Gigaohm single-channel recording from isolated Hermissenda crassicornis type B photoreceptors.

Invertebrate photoreceptor electrophysiology has mainly been studied with intracellular microelectrodes. Single-channel recording has proved to be difficult, possibly because of unusual plasma membrane configurations; in Limulus polyphemus successful gigaohm seals were obtained only after pronase treatment and brief sonication (Bacigalupo and Lisman, 1983). Here we describe an isolated Hermissenda eye preparation that permits single-channel recording (Hamill et al. 1981) from identified type B photoreceptors; this photoreceptor sub-type has been assiduously studied with intracellular microelectrodes, under both current-clamp and voltage-clamp conditions. Associative conditioning of Hermissenda crassicornis (in the paired light and rotation paradigm) is attributable in part to the reduction of potassium conductances in the type B photoreceptors (Alkon, 1984, 1987; Alkon et al. 1985). We have used the isolated eye preparation to characterize two types of potassium channels in photoreceptor plasma membranes. When microelectrodes are employed on Hermissenda photoreceptors, the preparations are treated with protease (Sigma type XXIV) to soften the connective tissue capsule that covers the photoreceptors, thus easing the insertion of microelectrodes (Alkon et al. 1984). Experiments with protease, collagenase and dispase showed that an appropriate enzyme cocktail for gigaohm seal formation was a mixture of protease and dispase (dispase, grade II was obtained from Boehringer Mannheim). The enzymes were dissolved in artificial sea water (NaCl 430; KC1 10; MgCl2 50; CaCl2 10; Hepes 10; pH7.4; concentrations in mmolP) to final concentrations of 2mgml~ protease and 20mgml~ dispase. The circumoesophageal nervous system (CONS) was removed from 2-4 cm long adult Hermissenda, obtained from Sea Life Supply, Sand City, CA. The animals were maintained at 14°C in sea water of defined composition (Tropic Marin, Marinus Inc., Long Beach, CA) for 3-14 days before the CONS were removed. Several CONS were incubated in the enzyme cocktail for 40-45 min at 23-25 °C, then rinsed with cold (10°C) artificial sea water (ASW). The eyes were microdissected from the CONS and transferred into fresh ASW in a plastic 35 mm sterile