Electrophysiological recordings in solitary photoreceptors from the retina of squid, Loligo pealei.

Abstract

A protocol was developed to isolate enzymatically photoreceptors from the retina of the squid, Loligo pealei. The procedure routinely results in a high yield of intact cells. Examination of solitary photoreceptors under Nomarski optics revealed that the fine morphological features described in anatomical studies of retinal sections are retained. The distal segment is up to 250 microns long, 4-7 microns wide, covered in part by short microvilli; the inner segment and the cell body, with the initial portion of the axon, are also clearly discernible in solitary cells. Suction electrode measurements performed from the cell body confirmed that responsiveness to light survived cell isolation. Macroscopic membrane currents were measured using the whole-cell tight-seal technique, and the perforated-patch method. Step depolarizations of membrane voltage administered in the dark elicited a slowly activating, sustained outward current. Light stimulation evoked an inward current graded with stimulus intensity; the peak current could amply exceed 1000 pA. Intense photostimulation gave rise to a prolonged inward aftercurrent that lasted for tens of seconds. On-cell patch recording along the intermediate segment and most of the smooth areas of the distal segment showed a large incidence of silent patches, with the occasional presence of voltage-dependent channels. On the other hand, channel activity could be recorded more frequently from electrode placements near the apical tip of the cell, where the presence of microvilli could be confirmed visually. Some patches were unresponsive to voltage stimulation applied in the dark but produced distinct bursts of channel openings after illumination. The feasibility of single-cell electrophysiology in isolated photoreceptors, together with the growing body of biochemical information on cephalopod preparations, makes squid an attractive model system to investigate the visual process in invertebrates using multiple experimental approaches.