Retinal Input to the Primate Lateral Geniculate Nucleus Revealed by Injection of a Different Label Into Each Eye

The primate lateral geniculate nucleus has long been a favorite structure among anatomists because of its striking lamination. It has been shown that each lamina receives input from a different eye using various single label techniques but never by double labeling. Here, we illustrate the organization of retinal inputs to the lateral geniculate nucleus by injection of cholera toxin-B conjugated to Alexa Fluor-488 into the right eye and cholera toxin-B conjugated to Alexa Fluor-594 into the left eye.

Photo and Video Essay Section Editors: Melissa W. Ko, Dean M. Cestari, Peter Quiros, Kimberly M. Winges, MD MD MD MD Retinal Input to the Primate Lateral Geniculate Nucleus Revealed by Injection of a Different Label Into Each Eye Zachary R. Spahr, BA, John R. Economides, PhD, Jonathan C. Horton, MD, PhD FIG. 1. Lateral geniculate nuclei labeled by CTB Alexa Fluor-488 (green) injected into the right eye and CTB Alexa Fluor-594 (red) injected into the left eye. In each lateral geniculate nucleus, the ipsilateral eye innervates laminae 2, 3, and 5, whereas the contralateral eye supplies laminae 1, 4, and 6. The ventral 2 laminae are magnocellular, receiving retinal input predominately from achromatic parasol cells. The dorsal 4 laminae are parvocellular, receiving retinal projections from red–green midget cells. Relative to parvo cells, magno cells have larger receptive fields and are capable of responding better to higher temporal frequencies and lower luminance contrast. Wedged between the 6 main laminae are scattered cell populations with disparate properties, some projecting directly to extrastriate cortex and others having blue–yellow (koniocellular) color opponent properties. CTB, cholera toxin-B. Abstract: The primate lateral geniculate nucleus has long been a favorite structure among anatomists because of its striking lamination. It has been shown that each lamina receives input from a different eye using various single label techniques but never by double labeling. Here, we illustrate the organization of retinal inputs to the lateral geniculate nucleus by injection of cholera toxin-B conjugated to Alexa Fluor-488 College of Medicine (ZRS), State University of New York, Upstate Medical University, Syracuse, New York; and Department of Ophthalmology (JRE, JCH), University of California, San Francisco, San Francisco, California. Supported by Grants EY029703 (J. C. Horton) and EY02162 (Vision Core Grant) from the National Eye Institute and by an unrestricted grant from Research to Prevent Blindness. The California National Primate Research Center is supported by a Base Grant from the NIH Office of the Director, OD011107. Mikayla Dilbeck assisted with histology. The authors report no conflicts of interest. Address correspondence to Jonathan C. Horton, MD, PhD, University of California, San Francisco, 10 Koret Way, San Francisco, CA 94143; E-mail: [e-mail] e596 into the right eye and cholera toxin-B conjugated to Alexa Fluor594 into the left eye. Journal of Neuro-Ophthalmology 2022;42:e596–e597 doi: 10.1097/WNO.0000000000001472 © 2022 by North American Neuro-Ophthalmology Society M inkowski discovered that crossed and uncrossed retinal ganglion cell axons terminate in separate laminae of the primate lateral geniculate nucleus. His decisive experiment was performed at the University of Zürich just over a century ago. He enucleated one eye of a macaque monkey and then, following a brief survival period, observed transsynaptic atrophy in Nissl-stained sections. Soon after, he extended his findings to humans by examining autopsy specimens from patients with a history of blindness in one eye (1). Spahr et al: J Neuro-Ophthalmol 2022; 42: e596-e597 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Photo and Video Essay The nearly absolute segregation of ocular inputs has made the lateral geniculate nucleus an inviting target for the testing of new neuroanatomical methods. For example, its laminar organization has been shown by 3[H]-proline autoradiography, cytochrome oxidase histochemistry, wheat germ agglutinin–horseradish peroxidase axon tracing, 2deoxyglucose metabolic labeling, neurotransmitter immunohistochemistry, and functional MRI (2–7). However, no prior study has used a separate anterograde tracer to label each retina’s inputs to both lateral geniculate nuclei in the primate brain. Following a protocol approved by the UCSF IACUC, cholera toxin-B subunit (CTB) was injected into the eyes of an adult macaque under general anesthesia with ketamine, supplemented by topical proparacaine. Cholera toxin-B subunit conjugated to Alexa Fluor-488 was injected into the right eye, and CTB conjugated to Alexa Fluor-594 was injected into the left eye (8,9). The injectate consisted of 500 mg of CTB Alexa Fluor dissolved in 50 mL of sterile normal saline containing 5% dimethyl sulfoxide. The injections produced no adverse reaction and visual function in each eye appeared unimpaired. A week later, the monkey was administered a euthanasia agent and perfused with normal saline followed by 4% paraformaldehyde. Sections were cut coronally at 40 mm with a freezing microtome and viewed for epifluorescence in a Zeiss Axiophot microscope. Photographs were taken with appropriate filter sets for each fluorophore, and global adjustments were made in brightness and contrast before merging the images in Adobe Photoshop. Of note, the lateral geniculate nuclei were examined in connection with a separate project investigating other, incompletely charted, retinal projections. The CTB Alexa Fluor labels show the axons of retinal ganglion cells terminating in discrete monocular laminae (Fig. 1). One could, of course, predict this innervation pattern from prior studies using different techniques that employed only a single label. There is beauty, nonetheless, in seeing the interleaved fluorophores dovetailed together in a pattern that rivals the finest joinery of a Parisian ébéniste. What purpose is served by anatomical segregation of the inputs from the right eye and the left eye that terminate in the lateral geniculate nucleus? It is generally assumed that strict lamination of inputs accounts for the fact that geniculate neurons respond to stimulation of only one eye. However, in the parvocellular laminae of the squirrel monkey (especially laminae 3 and 4), inputs from each eye Spahr et al: J Neuro-Ophthalmol 2022; 42: e596-e597 are intermingled extensively (10). Despite this interocular promiscuity, neurons retain their monocularity (11). It follows that mass segregation of retinal inputs is not necessary to generate monocular cells in the lateral geniculate nucleus. The reason for such striking lamination of the lateral geniculate nucleus in the macaque and the human remains unknown. There is stunning diversity among mammalian species in the organization of the lateral geniculate nucleus, making it difficult to extract simple lessons from its morphology (12). REFERENCES 1. Minkowski M. 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