Photoreceptor cells dissociated from the compound lateral eye of the horseshoe crab, Limulus polyphemus, II: Function

Abstract

A combination of enzymatic digestions and mechanical disruption was used to isolate photoreceptor cells from the compound lateral eye of the horseshoe crab, Limulus polyphemus. The cells were maintained in a culture medium and tested for function using whole-cell and cell-attached patch configurations of the gigaseal technique. The cells dissociated from the eye generated spontaneous voltage and current bumps in the dark, and depolarized in a graded fashion to increasing intensities of light over several decades, producing responses similar to those of cells in vivo. Currents evoked during voltage clamp were similar to those in ventral photoreceptor cells of Limulus, although transient currents in the dark-and light-activated currents were smaller in isolated lateral eye cells, perhaps because of the slow speed and spatial nonuniformity of the clamp in these large cells. In addition to isolated cells, dissociation of the compound eye produced small clusters of cells and isolated ommatidia which were also tested for function. Comparison of the electrical characteristics of isolated cells with those of cells in small clusters and in their ommatidial matrix suggests that the electrical junctions normally connecting photoreceptor cells within an ommatidium are functional in the latter groups, but not in isolated cells. Cell-attached patches of rhabdomeral membrane of isolated cells contained light-activated channels, resembling those observed in ventral photoreceptor cells, but no voltage-activated channels. Similar patches of arhabdomeral membrane contained voltageactivated channels, but no light-activated channels. We conclude that this preparation is suitable for studies of processes involved in generating the light response in invertebrate photoreceptor cells.

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

“…As reported in this paper, isolated retinular cells harvested from this dissociation technique have essentially naked R-and A-segments and possess very nearly normal ultrastructural properties including a healthy rhabdomere, making these cells perfect candidates for whole-cell recording techniques. Electrophysiological studies of these isolated photoreceptors presented in the companion paper (Hanna et al, 1993) reveal that these cells can remain functionally viable in an organ culture medium for up to 1 week and that excellent quality, high-resistance seals can be effected on these cells.…”

An enzymatically enhanced recording technique forLimulusventral photoreceptors: Physiology, biochemistry, and morphology

“…In this study, we set about to develop a different procedure for exposing the surface of the cell by disrupting the glial cell covering. Our starting point was the enzymatic dissociation technique developed by to produce isolated photoreceptors from the compound lateral eye which utilizes collagenase, papain, and trypsin rather than pronase (Jinks et al, 1993;Hanna et al, 1993). Our objective was to develop a procedure with a high yield of stable whole-cell recordings of transmembrane potentials that exhibited the appropriate physiological responses over a period of several hours.…”

The morphology and physiology of a “mini-ommatidium” in the median optic nerve of Limulus polyphemus

“…Although the recently developed dissociation techniques for the lateral eye Hanna et al, 1993) will enable detailed study of single retinular cells and single ommatidia, the likelihood of finding just one retinular cell still attached to an eccentric cell seems rather remote. On the other hand, if the glial disruption techniques that have been developed for the lateral eye Hanna et al, 1993) and the ventral eye (Zhang et al, 1994) can be adapted to the mini-ommatidium, two-electrode studies, including whole-cell recording with patch-clamp electrodes ought to be reasonably possible. The possibility of investigating the relationships between the receptor potential, the generator potential, and the resulting spike train under various conditions of light and darkness is appealing.…”

“…This preparation ought to prove useful for further investigation of the photoreceptor second-order cell connection in Limulus and whether or not the connection can be modified by light and efferent activity. Although the recently developed dissociation techniques for the lateral eye (Jinks et al, 1993;Hanna et al, 1993) will enable detailed study of single retinular cells and single ommatidia, the likelihood of finding just one retinular cell still attached to an eccentric cell seems rather remote. On the other hand, if the glial disruption techniques that have been developed for the lateral eye (Jinks et al, 1993;Hanna et al, 1993) and the ventral eye (Zhang et al, 1994) can be adapted to the mini-ommatidium, two-electrode studies, including whole-cell recording with patch-clamp electrodes ought to be reasonably possible.…”