Gordon L. Fain

Calcium and the mechanism of light adaptation in vertebrate photoreceptors.

Vertebrate photoreceptors transduce the absorption of light into a hyperpolarizing change in membrane potential. The mechanism of transduction is becoming fairly well understood and has been shown to occur via a G protein-coupled decrease in cyclic GMP. Attention is now turning to the way the enzymatic machinery in the outer segment of the photoreceptor cell is modulated during light adaptation. Recent studies show that light adaptation cannot occur if changes in the concentration of cytoplasmic free calcium in the outer segment are prevented, suggesting that calcium functions as a second messenger in sensitivity regulation.

GABA and glycine channels in isolated ganglion cells from the goldfish retina.

1. Adult goldfish retinas were enzymatically dissociated and ganglion cells were maintained in culture for periods of 1‐5 days. Ganglion cells could be identified by their morphology, and this identification was confirmed by retrograde transport of the fluorescent dye Fast Blue injected into the optic nerve stub. 2. All the ganglion cells tested responded to 30 microM‐GABA or 100 microM‐glycine between 2 and 30 h after enzymatic dissociation of the retina. 3. Whole‐cell responses to 30 microM‐GABA or glycine declined over a period of seconds during sustained applications of the agonists, probably as a result of desensitization. There was an irreversible decline in the peak whole‐cell response to repeated applications of 30 microM‐GABA unless the pipette‐filling solution contained 2 mM‐ATP, 4 mM‐Mg2+, 10 mM‐EGTA and no added Ca2+. Both GABA and glycine responses also showed an irreversible decline in outside‐out patches but, in this case, Mg2+, ATP, and very low Ca2+ failed to stabilize the response. 4. Whole‐cell currents activated by both GABA and glycine were demonstrated to be chloride‐selective by investigating the dependence of reversal potential (Vr) on internal chloride concentration ([Cl‐]i). For GABA responses, the dependence of Vr on [Cl‐]i could not be distinguished from that predicted by the Nernst relation. For glycine, deviations from Nernstian dependence were observed, but the permeability to Cl‐ was at least 20 times greater than to isethionate, SO4(2‐), or monovalent cations (Na+ and Cs+). 5. Bicuculline methochloride (10 microM) selectively blocked responses to 3‐30 microM‐GABA without affecting responses to 30 microM‐glycine. Bicuculline itself was not as selective. At agonist concentrations of 30 microM, 3 microM‐bicuculline partially blocked the response to GABA but not that to glycine, but bicuculline at 10 microM blocked responses to both GABA and glycine. Strychnine (0.3‐1 microM) blocked responses to 30 microM‐glycine but also competitively antagonized GABA responses. Picrotoxinin (10 microM) blocked responses to 3 microM‐GABA in some cells but also partially antagonized responses to 30 microM‐glycine. 6. GABA channels had at least two conductance states at 10‐12 degrees C in nearly symmetrical (141 mM in, 142 mM out) chloride. The slope conductance of the most frequently observed (main) state was 16 +/‐ 2 pS. The reversal potential for the main state was not significantly different from the chloride equilibrium potential (0 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of sodium replacement on the responses of toad rods

1. We have investigated the effects of Na+ substitution on the membrane potential and light responses of rods in the superfused retina of the toad, Bufo marinus.

The effects of low calcium and background light on the sensitivity of toad rods

1. We have examined the effects of decreases in extracellular Ca2+ concentration on the intracellularly recorded light responses of rods from the toad, Bufo marinus. In agreement with previous results (Brown & Pinto, 1974; Lipton, Ostroy & Dowling, 1977), Ca2+ concentrations below 10−6 M produced a depolarization of rod resting membrane potential of approximately 30‐40 mV and a corresponding increase in the maximum amplitude of the rods light responses, so that saturating flashes in normal and low Ca2+ Ringer produced hyperpolarizations to approximately the same membrane potential.

Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture.

1. The whole‐cell recording mode of the patch‐clamp technique was used to investigate the presence of voltage‐activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization‐activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization‐activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)‐independent. 5. The hyperpolarization‐activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below ‐120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage‐dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)‐dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials.

Mechanisms of synaptic transmission in the retina

This paper reviews the mechanisms of transmitter release, the kinetics of synaptic transfer, the mechanisms for the production of conductance changes by transmitters, and the nature of the conductance changes at synapses in vertebrate retina. A method for the culturing of adult retinal cells is described, together with preliminary experiments on the identification of cells in culture.

Whole-cell currents activated at nicotinic acetylcholine receptors on ganglion cells isolated from goldfish retina

We have recorded whole-cell membrane currents in response to exogenously applied acetylcholine (ACh), nicotine, and 1,1 dimethyl-4-phenyl piperazinium iodide on retinal ganglion cells enzymatically dissociated from goldfish retina. Agonist applications induced nicotinic-type responses in a majority of cells when cells were isolated under optimal conditions. Currents were reminiscent of nicotinic-type ganglionic responses. Dose-response measurements of ACh-induced currents indicated an EC50 of 52 microM and a Hill coefficient of 0.6. Currents were selective for Na+ over Cl- and were highly inwardly rectifying. Responses were blocked reversibly by d-tubocurarine, hexamethonium chloride, and N-methyl-D-glucamine. In 50% of the cases, alpha-bungarotoxin reversibly blocked the current induced by ACh application. The blocking action of mecamylamine was irreversible and independent of the presence of agonist but was more effective in the presence of ACh. We conclude that functional nicotinic ACh receptors exist on most goldfish retinal ganglion cells.

Chapter 19 Calcium and the Mechanism of Light Adaptation in Rods

Publisher Summary This chapter discusses the evidence that sensitivity is regulated by a diffusible messenger. Although there is now nearly universal agreement that an internal messenger is necessary for excitation, it is perhaps less widely known that a diffusible substance also regulates the sensitivity of the receptor during light adaptation. The chapter presents experiments that show that sensitivity is not normally controlled by increases in Ca2+. The chapter describes the effects of exposing rods to low-Ca2+ solutions and discusses some of the implications of the experiments that exclude for toad rods some of the models for light adaptation in receptors that have been previously proposed.