Reports. Table I. Identification of specimen studied. Ultrastructural description of a "cylinder organelle" in the outer plexiform layer

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1 Reports Ultrastructural description of a "cylinder organelle" in the outer plexiform layer of human retinas. JOSEPH CRAT, DANIEL M. ALBERT, AND TED W. REID. A cylinder-like organelle ("cylinder organelle") present in the outer plexiform layer of the retina and apparently confined to the horizontal cell processes is described. This structure was studied in detail in three normal human retinas from eyes enucleated for ciliary body melanomas. The cylinder organelle, when cut longitudinally, showed an electron-lucent area bound by two bands of electron-dense filaments. In transverse section, the cylinder organelle revealed a circular profile of electron-dense crisscrossing filaments about an electron-lucent matrix. Since this structure was only found in the rod spherule synaptic complex, it is hypothesized that it may be involved, in transmitting impulses. The purpose of this paper is to describe cylinder-like structures (hereafter termed cylinder organelles) found in the synaptic complex of the rod. The ultrastructural characteristics and location of this organelle have been studied. This structure has received only incidental mention in previous examinations of the human retina 1 ' - and has been noted in one report in a dog retina. 3 This cylinder organelle is morphologically distinct from various other organelles reported in retina cell processes. 1 " 7 Materials and methods. The retinas of three of four human eyes examined by electron microscopy contained the cylinder organelle described in this report. Identification of the eyes is given in Table I. Immediately following enucleation, portions of retina from between the posterior pole and equator which clinically and grossly appeared normal, were removed and placed in 3 per cent glutaraldehyde in 0.1 M cacodylate buffer at ph 7.4. The retinas were divided while in glutaraldehyde into 2 to 3 mm. portions and fixed for a total of one hour. The specimens were then washed in 0.1 M. cacodylate buffer at ph 7.4 for one hour with continuous agitation in the Reichert EM tissue processor. The specimens were postfixed in 2 per cent osmium tetroxide in 0.1 M cacodylate buffer. The dehydration, en bloc uranyl acetate staining, and initial plastic infiltration were carried out in the Reichert EM tissue processor, following which the specimens were removed and embedded in Epon-812. Ultrathin sections were cut with a DuPont Diamond Knife on an LKB Ultramicrotome and stained 923 Table I. Identification of specimen studied Initials and accession No. MK Y235 AH Y243 MB Y245 DC Age Sex Reason for enculeation spindle B-cell type epithelioid cell type mixed cell type Ependymoblastoma with lead citrate. The sections were examined with the Siemens Elmiskop 1 electron microscope using an 80 kv. accelerating voltage and a 50 U. objective aperture. They were optically enlarged for the final desired size. Results. Electron microscopic examination revealed the samples of the four specimens to be well preserved. The ultrastructural findings in the outer nuclear and outer plexiform layers conformed to the previous descriptions and the usual cytoplasmic structures were identified. Kolmer's crystalloid was present in the horizontal cells. Most of the cell processes showed the typical structural organelles including well-fonned synaptic vesicles, coated vesicles, synaptic ribbons, arciform densities, mitochondria, smooth endoplasmic reticulum, and occasional membranebound dense granules together with filaments. An additional finding was that of cylinder-like structures in retina horizontal cell processes (igs. 1 through 3). After the structure was initially observed (re: Table I, MK Y235) approximately one hundred additional sections were examined from this case. About 12 other cylinder organelles were observed at this time. Subsequently, approximately 100 sections of each of three additional retinas (re: Table I) were similarly examined and, in two of the three (AH Y243 and MB Y245), cylinder organelles were easily identified in multiple areas. Several variations in the structure of the cylinder organelles were observed in the transverse sections, including small ring forms (ig. 2, A) and a relatively more solid appearing type (ig. 2, A). On consecutive sections of some cell processes two separate (ig. 2, B) organelles were seen. Well preserved cylinder organelles are seen in ig. 3 with transverse and longitudinal sections juxtaposed. The overall length of the cylinder organelles is approximately 1.7 n, while the overall width of the above is about 0.7 p. (ig. 3). In longitudinal form the cylinder organelles reveal an electron-

2 924 Reports hioestigatioc Ophthalmology December 1975 ig. 1. A, general view of a rod spherule showing the horizontal cell processes and rudiments of the cylinder organelle (arrow). The nucleus is also seen (N) {x8,000). B, higher magnification of the beginning of a longitudinal section of the cylinder organelle. Synaptic vesicles (SV), synaptic ribbon (SR), and the synaptic cleft is indicated at the arrows (x36,000). C, oblique section of the cylinder-like organelles (x33,000).

3 Volume 14 Number 12 Reports 925 ig. 2. A, transverse section of several incomplete ring forms (small arrows) and a complete form (large arrow) of the cylinder organelle seen in the horizontal cell processes. Also seen are synaptic ribbon (SR), synaptic cleft (arrow) and the arciform density (AD) (x54,000). B, transverse section of the cylinder organelle seen in two different cell processes (x33,000).

4 926 Reports Investigative Ophthalmology December 1975 t ig. 3. Transverse and longitudinal section of the cylinder organelle seen in the horizontal cell processes within the rod spherule showing the electron-lucent matrix (M) and bands of filaments (B). Also seen are synaptic ribbons (SR), synaptic vesicles (SV), arcifomi density, and coated vesicles are indicated at the arrows {x42,000). lucent matrix having a diameter of 2,400 A bounded by two outer bands of electron-dense filament-like structures, each band measuring 2,400 A across {ig. 3). One filament within the band measures 130 A in diameter. The fila- ments are separated from each other by a less electron-dense layer of about 230 A. In transverse section, the cylinder structure as seen in ig. 3 has a "doughnut" appearance, with striations of small filaments or tubes intermeshed

5 Volume 14 Number 12 Reports 927 around the central matrix. The outer diameters of the circular profile of filaments seen surrounding the electron-lucent matrix in transverse sections were the same as in the longitudinal section, with the single exception of the smaller incomplete forms (ig. 2, A). The outer diameters in this case were approximately 740 A. It is interesting to note that the cylinder structures were always enveloped by the cell processes. Discussion. The organization of the vertebrate retina has become increasingly well recognized in recent years. 1 ' " The present paper describes in detail for the first time the ultrastructural characteristics of cylinder-like structures (termed the cylinder organelle) present in the horizontal cell processes. Previous reports of this structure in the human retina have included only a partial description of its ultrastructural detail. 1-2 Certain other specialized structures in this area have been described but appear morphologically distinct from the cylinder organelle: Evans 1 found parallel fibers of electron-dense material in chick rod endings. In 1967, Matsusaka 5 described a structure in the synaptic cytoplasm of chick retina cone. He found stacks of paired membranes and called them lamellar bodies. In serial sections, Mountford" described a structure in the receptorbipolar synaptic area of the guinea pig retina and described it as spindle-shaped with cross striations. Pedler and Tilly 7 observed a banded structure in Rhesus macaque monkeys; they reported that it extended for approximately 37 sections, having a length of approximately 3 /*. In a study by Leuenberger s paracrystalline inclusions were observed within the inner segments and the synaptic pedicles of the human photoreceptor cells. These inclusions are formed by microfilaments, 150 A in diameter and showing regular spacing. We would speculate that the cylinder structure apparently has gone unnoticed in previous studies, perhaps in part because of different orientations of the specimen block. The structure may also be more observable under certain pathologic conditions, and there may be some significance in the fact that all of the present specimens having the cylinder structure were from eyes with melanomas. In the present cases, however, the melanomas were distant from the areas where the cylinder organelles were seen. We do not believe the cylinder organelles to be artifacts, as we have not seen it in many other animal or human retinas, other ocular tissues or tumors similarly processed. At the present time the origin and function of the cylinder-like structure is unknown. Its close physical relationship with the synaptic vesicles might suggest that it is involved in the synaptic functions of the retina rod cells or horizontal cell processes. rom the Section of Tumor Research and Pathology, Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Conn. Supported in part by National Institutes of Health Crant EY Submitted for publication Aug. 12, Reprint requests: J. Craft, Department of Ophthalmology and Visual Science, Yale University School of Medicine, 333 Cedar St., New Haven, Conn REERENCES 1. Hogan, M. J., Alvarado, J. A., and Weddell, ]. E.: Histology of the Human Eye, Philadelphia, 1971, W. B. Saunders Company. 2. Radnot, M., and Lovas, B.: Die Ultrastruktur der Photoreceptor-Synapsen in einem alle von Sehnervenatrophie, Albrecht v. Craefes Arch. Klin. Exp. Ophthalmol. 173: 56, Hebel, R.: Uber ein Korperchen mit regelmassiger Innenstruktur in einer. Synapse der ausseren plexiformen Schicht des Hundeauges, Albrecht v. Graefes Arch. Klin. Exp. Ophthalmol. 180: 38, Evans, E. M.: On the infrastructure of the synaptic region of visual receptors in certain vertebrates, Z. Zellforsch. Mikrosk. Anat. 71: 499, Matsusaka, T.: Lamellar bodies in the synaptic cytoplasm of the accessory cone from the chick retina as revealed by electron microscopy, J. Ultrastr. Res. 18: 55, Mountford, S.: ilamentous organelles in receptor-bipolar synapses of the retina, J. Ultrastr. Res. 10: 207, Pedler, C. M. H., and Tilly, R.: Eye Structure, Eleventh Symposium, Stuttgart, 1965, Schattaver Verlag. 8. Leuenberger, P. M.: Mikrofibrillare Kristallartige Einschliisse in den Photoreceptorsynapsen der menschlichen Netzhaut, J. Microscopic 15: 79, Dowling, J. E.: Organization of vertebrate retinas, INVEST. OPHTHALMOL. 9: 655, Crossing axons in the third nerve nucleus. DON C. BIENANG. The research presented in this paper studied the pathway taken by the crossed fibers of the third nerve nucleus in an animal whose nucleus has been well mapped and found to correlate well with higher mammals and man. Autoradiography using tritiated amino acid labeled the cell bodies and axons of the left side of the oculomotor nucleus of the cat. Axons so labeled could be seen emerging from the ventral portion of the left nucleus through the median longitudinal fasciculus (mlf) to join the left oculomotor nerve. Labeled axons were also seen to emerge from the medial border of the caudal left nucleus, cross the midline, and pass through the right nucleus and the right mlf to pin the right oculomotor nerve. These latter axons must be the crossed axons of the superior