2.1 animals Male and female adult horseshoe crabs Limulus polyphemus collected at Cape Cod and maintained at the Institute for Sensory Research in Syracuse, NY. The animals were kept in a marine aquarium with artificial circulated seawater under skylights. The carapace size ranged from 15 to 20 cm across. 2.2 Immunocytochemistry A scalpel was used to dissect the lateral eyes free from the animal and the sample was then immersed in an ice cold methanol/formaldehyde solution (3.7% formaldehyde and 80% methanol). The sample was fixed in the solution held at 4 C overnight. The tissue was rehydrated through graded methanol solutions and placed on a dissecting microscope. A scalpel and tweezers were used to remove the neural plexis and retinal epidermis. Single ommatidia were then removed from the cornea using tweezers and mounted on gelatin-coated glass microscope slides. An antibody produced in rabbit and diluted to ratios between 1:100 to 1:1000 directed against opsin was used. An antibody produced in mouse and diluted to ratios between 1:50 to 1:200 directed against arrestin was used. The secondary antibodies used were fluorescein isothiocyanate-conjugated, tetrarhodamine isothiocyanate-conjugated, or Texas Red-conjugated antibodies directed against mouse IgG or rabbit IgG were used at a dilution of 1:50 4. Slides were washed in 0.1 M phosphate buffer (PB), and then the sections were incubated for 1 hour at room temperature in the primary antibody diluted in 0.1 M PB and 0.3% Triton-X 100. After three 10 minute washes in 0.1 M PB, the samples were incubated for 1 hour at room temperature in the secondary antibody diluted in 0.1 M PB and 0.3% Triton-X 100. After three more 10 minute washes, ommatidia were mounted with an aqueous nonfluorescent mounting medium and covered. 2.3 confocal microscopy Samples were examined with a confocal fluorescence microscope and signal-averaged. The confocal microscope took a z-series and digital images were produced and imported into Adobe Photoshop Results The ommatidia fluoresced both red and green indicating that both the opsin and arrestin were labeled. When a z-series of each ommatidium was constructed, all samples stained well for the outer sections Fig 3A, 3B, 3E, and 3F), but not for the central sections (Fig 3C, and 3D).

4 (A) (B) (C) (D) (E) (F) Figure 3. Six digital images of a whole ommatidium with a horizontal cut. Figure 3 Six digital images of anti-opsin fluorescence labeling captured by a confocal microscope. Red fluorescence indicates opsin staining. A-F are computerized cross sections from the bottom of the ommatidium to the top, respectively. The dark gap in the center of the sample is where the sample was divided using a scalpel on the dissecting microscope. 4. Conclusion It was hypothesized that the need for cutting sections and making reconstructions could be eliminated by performing immunocytochemistry and confocal microscopy on whole ommatidia. The results of this experiment disproved the hypothesis. Immunolabeling of opsin and arrestin appeared to work because both red and green colors fluoresced. The labeling is strongest for the outer-most sections of the z-series. The staining fades from the outer sections inward. These results suggest that there is less rhabdom in the center of the ommatidium compared to the outer region. Previous experiments have shown strong evidence supporting the migration of all rhabdom from the center of the ommatidia outward. Rhabdom originates in the center of the ommatidia and this is where a majority of rhabdom exists 5. The fact that there is less labeling of opsin in the center is most likely due to the opaque screening pigment in the ommatidium interfering with the confocal microscopy. The whole mount technique is not viable for studying rhabdom shedding. 5. Acknowledgements The author wishes to express appreciation to Dr. Steve Chamberlain, Bob Sacunas, Greg Rommel, Mark Choi, Arthur Pangaio, Howard Hughes Medical Institute, Union College, and Syracuse University. 6. References 1. Steven C. Chamberlain, McGraw-Hill Yearbook of Science and Technology (New York: McGraw-Hill Book Company, 1988). 2226

5 2. Sacunas, R.B., Papuga M.O., Malone M.A., Pearson A.C., Marjanovic M., Stroope D.G., Weiner W.W., Chamberlain S.C. Batelle B.A. Multiple Mechanisms of Rhabdom Shedding in the Lateral Eye of Limulus polyphemus. The Journal of Comparative Neurology (2002): 449: Battelle B-A, Dabdoub A., Malone M.A., Andrews A.W., Cacciatore C., Calman B.G., Smith W.C., Payne R. Immunocytochemical localization of Opsin, Visual Arrestin, Myosin III and Calmodulin in Limulus Lateral Eye Retinular Cells and Ventral Photoreceptors. J Comp Neurol (2001): 435: Batelle B-A, Andrews A.W., Kempler K.E., Edwards S.C., Smith W.C. Visual Arrestin in Limulus is phosphorylated at Multiple Sites in the Light and in the Dark. Visual Neuroscience (2002b): 17: Chamberlain, S.C., Barlow R.B. Light and Efferent Activity Control Rhabdom Turnover in Limulus Photoreceptors. Science (1979): 206: