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Photoreceptor cells dissociated from the compound lateral eye of the horseshoe crab, Limulus polyphemus, I: Structure and ultrastructure

Published online by Cambridge University Press:  02 June 2009

Robert N. Jinks ,
W. J. Brad Hanna ,
George H. Renninger  and
Steven C. Chamberlain
Robert N. Jinks
Affiliation:
Institute for Sensory Research, Syracuse University, Syracuse Department of Bioengineering and Neuroscience, Syracuse University, Syracuse
W. J. Brad Hanna
Affiliation:
Biophysics Interdepartmental Group, Department of Physics, University of Guelph, Guelph
George H. Renninger
Affiliation:
Biophysics Interdepartmental Group, Department of Physics, University of Guelph, Guelph
Steven C. Chamberlain
Affiliation:
Institute for Sensory Research, Syracuse University, Syracuse Department of Bioengineering and Neuroscience, Syracuse University, Syracuse
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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

DissociationCollagenaseTrypsinPapainIsolated photoreceptors/ommatidiaRhabdomHoffman microscopy
Type
Research Articles
Information
Visual Neuroscience , Volume 10 , Issue 4 , July 1993 , pp. 597 - 607
Copyright
Copyright © Cambridge University Press 1993

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