Volume 14 Reports 927 Number 12

around the central matrix. The outer diameters From the Section of Tumor Research and Pa- of the circular profile of filaments seen surround- thology, Department of Ophthalmology and Visual ing the electron-lucent matrix in transverse sec- Science, Yale University School of Medicine, New tions were the same as in the longitudinal sec- Haven, Conn. Supported in part by National Institutes of Health Crant EY 00108-06. Submitted tion, with the single exception of the smaller for publication Aug. 12, 1975. Reprint requests: incomplete forms (Fig. 2, A). The outer diameters J. Craft, Department of Ophthalmology and in this case were approximately 740 A. It is Visual Science, Yale University School of Medi- interesting to note that the cylinder structures cine, 333 Cedar St., New Haven, Conn. 06510. were always enveloped by the cell processes. Discussion. The organization of the vertebrate REFERENCES retina has become increasingly well recognized 1 1. Hogan, M. J., Alvarado, J. A., and Weddell, in recent years. ' " The present paper describes ]. E.: Histology of the Human Eye, Phila- in detail for the first time the ultrastructural delphia, 1971, W. B. Saunders Company. characteristics of cylinder-like structures (termed 2. Radnot, M., and Lovas, B.: Die Ultrastruktur the cylinder organelle) present in the horizontal der Photoreceptor-Synapsen in einem Falle cell processes. Previous reports of this structure von Sehnervenatrophie, Albrecht v. Craefes in the human retina have included only a partial Arch. Klin. Exp. Ophthalmol. 173: 56, 1967. description of its ultrastructural detail.1- 2 3. Hebel, R.: Uber ein Korperchen mit regel- Certain other specialized structures in this area massiger Innenstruktur in einer. Synapse der have been described but appear morphologically ausseren plexiformen Schicht des Hundeauges, 1 Albrecht v. Graefes Arch. Klin. Exp. Ophthal- distinct from the cylinder organelle: Evans found mol. 180: 38, 1970. parallel fibers of electron-dense material in chick 5 4. Evans, E. M.: On the infrastructure of the rod endings. In 1967, Matsusaka described a synaptic region of visual receptors in certain structure in the synaptic cytoplasm of chick retina vertebrates, Z. Zellforsch. Mikrosk. Anat. 71: cone. He found stacks of paired membranes and 499, 1966. called them lamellar bodies. In serial sections, 5. Matsusaka, T.: Lamellar bodies in the synaptic Mountford" described a structure in the receptor- cytoplasm of the accessory cone from the bipolar synaptic area of the guinea pig retina chick retina as revealed by electron microscopy, J. Ultrastr. Res. 18: 55, 1967. and described it as spindle-shaped with cross 7 6. Mountford, S.: Filamentous organelles in re- striations. Pedler and Tilly observed a banded ceptor-bipolar synapses of the retina, J. Ultrastr. structure in Rhesus macaque monkeys; they re- Res. 10: 207, 1964. ported that it extended for approximately 37 sec- 7. Pedler, C. M. H., and Tilly, R.: Eye Structure, tions, having a length of approximately 3 /*. Eleventh Symposium, Stuttgart, 1965, Schat- In a study by Leuenbergers paracrystalline inclu- taver Verlag. sions were observed within the inner segments 8. Leuenberger, P. M.: Mikrofibrillare Kristal- and the synaptic pedicles of the human photo- lartige Einschliisse in den Photoreceptorsynap- sen der menschlichen Netzhaut, J. Microscopic receptor cells. These inclusions are formed by 15: 79, 1972. microfilaments, 150 A in diameter and showing 9. Dowling, J. E.: Organization of vertebrate regular spacing. retinas, INVEST. OPHTHALMOL. 9: 655, 1970. 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 Crossing in the third . more observable under certain pathologic con- DON C. BIENFANG. ditions, and there may be some significance in the fact that all of the present specimens having The research presented in this paper studied the cylinder structure were from eyes with mela- the pathway taken by the crossed fibers of the nomas. In the present cases, however, the mela- third nerve nucleus in an animal whose nucleus nomas were distant from the areas where the has been well mapped and found to correlate well cylinder organelles were seen. We do not believe with higher mammals and man. Autoradiography the cylinder organelles to be artifacts, as we have using tritiated amino acid labeled the cell bodies not seen it in many other animal or human and axons of the left side of the oculomotor retinas, other ocular tissues or tumors similarly nucleus of the cat. Axons so labeled could be seen processed. emerging from the ventral portion of the left nucleus through the median longitudinal fasciculus At the present time the origin and function of (mlf) to join the left . Labeled the cylinder-like structure is unknown. Its close axons were also seen to emerge from the medial physical relationship with the synaptic vesicles border of the caudal left nucleus, cross the midline, might suggest that it is involved in the synaptic and pass through the right nucleus and the right functions of the retina rod cells or horizontal mlf to pin the right oculomotor nerve. These latter cell processes. axons must be the crossed axons of the superior

Downloaded from iovs.arvojournals.org on 09/28/2021 928 Reports Investigative Ophthalmology December 1975

rectus and levator palpebrae subnuclei. Since the head holder and an occipital craniotomy was per- path of these crossed axons is through the caudal formed. A small amount of the tip of the bony portion of the nucleus of the opposite side, the tentorium was removed to allow the stereotactic destruction of one lateral half of the octdomotor placement of a glass micropipette into the left nucleus would result in a bilateral palsy of the third . The micropipettes had crossed subnuclei. Bilateral palsy of the superior previously been loaded with 1 to 2 juCi of des- rectus and bilateral assymetrical palsy of the sicated tritiated proline or leucine. The dry amino levator palpebrae muscles would result. acid was redissolved in a small amount of normal saline at the time of the experiment. The bolus Previous investigations in the cat and the mon- of approximately 0.5 to 1.0 /tliters of solution was key have established that the branch of the third injected into the region of the left third nerve nerve to the superior rectus consists entirely of nucleus through the hydraulic pressure created by crossed fibers, and that the branch to the levator a Hamilton syringe connected to the micropipette palpebrae consists of both crossed and uncrossed with polyethylene tubing. 1 3 fibers. " This suggests that all the axons from the The scalp wound was sutured and the animals superior rectus and some from the levator were killed after one week. The were fixed palpebrae must cross the mesencephalic midline. in 10 per cent formaldehyde buffered to pH 7.4, The location of this crossing and the subsequent and the stem was processed for paraffin path taken by these crossed fibers has not been sectioning. Sections were cut at 10 micra and one well studied though some suggestive information section of every twenty was mounted. The is available.4' 5 mounted sections were processed down to the Stains for central nervous system myelin or for water stage and then dipped in % strength Kodak neuron fibers easily demonstrate an abundance of NTB-2 photographic emulsion. They were stored nerve fibers crossing the midline between the in dry, light-free boxes in a freezer at —13° C. paired third nerve nuclei of the cat. Though for six weeks and developed in Dektol. The sec- present throughout the rostro-caudal extent of the tions were lightly stained with toluidine blue. nucleus, they are most prominent in the caudal Results. In three of the five cats the caudal one-third, suggesting that they are axons of the portion of the third nerve nucleus was labeled. superior rectus, inferior oblique, and levator These three cats demonstrated radiolabeled axons palpebrae subnuclei located there.2- 3 that originated in the left third nerve nucleus, However, Cajal'1 pointed out on the basis of crossed the midline, and entered the right third Golgi studies of the third nerve nucleus that there nerve after passing through the right third nerve are two other potential sources for crossing fibers nucleus. Since proline seemed to have a greater in this nucleus, namely axons of afferents to the tendency to label small cells than leucine, and nucleus, e.g., from the ,7 and hence showed less contrast, the results were more dendrites of the cell bodies of the third nerve striking with leucine. This differential uptake has nucleus. been noted in other areas of the brain.9 With Positive identification of these crossed fibers as both amino acids, an abundance of radioactive the axons of the cell bodies of the opposite side fibers can be shown exiting from the ventral por- could be made with a whole neuron marking tech- tion of the left nucleus destined for the muscles nique such as autoradiography. innervated by uncrossed axons. On the opposite Radioactive amino acids, when injected in the side, exiting from the ventral portion of the right vicinity of neurons, are taken up by the soma, nucleus are radioactive fibers that have had their incorporated into the protein of the cell, and origin in cell bodies on the left side of the mid- ultimately distributed along the course of the line (Figs. 1 and 2). entire cell thus labeling the cell body and the The details of these crossing fibers is shown in course of its .s This new tracing device offers Fig. 3. It should be noted that these radioactive great promise for neuro-anatomic mapping since axons cross the midline in the dorsoventral center the amino acid is not taken up by axons passing of the nucleus and plunge into the mass of cell through the region of the injection but only by bodies of the opposite side before exiting through cell bodies at the site of the injection. The cells the medial longitudinal fasciculus to form the that incorporate the radioactive amino acid iden- intramedullary portion of the third nerve. The tify themselves by labeling their cell bodies. level at which these crossing fibers are seen Technique. Five adult cats weighing from two passing through the right third nerve nucleus to four kilograms were used. The cats were pre- would correspond to the location of the superior medicated with 50 mg. of pentobarbital given rectus, inferior oblique, and part of the levator intraperitoneally. Atropine sulphate, 0.1 mg. was palpebrae subnuclei. given subcutaneously and the animals were deeply Indirect evidence to support these findings was anesthetized with intramuscular ketamine hydro- supplied by the two experiments which failed to chloride. The head was placed in a stereotactic inject the caudal third nerve nucleus. In one, only

Downloaded from iovs.arvojournals.org on 09/28/2021 Volume 14 Reports 929 Number 12

Fig. 1. Autoradiograph and toluidine blue, x50, the caudal third nerve nucleus in cross-section. The blackness overlying the large neuronal soma on the left side of the nucleus indicates they have incorporated the radioactive leucine. None of the neuronal soma in the right side of the nucleus are labeled. Axons which have incorporated radioactivity are seen streaming out of the left nucleus (arrow), passing through the right nucleus (arrow) and the right mlf to join the right third nerve. Uncrossed axons are seen exiting through the left mlf.

the rostral half of the third nerve nucleus was There are many articles in the French and German injected. No crossing fibers were seen and no literature of the late nineteenth and early radioactive fibers were seen in the contralateral twentieth century postulating the existence of third nerve. In the second, the periaqueductal grey crossed axons in the third cranial nerve and its matter and the left fourth nerve nucleus were nucleus. With the exception of the articles by injected but not the third nerve nucleus. Once Perlia,4 Van Gehuchten/1 and Cajal/1 they fail to again, no crossed fibers were seen in the third positively identify the course and location of these nerve nucleus or in the third nerve. Since these crossed fibers. Thus, although the proof of the latter two experiments bracket the area of interest origin of these crossed fibers from the superior they indicate that the crossed fibers observed rectus and levator palpebrae subnuclei could have did not come from cells in the vicinity of the been deduced from the fiber degeneration studies caudal half of the third nerve nucleus. of Perlia4 and Van Gehuchten5 or from the Golgi 0 Finally, no cells of the interstitial nucleus, the stains of Cajal, these were done in the rabbit or the vestibular nuclei were and mouse, respectively, and do not have as much labeled in any of the experiments. pertinence to the human as does the cat. Discussion. Positive identification of the source The fact that the axons of the superior rectus of the abundant axons crossing in the caudal third subnucleus of one side cross the midline and pass nerve nucleus rests on a whole neuron marking through the superior rectus subnucleus of the other technique. Since the autoradiographic injection can side implies that if one side of the third nerve only label an axon by first reaching its cell body, nucleus is destroyed there will result a bilateral the labeled axons exiting from the right ventral paralysis of superior rectus function. Since the third nerve nucleus after crossing the midline axons of the levator palpebrae subnucleus are must have originated from cells in the left third mixed-crossed and uncrossed, the lids would be nucleus. No others cells with known projections expected to exhibit profound ipsilateral to the third nerve nucleus were ever labeled. and partial contralateral ptosis. The organization of the third nerve nucleus Since the anatomy of the third nerve nucleus of the cat has been the most thoroughly studied. in man resembles that of the cat with respect to

Downloaded from iovs.arvojournals.org on 09/28/2021 930 Reports Investigative Ophthalmology December 1975

/ J •*.»-* 'I

Fig. 2. Same as Fig. 1 except that radioactive proline was used. Many small cells (probably glia) as well as the large cells are labeled in the left side of the third nerve nucleus. No large cells of the right nucleus are labeled. Labeled crossed axons are seen exiting from the right ventral portion of the nucleus (arrow) through the mlf.

Fig, 3. Detail of Fig. 1 xl20. The radioactive change over the neuronal soma in the right side of the nucleus is no greater than the background in contrast to the neurons of the left side (arrow). Midline indicated by vertical lines.

Downloaded from iovs.arvojournals.org on 09/28/2021 Volume 14 Reports 931 Number 12

the location of as yet unidentified crossed axons,10 mus with this defect suggests that a similar path- the clinical neurologic findings following destruc- way defect without pigmentation anomalies, may tion of one lateral half of the third cranial nerve be the cause of much human . Creel, nucleus may be similar. Witkop, and King8 have used evoked potential methods to show that such a pathway defect From the Howe Laboratory of Ophthalmology, likely occurs in the human albino. While un- Massachusetts Eye and Ear Infirmary, 243 Charles published control experiments verified their results St., Boston, Mass. 02114. This work was supported on human albinos, no such defect has been found by NEI National Education Institute Grant No. EY 01 370-01. Submitted for publication July 2, in the normally-pigmented human squinter. It is 1975. Reprint requests: Dr. Bienfang. concluded that the visual pathway anomaly is limited to albinism and is not a likely cause of Key words: , superior rectus, most human strabismus. levator palpebrae, autoradiography, axons. The causes of human squint (strabismus) are REFERENCES usually unknown, especially in terms of specific neurologic models. In a rather small percentage 1. Warwick, R.: Oculomotor Organization, pp. of cases, pathologic or mechanical factors can be 173-204. In "The Oculomotor System" Bender, shown to be the primary cause. In most cases, M. B., editor, New York, 1964, Harper and Row, pp. 173-204. however, no demonstrable pathologic or mechani- 2. Gacek, R. R.: Localization of neurons sup- cal involvement can be found which would be plying the in the kitten significant enough to cause strabismus. These using horseradish peroxidase, Exp. Neurol. cases usually fall under the broad classification 44: 381, 1974. known as "concomitant"0 squint; a classification 3. Bienfang, D. C: Location of the cell bodies which would include a possible cause of strabis- of the superior rectus and inferior oblique mus which is to be tested here. Since anatomic motoneurons in the cat, Exp. Neurol. 21: or physiologic research is reasonably quite limited 455, 1968. in human squinters, it is necessary to look toward 4. Perlia: Die Anatomie des Oculomotorius- animal studies for possible models of human centrums beim Menschen, Arch Ophthalmol. 35:-4: 287, 1889. strabismus. It is true, however, that naturally 5. Van Gehuchten, A.: Recherches sur l'origine occurring squint in binocular animals other than et le trajet intracerebral des Nerfs moteurs par man is virtually nonexistent, so any binocular la Methode de la degenerescence Wallerienne animal showing such a squint deserves considera- indirecte, Le Nevraxe, 5: 265, 1903. tion as a model for human squint. The Siamese 6. Ramon y Cajal, S.: Histologie du systeme cat is one such animal. nerveaux, Paris, 1909-1911, vol. 2, p. 237. In 1974, Guillery1 described the unusual visual 7. Cacek, R. R.: Anatomical Demonstration of pathway defect of Siamese cats in which ganglion the Vestibulo-Ocular Projections in the Cat, cell axons arising from the central temporal hemi- Laryngoscope 81: 1595, 1971. retinas of each eye cross at the optic 8. Cowan, W. M., Gottlieb, D. I., Hendrickson, instead of taking the normal ipsilateral course. A. E., et al.: The autoradiographic demon- Since all visual centers in the Siamese cat's brain stration of axonal connections in the central nervous system, Br. Res. 37, 21: 51, 1972. receive mismatched retinotopic inputs from the 9. Kuenzle, H., and Cuenod, M.: Differential eyes, the binocular vision necessary to maintain uptake of (3H) proline and (3H) leucine by ocular alignment would presumably not be avail- neurons: its importance for the autoradio- able, and squint would be predicted. Squint does graphic tracing of pathways. Br. Res. 62: in fact occur very commonly in Siamese cats, 213, 1973. and would seem to be of the concomitant type 10. Pearson, A. A.: The oculomotor nucleus in described above. Mechanical adhesions in the the human fetus, J. Comp. Neurol. 80: 47, periorbital adnexa do occur in some Siamese cats,- 1944. but they are the exception and are not frequent enough to explain the incidence of squint in Siamese cats. Electrophysiologic evidence for normal op- The Siamese cat would then seem to be a tic nerve fiber projections in normally potentially good model for concomitant human pigmented squinters. GLEN L. MCCOR- squint, but a difficulty in the viability of this MACK. model for the human arises from the fact that

The Siamese cat, a type of albino, has a visual "Concomitant strabismus can be defined as that class of pathway anomaly in which too many strabismus where the angle of deviation between the eyes does not change in the various directions of gaze. fibers cross at the , and also fre- While "accomodative" strabismus is technically included in this definition, this report is not directed toward accom- quently has strabismus. The correlation of strabis- odative strabismus.

Downloaded from iovs.arvojournals.org on 09/28/2021