Whole-cell recording of light-evoked photoreceptor responses in a slice preparation of the cuttlefish retina

Abstract

A new tissue slice preparation of the cuttlefish eye is described that permits patch-clamp recordings to be acquired from intact photoreceptors during stimulation of the retina with controlled light flashes. Whole-cell recordings using this preparation, from the retinas of very young Sepia officinalis demonstrated that the magnitude, latency, and kinetics of the flash-induced photocurrent are closely dependent on the magnitude of the flash intensity. Depolarizing steps to voltages more positive than -40 mV, from a membrane holding potential of -60 mV, induced a transient inward current followed by a larger, more sustained outward current in these early-stage photoreceptors. The latter current resembled the delayed rectifier (I(K)) already identified in many other nerve cells, including photoreceptors. This current was activated at -30 mV from a holding potential of -60 mV, had a sustained time course, and was blocked in a dose-dependent manner by tetraethylammonium chloride (TEA). The smaller, transient, inward current appeared at potentials more positive than -50 mV, reached peak amplitude at -30 mV and decreased with further depolarization. This current was characterized as the sodium current (I(Na)) on the basis that it was inactivated at holding potentials above -40 mV, was blocked by tetrodotoxin (TTX) and was insensitive to cobalt. Intracellular perfusion of the photoreceptors, via the patch pipette, demonstrated that U-73122 and heparin blocked the evoked photocurrent in a dose-dependent manner, suggesting the involvement of the phospholipase C (PLC) and inositol 1,4,5-triphosphate (InsP(3)), respectively, in the phototransduction cascade. Perfusion with cyclic GMP increased significantly the evoked photocurrent, while the inclusion of phorbol-12,13-dibutyrate reduced significantly the evoked photocurrent, supporting the involvement of cGMP and the diacylglycerol (DAG) pathways, respectively, in the cuttlefish transduction process.

Lost in phototransduction: a few facts and hypotheses on cephalopod photoresponse

“…An additional difference between vertebrate and cephalopod photoreceptors is that the latter are of the "on" depolarizing sort (50), while the former are hyperpolarized by light absorption (51). Indeed, photoexcited rhodopsin activates cGMP phosphodiesterase (PDE) via photoreceptor-specific GTP-binding protein, transducin, resulting in the closure of cyclic guanosine monophosphate (cGMP)-sensitive non-specific cation channels and subsequent hyperpolarization of rods and cones (51).…”

“…Although the biochemical steps leading to the engagement of both PLC subtypes have been firmly established, and both isozymes are considered essential to raise [Ca 2+ ] i and depolarize photoreceptor membrane upon light absorption, there are no reports on light-elicited elevation in [Ca 2+ ] i in cephalopod rhabdomeres. In addition, the "whole-cell" patch clamp analysis of the evoked photocurrent has been rather scarce (50,86; see Paragraph 5). In most invertebrates, including Limulus and Drosophila melanogaster, excitation of rhabdomeric photoreceptors is accompanied by a large intracellular Ca 2+ signal.…”

“…A novel tissue slice preparation obtained from the retinas of very young cuttlefish (Sepia officinalis) has recently provided valuable insights into the cephalopod light-induced current (LIC) (50). The "whole-cell" patchclamp analysis performed on this preparation has shown that LIC displays a current-to-voltage (IV) curve reversing between 0 and +20 mV (50), which indicates a non-specific cation conductance, similar to that found in Drosophila photoreceptors (0/+40 mV) (96). The Ca 2+ /Na + permeability ratio, whose value amounts to 100:1 in the fruit fly (52), however, has not been determined.…”

“…The Ca 2+ /Na + permeability ratio, whose value amounts to 100:1 in the fruit fly (52), however, has not been determined. As suggested by the IV-relation shape, cuttlefish photocurrents are inwardly directed at a holding membrane potential of -60 mV and are, thus, depolarizing (50). The LIC is prevented either by inhibiting PLC and InsP 3 Rs or by the application of DAG surrogates (50).…”

“…However, more recent work has demonstrated that store emptying is not responsible for TRP channel gating in Drosophila rhabdomeres: evidence is mounting that either DAG or its metabolites, such poly-unsaturated fatty acids (PUFA), might be responsible their activation in this system (93,98). Nevertheless, according to the results obtained by Chrachri, the cephalopod photoreceptors LIC is inhibited by the application of DAG surrogates (50). In light of the diverse mechanisms which underlie TRP channels gating in mammals (99), and of the observations reported in cuttlefish (50), it is conceivable to envisage one of the following mechanisms to operate in cephalopods: 1) sTRP directly interacts with intracellular InsP 3 Rs and channel activation is mediated through coupling to InsP 3 Rs (100); 2) sTRP belongs to the group of the hitherto unidentified InsP 3 -binding proteins and is directly activated by such phosphoinositide; and 3) sTRP is inhibited by PIP 2 in the dark, while light-induced PLC stimulation leads to phosphoinositide depletion and relieves the channel from PIP 2 -dependent block (101; see also 102).…”