Visual Neuroscience (1993), 10, 597-607. Printed in the USA. Copyright © 1993 Cambridge University Press 0952-5238/93 $5.00 + .00 Photoreceptor cells dissociated from the compound lateral eye of the horseshoe crab, Limulus polyphemus, I: Structure and ultrastructure ROBERT N. JINKS, 1 - 2 W. J. BRAD HANNA, 3 GEORGE H. RENNINGER, 3 AND STEVEN C. CHAMBERLAIN 12 1 Institute for Sensory Research, Syracuse University, Syracuse 2 Department of Bioengineering and Neuroscience, Syracuse University, Syracuse 'Biophysics Interdepartmental Group, Department of Physics, University of Guelph, Guelph (RECEIVED August 3, 1992; ACCEPTED November 1, 1992) Abstract Isolated photoreceptors are desirable for whole-cell and patch-clamp studies of functional properties of visual processes that cannot be clearly analyzed when the photoreceptors are coupled. The retina of the compound lateral eye of the horseshoe crab, Limulus polyphemus, was dissociated into individual retinular cells using an enzyme pretreatment consisting of collagenase, papain, and trypsin, and a two-stage mechanical dissociation. These photoreceptors are functionally viable in an organ culture medium for up to 1 week and possess naked arhabdomeral and rhabdomeral segment membranes which are easily accessible for whole-cell recordings. A dissection technique was also developed whereby the retinal epidermis and neural plexus, as well as the second-order eccentric cells, could be separated from the ommatidia of the compound lateral eye in one simple step, providing viable isolated ommatidia attached to the cornea. The enzyme pretreatment used for dissociating the retina was then used to remove the individual ommatidia from the corneal cones. Hoffman modulation contrast microscopy was used to develop a reliable method for sorting and collecting viable isolated retinular cells for morphological and electrophysiological studies. Morphological analysis using light microscopy and scanning and transmission electron microscopy revealed that isolated retinular cells are morphologically nearly identical to retinular cells in situ. Isolated retinular cells possess a normal rhabdomere with no apparent loss of microvillar membrane as a result of the isolation process. Ommatidia can presently be isolated with up to six retinular cells possessing essentially normal structure and ultrastructure including thick rays of rhabdom. Isolated ommatidia possess naked A-segment membranes which are also well suited for whole-cell recording techniques. Keywords: Dissociation, Collagenase, Trypsin, Papain, Isolated photoreceptors/ommatidia, Rhabdom, Hoffman microscopy Introduction The horseshoe crab, Limulus polyphemus, first used in the study of vision in the late Nineteenth Century continues to be an im- portant model system for the study of basic visual processes. The compound lateral eye of the horseshoe crab is composed of a cornea with cuticular cones radiating inward; each under- lain by an ommatidium and specialized retinal epidermis (Fahr- enbach, 1968). The ommatidium is the photoreceptive unit of the eye and is made up of photoreceptor cells (retinular cells) arranged like the wedges of an orange (Grenacher, 1879; Fahren- Reprint requests to: Robert N. Jinks, Institute for Sensory Research, Merrill Lane, Syracuse University, Syracuse, NY 13244-5290, USA. bach, 1969) around a central dendrite from the second-order eccentric cell (Fahrenbach, 1969). In the past, investigators have relied upon the large size of the individual facets (cuticular cones) of the compound lateral eye to isolate a light stimulus to a single ommatidium and record its output as the activity of a single fiber in the lateral optic nerve. Similarly, a large data base of the in situ functional prop- erties of the retinular cells was developed through intracellular recordings (e.g. Barlow & Kaplan, 1971,1977; Kaplan & Barlow, 1975). The ventral photoreceptor cell provides an alternative means for collecting basic data from individual photoreceptor cells (Millecchia et al., 1966; Clark et al., 1969). Intracellular recordings from these relatively large photoreceptors could be obtained much easier (Millecchia & Mauro, 1969a, b) than from the retinular cells of the lateral eye; however, they could not 597