PHOTORECEPTOR CELLS DISSOCIATED FROM THE COMPOUND LATERAL EYE OF THE HORSESHOE-CRAB, LIMULUS-POLYPHEMUS .1. STRUCTURE AND ULTRASTRUCTURE

Isolated photoreceptors are desirable for whole-cell and patch-clamp s tudies of functional properties of visual processes that cannot be cle arly analyzed when the photoreceptors are coupled. The retina of the c ompound lateral eye of the horseshoe crab, Limulus polyphemus, was dis sociated into individual retinular cells using an enzyme pretreatment consisting of collagenase, papain, and trypsin, and a two-stage mechan ical dissociation. These photoreceptors are functionally viable in an organ culture medium for up to 1 week and possess naked arhabdomeral a nd rhabdomeral segment membranes which are easily accessible for whole -cell recordings. A dissection technique was also developed whereby th e retinal epidermis and neural plexus, as well as the second-order ecc entric cells, could be separated from the ommatidia of the compound la teral eye in one simple step, providing viable isolated ommatidia atta ched to the cornea. The enzyme pretreatment used for dissociating the retina was then used to remove the individual ommatidia from the corne al 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. Morphologica l analysis using light microscopy and scanning and transmission electr on microscopy revealed that isolated retinular cells are morphological ly nearly identical to retinular cells in situ. Isolated retinular cel ls possess a normal rhabdomere with no apparent loss of microvillar me mbrane as a result of the isolation process. Ommatidia can presently b e isolated with up to six retinular cells possessing essentially norma l structure and ultrastructure including thick rays of rhabdom. Isolat ed ommatidia possess naked A-segment membranes which are also well sui ted for whole-cell recording techniques.